Dominique Lison

Abstract: IL-17-producing T lymphocytes play a crucial role in inflammation, but their possible implication in fibrosis remains to be explored. In this study, we examined the involvement of these cells in a mouse model of lung inflammation and fibrosis induced by silica particles. Upregulation of IL-17A was associated with the development of experimental silicosis, but this response was markedly reduced in athymic, gammadelta T cell-deficient or CD4(+) T cell-depleted mice. In addition, gammadelta T lymphocytes and CD4(+) T cells, but not macrophages, neutrophils, NK cells or CD8 T cells, purified from the lungs of silicotic mice markedly expressed IL-17A. Depletion of alveolar macrophages or neutralization of IL-23 reduced upregulation of IL-17A in the lung of silicotic mice. IL-17R-deficient animals (IL-17R(-/-)) or IL-17A Ab neutralization, but not IL-22(-/-) mice, developed reduced neutrophil influx and injury during the early lung response to silica. However, chronic inflammation, fibrosis, and TGF-beta expression induced by silica were not attenuated in the absence of IL-17R or -22 or after IL-17A Ab blockade. In conclusion, a rapid lung recruitment of IL-17A-producing T cells, mediated by macrophage-derived IL-23, is associated with experimental silicosis in mice. Although the acute alveolitis induced by silica is IL-17A dependent, this cytokine appears dispensable for the development of the late inflammatory and fibrotic lung responses to silica.

Abstract: OBJECTIVE: To conduct a systematic review and meta-analysis of published studies on the association between parental occupational exposure to pesticides and childhood leukaemia. METHODS: Studies were identified from a MEDLINE search through 31 July 2009 and from the reference lists of identified publications. Relative risk (RR) estimates were extracted from 25 studies published between 1985 and 2008. Meta-rate ratio estimates (mRR) were calculated according to fixed and random-effect meta-analysis models. Separate analyses were conducted after stratification for study design, definition of exposure (employment in a farm/agriculture assuming exposure to pesticides versus exposure to pesticides stipulated), exposed parent, window of exposure, type of leukaemia and biocide category. RESULTS: No statistically significant association between childhood leukaemia and parental occupation as farmers/agricultural workers was observed. When exposure to pesticides was stipulated, positive associations were reported for maternal exposure for all studies combined (mRR: 1.62; 95% CI: 1.22-2.16), in all exposure windows considered and for acute non-lymphocytic leukaemia (ANLL). There was no association with paternal exposure when combining all studies (mRR: 1.14; 95% CI: 0.76-1.69). However, significant increased risks were seen for paternal exposure, in some exposure windows as well as for the biocide category. CONCLUSIONS: The strongest evidence of an increased risk of childhood leukaemia comes from studies with maternal occupational exposure to pesticides. The associations with paternal exposure were weaker and less consistent. These results add to the evidence leading to recommend minimizing parental occupational exposure to pesticides. Our findings also support the need to rely more on studies that clearly stipulate exposure to pesticides rather than those that assume pesticide exposure because of farm/agriculture employment.

Abstract: PURPOSE: To evaluate the effectiveness of personal protective measures in a dismantling plant for chemical weapons from World War I of the Belgian Defence. METHODS: Seventeen NIOSH level B-equipped plant workers exposed to arsenic trichloride (AsCl(3)) in combination with phosgene or hydrogen cyanide (HCN) were compared to 24 NIOSH level C-protected field workers occasionally exposed to genotoxic chemicals (including AsCl(3)-phosgene/HCN) when collecting chemical ammunition, and 19 matched referents. Chromosomal aberrations (CA), micronuclei (MNCB and MNMC), sister chromatid exchanges (SCE) and high frequency cells (HFC) were analysed in peripheral blood lymphocytes. Urinary arsenic levels and genetic polymorphisms in major DNA repair enzymes (hOGG1(326), XRCC1(399), XRCC3(241)) were also assessed. RESULTS: SCE and HFC levels were significantly higher in plant-exposed versus referent subjects, but MNCB and MNMC were not different. MNCB, SCE and HFC levels were significantly higher and MNMC levels significantly lower in field-exposed workers versus referents. AsCl(3) exposure was not correlated with genotoxicity biomarkers. CONCLUSIONS: Protective measures for plant-exposed workers appear adequate, but protection for field-exposed individuals could be improved.

Abstract: We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.

Abstract: For the investigation of the interaction of nanoparticles with biomolecules, cells, organs, and animal models there is a need for well-characterized nanoparticle suspensions. In this paper we report the preparation of monodisperse dense amorphous silica nanoparticles (SNP) suspended in physiological media that are sterile and sufficiently stable against aggregation. SNP sols with various particle sizes (2-335 nm) were prepared via base-catalyzed hydrolysis and polymerization of tetraethyl orthosilicate under sterile conditions using either ammonia (Stober process (1) ) or lysine catalyst (Lys-Sil process (2) ). The series was complemented with commercial silica sols (Ludox). Silica nanoparticle suspensions were purified by dialysis and dispersed without using any dispersing agent into cell culture media (Dulbecco's Modified Eagle's medium) containing antibiotics. Particle sizes were determined by dynamic light scattering. SNP morphology, surface area, and porosity were characterized using electron microscopy and nitrogen adsorption. The SNP sols in cell culture medium were stable for several days. The catalytic activity of the SNP in the conversion of hydrogen peroxide into hydroxyl radicals was investigated using electron paramagnetic resonance. The catalytic activity per square meter of exposed silica surface area was found to be independent of particle size and preparation method. Using this unique series of nanoparticle suspensions, the relationship between cytotoxicity and particle size was investigated using human endothelial and mouse monocyte-macrophage cells. The cytotoxicity of the SNP was strongly dependent on particle size and cell type. This unique methodology and the collection of well-characterized SNP will be useful for further in vitro studies exploring the physicochemical determinants of nanoparticle toxicity.

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Abstract: Lung disorders induced by inhaled inorganic particles such as crystalline silica are characterized by chronic inflammation and pulmonary fibrosis. Here, we demonstrate the importance of type I interferon (IFN) in the development of crystalline silica-induced lung inflammation in mice, revealing that viruses and inorganic particles share similar signaling pathways. We found that instillation of silica is followed by the upregulation of IFN-beta and IRF-7 and that granulocytes (GR1(+)) and macrophages/dendritic cells (CD11c(+)) are major producers of type I IFN in response to silica. Two months after silica administration, both IFNAR- and IRF-7-deficient mice produced significantly less pulmonary inflammation and chemokines (KC and CCL2) than competent mice but developed similar lung fibrosis. Our data indicate that type I IFN contributes to the chronic lung inflammation that accompanies silica exposure in mice. Type I IFN is, however, dispensable in the development of silica-induced acute lung inflammation and pulmonary fibrosis.

Abstract: OBJECTIVE: To conduct a systematic review of published studies on the association between residential/household/domestic exposure to pesticides and childhood leukaemia, and to provide a quantitative estimate of the risk. METHODS: Publications in English were searched in MEDLINE (1966-31 December 2009) and from the reference list of identified publications. Extraction of relative risk (RR) estimates was performed independently by 2 authors using predefined inclusion criteria. Meta-rate ratio estimates (mRR) were calculated according to fixed and random-effect models. Separate analyses were conducted after stratification for exposure time windows, residential exposure location, biocide category and type of leukaemia. RESULTS: RR estimates were extracted from 13 case-control studies published between 1987 and 2009. Statistically significant associations with childhood leukaemia were observed when combining all studies (mRR: 1.74, 95% CI: 1.37-2.21). Exposure during and after pregnancy was positively associated with childhood leukaemia, with the strongest risk for exposure during pregnancy (mRR: 2.19, 95% CI: 1.92-2.50). Other stratifications showed the greatest risk estimates for indoor exposure (mRR: 1.74, 95% CI: 1.45-2.09), for exposure to insecticides (mRR: 1.73, 95% CI: 1.33-2.26) as well as for acute non-lymphocytic leukaemia (ANLL) (mRR: 2.30, 95% CI: 1.53-3.45). Outdoor exposure and exposure of children to herbicides (after pregnancy) were not significantly associated with childhood leukaemia (mRR: 1.21, 95% CI: 0.97-1.52; mRR: 1.16, 95% CI: 0.76-1.76, respectively). CONCLUSIONS: Our findings support the assumption that residential pesticide exposure may be a contributing risk factor for childhood leukaemia but available data were too scarce for causality ascertainment. It may be opportune to consider preventive actions, including educational measures, to decrease the use of pesticides for residential purposes and particularly the use of indoor insecticides during pregnancy.

Abstract: The metabolome is the set of small molecular mass organic compounds found in a given biological media. It includes all organic substances naturally occurring from the metabolism of the studied living organism, except biological polymers, but also xenobiotics and their biotransformation products. The metabolic fingerprints of biofluids obtained by mass spectrometry (MS) or nuclear magnetic resonance (NMR)-based methods contain a few hundreds to thousands of signals related to both genetic and environmental contributions. Metabolomics, which refers to the untargeted quantitative or semi-quantitative analysis of the metabolome, is a promising tool for biomarker discovery. Although proof-of-concept studies by metabolomics-based approaches in the field of toxicology and clinical chemistry have initially been performed using NMR, the use of liquid chromatography hyphenated to mass spectrometry (LC/MS) has increased over the recent years, providing complementary results to those obtained with other approaches. This paper reviews and comments the input of LC/MS in this field. We describe here the overall process of analysis, review some seminal papers in the field and discuss the perspectives of metabolomics for the biomonitoring of exposure and diagnosis of diseases.

Abstract: BACKGROUND: Efavirenz (EFV) is characterized by interindividual pharmacokinetic variability causing inconsistent clinical responses. Previous studies have identified some possible genetic determinants of the variability in plasma concentrations. However, their impact on EFV intracellular pharmacokinetics remains mostly unexplored. AIMS: To confirm previous observations concerning the influence of genetic polymorphisms on EFV plasma concentrations and to assess their effect on the intracellular pharmacokinetics of EFV. Materials & methods: EFV concentrations in plasma ([EFV](Cmin)) and in peripheral blood mononuclear cells ([EFV](CC)) were determined in 50 HIV-infected patients. Subjects were genotyped for 13 polymorphisms in 5 different genes (CYP2A6, CYP2B6, CYP3A5, UGT2B7 and ABCB1). Relationships between genetic status and [EFV](Cmin), [EFV](CC) or EFV accumulation in peripheral blood mononuclear cells (EFV accumulation ratio or accumulation ration [AR]) were then evaluated. RESULTS: CYP2B6 allelic status was associated with differences in [EFV](Cmin) but also in [EFV](CC). Patients carrying at least one mutated allele showed significantly higher [EFV](Cmin) and [EFV](CC) than homozygous wild-type (mutated homozygous [m/m] >heterozygous [wt/m]>homozygous wild-type [wt/wt], p<0.001). ABCB1 rs3842T>C was significantly associated with higher EFV AR (p = 0.032). Finally, the ABCB1 3435C>T SNP was associated with a lower increase in CD4-cell count after EFV therapy initiation. CONCLUSION: Our study corroborates previous findings indicating that knowledge of CYP2B6 genetic status should be taken into account for an EFV treatment. Our results also constitute the first demonstration of the significant influence of CYP2B6 genetic polymorphisms on [EFV](CC) and suggest that ABCB1 SNPs may also influence the clinical impact of EFV treatment.

Abstract: Identifying the physico-chemical characteristics of nanoparticles (NPs) that drive their toxic activity is the key to conducting hazard assessment and guiding the design of safer nanomaterials. Here we used a set of 17 stable suspensions of monodisperse amorphous silica nanoparticles (SNPs) with selected variations in size (diameter, 2-335 nm), surface area (BET, 16-422 m(2)/g) and microporosity (micropore volume, 0-71 microl/g) to assess with multiple regression analysis the physico-chemical determinants of the cytotoxic activity in four different cell types (J774 macrophages, EAHY926 endothelial cells, 3T3 fibroblasts and human erythrocytes). We found that the response to these SNPs is governed by different physico-chemical parameters which vary with cell type: In J774 macrophages, the cytotoxic activity (WST1 assay) increased with external surface area (alphas method) and decreased with micropore volume (r(2) of the model, 0.797); in EAHY926 and 3T3 cells, the cytotoxic activity of the SNPs (MTT and WST1 assay, respectively) increased with surface roughness and small diameter (r(2), 0.740 and 0.872, respectively); in erythrocytes, the hemolytic activity increased with the diameter of the SNP (r(2), 0.860). We conclude that it is possible to predict with good accuracy the in vitro cytotoxic potential of SNPs on the basis of their physico-chemical characteristics. These determinants are, however, complex and vary with cell type, reflecting the pleiotropic interactions of nanoparticles with biological systems.

Abstract: Prostaglandin (PG) D(2) exerts contrasting activities in the inflamed lung via two receptors, the D prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper 2 lymphocytes. DP activation is known mainly to inhibit proinflammatory cell functions. We tested the effect of a DP-specific agonist, (4S)-(3-[(3R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptanoic acid (BW245C), on pulmonary fibroblast functions in vitro and in a mouse model of lung fibrosis induced by bleomycin. DP mRNA expression was detected in cultured mouse lung primary fibroblasts and human fetal lung fibroblasts and found to be up- and down-regulated by interleukin-13 and transforming growth factor (TGF)-β, respectively. Although micromolar concentrations of BW245C and PGD(2) did not affect mouse fibroblast collagen synthesis or differentiation in myofibroblasts, they both inhibited fibroblast basal and TGF-β-induced proliferation in vitro. The repeated administration of BW245C (500 nmol/kg body weight instilled transorally in the lungs 2 days before and three times per week for 3 weeks) in bleomycin-treated mice significantly decreased both inflammatory cell recruitment and collagen accumulation in the lung (21 days). Our results indicate that BW245C can reduce lung fibrosis in part via its activity on fibroblast proliferation and suggest that DP activation should be considered as a new therapeutic target in fibroproliferative lung diseases.

Abstract: Macrophages phagocyte pathogenic microorganisms and orchestrate immune responses by producing a variety of inflammatory mediators. The cystic fibrosis (CF) transmembrane conductance regulator chloride channel has been reported to be of pivotal importance for macrophage functions. The exact phenotype and role of macrophages in CF is still unknown. Alveolar and peritoneal macrophages were monitored in CF mice homozygous for the F508 del mutation and in wild-type control animals. Classical (M1) and alternative (M2) macrophage polarization and responses to LPS from Pseudomonas aeruginosa were investigated, and the effect of azithromycin was examined in both cell populations. We show that alveolar macrophage counts were 1.7-fold higher in CF as compared with wild-type mice. The macrophage-related chemokine, chemokine C-C motif ligand (CCL)-2, was found to be at least 10-fold more abundant in the alveolar space of mutant mice. Cell count and CCL-2 protein levels were also increased in the peritoneal cavity of CF mice. Both M1 and M2 macrophage polarization were significantly enhanced in alveolar and peritoneal cells from F508del-CF mice as compared with control animals. LPS-stimulated expression of proinflammatory mediators, such as nitric oxide synthase-2, IL-1beta, and CCL-2, was increased, whereas anti-inflammatory IL-10 expression was decreased in CF macrophages. Azithromycin, added to cell cultures at 1 mg/liter, significantly reduced proinflammatory cytokine expression (IL-1beta, CCL-2, TNF-alpha) in M1-induced CF and wild-type alveolar macrophages. Our findings indicate that CF macrophages are ubiquitously accumulated, and that these cells are polarized toward classical and alternative activation status. Azithromycin down-regulates inflammatory cytokine production by M1-polarized CF alveolar macrophages.

Abstract: BACKGROUND: False-negative responses to specific inhalation challenge (SIC) with occupational agents may occur. We explored whether assessing changes in sputum cell counts would help improve the identification of bronchial reactivity to occupational agents during SICs. METHODS: The predictive value of the changes in sputum cell counts after a negative FEV(1) response to a first challenge exposure to an occupational agent was determined using the changes in airway calibre observed during repeated challenges as the 'gold standard'. The study included 68 subjects investigated for work-related asthma in a tertiary centre. After a control day, the subjects were challenged with the suspected occupational agent(s) for up to 2 h. All subjects who did not show an asthmatic reaction were re-challenged on the following day. Additional challenges were proposed to those who demonstrated a > or = 2% increase in sputum eosinophils or an increase in nonspecific bronchial hyperresponsiveness to histamine after the second challenge day. RESULTS: Six of the 35 subjects without changes in FEV(1) on the first challenge developed an asthmatic reaction on subsequent challenges. ROC analysis revealed that a >3% increase in sputum eosinophils at the end of the first challenge day was the most accurate parameter for predicting the development of an asthmatic response on subsequent challenges with a sensitivity of 67% and a specificity of 97%. CONCLUSIONS: An increase in sputum eosinophils is an early marker of specific bronchial reactivity to occupational agents, which may help to identify subjects who will develop an asthmatic reaction only after repeated exposure.

Abstract: Indium-Tin-Oxide (ITO) is a sintered mixture of indium- (In(2)O(3)) and tin-oxide (SnO(2)) in a ratio of 90:10 (wt:wt) that is used for the manufacture of LCD screens and related high technology applications. Interstitial pulmonary diseases have recently been reported in workers from ITO producing plants. The present study was conducted to identify experimentally the exact chemical component responsible for this toxicity and to address possible mechanisms of action. The reactivity of respirable ITO particles was compared with that of its single components alone or their unsintered 90:10 mixture (MIX) both in vivo and in vitro. For all endpoints considered, ITO particles behaved as a specific toxic entity. In vivo, after a single pharyngeal administration (2-20 mg per rat), ITO particles induced a strong inflammatory reaction. At day 3, the inflammatory reaction (cell accumulation, LDH and protein in bronchoalveolar lavage fluid) appeared more marked with ITO particles than with each oxide separately or the MIX. This inflammatory reaction persisted and even worsened after 15 days. After 60 days, this inflammation was still present but no significant fibrotic response was observed. The cytotoxicity of ITO was assessed in vitro in lung epithelial cells (RLE) and macrophages (NR8383 cell line). While ITO particles (up to 200 microg/ml) did not affect epithelial cell integrity (LDH release), a strong cytotoxic response was found in macrophages exposed to ITO, but not to its components alone or mixed. ITO particles also induced an increased frequency of micronuclei in type II pneumocytes in vivo but not in RLE in vitro, suggesting the preponderance of a secondary genotoxic mechanism. To address the possible mechanism of ITO toxicity, reactive oxygen species production was assessed by electron paramagnetic resonance spectrometry in an acellular system. Carbon centered radicals (COO-.) and Fenton-like activity were detected in the presence of ITO particles, not with In(2)O(3), SnO(2) alone, or the MIX. Because the unsintered mixture of SnO(2) and In(2)O(3) particles was unable to reproduce the reactivity/toxicity of ITO particles, the sintering process through which SnO(2) molecules are introduced within the crystal structure of In(2)O(3) appears critical to explain the unique toxicological properties of ITO. The inflammatory and genotoxic activities of ITO dust indicate that a strict control of exposure is needed in industrial settings.

Abstract: The effect that monodisperse amorphous spherical silica particles of different sizes have on the viability of endothelial cells (EAHY926 cell line) is investigated. The results indicate that exposure to silica nanoparticles causes cytotoxic damage (as indicated by lactate dehydrogenase (LDH) release) and a decrease in cell survival (as determined by the tetrazolium reduction, MTT, assay) in the EAHY926 cell line in a dose-related manner. Concentrations leading to a 50% reduction in cell viability (TC(50)) for the smallest particles tested (14-, 15-, and 16-nm diameter) ranging from 33 to 47 microg cm(-2) of cell culture differ significantly from values assessed for the bigger nanoparticles: 89 and 254 microg cm(-2) (diameter of 19 and 60 nm, respectively). Two fine silica particles with diameters of 104 and 335 nm show very low cytotoxic response compared to nanometer-sized particles with TC(50) values of 1095 and 1087 microg cm(-2), respectively. The smaller particles also appear to affect the exposed cells faster with cell death (by necrosis) being observed within just a few hours. The surface area of the tested particles is an important parameter in determining the toxicity of monodisperse amorphous silica nanoparticles.

Abstract: PURPOSE: trans,trans-Muconic acid (t,t-MA) is generally considered as a useful biomarker of exposure to benzene. However, because of its lack of specificity, concerns about its value at low level of exposure have recently been raised. The aim of this study was (a) to compare t,t-MA, S-phenylmercapturic acid (SPMA) and benzene (B-U) as urinary biomarkers of exposure to low levels of benzene in petrochemical workers and, (b) to evaluate the influence of sorbic acid (SA) and genetic polymorphisms of biotransformation enzymes on the excretion of these biomarkers. METHOD: A total of 110 workers (including 24 smokers; 2-10 cigarettes/day) accepted to take part in the study. To assess external exposure to benzene, air samples were collected during the whole working period by a passive sampling device attached close to the breathing zone of 98 workers. Benzene was measured in blood (B-B) samples taken at the end of the shift, and was considered as the reference marker of internal dose. Urine was collected at the end of the shift for the determination of B-U, SPMA, t,t-MA, SA and creatinine (cr). B-U and B-B were determined by head-space/GC-MS, SPMA and SA by LC-MS, t,t-MA by HPLC-UV. RESULTS: Most (89%) personal measurements of airborne benzene were below the limit of detection (0.1 ppm); B-B ranged from <0.10 to 13.58 mug/l (median 0.405 microg/l). The median (range) concentrations of the urinary biomarkers were as follows: B-U 0.27 microg/l (<0.10-5.35), t,t-MA 0.060 mg/l (<0.02-0.92), SPMA 1.40 microg/l (0.20-14.70). Urinary SA concentrations ranged between <3 and 2,211 microg/l (median 28.00). Benzene concentration in blood and in urine as well as SPMA, but not t,t-MA, were significantly higher in smokers than in non-smokers. The best correlation between B-B and urinary biomarkers of exposure were obtained with benzene in urine (microg/l r = 0.514, P < 0.001; microg/g cr r = 0.478, P < 0.001) and SPMA (microg/l r = 0.495, P < 0.001; microg/g cr r = 0.426, P < 0.001) followed by t,t-MA (mg/l r = 0.363, P < 0.001; mg/g cr r = 0.300, P = 0.002). SA and t,t-MA were highly correlated (r = 0.618, P < 0.001; corrected for cr r = 0.637). Multiple linear regression showed that the variation of t,t-MA was mostly explained by SA concentration in urine (30% of the explained variance) and by B-B (12%). Variations of SPMA and B-U were explained for 18 and 29%, respectively, by B-B. About 30% of the variance of B-U and SPMA were explained by B-B and smoking status. Genetic polymorphisms for biotransformation enzymes (CYP2E1, EPHX1, GSTM1, GSTT1, GSTP1) did not significantly influence the urinary concentration of any of the three urinary biomarkers at this low level of exposure. CONCLUSION: At low levels of benzene exposure (<0.1 ppm), (1) t,t-MA is definitely not a reliable biomarker of benzene exposure because of the clear influence of SA originating from food, (2) SPMA and B-U reflect the internal dose with almost similar accuracies, (3) genetically based inter-individual variability in urinary excretion of biomarkers seems negligible. It remains to assess which biomarker is the best predictor of health effects.

Abstract: BACKGROUND: Beneficial effects of azithromycin in cystic fibrosis (CF) have been reported, however, its mechanism of action remains unclear. The present study aimed at investigating the effect of azithromycin on CF airway epithelial cells. METHODS: Primary cultures of purified tracheal epithelial cells from F508del and normal homozygous mice were established. Responses to lipopolysaccharide from Pseudomonas aeruginosa (LPS, 0.1 microg/ml) on mRNA expression of neutrophil-related chemokines, pro- and anti-inflammatory cytokines were investigated in the presence or the absence of azithromycin (1 microg/ml). RESULTS: CF airway epithelial cells showed upregulation of MIP-2 and KC responses to LPS, and azithromycin failed to downregulate these responses. In contrast, in CF cells, azithromycin increased KC and TNF-alpha expression under non-stimulated and LPS-stimulated conditions, respectively. In non-CF cells, the macrolide potentiated the LPS response on MIP-2 and on IL-10. CONCLUSIONS: Airway epithelial cells contribute to the dysregulated immune processes in CF. Azithromycin rather stimulates cytokine expression in CF airway epithelial cells.

Abstract: Serum Clara cell protein (CC16) and surfactant-associated protein D (SP-D) were measured in 161 workers exposed to sulphur dioxide (SO(2)) in a non-ferrous smelter. Seventy workers from a blanket manufacture served as referents. Exposure to SO(2) and tobacco smoking were associated with a decrease of CC16 and an increase of SP-D in serum. Tobacco smoking and exposure SO(2) interacted synergistically to decrease serum CC16 but not to increase serum SP-D. While further illustrating the potential of serum CC16 and SP-D, our study confirms that SO(2) can cause airways damage at exposure levels below current occupational exposure limits.

Abstract: AIM: Lopinavir (LPV) is a potent protease inhibitor used in combination with low doses of ritonavir in the treatment of HIV-infected patients. LPV pharmacokinetics is characterized by a large interindividual variability requiring the use of therapeutic drug monitoring in different clinical situations. While the sources of this variability are still unknown, several genetic polymorphisms in biotransformation enzymes or transporter proteins involved in the metabolism and/or the distribution of LPV appear as good candidates. Therefore, the aim of the present study was to investigate the influence of selected genetic polymorphisms on LPV trough plasma concentrations ([LPV](Cmin)), LPV concentrations in peripheral blood mononuclear cells ([LPV](CC)) and the LPV accumulation ratio ([LPV](CC):[LPV](Cmin)). MATERIALS & METHODS: A total of 53 patients receiving Kaletra((R)) (Abbott Laboratories, IL, USA) (LPV+ritonavir) were genotyped for 14 different polymorphisms in biotransformation enzymes and transporter proteins. [LPV](Cmin), [LPV](CC) and [LPV](CC):[LPV](Cmin) were compared according to the patient's genotypes. RESULTS & CONCLUSION: The 4544G>A (rs8187710)polymorphism in ABCC2 was associated with a higher accumulation of LPV in peripheral blood mononuclear cells of HIV-treated patients. As already observed in previous studies, ABCB1 or CYP3A5 polymorphisms had no impact on [LPV](Cmin) and we did not detect any influence of these polymorphisms on [LPV](CC) and its accumulation in mononuclear cells. In conclusion, this pilot study suggests, for the first time, that the 4544G>A polymorphism in ABCC2 could explain a significant part of the interindividual variability in LPV pharmacokinetics. Further investigations are needed to confirm this association and to explore its real pharmacodynamic impact.

Abstract: Toxicological investigations of carbon nanotubes have shown that they can induce pulmonary toxicity, and similarities with asbestos fibers have been suggested. We previously reported that multiwall carbon nanotubes (MWCNT) induced lung inflammation, granulomas and fibrotic reactions. The same MWCNT also caused mutations in epithelial cells in vitro and in vivo. These inflammatory and genotoxic activities were related to the presence of defects in the structure of the nanotubes. In view of the strong links between inflammation, mutations and cancer, these observations prompted us to explore the carcinogenic potential of these MWCNT in the peritoneal cavity of rats. The incidence of mesothelioma and other tumors was recorded in three groups of 50 male Wistar rats injected intraperitoneally with a single dose of MWCNT with defects (2 or 20 mg/animal) and MWCNT without defects (20 mg/animal). Two additional groups of 26 rats were used as positive (2 mg UICC crocidolite/animal) and vehicle controls. After 24 months, although crocidolite induced a clear carcinogenic response (34.6% animals with mesothelioma vs. 3.8% in vehicle controls), MWCNT with or without structural defects did not induce mesothelioma in this bioassay (4, 0, or 6%, respectively). The incidence of tumors other than mesothelioma was not significantly increased across the groups. The initial hypothesis of a contrasting carcinogenic activity between MWCNT with and without defects could not be verified in this bioassay. We discuss the possible reasons for this absence of carcinogenic response, including the length of the MWCNT tested (< 1 mum on average), the absence of a sustained inflammatory reaction to MWCNT, and the capacity of these MWCNT to quench free radicals.

Abstract: This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm x 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25-125 ng/ml of cell extract by a 1/x(2) weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of +/-15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n=48) or with Stocrin (600 mg once a day, n=50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus (500 mg twice a day). The patients treated by Kaletra showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin showed mean CC of 2388.11 ng/ml while both patients under Aptivus showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.

Abstract: PURPOSE: The purpose of this paper is to review available cohort studies and to estimate quantitatively the association between occupational exposure in plants manufacturing pesticides and leukaemia. METHODS: Following a systematic literature search, relative risks were extracted from 14 studies published between 1984 and 2004. Fixed effect analyses were carried out as heterogeneity between studies was not detected. Meta-analyses were performed on the whole set of data and separate analyses were conducted for specific chemical classes of pesticides as well as type of leukaemia. RESULTS: The meta-rate ratio estimate for all studies was 1.43 (95% confidence interval [CI] 1.05-1.94). After stratification by chemical class, consistent increases in the risk of leukaemia were found in all groups but statistical significance was found only for phenoxy herbicides unlikely to have been contaminated with dioxins and furans. This last finding appears equivocal in view of the existing literature. The separate analysis conducted on leukaemias from the myeloid lineage showed the highest relative risk (6.99; 95% CI 1.96-24.90). There was no obvious indication of publication bias. CONCLUSION: The overall meta-analysis among pesticide manufacturing workers provides quantitative evidence to consider occupational exposure to pesticides as a possible risk factor for leukaemia but available data are too scarce for causality ascertainment. Epidemiological evidence did not allow identifying a specific pesticide or chemical class that would be responsible for the increased risk. Exposure to pesticides may be a significant risk factor for specifically developing myeloid leukaemia and there is a need for additional large well-conducted studies with clear definition of exposure and of leukaemia type(s).

Abstract: Organochlorines (polychlorinated biphenyls and dioxin-like compounds) are suspected to play a role in the etiopathogenesis of endometriosis. This hypothesis, based on experimental data, has been circulating for years in the scientific community and several epidemiologic surveys have attempted to obtain confirmatory human data. The purpose of this mini-review is to provide an overview of the twelve epidemiological studies that have assessed the relationship between endometriosis and organochlorine exposure. Several studies did not observe a significant association between peritoneal endometriosis and organochlorines. The deep nodular form of endometriosis appears associated with a higher serum level of both dioxin-like compounds and polychlorobiphenyls. The type of control women, the nature of the chemicals measured, and the definition of the disease could modulate the ability to detect the possible relationship between endometriosis and organochlorine exposure.

Abstract:

Abstract: Hard metals consist of tungsten carbide (WC) and metallic cobalt (Co) particles and are important industrial materials produced for their extreme hardness and high wear resistance properties. While occupational exposure to metallic Co alone is apparently not associated with an increased risk of cancer, the WC-Co particle mixture was shown to be carcinogenic in exposed workers. The in vitro mutagenic/apoptogenic potential of WC-Co in human peripheral blood mononucleated cells was previously demonstrated by us. This study aimed at obtaining a broader view of the pathways responsible for WC-Co induced carcinogenicity, and in particular genotoxicity and apoptosis. We analyzed the profile of gene expression induced in vitro by WC-Co versus control (24 h treatment) in human PBMC and monocytes using microarrays. The most significantly up-regulated pathways for WC-Co treated PBMC were apoptosis and stress/defense response; the most down-regulated was immune response. For WC-Co treated monocytes the most significantly up- and down-regulated pathways were nucleosome/chromatin assembly and immune response respectively. Quantitative RT-PCR data for a selection of the most strongly modulated genes (HMOX1, HSPA1A, HSPA1L, BNIP3, BNIP3L, ADORA2B, MT3, PLA2G7, TNFAIP6), and some additionally chosen apoptosis related genes (BCL2, BAX, FAS, FASL, TNFalpha), confirmed the microarray data after WC-Co exposure and demonstrated limited differences between the Co-containing compounds. Overall, this study provides the first analysis of gene expression induced by the WC-Co mixture showing a large profile of gene modulation and giving a preliminary indication for a hypoxia mimicking environment induced by WC-Co exposure.

Abstract: OBJECTIVES: To review epidemiological studies which led to a change in the classification of formaldehyde by the International Agency for Research on Cancer (IARC) in 2004 as well as studies published thereafter, with the objective to examine whether occupational exposure levels for formaldehyde should be adapted. METHOD: Cohort and case-control studies investigating the association between occupational exposure to formaldehyde and nasopharyngeal cancer (NPC) and reporting estimates of formaldehyde exposure as well as the most recent meta-analyses, published after 1994, were reviewed. RESULTS: Evidence of an association between occupational formaldehyde exposure and NPC appears debatable. Results of the cohort studied by Hauptmann et al. (Am J Epidemiol 159(12):1117-1130, 2004) were key findings in the IARC evaluation. In this study, mortality from NPC was elevated compared with that of the US general population. However, internal comparison analysis using alternative categorization revealed that none of the relative risk for NPC was statistically significantly increased in any category of exposure (Marsh and Youk in Regul Toxicol Pharmacol 42(3):275-283, 2005) and re-analyses of the data highlighted the inappropriateness of the exposure assessment used by Hauptmann et al. (Am J Epidemiol 159(12):1117-1130, 2004) and Marsh et al. (Regul Toxicol Pharmacol 47(1):59-67, 2007). Two other cohorts (Coggon et al. in J Natl Cancer Inst 95(21):1608-1615, 2003; Pinkerton et al. in Occup Environ Med 61(3)193-200, 2004) reported no increase in NPC. Two case-control studies brought some evidence of an increased risk of NPC but the assessment of exposure levels was uncertain. DISCUSSION: Human studies fail to raise a convincing conclusion concerning the carcinogenicity of formaldehyde and are not helpful to delineate a possible dose-response relationship. Experimental data indicate that in rats, the carcinogenic activity of formaldehyde is associated with cytotoxic/proliferative mechanisms. Therefore protecting from these effects associated with formaldehyde exposure should be sufficient to protect from its potential carcinogenic effects, if any in humans. CONCLUSION: Current occupational exposure levels to formaldehyde, set to protect against local irritation, should not be adapted.

Abstract: Experimental studies indicate that carbon nanotubes (CNTs) have the potential to induce adverse pulmonary effects, including alveolitis, fibrosis, and genotoxicity in epithelial cells. Here, we explored the physicochemical determinants of these toxic responses with progressively and selectively modified CNTs: ground multiwall CNTs modified by heating at 600 degrees C (loss of oxygenated carbon functionalities and reduction of oxidized metals) or at 2400 degrees C (annealing of structural defects and elimination of metals) and by grinding the material that had been heated at 2400 degrees C before (introduction of structural defects in a metal-deprived framework). The CNTs were administered intratracheally (2 mg/rat) to Wistar rats to evaluate the short-term response (3 days) in bronchoalveolar lavage fluid (LDH, proteins, cellular infiltration, IL-1beta, and TNF-alpha). The long-term (60 days) lung response was assessed biochemically by measuring the lung hydroxyproline content and histologically. In vitro experiments were also performed on rat lung epithelial cells to assess the genotoxic potential of the modified CNTs with the cytokinesis block micronucleus assay. The results show that the acute pulmonary toxicity and the genotoxicity of CNT were reduced upon heating but restored upon grinding, indicating that the intrinsic toxicity of CNT is mainly mediated by the presence of defective sites in their carbon framework.

Abstract: Information on the toxicity of carbon nanotubes is still fragmentary but indicates that these particles can induce adverse effects. We previously demonstrated in rats that, when purified multi-wall carbon nanotubes (MWCNT) reach the lung, they are biopersistent and induce lung inflammation as well as fibrosis. The present study was designed to address the genotoxic potential of this material in the same species. In vivo, micronuclei (MN) were assessed in type II pneumocytes 3 days after a single intra-tracheal administration of MWCNT (0.5 or 2 mg). We also used the cytokinesis-block micronucleus assay in rat lung epithelial cells exposed in vitro to MWCNT (10, 25, 50 mug/ml). Finally, we applied a human pancentromeric fluorescent probe (fluorescent in situ hybridization assay) to differentiate clastogenic and/or aneugenic mechanisms in a human epithelial cell line (MCF-7). In vivo, we found a significant and dose-dependent increase in micronucleated pneumocytes after a single administration of MWCNT ( approximately a 2-fold increase at the highest dose). In vitro, we observed a significant increase of MN in epithelial cells after exposure to MWCNT (up to a 2-fold increase at the cytotoxic dose of 50 mug/ml). Finally, we found that MWCNT induced both centromere-positive and -negative MN in MCF-7 cells. Overall, this study provides the first evidence of the potential of MWCNT to induce clastogenic as well as aneugenic events.

Abstract: Carbon nanotubes (CNT) have been reported to elicit toxic responses in vitro and in vivo, ascribed so far to metal contamination, CNT length, degree of oxidation, or extent of hydrophilicity. To examine how structural properties may modulate the toxicity of CNT, one preparation of multiwall CNT has been modified (i) by grinding (introducing structural defects) and subsequently heating either in a vacuum at 600 degrees C (causing reduction of oxygenated carbon functionalities and reduction of metallic oxides) or in an inert atmosphere at 2400 degrees C (causing elimination of metals and annealing of defects) and (ii) by heating at 2400 degrees C in an inert atmosphere and subsequently grinding the thermally treated CNT (introducing defects in a metal-deprived carbon framework). The presence of framework and surface defects, metals, and oxygenated functionalities was monitored by means of a set of techniques, including micro-Raman spectroscopy, adsorption calorimetry, X-ray photoelectron spectroscopy, inductively coupled plasma mass spectrometry, and atomic emission spectroscopy. Contrary to traditional toxicants, such as asbestos, CNT may quench rather than generate oxygenated free radicals. The potential of the modified CNT to scavenge hydroxyl radicals was thus evaluated by means of electron spin resonance spectroscopy (spin trapping). The original ground material exhibited a scavenging activity toward hydroxyl radicals, which was eliminated by heating at 2400 degrees C but restored upon grinding. This scavenging activity, related to the presence of defects, appears to go paired with the genotoxic and inflammatory potential of CNT reported in the companion paper. Thus, defects may be one of the major factors governing the toxic potential of CNT.

Abstract: Because of their small size and large specific surface area (SA), insoluble nanoparticles are almost not affected by the gravitational force and are generally formulated in stable suspensions or sols. This raises, however, a potential difficulty in in vitro assay systems in which cells adhering to the bottom of a culture vessel may not be exposed to the majority of nanoparticles in suspension. J. G. Teeguarden et al., 2007, Toxicol. Sci. 95, 300-312 have recently addressed this issue theoretically, emphasizing the need to characterize the effective dose (mass or number or SA dose of particles that affect the cells) which, according to their model based on sedimentation and gravitation forces, might only represent a very small fraction of the nominal dose. We hypothesized, in contrast, that because of convection forces that usually develop in sols, the majority of the particles may reach the target cells and exert their potential toxicity. To address this issue, we exposed three different cell lines (A549 epithelial cells, EAHY926 endothelial cells, and J774 monocyte-macrophages) to a monodisperse suspension of Stöber silica nanoparticles (SNP) in three different laboratories. Four different end points (lacticodehydrogenase [LDH] release, LDH cell content, tetrazolium salt (MTT), and crystal violet staining) were used to assess the cell response to nanoparticles. We found, in all cell lines and for all end points, that the cellular response was determined by the total mass/number/SA of particles as well as their concentration. Practically, for a given volume of dispersion, both parameters are of course intimately interdependent. We conclude that the nominal dose remains the most appropriate metric for in vitro toxicity testing of insoluble SNP dispersed in aqueous medium. This observation has important bearings on the experimental design and the interpretation of in vitro toxicological studies with nanoparticles.

Abstract: Noise is the most common preventable cause of irreversible sensorineural hearing loss. During recent years, the results of experimental and human investigations have raised the level of concern about the potential ototoxicity of chemical agents and their interaction with noise. European Directive 2003/10/EC on the minimum health and safety requirements regarding the exposure of workers to the risks arising from noise specifies that the employer shall give particular attention, when carrying out the risk assessment, to, among others, any effects on workers' health and safety resulting from interactions between noise and work-related ototoxic substances. There is, however, currently very little awareness in the occupational health community of the chemical hazards to hearing. The main objective of this review was to analyze the available scientific literature on the ototoxic effects of styrene and toluene, in order to examine dose-response/effect relationships and the relevance of the prevention strategy for people exposed to these solvents. While both solvents appear clearly ototoxic in rats, human data are less straightforward and the existing evidence does not allow characterization of the dose-response/effect relationships; further research is needed. However, once hearing loss is incurred, it is irreversible, and one should be alert to the possible hearing loss induced by toluene and styrene and to the possible additive, potentiating, or synergistic ototoxic effects in case of combined exposure to several chemicals and in case of combined exposure to noise and chemical substances.

Abstract: Increased IL-9 expression, either systemically or under the control of lung-specific promoter, induces an asthma-like phenotype, including mucus overproduction, mastocytosis, lung eosinophilia, and airway hyperresponsiveness. These activities correlate with increased production of other Th2 cytokines such as IL-4, IL-5, and IL-13 in IL-9 Tg mice. To determine the exact role of IL-13 in this phenotype, mice overexpressing IL-9 were crossed with IL-13-deficient mice. In these animals, IL-9 could still induce mastocytosis and B lymphocyte infiltration of the lungs. Although IL-9-induced eosinophilia in the peritoneal cavity was not diminished in the absence of IL-13, IL-13 was required for IL-9 to increase eotaxin expression and lung eosinophilia. Mucus production and up-regulation of lung epithelial genes upon IL-9 overexpression were completely abolished in the absence of IL-13. Using hemopoietic cell transfer experiments with recipients that overexpressed IL-9 but were deficient in the IL-9 receptor (IL-9R), we could demonstrate that the effect of IL-9 on lung epithelial cells is indirect and could be fully restored by transfer of hemopoietic cells expressing IL-9R. Mucus production by lung epithelial cells was only up-regulated when hemopoietic cells simultaneously expressed functional IL-9R and IL-13 genes, indicating that IL-13 is not a cofactor but a direct mediator of the effect of IL-9 on lung epithelial cells. Taken together, these data indicate that IL-9 can promote asthma through IL-13-independent pathways via expansion of mast cells, eosinophils, and B cells, and through induction of IL-13 production by hemopoietic cells for mucus production and recruitment of eosinophils by lung epithelial cells.

Abstract: Peritoneal endometriosis (PE) and deep endometriotic nodules (DEN) are gynecological diseases recently shown to be associated with elevated serum concentrations of organochlorines. The objective of the present study was to compare risk factors associated with both forms of the disease, with a particular attention to potential sources of organochlorine exposure. This matched case-control study with prospective recruitment included 88 triads (PE-DEN-control). All women were face-to-face interviewed with a standardized questionnaire, and serum dioxin and polychlorinated biphenyl measurements were available for 58 of them. Alcohol consumption (odds ratio (OR): 5.82 [confidence interval at 95% (95%CI) 1.20-28.3]) in DEN and low physical activity at work for DEN (OR: 4.58 [95%CI 1.80-11.62]) and PE (OR: 5.61 [95%CI 1.90-16.60]) were traced as significant risk factors. Organochlorine-related factors (use of tampons, occupational or environmental exposure) were not related to the disease. The current consumption of foodstuffs that were more likely to contribute to organochlorine body burden did not differ among the groups. Only some of these fatty foodstuffs (marine fish, pig meat) were traced by multiple regression analysis as significant determinants of organochlorine body burden, explaining only a small fraction (20%) of the interindividual variation of organochlorine body burden. We conclude that PE and DEN share similar patterns of risk or protective factors.

Abstract: It has been proposed that the development of lung fibrosis is associated with a T helper type 2 response, mainly characterized by IL-4 and IL-13 production. We investigated the potential role of type 2 immune polarization in the silicotic process and examined the pulmonary response to silica particles in mice genetically deficient for IL-4. We found that IL-4(-/-) mice were not protected against the development of silicosis, suggesting that IL-4 is not essential for the development of this fibrotic disease. By evaluating the intensity of silica-induced lung fibrosis in mice deficient for IL-4 receptor alpha (IL-4Ralpha), we showed that the establishment of pulmonary fibrosis was independent of both IL-4 and IL-13. Strong impairment of the type 2 immune response (IgG(1)) in the lungs of IL-4(-/-) and IL-4Ralpha(-/-) mice did not affect the development of the disease. Measurement of IL-13alpha2 receptor expression and IgG(2a), IL-12p70, and IFN-gamma levels in silica-treated IL-4(-/-) and IL-4Ralpha(-/-) animals showed that the development of silicosis was not related to an IL-13 signaling pathway or a switch to a type 1 response in deficient animals. Our data clearly indicate that the type 2 immune response associated with silicosis in mice is not required for the development of this inflammatory and fibrotic disease.

Abstract: Ethylene oxide is considered as a human carcinogen. A biomarker of exposure would be a useful instrument to assess the risk in occupationally exposed workers. This cross-sectional study aimed at examining (a) whether the urinary excretion of a metabolite of ethylene oxide, 2-hydroxyethyl mercapturic acid (HEMA), could be used for monitoring occupational exposure and (b) whether glutathione S-transferase (GST) and epoxide hydrolase genotypes influenced biological monitoring. Exposure to ethylene oxide was measured by personal sampling in 80 hospital workers (95% of those eligible). HEMA concentrations were determined in three urine samples (baseline, end of shift, and next morning) by liquid chromatography with tandem mass spectrometry. GSTs (GSTT1, GSTM1, and GSTP1) and epoxide hydrolase (EPHX1) were also genotyped. The influence of exposure, genotypes, and several other factors was examined in multiple regression analyses. Exposure was always <1 parts per million. On a group basis, exposure and a non-null GSTT1 genotype increased the HEMA concentrations in the urine sample collected at the end of the shift and these factors remained statistically significant after considering possible confounding or modifying factors.

Abstract: OBJECTIVE: Tacrolimus is an immunosuppressive drug widely used in hepatic transplantation to avoid graft rejection. Its pharmacokinetics is characterized by a large interindividual variability requiring the use of therapeutic drug monitoring in daily clinical practice. Some genetic polymorphisms in biotransformation enzymes or transporter proteins, such as CYP3A5 and P-glycoprotein (ABCB1), in donors and/or recipients, appear as important determinants of the Tac blood pharmacokinetics. A recent study has shown that Tac hepatic tissue concentrations vary greatly among patients and are well correlated with graft outcome. The aim of our study was to investigate the effect of genetic polymorphisms in biotransformation enzymes (CYP3A5 and CYP3A7) or in their regulatory protein pregnane X receptor as well as in transporter proteins (ABCB1 and OATP-C) on Tac pharmacokinetics in liver transplant patients and more specifically on Tac hepatic concentrations. METHODS: One hundred and fifty liver donors were genotyped for 13 different polymorphisms. Tac blood and hepatic concentrations were compared according to hepatic genotypes. RESULTS AND CONCLUSION: We confirmed that Tac dose requirement (on the basis of blood therapeutic drug monitoring) was higher among patients expressing hepatic CYP3A5 (at least one CYP3A5*1 allele) compared with patients who did not (CYP3A5*3/*3). Hepatic expression of CYP3A5, however, did not seem to influence Tac hepatic concentrations. In contrast, ABCB1 genetic polymorphisms significantly influenced Tac hepatic concentrations, whereas their impact on blood concentrations seemed negligible. Among these ABCB1 polymorphisms, the 1199G>A and 2677G>T/A single nucleotide polymorphisms seemed to reduce the activity of P-gp on Tac. As Tac hepatic concentrations have been significantly related to the graft outcome, it might be interesting, in the future, to genotype donors for ABCB1 polymorphisms to better individualize the Tac immunosuppressive therapy in hepatic transplantation.

Abstract: IL-9 overexpression protects against alveolar fibrosis induced by crystalline silica particles. This cytokine is also involved in allergic asthma. In the present study, we examined the effect of IL-9 overexpression on the subepithelial fibrotic response, a feature of asthmatic remodeling, induced by chronic exposure to Alternaria alternata extract. IL-9-overexpressing mice (Tg5) and their wild-type counterparts (FVB) were intranasally exposed to A. alternata extract or PBS (controls) twice a week during 3 mo. At the end of the allergic challenge, enhanced pause (Penh) measured in response to methacholine and fibrotic parameters, such as collagen and fibronectin lung content, were significantly higher in Tg5 compared with FVB. Staining of lung sections with Masson's Trichrome also showed more collagen fibers in peribronchial areas of treated Tg5 mice. A similar recruitment of inflammatory cells was observed in challenged FVB and Tg5 mice, except for eosinophils, which were significantly more abundant in the lung of Tg5. High serum levels of IgE and IgG1 in both strains indicated that FVB and Tg5 developed a strong type 2 immune response. The concentration of the eosinophil chemoattractant RANTES and the profibrotic mediator connective tissue growth factor (CTGF) was higher in the BAL of challenged Tg5 than FVB. These results demonstrate a profibrotic role of IL-9 in an airway remodeling model, possibly involving eosinophils and CTGF. These data also highlight a dual role of IL-9 in lung fibrosis, being anti- or profibrotic depending on the alveolar or airway localization of the process, respectively.

Abstract: OBJECTIVE: To conduct a systematic review and meta-analyses of published studies examining the association between myeloid leukemias (ML) and occupational pesticide exposure. METHODS: Studies were identified from a MEDLINE search through 31 May 2006 and from the reference lists of identified publications. Studies were summarized and evaluated for publication bias. Relative risk estimates for ML were extracted from 17 cohort and 16 case-control studies published between 1979 and 2005. Fixed- or random-effect meta-analysis models were used depending on the presence of heterogeneity between studies. Separate analyses were conducted after stratification for study design, occupational group, ML subtype or gender. RESULTS: The overall meta-rate ratio estimate (meta-RR) for the cohort studies was 1.21 (95% confidence interval [CI] 0.99-1.48). Substantial heterogeneity existed among cohort studies (p=1.064 x 10(-5)), mainly reflecting the varying occupational categories examined. The meta-RR was 6.32 (95% CI: 1.90-21.01) for manufacturing workers and 2.14 (95% CI: 1.39-3.31) for pesticide applicators. After stratification of cohort studies by specific ML subtype, an increased risk of acute myeloid leukemia (AML) was found (meta-RR: 1.55; 95% CI: 1.02-2.34). No significant heterogeneity was detected among case-control studies and an increased risk of chronic myeloid leukemia (CML) was found among men (meta-RR: 1.39; 95% CI: 1.03-1.88) and farmers or agricultural workers (meta-RR: 1.38; 95% CI: 1.06-1.79). CONCLUSION: The strongest evidence of an increased risk of ML comes from manufacturing workers and pesticide applicators. Further studies will be needed to correlate reliable exposure data within these specific occupational groups with well-defined subtypes of leukemia to refine this assessment.

Abstract: Carbon nanotubes (CNTs) currently attract intense research efforts because of their unique properties which make them suitable for many industrial applications. When inhaled, CNTs constitute a possible hazard to human health. Several studies have shown that when instilled in the lung of experimental animals, CNTs induced an inflammatory and fibrotic response similar to that caused by other toxic particles which might be the result of oxidative stress caused by particle- and/or cell-derived free radicals. There is, however, no direct experimental evidence of a capacity of carbon nanotubes to generate directly free radicals. Here we report that multiwall carbon nanotubes (MWCNT) in aqueous suspension do not generate oxygen or carbon-centered free radicals in the presence of H2O2 or formate, respectively, as detected with the spin-trapping technique. Conversely, we observed that, when in contact with an external source of hydroxyl or superoxide radicals, MWCNT exhibit a remarkable radical scavenging capacity. It is therefore possible that the inflammatory reaction reported in vivo must be ascribed to MWCNT features other than particle-derived free radical generation.

Abstract: Styrene oxide (SO), ethylene oxide (EO) and gamma-radiation (G) are agents with a well-described metabolism and genotoxicity. EPHX1 and GSTs play an important role in the detoxification of electrophiles and oxidative stress. Enzymes involved in base excision repair (hOGG1, XRCC1), in rejoining single strand breaks (XRCC1) and in repair of cross-links and chromosomal double strand breaks (XRCC3) might have an impact on genotoxicity as well. In this study we assessed the dose-dependent effect of genetic polymorphisms in biotransforming (EPHX (Tyr113/His113 and His139/Arg139), GSTP1 (Ile105/Val105), GSTM1 and GSTT1) and DNA repair enzymes (hOGG1 (Ser326/Cys326), XRCC1 (Arg194/Trp194, Arg280/His280, Arg399/Gln399), XRCC3 (Thr241/Met241)) on the induced genotoxicity. Peripheral blood mononuclear cells from 20 individuals were exposed to 3 doses per agent (+control). Genotoxicity was evaluated by measuring comet tail length (TL) and micronucleus frequencies in binucleated cells (MNCB). Dose-dependent DNA damage was found for all agents and end-points, with the exception of MNCB induced by EO. Repeated measure ANOVA revealed a significant contribution of hOGG1 and XRCC3 genotypes to the inter-individual variability of TL and MNCB in cells exposed to EO and G. Homozygous hOGG1326 wild cells showed significantly lower EO-induced TL than the heterozygous cells. Significantly higher TL and MNCB were found in EO-exposed cells carrying the XRCC3(241)Met variant and the influence on TL was more pronounced at higher dose. In G-irradiated cells, TL was significantly higher in the hOGG1326 homozygous wild types compared with mutated genotypes. The influence of hOGG1326 on TL was borderline dose-dependent. We conclude that the influence of genetic polymorphisms of enzymes involved in DNA repair on induced genotoxicity depends on exposure dose.

Abstract: We found, by reverse transcription--real time--polymerase chain reaction, that the expression of aromatase (CYP19) in ovarian, peritoneal endometriosis, and deep endometriotic nodules is significantly different, which strengthens the theory of three distinct clinical entities. Compared with peritoneal endometriosis, ovarian endometriosis exhibits an 8-fold higher expression of aromatase, which suggests that aromatase inhibitors may be particularly active in this form of endometriosis.

Abstract: BACKGROUND: The lead mobilization test reflects the mobilizable and likely toxicologically active fraction of the lead body burden. We propose a safe and convenient protocol for this test, to assess concomitant copper and zinc excretion and to determine the size of the chelatable lead pool in nonoccupationally exposed adults. METHODS: The study population included 80 white adults: 40 controls [median blood lead concentration (PbB), 25 microg/L] and 40 lead-exposed individuals (315 microg/L). After collection of 4- and 24-h baseline urine specimens and a blood sample, dimercaptosuccinic acid (DMSA) was administered orally (1 g), and additional 4- and 24-h urine specimens were obtained. Determinants of the chelatable urinary lead (DMSA-PbU) were traced by linear regression analysis. RESULTS: Urinary DMSA and lead excretion peaked within 2-3 h after DMSA administration. The amounts of DMSA, lead, copper, and zinc recovered in the 4-h urinary collections were highly correlated with those in 24-h collections (r = 0.857, 0.859, 0.958, and 0.757, respectively). At PbB concentrations >300 microg/L, the relationship between DMSA-PbU and PbB showed a steep increase and a widespread dispersion of DMSA-PbU around the regression line. After DMSA, copper and zinc excretion rates were increased up to 91- and 33-fold, respectively. No side effects were reported after DMSA. CONCLUSIONS: Determination of DMSA-PbU in a 4-h collection after DMSA is convenient, apparently safe, and inexpensive. An upper reference limit value of 22 microg/4 h is proposed for Belgian reference individuals. The diagnostic value of DMSA-PbU is likely to be contributive for PbB >300 microg/L.

Abstract: We previously showed that overexpression of IL-9 controls lung fibrosis induced by silica particles in mice (Arras and colleagues; Am J Respir Cell Mol Biol 2001;24:368-375). This protection was associated with an expansion of lung B lymphocytes. To explore the contribution of these cells in the protective effect of IL-9, we crossed IL-9 transgenic (IL-9+) and B-deficient (B-) mice. The antifibrotic effect of IL-9 was abolished in mice deficient in B lymphocytes (B-IL-9+) and restored by reconstituting these mice with B lymphocytes. The expression of the antifibrotic mediator prostaglandin (PG)E2 was markedly increased in the lung of IL-9+ mice at baseline, and similarly high levels were found in both wild-type and transgenic strains upon silica treatment. This PGE2 expression was completely abolished in B- mice, both at baseline and upon silica administration. In vitro, alveolar and peritoneal macrophages from IL-9+ mice had an increased capacity to produce PGE2 in response to LPS or silica. This capacity was markedly reduced in macrophages obtained from B- mice and restored by co-incubating macrophages with B lymphocytes from IL-9+ mice. The increased PGE2 response of IL-9+ macrophages was dependent on cyclooxygenase 2 expression, based on transcript analysis and inhibition by NS398. We conclude that B lymphocytes are essential for the protection against lung fibrosis and macrophage overexpression of PGE2 in IL-9 transgenic animals.

Abstract: OBJECTIVES: Cadmium (Cd) and lead (Pb) have been demonstrated to exert endocrine disrupting activities. Their possible role in endometriosis, an oestrogen-dependent disease, is unknown. METHODS: We compared cadmium urinary excretion (CdU) and blood concentration of cadmium (CdB) and lead (PbB) in 119 patients with peritoneal endometriosis and/or deep endometriotic (adenomyotic) nodules of the rectovaginal septum and 25 controls. RESULTS: The mean levels of cadmium in urine and blood did not differ among the groups. Women suffering from endometriotic diseases showed lower levels of PbB than controls. CONCLUSIONS: These data do not support a role for cadmium in the onset or the growth of endometriotic diseases but suggest a possible relationship with lead.

Abstract: PURPOSE: The purpose of the present paper is to review cohort studies that examined the occurrence of prostate cancer in pesticide manufacturing workers in order to undertake a qualitative and quantitative evaluation of the risk as well as to assess the level of epidemiological evidence for each class of chemical compounds. METHODS: Following a systematic literature search, relative risk (RR) estimates for prostate cancer were extracted from 18 studies published between 1984 and 2004. All studies were summarised and evaluated for homogeneity and publication bias. As no significant heterogeneity was detected, combined RR estimators were calculated using a fixed effect model. Meta-analyses were performed both on the whole set of data and for each chemical class separately. RESULTS: The meta-rate ratio estimate for all studies was 1.28 [95% confidence interval (CI) 1.05-1.58]. After stratification by specific chemical class, consistent increases in the risk of prostate cancer were found in all groups but statistical significance was found only for accidental or non-accidental exposure to phenoxy herbicides contaminated with dioxins and furans. There was no obvious indication of publication bias. CONCLUSION: The overall meta-analysis provides additional quantitative evidence consistent with prior reviews focusing on other groups exposed to pesticides (farmers, pesticide applicators). The results again point to occupational exposure to pesticides as a possible risk factor for prostate cancer but the question of causality remains unanswered. Epidemiological evidence did not allow identifying a specific pesticide or chemical class that would be responsible for the increased risk but the strongest evidence comes from workers exposed to phenoxy herbicides possibly in relation with dioxin and/or furan contamination.

Abstract: Genetic polymorphisms in biotransformation enzyme CYP3A5 (6986G > A, CYP3A5*3; 14690A > G, CYP3A5*6) and drug transporter ABCB1 (1236C > T; 2677G > T/A; 3435C > T) are known to influence tacrolimus (Tac) dose requirements and trough blood levels in stable transplant patients. In a group of 19 volunteers selected with relevant genotypes among a list of 221 adult renal transplant candidates, we evaluated whether consideration of CYP3A5 and ABCB1 genetic polymorphisms could explain the interindividual variability in Tac pharmacokinetics after the first administration of a standard dose (0.1 mg/kg body weight twice a day). Lower area under the time versus blood concentration curves (AUC) or lower trough concentrations were observed among CYP3A5 expressors (n = 9) than among nonexpressors (n = 10) using two different analytical methods for Tac determination (liquid chromatography with tandem mass spectrometry (LC-MS/MS) and immunoassay). The median AUC(0-infinity) was 2.6- and 2.1-fold higher in nonexpressors for LC-MS/MS and immunologic methods, respectively. No difference was observed in Tac pharmacokinetic parameters in relation to ABCB1 polymorphisms. In conclusion, our study confirms the very significant effect of CYP3A5 polymorphism early after the first administration of Tac. It also provides a strong argument for a doubling of the loading dose in patients early identified a priori on the transplantation list as possessing at least one CYP3A5*1 allele.

Abstract: BACKGROUND: Inflammation plays a critical role in lung disease development and progression in cystic fibrosis. Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are poorly understood. We tested the hypothesis that azithromycin modulates lung inflammation in cystic fibrosis mice. METHODS: We monitored cellular and molecular inflammatory markers in lungs of cystic fibrosis mutant mice homozygous for the DeltaF508 mutation and their littermate controls, either in baseline conditions or after induction of acute inflammation by intratracheal instillation of lipopolysaccharide from Pseudomonas aeruginosa, which would be independent of interactions of bacteria with epithelial cells. The effect of azithromycin pretreatment (10 mg/kg/day) given by oral administration for 4 weeks was evaluated. RESULTS: In naive cystic fibrosis mice, a spontaneous lung inflammation was observed, characterized by macrophage and neutrophil infiltration, and increased intra-luminal content of the pro-inflammatory cytokine macrophage inflammatory protein-2. After induced inflammation, cystic fibrosis mice combined exaggerated cellular infiltration and lower anti-inflammatory interleukin-10 production. In cystic fibrosis mice, azithromycin attenuated cellular infiltration in both baseline and induced inflammatory condition, and inhibited cytokine (tumor necrosis factor-alpha and macrophage inflammatory protein-2) release in lipopolysaccharide-induced inflammation. CONCLUSION: Our findings further support the concept that inflammatory responses are upregulated in cystic fibrosis. Azithromycin reduces some lung inflammation outcome measures in cystic fibrosis mice. We postulate that some of the benefits of azithromycin treatment in cystic fibrosis patients are due to modulation of lung inflammation.

Abstract: BACKGROUND: Cytochrome P450 3A5 (CYP3A5) and ABCB1 polymorphisms have been shown to influence tacrolimus (Tc) blood concentrations in the stable phase after organ transplantation. We hypothesized that Tc pharmacokinetics may be affected by genetic mutations subsequent to starting doses. METHODS: We retrospectively analyzed data from a cohort of 59 kidney transplant recipients, in whom CYP3A5 (intron 3) and ABCB1 (exons 12, 21 and 26) genotypes were correlated to dose- and weight-standardized Tc trough concentrations obtained after initial Tc doses. Renal function, expressed as glomerular filtration rate (GFR) (MDRD equation), on days 7 and 14 after transplantation was evaluated and its relationship with Tc concentrations was analyzed. RESULTS: Dose- and weight-standardized Tc trough concentrations were lower in patients carrying the CYP3A5 *1 allele (p<0.01). There was no statistically significant association with ABCB1 polymorphisms. In a multivariate analysis, both the presence of at least one CYP3A5 *1 allele (p=0.006) and age at the time of transplantation (p=0.010) were significant independent variables affecting Tc trough blood concentrations standardized to the first dosages (model r2=0.23). GFR was not affected by Tc concentrations. CONCLUSIONS: Prospective trials are needed to prove that a genetic approach to Tc pharmacokinetics and its related side effects during the early period after grafting may improve patient outcome.

Abstract: Dioxin-like compounds (DLCs) are suspected etiological factors of endometriosis but their potential mechanisms of action remain elusive. Because endometriosis is an estrogen-dependent disease and since aromatase (CYP19), a key enzyme in estrogen biosynthesis, was recently demonstrated to be expressed in endometriotic lesions, we hypothesized that dioxin-like compounds could modulate local estrogen production through an up-regulation of aromatase. We tested this hypothesis by examining the correlation between serum DLC levels and CYP19 expression in endometriotic tissue obtained from 47 patients with peritoneal, ovarian endometriosis and/or deep endometriotic nodules of the rectovaginal septum. Aromatase expression was assessed by real-time RT-PCR in biopsied endometriotic tissues [peritoneal (n=19), ovarian (n=17) endometriosis and deep endometriotic nodules of the recto-vaginal septum (n=29)]. The relationship between aromatase expression and DLCs was traced by simple regression analysis. DLCs did not appear to be significant determinants of aromatase expression. CYP1A1 expression, measured as a positive control, was found associated with current smoking but not with DLCs. We conclude that DLCs do probably not facilitate the growth of endometriotic lesions by up-regulating the local expression of aromatase.

Abstract: OBJECTIVE: Interpretation of urinary arsenic measurements is sometimes difficult because of the absorption of seafood that contains trimethylated arsenic forms (arsenobetaine and arsenocholine). The objective of this study was to develop a rapid and robust technique for the measurement of the sum of inorganic arsenic metabolites. METHODS: Measurement of arsenic was performed in urine after hydride generation in acid medium. Using atomic fluorescence spectrometry (AFS) as the detection system, we developed a rapid (one determination in less than 2 min) technique using 50 microl urine without pre-treatment. Standardisation was done externally with a mixed standard solution containing inorganic trivalent arsenic (As(i) (III)), inorganic pentavalent arsenic (As(i) (V)), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) (15/15/12.5/57.5). RESULTS: Samples distributed in the frame of an international comparison programme were used to assess accuracy of the AFS procedure, which gives a linear response up to 50 microg/l and proves more precise [coefficient of variation (CV)< 4% at 5 microg/l] and sensitive than the atomic absorption spectroscopy (AAS) technique using a quartz cell. An additional adaptation that allows the detection of non-directly reducible arsenic forms has also been validated for samples with high arsenic concentrations. CONCLUSIONS: The present study demonstrates the superiority of AFS over atomic absorption spectrometry (AAS) in arsenic determination and the interest of online mineralisation prior AFS detection for the determination of arsenic concentration in urine.

Abstract: Identification of genetic polymorphisms responsible for reduced DNA repair capacity may allow better cancer prevention. We examined whether variations in genes involved in base-excision (hOGG1, XRCC1) and double strand break (XRCC3) DNA repair contribute to inter-individual differences in genotoxic effects induced in the lymphocytes of 21 cobalt (Co) exposed, 26 hard metal (WC-Co) exposed and 26 matched control male workers. Genotyping was performed by PCR-RFLP. DNA single strand breaks and alkali-labile sites were measured by the alkaline Comet assay. Chromosomal rearrangements resulting from chromosome loss or acentric fragments were assessed as micronucleated mononucleates (MNMC) and binucleates (MNCB) with the cytokinesis-block micronucleus test. Urinary 8-hydroxydeoxyguanosine (8-OHdG) levels were used as an indicator of systemic oxidative DNA damage. A significantly higher frequency of MNMC was observed in WC-Co exposed workers with variant hOGG1(326) genotype. Multivariate analysis performed with genotypes, age, exposure status, type of plant, smoking and their interaction terms as independent variables indicated that MNMC and Comet tail DNA (TD) were influenced by genetic polymorphisms. In the exposed and total populations, workers variant for both XRCC3 and hOGG1 had elevated MNMC frequencies. Further studies will demonstrate whether genotyping for hOGG1 and XRCC3 polymorphisms is useful for a better individual monitoring of workers.

Abstract:

Abstract: Chronic inflammation and proinflammatory cytokines as well as T helper type 2 (Th2) cytokines have been involved in the pathogenesis of pulmonary injury and lung fibrosis. The actual role of IL-10 in lung fibrosis is still unclear because this cytokine has been identified as Th2 but possesses strong anti-inflammatory properties. To better dissect the potential role of IL-10 in silica-induced lung fibrosis, IL-10 was overexpressed in the lung of mice by adenoviral gene transfer during the inflammatory (administered at day -1) or the fibrotic (administered at day +30) stages of the disease. Pulmonary overexpression of IL-10 during both silica-induced lung inflammation and fibrosis exacerbated the fibrotic lesions as estimated by the measurement of hydroxyproline and other biochemical and histological markers. Increased expression of IL-10 significantly enhanced the number of lung lymphocytes and bronchoalveolar lavage fluid IgG1 but not IgG2a levels, indicating the induction of a Th2-like immune response. In addition, the production of the profibrotic Th2 cytokines IL-4 and IL-13 was also significantly increased upon IL-10 overexpression. No difference in transforming growth factor-beta or PGE(2) production was noted after adenoviral IL-10 treatment of silica-treated mice. Together, these data indicate that the increased expression of IL-10 significantly contributed to silica-induced lung fibrosis by exacerbating the Th2 response and the production of the profibrotic cytokines IL-4 and IL-13.

Abstract:

Abstract: The induction of cytochrome P4502E1 (CYP2E1) is believed to play a role in the development of fibrosis in hepatitis C patients. However, information about CYP2E1 activity in chronic hepatitis C patients is fragmentary and the relationship between CYP2E1 activity and mRNA expression is unknown in this disease. The purpose of this study was (a) to characterise CYP2E1 activity in those patients and (b) to analyse its relationship with CYP2E1 mRNA expression in the liver and in peripheral blood lymphocytes (PBLs), previously proposed as a surrogate to assess changes in CYP2E1 activity. Fourteen chronic hepatitis C patients were submitted to a routine transcutaneous liver biopsy. CYP2E1 activity was assessed by using chlorzoxazone (CZX) pharmacokinetic parameters and hepatic and PBLs CYP2E1 mRNA expression was measured by real-time RT-PCR. The mean oral clearance of CZX (CLT: 21.5+/-10.1L/h) was within the normal range and the chlorzoxazone metabolic ratio (CMR) at t = 2 h was closely related to other CZX pharmacokinetic parameters. None of the pharmacokinetic parameters did significantly correlate with CYP2E1 mRNA, neither in the liver nor in PBLs. Furthermore, there was no significant relationship between CYP2E1 mRNA levels in paired liver and PBL samples. Our data indicate that early stages of chronic hepatitis C are not associated with CYP2E1 induction. In this disease, the determination of the CMR at t = 2 h represents a reliable index to assess CYP2E1 activity. The measurement of CYP2E1 expression, at the mRNA level, in PBLs or in liver is not useful for that purpose.

Abstract: IL-9 is a Th2 cytokine that exerts pleiotropic activities, and might be involved in the regulation of lung inflammatory processes. To characterize the activity of IL-9 on lung injury, we compared the pulmonary responses to bleomycin (blm) in IL-9 transgenic (Tg5) and wild-type (FVB) mice. Following intratracheal instillation of lethal doses of blm, the mortality rate was markedly reduced in Tg5 mice compared to their wild-type counterparts (ie, 25% mortality for Tg5 versus 85% for FVB mice, 21 days after instillation of 0.05U blm/mouse). Histological and biochemical analyses showed that blm induced less lung injury and less epithelial damage in Tg5 as compared to FVB animals. This protection of Tg5 mice was accompanied by an expansion of eosinophils and B cells in the lungs. In addition, TGF-beta and prostaglandin-E2 (PGE2) levels in broncho-alveolar lavage fluid were also increased in transgenic mice. The contribution of B cells and eosinophils to the protective mechanism did not appear essential since eosinophil-deficient (IL-5 KO) and B-deficient (muMT) mice overexpressing IL-9 were also resistant to high doses of blm. We could rule out that TGF-beta was a key factor in the protective effect of IL-9 by blocking this mediator with neutralizing antibodies. Indomethacin treatment, which inhibited PGE2 production in both strains, suppressed the protection in Tg5 mice, supporting the idea that IL-9 controls blm-induced lung injury through a prostaglandin-dependent mechanism.

Abstract: A minireview is presented concerning the use of mercapturic acids as biological exposure index for electrophilic chemicals. Besides pure analytical aspects, this minireview considers possible issues in relation to (a) the added value of mercapturic acids as compared to other well validated biomarkers of exposure and (b) the high inter-individual variability in mercapturic acids excretion. Recent field and/or experimental studies confirm the usefulness of mercapturic acids as biological exposure index for electrophilic chemicals and suggest the interest of a toxicogenetic approach for a better interpretation of the results of biological monitoring.

Abstract: BACKGROUND: It has been generally well accepted that chronic inflammation is a necessary component of lung fibrosis but this concept has recently been challenged. METHODS: Using biochemical, histological, immunohistochemistry, and cellular analyses, we compared the lung responses (inflammation and fibrosis) to fibrogenic silica particles (2.5 and 25 mg/g lung) in Sprague-Dawley rats and NMRI mice. RESULTS: Rats treated with silica particles developed chronic and progressive inflammation accompanied by an overproduction of TNF-alpha as well as an intense lung fibrosis. Dexamethasone or pioglitazone limited the amplitude of the lung fibrotic reaction to silica in rats, supporting the paradigm that inflammation drives lung fibrosis. In striking contrast, in mice, silica induced only a limited and transient inflammation without TNF-alpha overproduction. However, mice developed lung fibrosis of a similar intensity than rats. The fibrotic response in mice was accompanied by a high expression of the anti-inflammatory and fibrotic cytokine IL-10 by silica-activated lung macrophages. In mice, IL-10 was induced only by fibrotic particles and significantly expressed in the lung of silica-sensitive but not silica-resistant strains of mice. Anti-inflammatory treatments did not control lung fibrosis in mice. CONCLUSION: These results indicate that, beside chronic lung inflammation, a pronounced anti-inflammatory reaction may also contribute to the extension of silica-induced lung fibrosis and represents an alternative pathway leading to lung fibrosis.

Abstract: BACKGROUND: CYP3A5 and MDR1 polymorphisms have been shown to influence tacrolimus blood concentrations and dose requirements. The aim is to determine whether these polymorphisms also affect sirolimus trough concentrations and dose requirements after kidney transplantation. METHODS: Eighty-five renal transplant recipients receiving sirolimus were included. Twenty-four were treated with a combined sirolimus-tacrolimus regimen. Eighty-one patients received steroids. Sirolimus and tacrolimus were adjusted to a target therapeutic window. CYP3A5 (intron 3) and MDR1 (exons 12, 21, 26) genotypes were correlated to the adjusted trough concentrations and dose requirements for both sirolimus and tacrolimus. RESULTS: There were no significant correlation between adjusted sirolimus trough concentrations or dose requirements and genetic polymorphisms. In a multiple regression model, adjusted-prednisone dose was involved with a positive or negative effect when considering sirolimus dose requirements or adjusted concentrations, respectively. In the subgroup of patients treated by tacrolimus and sirolimus, adjusted tacrolimus doses were higher in patients carrying at least one CYP3A5 *1 allele (median 0.083 vs. 0.035 mg/kg for CYP3A5*3/*3 patients, P<0.05). Adjusted-prednisolone dose and CYP3A5 polymorphism explained up to 61% of the variability in tacrolimus dose requirements. CONCLUSIONS: Unlike tacrolimus, sirolimus adjusted trough concentrations and dose requirements seem not affected by CYP3A5 and MDR1 polymorphisms. Adjusted-prednisone dose has a significant impact on tacrolimus and sirolimus dose requirements.

Abstract: OBJECTIVE: To investigate the possible association between the body burden of dioxin-like compounds and endometriotic disease. DESIGN: Case-control study. SETTING: Gynecology ward in a university hospital. PATIENT(S): Seventy-one women with peritoneal endometriosis (n = 25) or deep endometriotic (adenomyotic) nodules (n = 25) and controls (n = 21). INTERVENTION(S): Collection of 200 mL of blood (fasted) and face-to-face interview. MAIN OUTCOME MEASURE(S): Assessment of dioxin (PCDD), furan (PCDF), and dioxin-like PCB serum concentrations (picograms toxic equivalent [TEQ]/g lipids). RESULT(S): Age and body mass index were traced by linear multiple regression as determinants of total TEQ levels. After standardization for these variables (30 years and 22.5 kg/m2), the mean TEQ levels were 24.21 (controls), 30.62 (peritoneal endometriosis), and 37.60 (deep endometriotic [adenomyotic] nodules) pg TEQ/g lipids. Logistic regression analysis indicated a significantly increased risk of deep endometriotic (adenomyotic) nodules (odds ratio [OR], 3.3; 95% confidence interval [CI], 1.4-7.6) for an increment of 10 pg in total TEQ levels/g lipids. An increased risk was also found for peritoneal endometriosis (OR, 1.9; 95% CI, 0.9-3.8) for total TEQ levels and for dioxins alone (OR, 3.2; 95% CI, 1.0-9.9). CONCLUSION(S): The results provide the first epidemiological evidence of an association between increased PCDD/PCDF and PCB body burden and endometriosis.

Abstract: Carbon nanotubes focus the attention of many scientists because of their huge potential of industrial applications, but there is a paucity of information on the toxicological properties of this material. The aim of this experimental study was to characterize the biological reactivity of purified multi-wall carbon nanotubes in the rat lung and in vitro. Multi-wall carbon nanotubes (CNT) or ground CNT were administered intratracheally (0.5, 2 or 5 mg) to Sprague-Dawley rats and we estimated lung persistence, inflammation and fibrosis biochemically and histologically. CNT and ground CNT were still present in the lung after 60 days (80% and 40% of the lowest dose) and both induced inflammatory and fibrotic reactions. At 2 months, pulmonary lesions induced by CNT were characterized by the formation of collagen-rich granulomas protruding in the bronchial lumen, in association with alveolitis in the surrounding tissues. These lesions were caused by the accumulation of large CNT agglomerates in the airways. Ground CNT were better dispersed in the lung parenchyma and also induced inflammatory and fibrotic responses. Both CNT and ground CNT stimulated the production of TNF-alpha in the lung of treated animals. In vitro, ground CNT induced the overproduction of TNF-alpha by macrophages. These results suggest that carbon nanotubes are potentially toxic to humans and that strict industrial hygiene measures should to be taken to limit exposure during their manipulation.

Abstract: Toxic causes of seizures are numerous: alcohol and other substances of abuse, drugs, and industrial and household products. However, in the absence of a clearly suggestive history and/or associated symptoms and signs, identification of the toxic origin of new-onset seizures may be extremely difficult. We report here the case of a patient admitted in our hospital after a single generalized tonic-clonic seizure. The remarkable coincidence that a colleague of his, with whom he was working to clean the same workshop, had been admitted 1 week earlier for respiratory distress, coma, and de novo nonconvulsive focal status epilepticus, led us to consider a possible toxicologic etiology. Urine analysis revealed a high nickel concentration, suggestive of acute nickel poisoning.

Abstract: BACKGROUND: Lung fibrosis is characterized by tissue remodeling resulting from an imbalance between synthesis and degradation of extracellular organic matrices. To examine whether cathepsin(s) (Cat) are important in the development of pulmonary fibrosis, we assessed the expression of four Cat known for their collagenolytic activity in a model of silica-induced lung fibrosis. METHODS: Different strains of mice were transorally instilled with 2.5 mg crystalline silica or other particles. Cat expression (Cat K, S, L and B) was quantified in lung tissue and isolated pulmonary cells by quantitative RT-PCR. In vitro, we assessed the effect of different cytokines, involved in lung inflammatory and fibrotic responses, on the expression of Cat K by alveolar macrophages and fibroblasts. RESULTS: In lung tissue, Cat K transcript was the most strongly upregulated in response to silica, and this upregulation was intimately related to the fibrotic process. In mouse strains known for their differential response to silica, we showed that the level of Cat K expression following silica treatment was inversely related to the level of TGF-beta expression and the susceptibility of these strains to develop fibrosis. Pulmonary macrophages and fibroblasts were identified as Cat K overproducing cells in the lung of silicotic mice. In vitro, Cat K was downregulated in mouse and human lung fibroblasts by the profibrotic growth factor TGF-beta1. CONCLUSION: Altogether, these data suggest that while Cat K may contribute to control lung fibrosis, TGF-beta appears to limit its overexpression in response to silica particles.

Abstract: We previously described a reduction of silica-induced lung fibrosis in interleukin-10-deficient mice (IL-10-/-) (Huaux and colleagues; Am. J. Respir. Cell Mol. Biol. 1998;18:51-59). In the present study, we further dissect the exact functions of IL-10 in experimental silicosis. The reduced lung fibrotic response to silica in IL-10-/- mice was accompanied by a marked recruitment of TH1 CD4+ lymphocytes. However, treatment with anti-CD4 antibodies reduced silica-induced lung fibrosis in both IL-10-/- and IL-10+/+ mice, suggesting that this T cell population actually contributes to the extension of the fibrotic lesions in a manner that is independent of IL-10. In IL-10-/- mice, silica-induced lung production of the profibrotic mediator transforming growth factor (TGF)-beta1 and the antifibrotic eicosanoid PGE2 were reduced and increased, respectively, relative to that in IL-10+/+ mice. In addition, in vitro experiments indicated that recombinant IL-10 upregulated TGF-beta1 expression in alveolar macrophages while in contrast it downregulated PGE2 production and cyclooxygenase-2 expression in both lung fibroblasts and macrophages. Thus the net profibrotic activity of IL-10 in vivo appears to be mediated by its ability to stimulate the expression of the profibrotic cytokine TGF-beta1 while suppressing the expression of cyclooxygenase-2 and thus production of the antifibrotic eicosanoid PGE2. These effects appear to be independent of the enhanced lung CD4+ T-lymphocytosis observed in IL-10-deficient mice.

Abstract: PURPOSE: The purpose of this study was to determine the immunization efficacy of antigen and DNA vaccines after delivery to the lung, to assess the integrity of the pulmonary tissue after vaccination, and to elucidate mechanisms involved in the induction of immunity. METHODS: Ovalbumin, the plasmid encoding ovalbumin, the hepatitis B surface antigen (HBsAg), or plasmid encoding HBsAg were intratracheally instilled or injected in quadriceps in mice. The immune response and its Th polarization were analyzed over time. Markers of inflammation were measured in bronchoalveolar lavage, and lung histology was performed. The fate of ovalbumin following intratracheal instillation was studied. RESULTS: According to the vaccine, the pulmonary route produced stronger or equivalent humoral and cellular responses systemically and locally in the lung as compared to injection. The IgG subclasses and cytokine pattern indicate that the immunity was preferentially polarized toward the Th2 and Th1 type for antigen and DNA immunization, respectively. Ovalbumin penetrated the respiratory tissue and blood poorly after intratracheal instillation, suggesting that the immune response was triggered at airway surfaces. Overall, vaccines delivered to the lung did not induce any local sign of inflammation. CONCLUSIONS: Pulmonary administration of vaccines might be a promising alternative to conventional vaccination by injection.

Abstract: The sum of bulk polychlorobiphenyl levels was significantly higher in women with rectovaginal adenomyosis than in women with endometriosis and controls.

Abstract: The objective of the study was to examine the influence of cobalt exposure on lung function changes in workers from a cobalt-producing plant in a health monitoring program implemented between 1988 and 2001. A total of 122 male workers with at least 4 (median = 6) lung function tests (FEV(1) and FVC) during the follow-up period were assessed longitudinally. Cobalt exposure significantly decreased over the follow-up period, as reflected by the measurements in air and urine. The possible association of spirometric changes with cobalt exposure was examined by a random coefficients model, taking into account other potential influential variables, such as smoking, age, previous respiratory illness, exposure to other lung toxicants, or the presence of glutamate in position 69 in the HLA-DP beta-chain, an HLA polymorphism possibly associated with hard-metal-induced lung diseases. The main finding of the follow-up study was that cobalt exposure contributed to a decline in FEV(1) over time, and only in association with smoking. No influence of glutamate in position 69 in the HLA-DP beta-chain polymorphism was detected. Although the amplitude of the additional FEV(1) decrement associated with smoking exposure was relatively small (< 20%) compared with the expected decline in a non-cobalt-exposed smoker, the results indicate that further efforts to reduce cobalt exposure and to encourage workers to quit smoking are still warranted.

Abstract: The present study aimed at comparing in vitro the apoptogenic properties of metallic cobalt (Co), tungsten carbide (WC) and tungsten carbide-cobalt (WC-Co) in conditions known to cause genotoxicity. Human peripheral blood mononucleated cells were incubated with 2.0-6.0 microg/ml of Co alone or mixed with WC particles and 33.3-100.0 microg/ml WC alone for up to 24 h. Under these culture conditions the majority (60%) of the cobalt metal particles were almost immediately solubilised in the culture medium, while WC remained under the form of particles that were progressively phagocytosed by monocytes. Apoptosis was assessed by Annexin-V staining, flow cytometry and analysis of DNA fragmentation by ELISA. Metallic Co-particles induced apoptosis in vitro. Furthermore, although so far considered as biologically inert, WC particles also induced apoptosis. When compared with its individual components WC-Co displayed an additive apoptotic effect in the DNA fragmentation assay. Apoptosis induced by WC particles was found largely dependent on caspase-9 activation and occurred presumably in monocytes, while that induced by Co involved both caspase-9 and -8 activation. The data suggest that apoptosis induced by the tested WC-Co mixture results from the additive effects of WC apoptosis induced in monocytes and Co-specific apoptosis in both monocytes and lymphocytes. The apoptogenic properties of these metals may be important in the mechanism of lung pathologies induced by the cobalt-containing particles.

Abstract: Cyclosporine and tacrolimus are immunosuppressive drugs largely used in renal transplantation. They are characterized by a wide inter-individual variability in their pharmacokinetics with a potential impact on their therapeutic efficacy or induced toxicity. CYP3A5 and P-glycoprotein appear as important determinants of the metabolism of these drugs. The objective of this study was to investigate the effect of CYP3A5 and MDR1 (ABCB1) polymorphisms on cyclosporine and tacrolimus dose requirements and trough blood concentrations in stable transplant patients. Stable renal transplant recipients receiving cyclosporine (n = 50) or tacrolimus (n = 50) were genotyped for CYP3A5*3 and *6, and MDR1 C1236T, G2677T/A and C3435T. Dose-adjusted trough blood levels (ng/ml per mg/kg body weight) as well as doses (mg/kg body weight) required to achieve target blood concentrations were compared among patients according to allelic status for CYP3A5 and MDR1. Dose-adjusted trough concentrations were three-fold and 1.6-fold higher in CYP3A5*3/*3 patients than in CYP3A5*1/*3 patients for tacrolimus and cyclosporine, respectively. In the case of tacrolimus, the difference was even more striking when considering CYP3A5*1/*1 patients showing dose-adjusted trough concentrations 5.8-fold lower than CYP3A5*3/*3 patients. For both drugs, no association was found between trough blood concentrations or dose requirement and MDR1 genotype. Multiple regression analyses showed that CYP3A5*1/*3 polymorphism explained up to 45% of the variability in dose requirement in relation to tacrolimus use. Given the importance of rapidly achieving target blood concentrations after transplantation, further prospective studies should consider the immediate post-graft period and assess the influence of this specific polymorphism. Beside non-genetic factors (e.g. steroids dosing, drugs interactions), CYP3A5 pharmacogenetic testing performed just before transplantation could contribute to a better individualization of immunosuppressive therapy.

Abstract: Environmental chemicals with oestrogen-like activity are suspected aetiological factors of endometriosis. In animal experiments, cadmium was recently shown to possess oestrogen-like properties. In the frame of a case-control study designed to investigate environmental risk factors for endometriosis, we compared cadmium urinary excretion (CdU) and blood concentration in 59 patients with peritoneal endometriosis, deep endometriotic (adenomyotic) nodules of the recto-vaginal septum and controls. After standardisation for age (30 years) and smoking status, the mean levels of cadmium in urine were (geometric mean [geometric S.D.]) 0.25 [1.50], 0.29 [1.76] and 0.26 [1.46] microg/g creatinine, respectively. Cadmium concentrations in blood did not differ among the three groups. These data, therefore, do not support a role for cadmium in the onset or the growth of endometriosis or deep endometriotic (adenomyotic) nodules of the recto-vaginal septum.

Abstract: A study on 44 workers exposed to styrene and 44 matched referents was performed in order to examine the influence of genetic polymorphisms in biotransformation and DNA repair enzymes on the levels of N-terminal hemoglobin adducts and genotoxicity biomarkers. Urinary mandelic acid concentration averaged 201.57 mg/g creatinine +/-148.32 in exposed workers, corresponding to a calculated average airborne styrene exposure of 9.5 ppm +/-9.6. Individuals with a high level of N-terminal valine adducts had higher levels of DNA damage, as evaluated by the Comet assay (r = 0.29, P = 0.008). Frequencies of micronucleated mononucleated lymphocytes (MNMC) (0.71 +/- 0.88 vs 0.11 +/- 0.20, P<0.0001), micronucleated binucleated lymphocytes (MNBC) (3.93 +/- 2.75 vs 2.65 +/- 1.94, p = 0.02) and micronucleated nasal epithelial cells (0.52 +/- 0.49 vs 0.23 +/- 0.31, p = 0.04) differed significantly between the exposed and referent groups. In the whole group of 88 individuals, higher frequencies of MNMC were found in individuals possessing the XRCC3 Met(241) allele and those individuals with the XRCC1 Gln( (399) ) allele showed higher frequencies of MNMC and MNCB. In vitro DNA repair capacity, as measured by residual DNA strand breaks in peripheral blood leukocytes after a styrene oxide challenge, was also influenced by styrene exposure, with an apparent induction of early repair mechanisms associated with the intensity of recent exposure and a reduction of late (24 h) repair capacity that was associated with the duration of employment. After 1 h of repair, lower levels of residual DNA damage were found in individuals possessing GSTT1 (P = 0.043). After 24 h of repair, lower residual DNA damage was found in individuals homozygous for XRCC1 Arg(194) (P = 0.013). Multivariate regression analysis indicated that the duration of exposure, smoking habits and polymorphisms of XRCC1 at codon 399 were important variables affecting the genotoxic responses. Our data suggest that DNA damage is formed in workers exposed to low concentrations of styrene, and that genotypes of metabolising and DNA-repair genes are important for the assessment of individual genotoxic risk to styrene. The in vitro DNA repair phenotype assay might be a valuable method to estimate the susceptibility of workers.

Abstract: Macrophages are characterized by a marked phenotypic heterogeneity depending on their microenvironmental stimulation. Beside classical activation (M1), it has been shown that macrophages could follow a different activation pathway after stimulation with interleukin (IL)-4 or IL-13 (M2). Recently, it has been postulated that those "alternatively activated" macrophages may be critical in the control of fibrogenesis. In an experimental model of silicosis, where pulmonary macrophages play a central role, we addressed the question of whether lung fibrosis development would be associated with alternative macrophage activation. As available markers for alternative macrophage activation, type-1 arginase (Arg-1), Fizz1, Ym1/2, and mannose receptor expression were evaluated at the mRNA and/or protein levels at different stages of the disease. Nitric oxide synthase-2 (NOS-2) expression was also examined to investigate the classical counterpart. We found that the expression of Arg-1, Fizz1, and NOS-2 in adherent bronchoalveolar lavage cells was highly up-regulated 3 days after silica administration but returned to control levels during the fibrotic stage of the disease (60 days). By comparing the early response to silica in C57BL/6 and BALB/c mice, we observed that the amplitude of Arg-1 mRNA up-regulation was not associated with the severity of lung fibrosis. Using a model of manganese dioxide particles (resolutive alveolitis), we showed that this early Arg-1 mRNA was not specific to a fibrogenic lung response. Our data indicate that the modifications of M1/M2 marker expression are limited to the early inflammatory stage of silicosis and that the establishment of a fibrotic process is not necessarily associated with M2 polarization.

Abstract: OBJECTIVE: In mice, the renal toxicity of arsenic (As) and cadmium (Cd) has been shown to be exacerbated by the simultaneous administration of both elements. To verify the existence of such an interaction in humans, cohorts slightly (Belgian) and moderately (Chinese) exposed to both elements were examined. METHODS: Biological indicators of exposure (Cd in urine and in blood; As in urine) and renal effect parameters (retinol binding protein (RBP); beta(2)-microglobulin (beta(2)M); albumin (ALB); N-acetyl-beta-glucosaminidase (NAG) in urine) were determined and their relationships studied by multiple regression analyses. RESULTS: Changes in renal effect parameters could be ascribed to Cd body burden. RBP and beta(2)M urinary concentration were influenced by exposure to As only in Chinese women, directly and in interaction with Cd exposure. CONCLUSION: A synergistic action of As on the tubular effects of Cd is observed in women moderately exposed to these elements and leads to RBP urinary excretion slightly above normal values.

Abstract: The aim of this work was to prepare and characterize inhalation dry powders of human parathyroid hormone (PTH), as well as to assess their efficacy for systemic delivery of the peptide and safety in rats. The powders were prepared by spray-drying using PTH, sugars, dipalmitoylphosphatidylcholine, and/or albumin. They presented an average primary particle diameter of 4.5 microm and tap density of 0.06 g/cm(3), a mass median aerodynamic diameter between 3.9 and 5.9 microm, and reached up to 98% emitted dose and up to 61% fine particle fraction in the multi-stage liquid impinger using a Spinhaler inhaler device. Varying the airflow rate from 30 to 100 L/min had limited influence on the aerodynamic behavior of the aerosols. The absolute PTH bioavailability was 21% after intratracheal administration of the powder formed of PTH/albumin/lactose/dipalmitoylphosphatidylcholine and 18% after subcutaneous injection in rats. Equilibrium dialysis revealed a 78% binding of PTH to albumin and the withdrawal of albumin from the powder increased absolute bioavailability after inhalation from 21 to 34%. No acute inflammation appeared in the lung up to 48 h after a single inhalation. The increased bioavailability of the optimized powder aerosol of PTH makes it a promising alternative to subcutaneous injection.

Abstract: Chromium-based catalysts are used for the synthesis of polyethylene, but little is known about the hazard and biomonitoring possibilities of this type of chromium for workers who may be occupationally exposed to such compounds. Therefore, the bioavailability and toxicokinetics of chromium were studied in male Wistar rats after a single intratracheal instillation (2 ml/kg body weight) of various doses (1, 5, or 25 mg/kg body weight) of the catalyst (approximately 1% chromium bound to an amorphous silica matrix), either before (CAT-Cr[III]) or after (CAT-Cr[VI]) heat treatment. The results were compared with those of equivalent amounts of two chromium salts (CrCl(3) and K(2) Cr(2) O (7). Each dose group was composed of three rats. The concentration of chromium was determined by atomic absorption spectrometry in urine (collected daily for 7 d) and in plasma, erythrocytes, lung, and liver tissue obtained 2 d (only highest concentration) and 7 d after dosing. On d 2, a significant increase in lung weight was found in the animals treated with the highest dose of the hexavalent Cr products. On d 7, on the basis of body weights, lung weights, and lung histology, there was no overt toxicity, except after the highest dose of CAT-Cr(VI). The elimination of all forms of chromium was apparently monoexponential, with calculated half-life elimination times in urine of 4-11 h for Cr(III) (CAT-Cr[III] and CrCl3 ) and 8-21 h for Cr(VI) (CAT-Cr[VI] and K(2) Cr(2) O(7). On d 2, the erythro-cytes Cr concentrations were significantly higher for the hexavalent Cr products than for the trivalent Cr products. After 7 d, the erythrocytes Cr concentrations were significantly increased above control values (3 microg/L) only in rats treated with the 2 highest doses of Cr( VI) compounds (12 and 64 microg/L for K(2) Cr(2) O(7), and 14 and 79 microg/L for CAT-Cr[VI]). The present study shows that intratracheally instilled Cr(VI) and Cr(III) have different toxicokinetic profiles and that the Cr(VI) catalyst has the same bioavailability and excretion kinetics as a water-soluble Cr(VI) salt. Exposure to chromium compounds could be monitored by measuring Cr concen-trations in urine (shortly after exposure) and in erythrocytes (also at later time points after high Cr[VI] exposure).

Abstract: The involvement of cytochrome P450 2E1 (CYP2E1) in the metabolism of N-methyl-2-pyrrolidone (NMP) was studied with three experimental approaches: in the rat, in vitro in human microsomes, and in human volunteers. NMP was administered dermally (40 mg/kg) to OFA rats to examine the influence of CYP2E1 inhibition (5 mg/kg diethyldithiocarbamate, DETC, 30 min before) and CYP2E1 induction (after 4 days of fasting). The main NMP metabolite 5-hydroxy- N-methylpyrrolidone (5HNMP) in the urine fractions collected during the following 48 h was analysed by gas chromatography-mass spectrometry. CYP2E1 inhibition led to a statistically significant retardation of 5HNMP excretion in urinary fractions collected during the first 12 h. In the group of fasted rats, a two-fold increase of CYP2E1 activity was observed in comparison with the control group. During the first 6 h after dermal administration of NMP to fasted rats, about 33% of the dose was excreted in urine versus 22% in controls. In vitro, NMP (15 mM) was incubated (up to120 min) with human liver microsomes and the formation of 5HNMP followed Michaelis-Menten kinetics with V(max) of 1.1 nmol/min per mg protein and K(m) of 2.4 mM. The formation of 5HNMP was inhibited by 35% in the presence of a monoclonal antibody against CYP2E1, but not by CYP1A2 antibody. In a dermal application experiment, 12 humans volunteers were exposed by means of a dermal patch to 300 mg NMP; five urine fractions were collected during the 48 h following the onset of application in order to measure the major metabolites 5HNMP and 2-hydroxymethylsuccinimide (2HMSI). Before NMP application, a blood sample was collected for the quantification of CYP2E1 mRNA in peripheral blood lymphocytes (PBLs). The mean dermal absorption of NMP was 67.9%. The highest amount of 5HNMP was excreted in urine in the fraction collected between 6-12 h (12.6% of dose), while 2HMSI peaked in fractions 12-24 h and 36-48 h (3.3 and 3.2% of dose, respectively). A significant relationship was found between CYP2E1 mRNA content in PBLs and the amount of both the metabolites excreted in urine within 24 h ( r(2)=0.54, P<0.01). It is concluded that CYP2E1 is involved in the first steps of NMP metabolism in the rat and, to a lesser extent, in humans. Since large variations in CYP2E1 activity exist in the human population (at least 5-fold range), it seems justified to take into account the activity of this enzyme in an individual for an accurate interpretation of biological monitoring of exposure to NMP when relying on 5HNMP and/or 2HMSI determination in urine.

Abstract: Occupational exposure to hard metal dust, consisting of tungsten carbide (WC) and metallic cobalt particles (Co), is associated with an increased risk of lung cancer, while no increased risk was observed in workers exposed to Co alone. In vitro, in human peripheral blood mononucleated cells (PBMC), we previously demonstrated that WC-Co is more genotoxic than Co and WC alone. A possible mechanism underlying this higher genotoxicity is a specific physicochemical interaction between Co and WC particles leading to the enhanced short-term formation of active oxygen species. The aim of this study was to evaluate the in vitro genotoxicity of other combinations of Co with metal carbide particles in comparison with WC-Co. The ability of Cr(3)C(2), Mo(2)C and NbC and of their powder mixtures with Co to induce DNA strand breaks and alkali-labile sites was assessed by the alkaline Comet assay and their potential to induce chromosome(/genome) mutations by the cytokinesis-block micronucleus test on human PBMC from two donors. PBMC were treated in vitro for 15 min, 24 h after the onset of PHA stimulation. In the micronucleus test, while the metal carbides alone did not increase the micronucleus frequency, Co alone and the four tested carbide-Co mixtures induced a statistically significant concentration-dependent increase in micronucleated binucleates. In addition to WC, NbC and Cr(3)C(2) particles were able to interact with Co, producing a higher mutagenic effect than the individual metal particles. Mo(2)C particles did not display interactive mutagenicity with Co in the micronucleus test, possibly related to their small specific surface area, compactness and/or spherical shape. With the Comet assay, applied directly at the end of the treatment, less clear results, due to inter-experimental and inter-donor variation, were obtained. These data indicate that particular interaction of a metal carbide with Co leading to enhanced mutagenicity is not specific for WC.

Abstract: Cadmium (Cd) and its compounds were classified as "carcinogenic to humans (Group 1)" by IARC in 1993. The observation of an increased number of lung cancers in a U.S. cohort of cadmium-exposed workers and the finding of tumors in animals exposed to various cadmium compounds apparently played an important role in this assessment. Since this evaluation, several cohorts of cadmium exposed workers have been updated and some additional data regarding environmental exposure to cadmium and cancer risk have been published. The main purpose of this systematic review was to examine whether inclusion of the studies that were not available for the 1993 evaluation might change the overall assessment of the carcinogenic potential of cadmium compounds. A second objective was to examine whether the recent studies are qualitatively better than the older ones and whether they should receive more weight in this assessment. A third issue was to investigate whether a competing effect between nonmalignant respiratory disease (NMRD) and lung cancer may have affected the results for lung cancer in occupationally exposed cohorts. Overall, considering the results of the most recent studies does not suggest that the effect of cadmium on lung cancer increases with improvement of the study design but points to a lower relative risk in the groups exposed to cadmium in the absence of arsenic and nickel. No evidence was found to support the hypothesis that NMRD represents a competing cause of death reducing the mortality from lung cancer. The association between cadmium exposure and prostate cancer was not confirmed in the latest available updates. Studies in environmentally exposed populations do not indicate an increased relative risk of cancer.

Abstract: The purpose of this review is to summarise the data concerning genotoxicity and carcinogenicity of Co and Sb. Both metals have multiple industrial and/or therapeutical applications, depending on the considered species. Cobalt is used for the production of alloys and hard metal (cemented carbide), diamond polishing, drying agents, pigments and catalysts. Occupational exposure to cobalt may result in adverse health effects in different organs or tissues. Antimony trioxide is primarily used as a flame retardant in rubber, plastics, pigments, adhesives, textiles, and paper. Antimony potassium tartrate has been used worldwide as an anti-shistosomal drug. Pentavalent antimony compounds have been used for the treatment of leishmaniasis. Co(II) ions are genotoxic in vitro and in vivo, and carcinogenic in rodents. Co metal is genotoxic in vitro. Hard metal dust, of which occupational exposure is linked to an increased lung cancer risk, is proven to be genotoxic in vitro and in vivo. Possibly, production of active oxygen species and/or DNA repair inhibition are mechanisms involved. Given the recently provided proof for in vitro and in vivo genotoxic potential of hard metal dust, the mechanistic evidence of elevated production of active oxygen species and the epidemiological data on increased cancer risk, it may be advisable to consider the possibility of a new evaluation by IARC. Both trivalent and pentavalent antimony compounds are generally negative in non-mammalian genotoxicity tests, while mammalian test systems usually give positive results for Sb(III) and negative results for Sb(V) compounds. Assessment of the in vivo potential of Sb2O3 to induce chromosome aberrations (CA) gave conflicting results. Animal carcinogenicity data were concluded sufficient for Sb2O3 by IARC. Human carcinogenicity data is difficult to evaluate given the frequent co-exposure to arsenic. Possible mechanisms of action, including potential to produce active oxygen species and to interfere with DNA repair systems, still need further investigation.

Abstract: OBJECTIVE: Cytochrome P4502E1 (CYP2E1) is expressed in human peripheral blood lymphocytes (PBLs), and previous reports have suggested the possibility of using this readily available tissue as a reporter of CYP2E1 status. To further explore the relevance of this approach we assessed CYP2E1 expression in PBLs in two contrasting conditions, chronic hepatitis C and insulin-dependent diabetes (IDD), illustrating an organ and a systemic disease, respectively. METHODS: Total RNA was isolated from extracted PBLs (hepatitis C patients + IDD) and by percutaneous needle biopsy (hepatitis C patients only). Gene expression for CYP2E1 was determined by real-time reverse-transcription polymerase chain reaction. Histological changes in liver tissue were assessed according to Ludwig's criteria. RESULTS: In patients with chronic hepatitis C a clear relationship was found between CYP2E1 expression in the liver and the progression of hepatic disease (both lobular inflammation and fibrosis indices), and observed variations were consistent with the preferential distribution of CYP2E1 in the lobular zone. No effect of the liver disease was, however, found at the PBL level. A statistically significant increase in mean CYP2E1 expression level was observed in the lymphocytes from poorly controlled IDD subjects compared to controls. CONCLUSIONS: Taken together, our data indicate that the measurement of CYP2E1 expression in PBLs is not useful in liver diseases. However, in a systemic condition (IDD) this measurement can be proposed for monitoring the CYP2E1 induction in a relatively noninvasive manner. This tool should therefore be further validated in clinical field or experimental studies for CYP2E1 phenotyping purposes.

Abstract: This study investigated cytokine protein levels (interleukin-12 p70 [IL-12p70], p40, and IL-10) in bronchoalveolar lavage fluid (BALF) from patients with pulmonary sarcoidosis (n = 59), healthy control subjects (n = 17), and patients with idiopathic pulmonary fibrosis (IPF) (n = 30). The relationship between cytokine levels and clinical course of sarcoidosis was also examined. Overall, p40 was far more abundant than IL-12p70. p40 levels (pg/ml, mean +/- SEM) were significantly higher in the BALF from patients with sarcoidosis (2.97 +/- 3.69) than in IPF patients (0.83 +/- 1.57) and healthy subjects (0.78 +/- 1.00). Size exclusion chromatography indicated that p40 detected in BALF from sarcoidosis patients corresponded to p40 monomers or (p40)(2) homodimers. Further, p40 levels were associated with (paralleled) the clinical course of sarcoidosis, with the highest levels detected in BALF from patients with persistent disease. Higher p40 levels were also found in the BALF from sarcoid patients who required corticosteroid treatment compared with patients with spontaneous regression (3.51 +/- 3.83 vs. 2.01 +/- 3.43, p = 0.03). IL-10 concentrations paralleled p40 changes. No similar association was found for IL-12p70 levels. In conclusion, this report shows that the BALF from patients with sarcoidosis contains elevated levels of p40, (p40)(2), and IL-10 protein but not of IL-12p70. The present data also suggest that BALF p40 concentrations may be indicative of the sarcoidosis clinical course.

Abstract:

Abstract: Inhalation of hard metal dust (WC-Co particles) has been associated with an increased risk for lung cancer in occupational settings. In vitro, WC-Co was genotoxic in human lymphocytes producing DNA strand breaks and micronuclei. The aim of the present study was to evaluate the in vivo genotoxic effects of WC-Co dust in rat type II pneumocytes. DNA breaks/alkali-labile sites (alkaline comet assay) and chromosome/genome mutations (micronucleus test) were assessed after a single intra-tracheal (i.t.) instillation of WC-Co, including dose-effect and time trend relationships. In addition, the alkaline comet assay was performed on cells obtained after broncho-alveolar lavage (BAL) and on peripheral blood mononucleated cells (PBMC). As pulmonary toxicity parameters, protein content, lactate dehydrogenase activity, total and differential cell count in BAL fluid were evaluated in parallel. In type II pneumocytes, WC-Co induced a statistically significant increase in tail DNA (12 h time point) and in micronuclei (72 h) after a single treatment with 16.6 mg WC-Co/kg body wt, a dose that produced mild pulmonary toxicity. This observation provides the first evidence of the in vivo mutagenic potential of hard metal dust. In PBMC, no increase in DNA damage or micronuclei was observed. This study indicates the potential to detect chromosome/genome mutations (micronuclei) in relevant target cells (type II pneumocytes) after i.t. instillation of a particle mixture.

Abstract: The present paper describes the outbreak of health complaints that occurred in Belgium, in June 1999, among schoolchildren and members of the general public in relation to the consumption of Coca-Cola and other soft drinks. The outbreak took place in the wake of a major food crisis, caused by PCB/dioxin contamination of animal feed, that had erupted shortly before. The clinical features (absence of serious poisoning) and epidemiological characteristics of the Coca-Cola outbreak pointed to mass sociogenic illness, and no subsequent toxicological or other data have refuted this hypothesis.

Abstract: The p40 subunit of IL-12 (IL-12p40), but not the heterodimeric form IL-12p70, is secreted during the development of silica-induced lung fibrosis in C57BL/6 mice. To delineate the contribution of IL-12p40 to the lung inflammatory and fibrotic processes, we compared the pulmonary responses with silica particles of IL-12p35-deficient mice (IL-12p35(-/-), able to produce IL-12p40) and IL-12p40-deficient mice (IL-12p40(-/-)). IL-12p35(-/-) and IL-12p40(-/-) animals developed strikingly contrasting responses to silica in comparison with wild-type C57BL/6 mice. Although the IL-12p40(-/-) mice exhibited limited inflammatory and fibrotic reactions, the IL-12p35(-/-) mice presented a robust and well-developed pulmonary inflammation and fibrosis. Furthermore, the silica-induced increase in lung IL-12p40 content was significantly higher in IL-12p35(-/-) mice than in wild-type controls, and was associated with extensive lung fibrosis and pulmonary macrophage infiltration. The contrasting responses observed between these two IL-12 subunit-deficient murine strains were not accompanied by a strict type 1 or type 2 polarization as estimated by the measurements of lung IFN-gamma/IgG2a and IL-4/IgG1 content. In vitro proliferation, type I collagen expression, as well as myofibroblast differentiation of purified pulmonary fibroblasts were not affected by treatment with exogenous rIL-12p40. In vivo, supplementation with rIL-12p40 restored the impaired pulmonary fibrotic response and macrophage accumulation in silica-treated IL-12p40(-/-) mice, and also promoted fibrosis and macrophage influx in wild-type mice. Together, our data suggest that IL-12p40 plays an important role in silica-induced pulmonary inflammation and fibrosis, possibly by exacerbating macrophage recruitment.

Abstract: OBJECTIVES: To analyse the relationship between cytochrome P4502E1 (CYP2E1) activity as assessed by the chlorzoxazone metabolic ratio (CMR) and frequent CYP2E1 genotypes ( CYP2E1*5B, *6, *1B and *1D) and to assess the value of CMR in refining the biomonitoring of exposure to styrene. METHODS: Thirty-one workers from a fibreglass-reinforced plastics factory took part in the study. Ambient styrene concentration was determined during the whole workshift by passive sampling. Each worker received a 500-mg chlorzoxazone (CZX) tablet at the beginning of the workday, and blood was taken after 2 h for CMR and CYP2E1 genotypes determination. Urine was collected at the end of the shift for the determination of styrene-specific metabolites. RESULTS: While the only worker heterozygous for CYP2E1*1D allele presented the highest value of CMR, a trend to lower CMR value for individuals possessing at least one mutant CYP2E1*6 allele compared with homozygous wild type was observed. The integration of CMR value as an independent variable to explain inter-individual variability in urinary metabolite excretion was not conclusive. CONCLUSION: Although, in the present population of workers, the CMR test was able to detect a slight influence of some genotypes on the activity of the CYP2E1 enzyme, it must be recognised that this method is not appropriate for refining the biological monitoring of industrial compounds that are metabolised by CYP2E1.

Abstract: BACKGROUND: Exposure to cadmium fumes or dusts has been associated with an increased risk of lung cancer and the characterisation of the genotoxic potential of cadmium compounds is, among other possible mechanisms, an important element in the assessment of the carcinogenic hazard of the element. While there is some evidence that in experimental systems, cadmium compounds may exert genotoxic effects, the results of the epidemiological studies having examined cytogenetic endpoints in humans exposed to cadmium appear conflicting. Therefore, a systematic review was undertaken to assess whether a cytogenetic effect of cadmium exposure is supported by the studies with the strongest design. METHODS: The relevant literature was identified through several databases and assessed with a check-list by two reviewers. Causes of heterogeneity between studies were looked for. Results were extracted and the strength of the evidence was evaluated with causality criteria. RESULTS: No studies met the criteria for being considered as very convincing. Several factors were identified that could explain contradictory findings (small sample size, selection bias, insufficient characterisation of exposure, lack of consideration of confounders) but their actual impact could not be conclusively assessed with the published information. Importantly, it should be recognised that the absence of a clear mechanism for the cytogenetic action of cadmium compounds did not allow to select the most appropriate endpoint to be examined. CONCLUSIONS: No clear association between cadmium exposure and cytogenetic endpoint appeared but no definite conclusion can be drawn from the existing studies in humans. Future research efforts should mainly focus on experimental studies to understand how cadmium compounds could produce genotoxic/carcinogenic effects, in order to target the most relevant endpoint to be examined in humans.

Abstract: The aim of this work was to validate a sensitive method for quantitative analysis of 5-hydroxy-N-methylpyrrolidone (5-HNMP) in urine. This compound has been recommended as a marker for biological monitoring of N-methylpyrrolidone (NMP) exposure. Different solvents and alternative methods of extraction including liquid-liquid extraction (LLE) on Chem Elut and solid-phase extraction (SPE) on Oasis HLB columns were tested. The most efficient extraction of 5-HNMP in urine was LLE with Chem Elut columns and dichloromethane as a solvent (consistently 22% of recovery). The urinary extracts were derivatized by bis(trimethylsilyl)trifluoroacetamide and analysed by gas chromatography-mass spectrometry (GC-MS) with tetradeutered 5-HNMP as an internal standard. The detection limit of this method is 0.017 mg/l urine with an intraassay precision of 1.6-2.6%. The proposed method of extraction is simple and reproducible. Four different m/z signal ratios of TMS-5-HNMP and tetralabelled TMS-5-HNMP have been validated and could be indifferently used in case of unexpected impurities from urine matrix.

Abstract: In the field of occupational and/or environmental toxicology, the measurement of specific metabolites in urine may serve to assess exposure to the parent compounds (biological monitoring of exposure). Styrene is one of the chemicals for which biological monitoring programs have been validated and implemented in environmental and occupational medicine. However, inter-individual differences in the urinary excretion exist both for the main end-products (mandelic acid and phenylglyoxylic acid) and for its specific mercapturic acids (phenylhydroxyethylmercapturic acids, PHEMA). This limits to a certain extent the use of these metabolites for an accurate assessment of styrene exposure. In a group of 26 volunteers selected with relevant genotypes, and exposed to styrene vapours (50 mg/m3, 8 h) in an inhalation chamber, we evaluated whether genotyping or phenotyping relevant drug-metabolizing enzymes (CYP2E1, EPHX1, GSTM1, GSTT1 and GSTP1) may help to explain the observed inter-individual variability in the urinary metabolite excretion. Peripheral blood lymphocytes were used for genotyping and as reporter cells for the phenotyping of CYP2E1 and EPHX1. The GSTM1 genotype was clearly the most significant parameter explaining the variance in urinary PHEMA excretion (6-fold lower in GSTM1 null subjects; P < 0.0001) so that systematic GSTM1 genotyping should be recommended routinely for a correct interpretation of PHEMA urinary levels. Variant alleles CYP2E1*6 (7632T>A) and His113EPHX1 were associated with a significant reduction of, respectively, the expression (P = 0.047) and activity (P = 0.022) of the enzyme in peripheral blood lymphocytes. In combination with GSTM1 genotyping, the phenotyping approach also contributed to improve the interpretation of urinary results, as illustrated by the combined effect of CYP2E1 expression and GSTM1 allelic status that explained 77% of the variance in PHEMA excretion and allows the recommendation of mercapturates as specific and reliable biomarkers of exposure to styrene.

Abstract: Cytokines are important mediators of the pathogenesis of lung fibrosis. The experimental studies carried out in our laboratory have used a murine model of lung silicosis to explore the activity of interleukin-9, -10 and -12. The data presented in this short overview illustrate the pro-fibrotic activity of anti-inflammatory cytokines (IL-10), and the role of type 2 immune polarisation, as illustrated by the paradoxical effect of IL-9 and the activity of IL-12 p40 subunit.

Abstract: HCFC-123 (2,2-dichloro-1,1,1-trifluoroethane), a substitute for the banned chlorofluorocarbons (CFCs), is a structural analogue of the well-known hepatotoxicant halothane. The objectives of these experiments were to investigate (1) whether, like halothane, multiple exposure increases the risk of HCFC-123-induced liver toxicity, and (2) whether ethanol, a potent CYP2E1 inducer, potentiates the liver toxicity of HCFC-123. In experiment 1, male Hartley guinea-pigs were exposed twice a week to 5000 ppm HCFC-123 (4 h) during 3 weeks followed by 2 weeks recovery, and then re-exposed or not during 4 h to 5000 ppm HCFC-123. A group with a single exposure to 5000 ppm HCFC-123 and a control group were also included. In experiment 2, guinea-pigs received 5 or 10% ethanol in drinking water during 12 days before a single 4-h exposure to 5000 ppm HCFC-123. A group receiving 10% only, a group exposed once to 5000 ppm HCFC-123 but not pre-treated with ethanol and a control group were also included. In both experiments, the liver toxicity was assessed, 24 h post-exposure, by the serum activities of alanine aminotransferase (ALT) and isocitrate dehydrogenase (ICDH) as well as by histopathology. In experiment 2 the urinary excretion rate of the main metabolites trifluoroacetic acid (TFA) and chlorodifluoroacetic acid (CDFA) was assessed and CYP2E1 activity was measured by the chlorzoxazone metabolic ratio. Multiple exposure to 5000 ppm HCFC-123 did not cause greater liver damage than a single exposure (ALT, ICDH 3-fold control values). At this level of exposure the liver lesions were totally reversible within two weeks. Ethanol consumption produced CYP2E1 induction, increased urinary excretion of both HCFC-123 metabolites (more than 2-fold the rate measured in the non-induced group) and markedly increased the liver toxicity of HCFC-123 as shown by the serum liver enzyme activities (ALT 8.5-fold increase, ICDH 13-fold increase), and the histopathology. The necrosis was predominantly localised in the intermediate zone of the hepatic lobules with vacuolisation of the centrilobular zones. The effects associated with 10% ethanol pre-treatment were less marked than those observed with ethanol 5% and could be explained by the remaining blood ethanol levels causing an inhibition of HCFC-123 biotransformation. Significant correlations were obtained between the serum enzyme activities, the histopathology, the excretion rate of the metabolites and CYP2E1 activity. It can be concluded that (1) multiple exposure to HCFC-123 did not increase the liver toxicity of HCFC-123 in this experimental model, and (2) chronic ethanol consumption, known to be CYP2E1 inducer, strongly enhanced the biotransformation of HCFC-123 and its liver toxicity.

Abstract:

Abstract: We examined the effect of interleukin (IL)-9, a cytokine active on B and T lymphocytes and associated with bronchial asthma, on the development of lung fibrosis induced by crystalline silica particles. Therefore, we compared the response to silica (1 and 5 mg/animal, intratracheally) in transgenic mice that constitutively express high levels of IL-9 (Tg5) and their wild-type counterparts (FVB). At 2 and 4 mo after treatment with silica, histologic examination and measurement of lung hydroxyproline content showed that the severity of fibrosis was significantly less important in Tg5 mice than in their wild-type counterparts. Intraperitoneal injection of IL-9 in C57BL/6 mice also reduced the amplitude of silica-induced lung fibrosis. The reduction of lung fibrosis by IL-9 was associated with a significant expansion of the B-lymphocyte population, both in bronchoalveolar lavage (BAL) and in the pulmonary parenchyma. In wild-type animals, silica-induced fibrosis correlated with markers of a T helper 2-like response such as upregulation of IL-4 levels in lung tissue and an increased immunoglobulin (Ig) G1/IgG2a ratio in BAL. Immunohistochemical studies demonstrated that the upregulation of IL-4 associated with the development of fibrosis was mainly localized in inflammatory alveolar macrophages. In transgenic mice, the level of IL-4 in lung homogenates was not significantly affected by silica treatment, and a reduced IgG1/IgG2a ratio was observed upon treatment with silica. The levels of interferon-gamma were significantly decreased after silica treatment in both strains. Together, these observations point to an antifibrotic effect of IL-9 in pulmonary fibrosis associated with a limitation of the type 2 polarization which accompanies lung fibrosis.

Abstract: 2,2-Dichloro-1,1,1-trifluoroethane (HCFC-123) has been developed as a substitute for ozone-depleting chlorofluorocarbons (CFCs). It is a structural analogue of halothane and similarities in the metabolic pathways and liver toxicity of both compounds have been described. The present study was initiated after an accidental outbreak of hepatitis in an industrial setting to examine whether concomitant exposure to 2-chloro-1,1,1,2-tetrafluoroethane (HCFC-124), which is not hepatotoxic, could enhance the liver toxicity of HCFC-123. Male Hartley guinea-pigs were exposed for 4 h to 5,000 ppm HCFC-123 alone or blended with 5,000 ppm HCFC-124, either once (single exposure) or on 5 consecutive days (repeated exposure). The animals were killed either 24 or 48 h after the last exposure. A transient cytolytic action of HCFC-123 was evident by increased mean serum levels of alanine aminotransferase at 24 h and isocitrate dehydrogenase at 24 and 48 h, both after a single or repeated exposure. The liver toxicity of HCFC-123 was confirmed by pathological examination of liver tissue, which showed mild (foci of necrotic hepatocytes) to moderate (multifocal random degeneration and necrosis) damage. Steatosis was also observed and was more pronounced after repeated exposure than after single. One animal out of 6 that were repeatedly exposed to the blend and sacrificed at 24 h showed liver lesions similar to halothane hepatitis. Although a few other animals responded markedly in the blend-treated group, on average, no significant difference in the biochemical or pathological lesions was found between the groups treated with HCFC-123 alone or with the blend. Urinary excretion of trifluoroacetic acid and chlorodifluoroacetic acid increased dose-dependently upon exposure to HCFC-123 and indicated accumulation after repeated exposure. No difference in metabolite excretion was found between animals treated with HCFC-123 alone or blended with HCFC-124. Treatment with HCFC-123 depleted hepatic glutathione levels by about 40 and 25% after single and repeated exposure, respectively; the amplitude of this reduction was not modified by co-exposure to HCFC-124. In conclusion, this study confirmed the hepatotoxicity of HCFC-123, based on biochemical, histopathological and metabolite studies, and found only very limited indication of a potentiation by HCFC-124 of this hepatotoxic effect.

Abstract: OBJECTIVE: To integrate recent understandings of the mechanisms of genotoxicity and carcinogenicity of the different cobalt compounds. METHOD: A narrative review of the studies published since the last IARC assessment in 1991 (genotoxicity, experimental carcinogenesis, and epidemiology). RESULTS: Two different mechanisms of genotoxicity, DNA breakage induced by cobalt metal and especially hard metal particles, and inhibition of DNA repair by cobalt (II) ions contribute to the carcinogenic potential of cobalt compounds. There is evidence that soluble cobalt (II) cations exert a genotoxic and carcinogenic activity in vitro and in vivo in experimental systems but evidence in humans is lacking. Experimental data indicate some evidence of a genotoxic potential for cobalt metal in vitro in human lymphocytes but there is no evidence available of a carcinogenic potential. There is evidence that hard metal particles exert a genotoxic and carcinogenic activity in vitro and in human studies, respectively. There is insufficient information for cobalt oxides and other compounds. CONCLUSION: Although many areas of uncertainty remain, an assessment of the carcinogenicity of cobalt and its compounds requires a clear distinction between the different compounds of the element and needs to take into account the different mechanisms involved.

Abstract: OBJECTIVES: This study examined whether consideration of the *1C/*1D CYP2E1 insertion polymorphism is important for interpreting the biological monitoring of exposure to N,N-dimethylformamide (DMF) in Japanese workers. METHODS: The insertion genotype, airborne DMF exposure on the last day of a work week, and NMF in urine sampled just after the last workshift of the week were determined in 44 male and female Japanese workers. RESULTS AND CONCLUSIONS: The allelic frequency of this CYP2E1 polymorphism was 0.261 in this Japanese population of workers. The CYP2E1 insertion polymorphism did not contribute to NMF levels even after consideration of BMI or alcohol intake. The results indicate that CYP2E1 insertion polymorphism does not appear to be an important determinant for the interpretation of biological exposure to DMF by the measurement of urinary NMF.

Abstract: The purpose of this review is to describe the present state of knowledge regarding host susceptibility factors that may determine the occurrence, development and severity of interstitial lung disease (ILD) caused by exogenous agents. First, host susceptibility may pertain to differences in the delivery and/or persistence of the noxious agent in the lung. The deposition and clearance of inhaled particles or fibres may vary depending on innate anatomical or physiological characteristics, and on acquired changes, such as nasal disease or smoking-induced alterations. Genetically- or environmentally-induced interindividual differences in the expression of pulmonary biotransformation enzymes may form the basis for, or contribute to the risk of, drug-induced interstitial lung disease. Secondly, there are genetic and acquired variations in various enzymatic and nonenzymatic defence systems that protect cells and tissues against oxidative stress, which is often involved in the pathogenesis of interstitial lung disease caused by particles, fibres, metals, organic agents and drugs. Thirdly, the occurrence of immunological sensitization is dependent on both genetic and environmental factors. This has been demonstrated in chronic beryllium lung disease and in hypersensitivity pneumonitis. Fourthly, the propensity of individuals to develop particular types of inflammation, such as granulomas, is probably under genetic control. The regulation and resolution of inflammation and fibrogenesis caused by dust particles are also partly determined by genetic factors, involving cytokine networks and growth factors. In conclusion, although the issue of genetics pervades the entire discussion of host susceptibility, genes are not the only determinants of health and disease. Environmental factors may be equally important in shaping host susceptibility. Therefore, research must be focused on both the genetic bases and the environmental determinants of interstitial lung disease, in order to provide mechanism-based prevention strategies, early detection of, and improved therapy for these conditions.

Abstract:

Abstract: On the basis of studies of the prevalence of skin cancer among users of As-rich well water in Taiwan, WHO experts recommended in 1984 a maximum As concentration of 50 microg/litre in drinking water. Since that time, a plethora of non-cancer as well as cancer effects has been observed in several other populations sustaining a chronic exposure to various As concentrations in drinking water. This prompted a revision of the standard and a provisional guideline of 10 microg/litre was recommended in 1993. While the uncertainty linked to the statistical inferences leading to the guideline are reduced by the fact that they are directly estimated from human data and result from extrapolations made relatively close to observed exposure levels, developed guideline depends strongly on the choice of the dose-response model (linear, quadratic, hockey-stick) and the accuracy of the exposure data. The potential exposure to As sources other than drinking water, dietary habits and genetic characteristics of the populations may also make more difficult the inference of a recommendation for As concentration in drinking water. Owing to the huge cost of strongly reducing the current As in water standard, many efforts are presently made to clarify the quantitative aspects of As-induced cancers, particularly at low dose levels. New data on the metabolism and carcinogenic mechanism of As in humans along with the results of epidemiological studies presently under way in several countries will help to reduce the uncertainty in the risk assessment of As.

Abstract: OBJECTIVES: To assess occupational exposure to inorganic germanium (Ge) in workers from a producing plant, and to assess the health of these workers, with a special focus on respiratory, kidney, and liver functions. METHODS: Cross sectional study of 75 workers exposed to Ge and 79 matched referents. Exposure was characterised by measuring air and urine concentrations of the element during a typical working week, and health was assessed by a questionnaire, clinical examination, lung function testing, chest radiography, and clinical chemistry in serum and urine, including high and low molecular weight urinary proteins. RESULTS: Airborne concentrations of Ge (inhalable fraction) ranged from 0.03 to 300 micrograms/m, which was reflected by increased urinary excretion of Ge (0.12-200 micrograms/g creatinine, after the shift at the end of the working week). Lung, liver, and haematological variables were not significantly different between referents and workers exposed to Ge. A slightly higher urinary concentration of high molecular weight proteins (albumin and transferrin) was found in workers exposed to Ge, possibly reflecting subclinical glomerular changes. No relation was found between the intensity or duration of exposure and the urinary concentration of albumin. No difference between referents and workers exposed to Ge was found for other renal variables. CONCLUSIONS: Measurement of urinary Ge can detect occupational exposure to inorganic Ge and its compounds. It is prudent to recommend the monitoring of renal variables in workers exposed to Ge.

Abstract:

Abstract: Mortality studies have shown that, in the past, lung cancer occurred after exposure to mixtures of cobalt metal and metallic carbide particles, the main constituents of hard metals, but apparently not when exposure was to cobalt alone. The major objective of this biomonitoring study was to assess genotoxic effects as a measure for carcinogenic risk in workers from cobalt refineries and hard metal plants currently exposed to the threshold limit value/time-weighted average (TLV-TWA) for cobalt-containing dust. The study comprised three groups of workers: 35 workers exposed to cobalt dust from three refineries, 29 workers exposed to hard metal dust from two producing plants, and 35 matched control subjects recruited from the respective plants. The study design integrated complementary methodologies to assess biomarkers of effects that represent both initial DNA damage (8-hydroxydeoxyguanosine [8-OHdG] in urine and comet assay on lymphocytes) and definitive chromosome breakage/loss (micronuclei in lymphocytes). Cobalt and cotinine were determined in urine as a measure for cobalt exposure and recent smoking, respectively. No significant increase of genotoxic effects was detected in workers exposed to cobalt-containing dust as compared to controls. No difference in any genotoxicity biomarker was found between workers exposed to cobalt and hard metal dusts. Multiple regression analysis indicated that workers who smoked and were exposed to hard metal dusts had elevated 8-OHdG and micronuclei values. Because this observation is in line with a previous epidemiological study of an increased risk of dying from lung cancer in workers from the hard metal industry who smoked, it is concluded that this specific occupational group needs closer medical surveillance.

Abstract:

Abstract: Interleukin (IL)-12 is a cytokine produced principally by activated macrophages which is involved in control of the T-helper 1/T-helper 2 cell (Th1/Th2) polarization of immune responses. To examine its potential involvement in the development of lung fibrosis, we examined the expression (protein, messenger RNA [mRNA]) of IL-12 (p70) and of its subunits (p40 and p35) in lung homogenates, bronchoalveolar lavage fluid (BALF), and bronchoalveolar lavage (BAL) cell cultures in mouse models of resolutive alveolitis (RA) and fibrosing alveolitis (FA) induced by inorganic particles (manganese dioxide [MnO2] and crystalline silica, respectively). The administration of tungsten carbide (WC), which behaved as an innocuous dust for the lung, served as a negative control condition. The FA was specifically accompanied by a Th2-like polarization characterized by high levels of immunoglobulin (Ig)G1 in BALF and by a protracted overproduction of both p40 protein and mRNA, but not by the biologically active form of IL-12 (p70). In the RA model, the p40 response was only transient, and a Th1-like response was reflected by increased levels of interferon (IFN)-gamma and dominant levels of IgG2a in BALF. Taken together, these findings suggest that production of the p40 subunit of IL-12 and Th2 polarization play important roles in lung inflammatory and fibrotic responses to inhaled inorganic particles.

Abstract: We have found reduced activity of tumor necrosis factor (TNF)-alpha accompanying resolving and fibrosing alveolitis induced in NMRI mice by mineral particles (MnO2 and SiO2, respectively), which is in apparent contradiction to the well-recognized proinflammatory and profibrotic activities of this cytokine. The objective of this study was to examine the mechanisms involved in this paradoxical response in NMRI mice. Although lung tissue messenger RNA (mRNA) levels for TNF-alpha were transiently (up to 15 d) and persistently (up to 120 d) upregulated in the resolving and fibrosing models, respectively, these changes were not accompanied by a parallel release of TNF-alpha protein, which was respectively transiently and persistently downregulated in bronchoalveolar lavage fluid and bronchoalveolar lavage cell cultures. The downregulation of the TNF-alpha protein was concurrent with the accumulation of recruited polymorphonuclear neutrophils (PMNs) in alveoli, and coculture experiments showed that PMN explanted from the lungs of mice treated with silica particles were able to downregulate the expression of TNF-alpha protein by naive alveolar macrophages. In addition, PMN depletion prevented the downregulation of TNF-alpha induced by silica, further establishing the role of PMNs in the downregulation of TNF-alpha. The possible degradation of TNF-alpha by proteolytic enzymes could be excluded. Marked increases in soluble p55 and p75 TNF receptors (sTNF-R), as well as in interleukin (IL)-10, paralleled the downregulation of TNF-alpha protein. The role of these mediators in the observed reduction of TNF-alpha activity was confirmed by immunoneutralizing the activity of p55 and p75 sTNF-R and by using IL-10-deficient animals. Because IL-10 also exerts profibrotic activity in addition to its antiinflammatory activity, the protracted overproduction of IL-10 observed in fibrosing alveolitis may help the understanding of why, in NMRI mice treated with silica particles, lung fibrosis develops in association with a downregulation of TNF-alpha.

Abstract: The objective of this study was to assess the effect of two arsenic-containing particles, coal fly ash (FA) and copper smelter dust (CU), on lung integrity and on the ex vivo release of tumor necrosis factor alpha (TNF-alpha) by alveolar phagocytes. Particle effects were compared in nonoverload condition on the basis of a low but identical volume load and arsenic content intratracheally instilled in the mouse lung (273 nl/mouse and 186 ng arsenic/mouse; FAL and CUL groups). Other mice received 600 ng arsenic/mouse in amounts of particles leading to different volume loads (FAH and CUH groups: 880 and 273 nl/mouse, respectively). Animals were sacrificed at 1, 6, 30, or 120 d (FAL and CUL groups) or at 6 and 120 d posttreatment (FAH and CUH groups). Biochemical markers and inflammatory cell number and type were analyzed in bronchoalveolar lavage, ex vivo TNF-alpha production by alveolar phagocytes was assessed, and measurement of arsenic lung content and histopathological examinations were performed. Our results show that coal fly ash and copper smelter dust bear distinct inflammatory properties. At the end of the observation period (d 120), the high CU dose (CUH) produced a fibrotic reaction whereas the high dose of FA particles (FAH) generated a delayed and persistent lung inflammatory reaction associated with lymphoid noduli. Marked differences in TNF-alpha production were observed within the CU and FA groups. CU particles, conceivably through their metal content, decreased TNF-alpha production by alveolar phagocytes. Due to their low arsenic content, considerably higher FA particle doses needed to be administered to produce an inhibition of TNF-alpha production. Since high doses of FA (FAH) caused an overload condition, our results do not allow us to decide whether FA-mediated TNF-alpha reduction is due to the load administered or to the metallic content.

Abstract: BACKGROUND: On March 21, 1998, the Regional Health Authority of Bobo-Dioulasso, Burkina Faso, asked the Centre Muraz to investigate an unexplained outbreak of epidemic fatal encephalopathy (EFE). We aimed to identify the cause of this epidemic. METHODS: We identified cases retrospectively through review of health-service records and interviews of family members, village chiefs, and local healers. Active surveillance was started in administrative divisions within the study area in April, 1998, to identify further EFE cases. We did a case-control study of households to investigate the risk from various environmental and health factors. Blood and urine samples were collected if possible and urine dicarboxylic acid concentrations measured by gas chromatography. FINDINGS: 29 cases of EFE were identified from January to May, 1998. Estimated age-specific attack rates (2-6 years) ranged from 31 to 847 per 100,000 population (p<0.001). The most common symptoms were hypotonia, vomiting, convulsions, and coma. All children died in 2-48 h. The only factor associated with EFE was the presence of ackee trees (Blighia sapida) within 100 m of households (odds ratio 5.1 [95% CI 1.8-14.7] p=0.001). Poisoning with unripe ackee fruits was suggested by urine concentrations of dicarboxylic acids four to 200 times higher in cases (n=2) than in controls (n=3). CONCLUSION: Consumption of unripe ackee fruit probably caused this epidemic and may lead to a substantial number of unexplained deaths in preschool children in west Africa every year. Educational campaigns have the potential to prevent these deaths.

Abstract: In 1987, a cross-sectional study in a dry-alkaline battery plant in Belgium revealed subclinical neurobehavioral dysfunctions associated with inhalation exposure to manganese dioxide (MnO2) particulate. The overall geometric mean of the time-weighted average concentration of manganese (Mn) in "total" dust (MnT) amounted, at that time, to 1 mg Mn/m3 and the duration of exposure was 5.5 years on average. An 8-year longitudinal investigation was conducted in this cohort (n = 92) in order to find out whether early effects on eye-hand coordination (EHC), hand steadiness (HST), and simple visual reaction time (VRT) were reversible when the airborne manganese concentration at the workplace was abated. During the observation period from 1988 to 1995, MnT monitoring was implemented on a monthly basis producing more than 1300 personal air samples, EHC tests were given yearly to assess the precision of the hand-forearm movement (PN1), and HST and VRT tests were carried out yearly since 1991. By the end of the study, the cohort size had dropped to 34 subjects. The model of unbalanced repeated measurements with unstructured covariance matrix and a time-varying covariate (log MnT) was the most appropriate to analyze the data. Wald chi 2 statistic was used for testing time-trends. The reduction of MnT over time was significantly associated with an improvement of the PN1 values (total cohort: Wald chi 2 = 8.5, p = 0.004; beta log MnT = -6.098 +/- 2.096). Like in the total cohort, time-trends were also found in the three exposure subgroups which could be identified in the cohort (average MnT over 1987-1992 were about 400, 600, and 2000 micrograms Mn/m3 for the low, medium, and high exposure subgroups, respectively). Only in the low exposure subgroup the PN1 value normalized when MnT (provisional estimates) decreased from about 400 to 130 micrograms Mn/m3 by the end of the study. Solely the reduction in MnT explained these findings on PN1, while a "healthy-worker-effect" mechanism was unlikely to have operated. The prognosis for the medium and high exposure subgroups remains uncertain as the improvement of their EHC performance may have been affected by past MnO2 exposure to such an extent that the persistence of a partial loss of EHC ability is suggested. The time courses of the HST and VRT test results, however, indicated the absence of any improvement, suggesting irreversible impairment of hand stability (postural tremor) and simple visual reaction time. A separate examination in a group of 39 control subjects, re-tested 10 years after the first test in 1987, virtually precluded age as confounding factor in this prospective study. The findings of the longitudinal study are corroborated by the outcome of a separate follow-up study in a group of 24 ex-Mn employees, who showed in 1996 a significant improvement of eye-hand coordination after at least three years with no MnO2 exposure; as to HST and VRT, there was no significant change in the deficit of these two neurobehavioral markers.

Abstract: Uncovering the exact cause of polyneuropathies seems to be impossible in up to 24% of the cases. Experimental studies have shown that cadmium (Cd), which is a well-known occupational and environmental hazard, can be a potent neurotoxicant for the peripheral nervous system. Moreover, Cd has a half-life of more than 15 years in humans. We hypothesize that older workers may be more susceptible to an increased Cd body burden, and may develop a peripheral polyneuropathy (PNP) over time. A blinded epidemiological survey was performed in 13 retired, long-term Cd-exposed workers and 19 age-matched controls. Historical Cd biomonitoring data were available over the last two decades. A neurological clinical examination, nerve conduction studies, and needle EMG were performed, and a standardized questionnaire was given to evaluate polyneuropathy complaints. If two of the following four criteria, i.e. complaints of polyneuropathy, neurophysiological changes compatible with polyneuropathy, distal symmetrical areflexia, or distal symmetrical anesthesia for vibration sense, temperature or blunt-sharp discrimination were present, the diagnosis of PNP was made. Two (11%) of the control and seven (54%) of the retired Cd workers met the PNP criteria OR: 9.92 (95%CI 1.60-61.6), Fisher exact test p=0.015. The existence of a polyneuropathy was related to the level of the Cd body burden as reflected by urinary Cd multiple logistic regression p=0.016, OR=1.26, (95%CI, 1.04-1.51), but not to blood lead (p=0.352). Our findings favour the hypothesis of a promoting role of increased cadmium body burden in the development of PNP at older age.

Abstract:

Abstract: Although the morphological description of sulfur mustard (SM) injury is well characterised, little is known of the molecular mediators involved in cutaneous toxicity. Since infiltration by lymphocytes and PMNs represents one of the very first events observed in vivo upon exposure to SM, this study examined whether SM exposure can modify the expression by cultured human keratinocytes of interleukin-8, one of the most important chemoattractants for polymorphonuclear leukocytes (PMNs) in humans. Conditioned medium harvested from control keratinocyte cultures showed a gradual accumulation of this cytokine over time followed by a levelling off after 12 hours. Upon treatment with 10(-6) and 10(-5) M SM, no significant difference compared to the control situation was observed. After 6 h, a significantly higher amount of IL-8 was secreted by human keratinocytes treated with 10(-4) M SM and the accumulation of the cytokine persisted up to 24 h after exposure. The expression of IL-8 mRNA was assessed semi-quantitatively (RT-PCR) at the same time points in control and SM-treated (10(-4) M) human keratinocytes. When compared to control cultures, a clear upregulation of IL-8 mRNA levels was observed 6 and 12 h after SM exposure, which is consistent with the secretion pattern of the protein. The present observation indicates that increased secretion of IL-8 by human keratinocytes represents an early event of the inflammatory reaction following SM which is coherent with the reported delay in the recruitment of lymphocytes and PMNs observed in vivo.

Abstract: A detailed characterisation of the biotransformation pathways followed by the chemical of interest is, evidently, of primordial importance to design reliable biomonitoring programs. This short overview discusses some recent studies that are of potential interest for the development of new avenues of research. Four topics are addressed: tissue localisation of the metabolic process; importance of speciation; metabolic interactions and polymorphism and interindividual variability. While some of these studies offer new data that is directly useful for practical application, others raise mainly questions and indicate that there is still a large effort of research to be performed to improve the design and the interpretation of biomonitoring programs.

Abstract: BACKGROUND: The clinical relevance of renal effects of cadmium in people exposed in the environment remains uncertain. This study examined the evolution of renal effects observed in a population exposed to cadmium in the environment. METHODS: 208 men and 385 women surveyed in 1985-89 (Cadmium in Belgium study [Cadmibel]; baseline) were re-examined on average 5 years later (Public health and environmental exposure to cadmium study [PheeCad]; follow-up). Urinary and blood cadmium and markers of renal tubular dysfunction and glomerular effects were measured. The association between cadmium body burden and renal factors was examined by multivariate logistic and linear regression. FINDINGS: In men, mean urinary cadmium excretion and blood cadmium concentration measured at follow-up were 7.5 nmol/24 h (SD 1.9) and 6.1 nmol/L (2.2), reductions of 16% and 35% from baseline, respectively. In women, the corresponding values were 7.6 nmol/24 h (1.9) and 7.8 nmol/L (2.1), reductions of 14% and 28% from baseline. No indication of progressive renal damage was found and the overall results suggest that the effects of low environmental exposure to cadmium on the kidney are weak, stable, or reversible. INTERPRETATION: Subclinical renal effects that have been reported in Belgium in patients with increased cadmium body burden are not associated with progressive renal dysfunction and most likely represent non-adverse manifestations.

Abstract: A fibrogenic sample of cristobalite dust, CRIS (crystalline silica of mineral origin), was heated to 1300 degrees C (CRIS-1300) to relate induced physicochemical modifications to cytotoxicity. Heating did not affect dust micromorphology and crystallinity, except for limited sintering and decreased surface area of CRIS-1300. Thermal treatments deeply affected surface properties. Electron paramagnetic resonance showed surface radicals progressively annealed by heating, mostly disappearing at >/=800 degrees C. Surface hydrophilicity or hydrophobicity, evaluated with water vapor adsorption, still showed some hydrophilic patches in CRIS-800, but CRIS-1300 was fully hydrophobic. Heating modified the biological activity of cristobalite. Cytotoxicity, tested on proliferating cells of the mouse monocyte macrophage cell line J774, showed that CRIS was cytotoxic and CRIS-800 was still cytotoxic, but CRIS-1300 was substantially inert. Cytotoxicity of CRIS to the rat lung alveolar epithelial cell line, AE6, as measured by colony forming efficiency, was greatly reduced for CRIS-800 and eliminated for CRIS-1300. The rate of lactate dehydrogenase release by rat alveolar macrophages was lowered for CRIS-800, and release was completely inactivated for CRIS-1300. The absence of surface radicals and the onset of hydrophobicity may both account for the loss of cytotoxicity upon heating. Differences observed between CRIS-800 and CRIS-1300, both fully deprived of surface radicals, indicate that hydrophobicity is at least one of the surface properties determining the cytotoxic potential of a dust.

Abstract: A successful prevention of renal diseases induced by occupational or environmental exposure to toxic metals such as mercury (Hg), lead (Pb), or cadmium (Cd) largely relies on the capability to detect nephrotoxic effects at a stage when they are still reversible or at least not yet compromising renal function. The knowledge of dose-effect/response relations has been useful to control nephrotoxic effects of these metals through a "biological monitoring of exposure approach". Chronic occupational exposure to inorganic mercury (mainly mercury vapor) may result in renal alterations affecting both tubules and glomeruli. Most of the structural or functional renal changes become significant when urinary mercury (HgU) exceeds 50 micrograms Hg/g creatinine. However, a marked reduction of the urinary excretion of prostaglandin E2 was found at a HgU of 35 micrograms Hg/g creatinine. As renal changes evidenced in moderately exposed workers were not related to the duration of Hg exposure, it is believed that those changes are reversible and mainly the consequence of recently absorbed mercury. Thus, monitoring HgU is useful for controlling the nephrotoxic risk of overexposure to inorganic mercury; HgU should not exceed 50 micrograms Hg/g creatinine in order to prevent cytotoxic and functional renal effects. Several studies on Pb workers with blood lead concentrations (PbB) usually below 70 micrograms Pb/dl have disclosed either no renal effects or subclinical changes of marginal or unknown health significance. Changes in urinary excretion+ of eicosanoids was not associated with deleterious consequences on either the glomerular filtration rate (GFR)--estimated from the creatinine clearance (C(Cr))--or renal hemodynamics if the workers' PbB was kept below 70 micrograms Pb/dL. The health significance of a slight renal hyperfiltration state in Pb workers is yet unknown. In terms of Pb body burden, a mean tibia Pb concentration of about 60 micrograms Pb/g bone mineral (that is 5 to 10 times the average "normal" concentration corresponding to a cumulative PbB index of 900 micrograms Pb/dL x year) did not affect the GFR in male workers. This conclusion may not necessarily be extrapolated to the general population, as recent studies have disclosed inverse associations between PbB and GFR at low-level environmental Pb exposure. A 10-fold increase in PbB (e.g., from 4 to 40 micrograms Pb/dL) was associated with a reduction of 10-13 mL/min in the C(Cr) and the odds ratio of having impaired renal function (viz. C(Cr) < 5th percentile: 52 and 43 mL/min in men and women, respectively) was 3.8 (CI 1.4-10.4; p = 0.01). However, the causal implication of Pb in this association remains to be clarified. The Cd concentration in urine (CdU) has been proposed as an indirect biological indicator for Cd accumulation in the kidney. Several biomarkers for detecting nephrotoxic effects of Cd at different renal sites were studied in relation to CdU. In occupationally exposed males, the CdU thresholds for significant alterations of renal markers ranged, according to the marker, from 2.4 to 11.5 micrograms Cd/g creatinine. A threshold of 10 micrograms Cd/g creatinine (corresponding to 200 micrograms Cd/g renal cortex: the critical Cd concentration in the kidney) is confirmed for the occurrence of low-molecular-mass proteinuria (functional effect) and subsequent loss of renal filtration reserve capacity. In workers, microproteinuria was found reversible when reduction or cessation of exposure occurred timely when tubular damage was still mild (beta(2)-microglobulinuria < 1500 micrograms/g creatinine) and CdU had never exceeded 20 micrograms Cd/g creatinine. As the predictive significance of other renal changes (biochemical or cytotoxic) is still unknown, it seems prudent to recommend that occupational exposure to Cd should not allow that CdU exceeds 5 micrograms Cd/g creatinine.(ABSTRACT TRUNCATED)

Abstract: A minireview is presented concerning the use of cotinine as a tobacco-smoke exposure index. First, general considerations about methods for the determination of urinary cotinine are presented. Besides pure analytical aspects, this minireview considers major problems encountered in the establishment of threshold values that can be used to distinguish not only smokers from nonsmokers but also nonsmokers exposed or not exposed to environmental tobacco smoke (ETS). In addition, the use of urinary cotinine is illustrated in several situations where smoking status assessment is of interest. Such situations include evaluation of the impact of smoking cessation programs, monitoring of pregnancy and of other groups at risk, assessment of occupational exposure to industrial pollutants, validation of phase I clinical trials, and the control of life insurance candidates. The specific problem of ETS exposure assessment is briefly mentioned.

Abstract: OBJECT: The dose-response relationship for lung carcinoma and other cancers at low doses of As is highly uncertain because it is based on modeling data collected in populations with a high daily intake of the element. The finding of a slightly increased exposure to arsenic in certain groups of the Belgian general population prompted us to examine whether this had repercussions on the causes of mortality. METHOD: Statistics of mortality by causes with a possible link to exposure to the element (standardized mortality ratio) were analyzed in groups of the Belgian population previously shown to have been exposed to As from natural (drinking water) and/or industrial (nonferrous metal smelter emissions) sources. RESULTS: A moderately increased absorption of As, leading to a 3- to 4- fold higher urinary excretion (35 micrograms/day as compared with 6-10 micrograms As/day in nonexposed subjects) did not enhance the mortality by diseases of the nervous system, liver and heart, and cancers. An increase in mortality by lung cancer, however, was observed in men but not women living around zinc smelters and might be related to past occupational exposure and/or smoking habits. CONCLUSION: A low to moderate level of environmental exposure to inorganic arsenic (0.3 microgram As/m3 of air; 20-50 micrograms As/l of drinking water) does not seem to affect the causes of mortality, suggesting in particular nonlinearity of the dose-response relationship for arsenic and cancer.

Abstract: There is evidence that, following exposure to crystalline silica, the release of several proinflammatory cytokines contributes to the induction of unbalanced inflammatory reaction leading to lung fibrosis. We have examined the potential contribution of interleukin-10 (IL-10), an anti-inflammatory cytokine, in the development of silicosis. In a mouse model of inflammatory lung reaction induced by intratracheal instillation of silica (0.5 mg and 5 mg DQ12/mouse), the levels of IL-10 protein (determined by ELISA) both in cells obtained after bronchoalveolar lavage (BAL) and in lung tissue homogenates were significantly increased when compared with controls. After in vitro lipopolysaccharide (LPS) stimulation (1 microg/ml), BAL cells obtained from silica-treated animals produced significantly more IL-10 protein and mRNA than cells obtained from control animals. To examine the role of IL-10 in the lung reaction induced by silica, IL-10-deficient animals were instilled with 5 mg of silica. Twenty-four hours after treatment, the amplitude of the inflammatory response (lactate dehydrogenase [LDH], protein and number of inflammatory cells in BAL) was significantly greater in IL-10-deficient animals than in the wild type. In contrast, the fibrotic response, evaluated by measuring lung hydroxyproline content and by histopathologic analysis 30 days after silica, was significantly less important in IL-10-deficient than in wild-type mice. Together, these data suggest that increased IL-10 synthesis induced by silica can limit the amplitude of the inflammatory reaction, but also contributes to amplify the lung fibrotic response.

Abstract: Altered expression of plasminogen activator inhibitors (PAIs) is of potential relevance to the process of lung fibrosis. To clarify the involvement of PAIs in interstitial lung diseases, we examined whether alterations in PAI-1 and PAI-2 were induced in response to a single intratracheal administration of a fibrosing dose of crystalline silica in mice (5 mg x animal(-1)). The time course of changes in PAI activity and PAI-1 protein were characterized in bronchoalveolar lavage fluid (BALF) and changes in PAI-1 and PAI-2 messenger ribonucleic acid (mRNAs) were monitored by reverse transcriptase polymerase chain reaction (RT-PCR) in BALF cells and lung tissue up to the fibrotic stage of the disease. Substantial levels of PAI activity were found in BALF of control animals, whereas no PAI-1 protein was detected. In response to silica treatment, we observed an acute increase of PAI activity and PAI-1 protein levels in BALF (day 1), associated with an induction of PAI-1 and PAI-2 mRNA levels in lung tissue. In alveolar macrophages, silica treatment induced a persistent upregulation of PAI-2 mRNA only. One month after silica treatment, PAI activity was undetectable in BALF while substantial PAI activity was still present in controls. At the same time point, sustained upregulation of PAI-1 and PAI- 2 mRNAs was, however, noted in lung tissue of animals treated with silica. These findings support the possible implication of PAIs in the remodelling process induced by silica in the lung.

Abstract: The lung plasminogen activator (PA) response was examined in four different models of particle-induced pulmonary lesions in NMRI mice (single intratracheal administration, 0.75 to 5 mg/mouse). Sequential changes in cellular (total and differential counts) and biochemical markers of alveolitis (lactate dehydrogenase [LDH], total proteins) were monitored in bronchoalveolar fluid (BALF) and the fibrotic lung response was assessed histologically. An intense but spontaneously resolving alveolitis was produced by manganese dioxide (MnO2) and a fibrosing alveolitis was elicited by crystalline silica (DQ12). Minimal and noninflammatory responses were obtained after instillation of titanium dioxide (TiO2) and tungsten carbide (WC), respectively. The comparison between the resolving and the fibrosing alveolitis model was especially taken into consideration in an attempt to identify fibrinolytic changes associated with the development of fibrosis. At the alveolitis stage, similarly increased BALF PA activities were measured in both the resolving and the fibrosing alveolitis models whereas only slight and no PA modifications were noted after administration of TiO2 and WC, respectively. Persistently (up to 120 d) increased BALF PA activity was selectively associated with the progression to fibrosis (DQ12), suggesting that PA is involved in the fibrotic process. ELISA measurements demonstrated that the changes in BALF PA activity were exclusively related to changes in urokinase (uPA), not tissue-type PA. A rapid and persisting (up to Day 30) upregulation of cell-associated PA activity occurred after DQ12, MnO2, and TiO2 treatment only. Cellular PA activity was however significantly higher in fibrogenic inflammatory cells recovered from DQ12 than from MnO2-treated mice suggesting that the intensity of cellular PA upregulation may represent an early indicator of the progression to fibrosis. The implication of urokinase in the pathogenesis of silica-induced fibrosis was demonstrated by the use of a uPA knockout mice. The acceleration of the fibrotic process in uPA-deficient compared with the wild type animals demonstrated the contribution of uPA to limit the fibrotic process.

Abstract: Impaired fibrinolytic activity and persistent fibrin deposits in lung tissue have been associated with lung fibrotic disorders. The present study examined the sources of plaminogen activator (PA) changes induced by a single intratracheal administration of silica particles (5 mg) in the mouse lung. We found in both control and silica-treated animals that amiloride almost totally abolished PA activity in bronchoalveolar lavage (BAL) fluid (BALF), indicating that initial upregulation (from day 1) as well as sustained PA activity (up to day 30) observed in response to silica is related to changes in urokinase-type PA (uPA). The upregulation of BALF uPA activity was associated with a marked and persistent increase in uPA mRNA levels in lung tissue. Changes in uPA expression were also reflected in the BAL cell fraction. A maximal and constant increase in cell uPA activity was associated with the early response to silica, whereas significant but lower upregulation was still noted at the fibrotic stage. From days 3 to 30, a progressive increase in uPA mRNA levels was noted in BAL inflammatory cells elicited by silica. Because the number of BAL neutrophils was strongly correlated with BALF and BAL cell-associated uPA activity, their involvement in uPA upregulation was addressed by inducing neutropenia with cyclophosphamide (200 mg/kg ip) before administration of the silica. Neutrophilic depletion did not, however, reduce, and even increased, the BAL cell-associated uPA activity. At the BALF level, neutropenia did not change PA activity in silica-treated mice, pointing to alveolar macrophages as the principal source of uPA in response to silica. Immunohistochemical stainings identified alveolar macrophages and pneumocytes as uPA-expressing cells in silica-treated animals (day 30). Intense and heterogenous staining was observed in silicotic nodules. These findings indicate that urokinase produced by alveolar macrophages is operative not only at the alveolitis stage but also later in the fibrotic process, produced by silica particles, supporting the role of uPA in fibrogenesis.

Abstract: Many organisms can easily dispose of toxic inorganic arsenic species through gradual methylation of the element and further urinary excretion. In order to clarify the urinary excretion of arsenobetaine observed in a human case of intoxication by arsine, the capacity of highly methylated arsenical synthesis has been investigated in rats acutely exposed during 1 h to increasing concentrations of the same gas [4 to 80 mg AsH3/m3]. Urinary metabolites of arsenic were determined with good agreement in two (Belgian and Italian) laboratories using two different analytical procedures. The sum of inorganic, mono- and dimethylated metabolites of arsenic in urine was shown to be related to the intensity of exposure to arsine. A biphasic relationship was observed: 1 h exposure to > 60 mg AsH3/m3 led to metabolite excretion which is roughly 10 times higher than for exposure levels below that limit, suggesting the saturation of a binding site reserve and the availability for metabolism of a greater proportion of the As absorbed above this threshold. Arsenobetaine production, if any, could only be detected when its presence in food was excluded; in addition, amounts appeared negligible and could be disregarded as a common arsenic metabolite in rats.

Abstract: Since tumor growth factor beta (TGF-beta) and its receptor are ubiquitously expressed and because latent TGF-beta cannot bind to the cell surface receptor, the ability of a cell to activate latent TGF-beta upon secretion represents an important regulatory mechanism of TGF-beta action. In vivo, the protease plasmin is considered to be one of the main enzymes operative in the proteolytic cleavage of the latency-associated peptide moiety from TGF-beta, which converts it into the biologically active form. The TGF-beta response was characterized in alveolar macrophages during pulmonary inflammation (d 3) and fibrosis (d 120) induced by a single intratracheal instillation of silica particles (5 mg/mouse). To appreciate the role of urokinase-type plasminogen activator (uPA) in the activation of TGF-beta, the production of total, active and latent TGF-beta by explanted alveolar macrophages was compared in uPA-deficient (uPA-/-) mice and their normal counterparts (uPA+/+). At d 3 and 120 after silica treatment, a significant increase in cell-associated PA activity was found in uPA+/+ mice compared to that of saline controls. As expected, this response was almost totally absent in uPA-/- mice. Alveolar macrophages from uPA+/+ controls were found to release TGF-beta mainly expressed in a biologically active form. In response to silica treatment, inflammatory cells were found to upregulate, especially at the fibrotic stage, their secretion of total and bioactive TGF-beta. No significant difference was found between uPA-/- and uPA+/+ silica-treated animals for the expression of total, active, or latent TGF-beta. Although it has previously been reported that macrophage surface activation of TGF-beta is dependent on both plasmin generation and uPA cell surface receptor, no evidence was found to support this hypothesis in the present study.

Abstract: The mechanisms of cobalt-induced pulmonary interstitial fibrosis and cancer are incompletely understood. DNA damage, either induced by genotoxic (direct or via oxygen radicals) or co-genotoxic (e.g. inhibition of DNA repair) processes may play an important role in the initiation of cancer. The alkaline comet assay provides a sensitive tool to investigate these two processes. Cobalt metal, a mixture of cobalt with tungsten carbide and cobalt chloride, were compared for their DNA-damaging capacity. Concentrations from 0 to 6.0 microg Co-equivalent/ml were tested. All three compounds were able to induce DNA damage in isolated human lymphocytes from three donors, in a dose- and time-dependent way. A relatively large interexperimental and interdonor variability in response was observed. This was ascribed to technical parameters and unidentified individual factors. This confirms the importance of repeating experiments using the same and different donors. The DNA-damaging potential of the cobalt-tungsten carbide mixture was higher than that of cobalt metal and cobalt chloride, which had comparable responses. No significant increase of DNA migration was observed when the DNA of cells treated with cobalt metal, cobalt-tungsten carbide or tungsten carbide were incubated with the oxidative lesion-specific enzyme formamidopyrimidine DNA glycosylase. This suggests that during the short treatment period no substantial oxidative damage to DNA was produced. Cobalt metal was able to inhibit the repair of methylmethanesulphonate-induced DNA damage. This was concluded from simultaneous exposure to cobalt and methyl methanesulphonate, post-incubation and post-treatment with 1.2 microg/ml cobalt of methyl methanesulphonate-treated cells.

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Abstract: The objective of this study was to examine the influence of specific surface area on the biological activity of insoluble manganese dioxide (MnO2) particles. The biological responses to various MnO2 dusts with different specific surface area (0.16, 0.5, 17 and 62 m2/g) were compared in vitro and in vivo. A mouse peritoneal macrophage model was used to evaluate the in vitro cytotoxic potential of the particles via lactate dehydrogenase (LDH) release. In vivo, the lung inflammatory response was assessed by analysis of bronchoalveolar lavage after intratracheal instillation in mice (LDH activity, protein concentration and cellular recruitment). In both systems, the results show that the amplitude of the response is dependent on the total surface area which is in contact with the biological system, indicating that surface chemistry phenomena are involved in the biological reactivity. Freshly ground particles with a specific surface area of 5 m2/g were also examined in vitro. These particles exhibited an enhanced cytotoxic activity, which was almost equivalent to that of 62 m2/g particles, indicating that undefined reactive sites produced at the particle surface by mechanical cleavage may also contribute to the toxicity of insoluble particles. We conclude that, when conducting studies to elucidate the effect of particles on the lung, it is important for insoluble particles such as manganese dioxide to consider the administered dose in terms of surface area (e.g. m2/kg) rather than in gravimetric terms (e.g. mg/kg).

Abstract: OBJECTIVES: (1) To assess the value of urinary butoxyacetic acid (BAA) measurement for the monitoring of workers exposed to low concentration of 2-butoxyethanol (BE); (2) to evaluate the in vivo effect of low occupational BE exposure on the erythrocyte lineage; and (3) to test the possible influence of genetic polymorphism for cytochrome P450 2E1 (CYP 2E1) on urinary BAA excretion rate. METHODS: Thirty-one male workers exposed to BE in a beverage package production plant were examined according to their external (BE) and internal (BAA) solvent exposure. The effect of this exposure on erythrocyte lineage [red blood cell numeration (RBC), hemoglobin (Hb), hematocrit (Htc), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), haptoglobin (Hp), reticulocyte numeration (Ret) and osmotic resistance (OR)], hepatic [aspartate aminotransferase (GOT), alanine aminotransferase (GPT)] and renal [plasmatic creatinine, urinary retinol binding protein (RBP)] parameters was also investigated. DNA purified from whole blood was used for CYP 2E1 genotyping. RESULTS: Average airborne concentration of BE was 2.91 mg/m3 (0.59 ppm) with a standard deviation of 1.30 mg/m3 (0.27 ppm). There was a relatively good correlation between external and internal exposure estimated by measuring BAA in post-shift urine samples (average 10.4 mg/g creatinine; r = 0.55; P = 0.0012). Compared with a matched control group (n = 21) exposed workers had a statistically significant decrease (3.3%; P = 0.03) in Hct while MCHC was increased (2.1%; P = 0.02). No significant difference was observed either in other erythroid parameters or in hepatic and renal biomarkers. One exposed individual exhibited a mutant allele with increased cytochrome P450 oxidative activity which coincided with a very low urinary BAA excretion. CONCLUSIONS: Single determination of BAA in post-shift urine samples can be used to assess exposure to low levels of BE. A slight but significant effect on erythroid parameters suggesting membrane damage was observed in exposed workers. The influence of the genetic polymorphism for CYP 2E1 deserves further investigation for the interpretation of urinary BAA measurements.

Abstract: The absorption and cerebral distribution of manganese (Mn) have been studied with respect to the route of administration and the chemical form of the Mn compound. Different groups of adult male rats received either MnCl2, 4H2O or MnO2 once a week for 4 weeks at a dose of 24.3 mg Mn/kg body wt. (b.w.) by oral gavage (g.) or 1.22 mg Mn/kg b.w. by intraperitoneal injection (i.p.) or intratracheal instillation (i.t.). Control rats were treated with 0.9% saline. Four days after the last administration the rats were killed and the concentration of Mn measured in blood, hepatic and cerebral tissues (cortex, cerebellum, and striatum). The liver Mn concentration was not affected by the treatments whatever the chemical form or the route of administration of the Mn compound. Administration of MnCl2 by g., i.p., and i.t. routes produced equivalent steady-state blood Mn concentrations (about 1000 ng Mn/100 ml), representing increases of 68, 59, and 68% compared with controls, respectively. Mn concentrations were significantly increased in the cortex but to a lesser extent (g., 22%; i.p., 36%; i.t., 48%) and were higher in the cerebellum after i.p. and i.t. administrations than after oral gavage. Rats treated i.t. with MnCl2 showed an elective increase of the striatal Mn concentration (205%). In contrast, MnO2 given orally did not significantly increase blood and cerebral tissue Mn concentrations; the low bioavailability is most likely due to the lack of intestinal resorption. Administration of MnO2 i.p. and i.t., however, led to significant increases of Mn concentrations in blood and cerebral tissues. These increments were not significantly different from those measured after MnCl2 administration, except for striatal Mn after i.t. which was markedly less (48%) after MnO2 administration. A comparison of the blood Mn kinetics immediately after g. and i.t. treatment with MnCl2 or MnO2 indicated that the higher elevation of blood Mn concentration (> 2000 ng Mn/100 ml) after i.t. administration of MnCl2 could account for the elective uptake of Mn in the striatum observed in repeated dosing experiments. It is concluded that the modulation of Mn distribution in brain regions according to the route of administration and the chemical form of the Mn compound may be explained on the basis of different blood Mn kinetics and regional anatomic specificities of the striatal region.

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Abstract: Hard metals (WC-Co) are made of a mixture of cobalt metal (Co, 5-10%) and tungsten carbide particles (WC, >80%). Excessive inhalation of WC-Co is associated with the occurrence of different lung diseases including an excess of lung cancers. The elective toxicity of hard metal is based on a physico-chemical interaction between cobalt metal and tungsten carbide particles to produce activated oxygen species. The aim of the present study was to assess the genotoxic activity of hard metal particles as compared with Co and WC alone. In human peripheral lymphocytes incubated with Co or WC-Co, a dose- and time-dependent increased production of DNA single strand breaks (ssb) was evidenced by alkaline single cell gel electrophoresis (SCGE) and modified alkaline elution (AE) assays. Addition of 1 M formate, a hydroxyl radical scavenger, had a protective effect against the production of ssb by both WC-Co or Co alone. On the basis of an equivalent cobalt-content, WC-Co produced significantly more ssb than Co. WC alone did not produce DNA ssb detectable by the AE assay, but results obtained with the SCGE assay may suggest that it either allows some uncoiling of the chromatin loops or induces the formation of slowly migrating fragments. Overall, this in vitro study is the first demonstration of the clastogenic property of cobalt metal-containing dusts. The results are consistent with the implication of an increased production of hydroxyl radicals when Co is mixed with WC particles. The SCGE results also suggest that WC may modify the structure of the chromatin, leading to an increased DNA sensitivity to clastogenic effects. Both mechanisms are not mutually exclusive and may concurrently contribute to the greater clastogenic activity of WC-Co dust. This property of WC-Co particles may account for the excess of lung cancers observed in hard metal workers.

Abstract: The expression of plasminogen activator (PA), a serine proteinase involved in the degradation of extracellular matrix proteins, has been investigated in 3T3 fibroblasts after in vitro exposure to sulphur mustard (SM). Expression of the cell-associated enzyme has been assessed with a synthetic substrate assay and at the mRNA level. Twenty-four hours after 100 microM SM, cell viability (monitored by MTT assay) was not significantly affected, but protein synthesis (tritiated leucine incorporation) was reduced to < 20% of the control value. PA activity was significantly increased compared to control cells with a 20-fold increase after 24 h. This up-regulation was independent of the cell density, occurred maximally between days 1 and 4 and persisted for at least 6 days after exposure. Lower concentrations of SM (< or = 10 microM) did not significantly affect PA activity. Northern blotting experiments revealed an increased expression of urokinase (u-PA) transcripts in cells treated with 100 microM SM, with a peak at 10 h after exposure. Conditioned culture medium from cell cultures treated with 100 microM SM did not affect the expression of PA activity in naive or SM-treated cultures. Thiodiglycol (100 microM), the main metabolite of SM, did not influence the expression of PA in the same system. Different compounds were tested for modulation of the PA upregulation after SM exposure. Nicotinamide (5 mM), vitamin D3 (10(-10)M), extracellular calcium (2 mM) or EGTA (5 mM) had no effect. Ryanodine (10 microM) amplified the PA up-regulation by a factor of 2 and vanadate (500 microM) reduced it by approximately 50%. Dexamethasone (1 microM) added directly after SM treatment almost completely prevented the induction of PA at both the protein and mRNA levels. Overall these results demonstrate an up-regulation of urokinase in 3T3 fibroblasts after treatment with SM, which is possibly mediated by intracellular calcium mobilization. Further studies are needed to identify the significance of this proteolytic response in the pathogenesis of blistering and/or DNA repair mechanisms.

Abstract: The study aimed at assessing the evolution of cadmium (Cd)-induced renal tubular dysfunction in Cd workers according to the severity of the microproteinuria observed at the time the exposure was substantially decreased. Male workers employed in the Cd production industry for whom formerly high exposure had markedly decreased by 1984 and for whom standardized medical data were available during two observation periods (1980-1984 and 1990-1992) were eligible for the study. A total of 32 Cd workers fulfilling this profile were divided into two groups on the basis of historical records of urinary Cd concentration (Cd-U) covering the period until 1984. The workers with Cd-U values of > 10 micrograms Cd/g creatinine were subdivided further on the basis of the urinary concentration of beta 2-microglobulin (beta 2 MG-U) measured during the first observation period (1980-1984). In each group, the tubular microproteinuria as reflected by beta 2 MG-U and the concentration of retinol-binding protein in urine as well as the internal Cd dose as reflected by the concentration of Cd in blood and urine were compared between the first and second (1990-1992) observation periods. Increased microproteinuria was often diagnosed in cases with Cd-U values of > 10 micrograms Cd/g creatinine. The evolution of tubular renal function has been found to depend on the extent of the body burden of Cd (as reflected by Cd-U) and the severity of the initial microproteinuria at the time high Cd exposure was reduced or ceased. When reduction of Cd exposure took place while beta 2 MG-U did not exceed the upper reference limit of 300 micrograms/g creatinine, the risk of developing tubular dysfunction at a later stage was likely to be low, even in cases with historical Cd-U values occasionally > 10 but always < 20 micrograms Cd/g creatinine. When the microproteinuria was mild (beta 2 MG-U > 300 and < or = 1,500 micrograms/g creatinine) at the time exposure was reduced, and the historical Cd-U values had never exceeded 20 micrograms Cd/g creatinine, there was indication of a reversible tubulotoxic effect of Cd. When severe microproteinuria (beta 2 MG-U > 1,500 micrograms/g creatinine) was diagnosed in combination with historical Cd-U values exceeding 20 micrograms Cd/g creatinine, Cd-induced tubular dysfunction was progressive in spite of reduction or cessation of Cd exposure.

Abstract: BACKGROUND: Hydrochlorofluorocarbons (HCFCs) are used increasingly in industry as substitutes for ozone-depleting chlorofluorocarbons (CFCs). Limited studies in animals indicate potential hepatotoxicity of some of these compounds. We investigated an epidemic of liver disease in nine industrial workers who had had repeated accidental exposure to a mixture of 1,1-dichloro-2,2,2-trifluoroethane (HCFC 123) and 1-chloro-1,2,2,2-tetrafluoroethane (HCFC 124). All nine exposed workers were affected to various degrees. Both compounds are metabolised in the same way as 1-bromo-1-chloro-2,2,2-trifluoroethane (halothane) to form reactive trifluoroacetyl halide intermediates, which have been implicated in the hepatotoxicity of halothane. We aimed to test whether HCFCs 123 and 124 can result in serious liver disease. METHODS: For one severely affected worker liver biopsy and immunohistochemical stainings for the presence of trifluoroacetyl protein adducts were done. The serum of six affected workers and five controls was tested for autoantibodies that react with human liver cytochrome-P450 2E1 (P450 2E1) and P58 protein disulphide isomerase isoform (P58). FINDINGS: The liver biopsy sample showed hepatocellular necrosis which was prominent in perivenular zone three and extended focally from portal tracts to portal tracts and centrilobular areas (bridging necrosis). Trifluoroacetyl-adducted proteins were detected in surviving hepatocytes. Autoantibodies against P450 2E1 or P58, previously associated with halothane hepatitis, were detected in the serum of five affected workers. INTERPRETATION: Repeated exposure of human beings to HCFCs 123 and 124 can result in serious liver injury in a large proportion of the exposed population. Although the exact mechanism of hepatotoxicity of these agents is not known, the results suggest that trifluoroacetyl-altered liver proteins are involved. In view of the potentially widespread use of these compounds, there is an urgent need to develop safer alternatives.

Abstract: Although it is well known that micronuclei may arise from either DNA breakage leading to acentric chromosome fragments or from chromosome/chromatid lagging in anaphase, the ratio between the amount of DNA breakage induced and the frequency of micronuclei expressed in the following interphase is unclear. With the development of the alkaline single cell gel electrophoresis assay, which measures single strand and/or double strand breaks in a cell by cell approach, it is new possible to address this question at the cellular level. We therefore compared the genotoxic potential of pure cobalt powder (Co) and a cobalt-containing alloy, cobalt-tungsten carbide (WC-Co), involved in specific lung disorders, in parallel with the alkaline single cell gel electrophoresis (SCGE) assay (comet assay) and the cytokinesis-blocked micronucleus (MN) test, both carried out in vitro on isolated human leukocytes. The comet assay indicated that the WC-Co mixture produced a higher level of DNA damage than Co alone; WC alone was not able to induce a dose-dependent DNA breakage effect as was seen for Co and WC-Co. Results from the MN test confirmed these observations. It was clear that the clastogenic property of Co-containing dust is significantly enhanced when the Co metal is mixed with WC and suggested that their physicochemical characteristics may act as one of the important parameters responsible for the increased incidence of lung cancers observed in the population of hard metal workers. In agreement with data obtained in the same laboratory on liposoluble chemicals (PCBs and chlorinated aliphatic hydrocarbons) and from the literature, the results indicate that both the comet assay and the micronucleus test were able to detect differences in the genotoxic potential of the compounds studied. Although the micronucleus test seemed to be less sensitive to assess a synergistic DNA damaging potential of the mixture involved, it detects chromosomal aberrations (chromosome/genome mutations) and not just repairable DNA breakage or alkali-labile sites. Combination of the comet assay and the in vitro MN test might therefore be recommended for genotoxins to understand the mechanisms underlying mutagenicity and to assess the lowest efficient dose.

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Abstract: We investigated the effect of intratracheally instilled coal fly ash (FA) and copper smelter dust (CU) on the lung integrity and on the ex vivo release of tumor necrosis factor alpha (TNF-alpha) by alveolar phagocytes. Groups of female NMRI mice received a single intratracheal administration of different particles normalized for the arsenic content (20 micrograms/kg body weight, i.e., 600 ng arsenic/mouse) and the particle load (100 mg/kg body weight, i.e., 3 mg/mouse). Mice received tungsten carbide (WC) alone (100 mg/kg), FA alone (100 mg/kg, i.e., 20 micrograms arsenic/kg), CU mixed with WC (CU, 13.6 mg/kg, i.e., 20 micrograms arsenic/kg; WC, 86.4 mg/kg) and Ca3(AsO4)2 mixed with WC (20 micrograms arsenic/kg; WC, 100 mg/kg). Animals were sacrificed at 1, 6, or 30 d posttreatment and analyzed by bronchoalveolar lavage for total protein (TP) content, inflammatory cell number and type, and TNF-alpha production. Additional mice were studied to evaluate particle retention by measuring total arsenic retention in the lung at appropriate times. Instillation of WC induced a mild and transient (d 1) inflammatory reaction characterized by an increase of TP and an influx of polymorphonuclear leukocytes in the alveolar compartment. Compared to WC, Ca3(AsO4)2 produced a significant increase of TP content in BALF. CU particles caused a severe but transient inflammatory reaction, while a persisting alveolitis (30 d) was observed after treatment with FA. Compared to control saline, a marked inhibition of TNF-alpha release was observed in response to LPS in all groups at d 1. Cytokine production was upregulated in WC- and Ca3(AsO4)1-treated animals after 6 and 30 d, respectively. However, a 90% inhibition of TNF-alpha production was still observed at d 30 after administration of CU and FA. Although arsenic was cleared from the lung tissue 6 d after Ca3(AsO4)2 administration, a significant fraction persisted (10-15% of the arsenic administered) in the lung of CU- and FA-treated mice at d 30. We hypothetize that suppression of TNF-alpha production is dependent upon the slow elimination of the particles and their metal content from the lung.

Abstract: The aim of this study is to assess the ability of three methods, alkaline elution (AE), nick translation (NT), and single-cell gel electrophoresis (SCGE), to detect DNA single-strand breaks (ssb) in human peripheral blood lymphocytes (HPBL) exposed in vitro to three genotoxic agents; gamma-rays, ethyl methanesulfonate (EMS) and benzo[a]pyrene diol epoxide (BPDE). The ultimate objective is to select the most feasible, sensitive, and reproducible method for the monitoring of populations exposed to genotoxic agents. AE and NT do not seem suitable assays. AE is able to detect DNA lesions induced by the three compounds, but only at relatively high doses (2 Gy, 5 mM EMS and 20 microM BPDE). With NT, DNA alterations induced by gamma-rays are not detected and ssb are only evidenced after exposure to EMS (80 mM), which already alters the viability of the lymphocytes. Nick translation is able to detect ssb induced by 10 microM BPDE. Compared to the other assays, the sensitivity of the SCGE assay is significantly higher since statistically significant changes were detected after incubation with 0.5 mM EMS and 1.25 microM BDPE. SCGE is a relatively simple method, not time-consuming and applicable to a large number of samples per working day. In conclusion, on the basis of the results of this in vitro comparison, SCGE seems a promising method for the monitoring of populations exposed to genotoxic chemicals.

Abstract: Occupational medicine and occupational health regulations in Belgium are succinctly presented. Since 1970 a minimum level of appropriate training has been required for conferral of a certificate in occupational medicine. At some universities this training is integrated into a larger programme which meets the requirements of EEC Directive 89/594. The current Belgian legislation relating to the prevention of occupational diseases and injuries is detailed in the Règlement pour la Protection du Travail, first published in 1946 and constantly updated. The occupational physician is supposed to provide advice on the risks to which workers are exposed and the adaptation of working conditions in accordance with the state of health or the abilities of the worker. Employers are obliged by law to cover the risks of accident by subscribing to a private insurance policy which covers any related costs. They also contribute financially to the Fonds des Accidents du Travail (Occupational Accidents Fund) and the Fonds des Maladies Professionnelles (Occupational Diseases Fund). Occupational diseases are recognised and may be financially compensated by the Fonds des Maladies Professionnelles.

Abstract: Enhancement of lung antioxidant capacity has been proposed in the therapy of acute lung injuries involving local accumulation of reactive oxygen species (ROS). We have studied in the female Sprague-Dawley rat the effect of intratracheal administration of catalase (CAT) on the acute lung response induced by different ROS generating systems. The lung response was assessed at several time intervals (60-360 min) by monitoring in bronchoalveolar fluid (BALF) the activity of lactate dehydrogenase and the levels of total protein, albumin, and glucose. While CAT (50,000 IU/rat) significantly reduced the biochemical changes induced by hydrogen peroxide produced by a glucose/glucose oxidase system, it markedly exacerbated the lesions induced by phorbol myristate acetate (PMA). Several observations indicate that a particular chemical species formed during the catalase inactivation process is responsible for this effect. Parallel to the development of the lung damage, we noted a rapid reduction of CAT activity (80%) in the BALF of animals treated with PMA and CAT. In vitro an inhibition of CAT activity was observed in the presence of a superoxide anion generating system, and this inhibition was prevented by superoxide dismutase (SOD). A dose of 10,000 IU superoxide dismutase did not prevent the development of the lung lesions induced by PMA plus CAT. Administered alone or in association with PMA, CAT inactivated by heat or 3-aminotriazole also caused severe lung damage. In conclusion, the present study indicates that exogenous catalase may not always protect against the inflammatory reaction resulting from an oxidative stress. In the presence of superoxide anions, catalase may aggravate the lesions, and this possibility should be kept in mind when considering an antioxidant therapy.

Abstract: To assess whether regular consumption of seafood, particularly fish and shellfish, by humans may lead to an overexposure to inorganic arsenic, a well-established human carcinogen, the urinary excretion of the relevant As metabolites (Asi, inorganic form; MMA, monomethylarsonic acid; DMA, dimethylarsinic acid) was compared in groups of subjects with different seafood consumption habits and in volunteers after ingestion of a known amount of seafood arsenicals. Studies of Italian cohorts, involving five groups of +/-30 subjects with different seafood consumption habits, and balance studies in Belgian volunteers failed to show a biologically significant absorption of inorganic arsenic either present as such in the food or formed from organoarsenicals during cooking or digestion. The results suggest that the digestion of some seafood, especially mussels, may increase the urinary excretion of the dimethylated arsenic metabolite. Therefore, the biological monitoring of exposure to inorganic arsenic in an industrial context should mainly rely upon specific measurement of the unmetabolized form when recent ingestion of seafood cannot be excluded.

Abstract: The etiology of male infertilities is largely undetermined, and our knowledge of exogenous factors affecting the male reproductive system is still limited. In particular, the role of specific environmental and occupational factors is incompletely elucidated. Various occupational (physical and chemical) agents have been shown to affect male reproductive functions in animals, but large differences in reproductive function and/or xenobiotic handling between species limit extrapolation to humans. When available, human data are often conflicting and, except in a few instances, usually refer to broad and heterogenous occupational categories or to groups of agents (e.g., solvents). It is often difficult to elucidate the role of a single agent because occupational exposure conditions are often complex and various confounding factors related to lifestyle (smoking, alcohol, and diet) or socioeconomic state may also affect sperm quality, fertility, or pregnancy outcomes. The objective of this work is to summarize the main epidemiological and, where relevant, experimental findings pertaining to agents (physical and chemical) encountered in the occupational environment that might affect the male reproductive system (sperm count, motility and morphology, libido, and fertility) and/or related pregnancy outcomes (spontaneous abortion, stillbirth, low birth weight, and birth defects and childhood malignancy in offspring). Some methodological issues related to research on the reproductive effects of toxicants are also discussed briefly.

Abstract: In the industry, the potential for exposure to cobalt metal dust is particularly important during the production of cobalt powder and the processing and use of hard metals and other cobalt-containing alloys. The different adverse health effects reported in these workers are reviewed. One of the main target organs is the respiratory tract, and this article concentrates on the lung parenchymal reactions induced by cobalt-containing dust. Clinical and epidemiological data indicate that this manifestation is rarely, if ever, induced by pure cobalt metal dust alone, but requires the concomitant inhalation of other compounds such as tungsten carbide in the hard metal industry (hard metal disease). Experimental studies demonstrate that cobalt metal and metallic carbides interact to produce an elective lung toxicity. Recent work on the mechanism of this interaction, which is based on the production of activated oxygen species, is reviewed. A practical implication in industrial hygiene should be that permissible exposure levels to Co dust might have to be different when exposure is to pure Co particles or an association with carbides.

Abstract: In recent years clinical, epidemiological and experimental evidence has accumulated indicating that cobalt metal particles, when inhaled in association with other agents such as metallic carbides (hard metals) or diamond dust, may produce an interstitial lung disease termed "hard metal disease" or "cobalt lung". This article summarizes the progress accomplished in our two laboratories to understand the pathogenesis of this disease. Gaps and weaknesses in our current knowledge have also been highlighted in order to suggest potential avenues for further research. Whilst animal models have proved useful for the demonstration of the toxic synergy between cobalt and carbides (e.g. tungsten carbide), most animal models have remained descriptive and have not provided information on the mechanism for this synergy. In particular, the bizarre multinucleated giant cells which are an important hallmark of the human disease, have not been reproduced consistently in experimental animals. Since cobalt is a known sensitizer, there may also be a need to develop experimental models to test the possible involvement of immunological mechanisms in the pathogenesis of the interstitial disease. In vitro systems including macrophage cell cultures and physico-chemical tests have been useful to investigate the mechanism underlying the toxic synergy. The recent finding that, in vitro, cobalt and metallic carbides interact with oxygen to produce toxic activated oxygen species opens a new avenue of research and may offer an alternative interpretation of the fact that only a limited proportion of exposed workers develop interstitial disease. Besides the possible involvement of immunological mechanisms, it may be speculated that individuals with a lower antioxidant defence are more susceptible to the toxic effect of activated oxygen species produced by cobalt-containing dusts from hard metal.

Abstract: In recent years, epidemiological evidence that exposure to toluene diisocyanate (TDI) is associated with adverse health effects has led to the development of useful analytical methods for the biological monitoring of TDI. In this paper, an HPLC method is presented that allows accurate determinations of toluenediamines (TDA), urinary metabolites of TDI, in hydrolysed human urine without complicated or time-consuming sample treatment. The procedure requires 5.0 ml of urine and involves the extraction with toluene of TDA and the hydrolysable conjugate fraction followed by further purification with a strong cation-exchange sorbent. Strongly alkaline conditions are chosen for the hydrolysis of urine samples and phenylene-1,3-diamine is used as internal standard to control the sample extraction and clean-up. Separation is performed on a base-deactivated octadecyl reversed-phase column by either ion-suppression or ion-pair chromatography. Chromatographic analysis is complete in less than 20 min and chromatograms with no interfering peaks are obtained. High sensitivity and selectivity are achieved by using electrochemical detection: 2,6- and 2,4-TDA can be detected at the 0.1 and 0.15 microgram l-1 levels, respectively. Absolute recoveries of the method tested with urine samples spiked at 10 micrograms l-1 with phenylene-1,3-diamine and from 1 to 25 micrograms l-1 with 2,6-and 2,4-TDA are greater than 87.6% and 88.3%, respectively. The assay is linear from 0 to 50 micrograms l-1. Within-run precisions evaluated on 10 urine samples ranging from 0 to 10 micrograms l-1 are 7.9% and 5.3% for 2,6- and 2,4-TDA, respectively. Results obtained with urine samples from 12 controls and 15 exposed workers from a flexible polyurethane foam factory indicate that the method is appropriate for the biological monitoring of occupational exposure to TDI.

Abstract: Epidemiological and clinical studies suggest that inhalation of cobalt metal dust (Co) mixed with tungsten carbide particles (WC), but not of cobalt dust alone, may cause interstitial pulmonary lesions (hard metal disease). In previous experimental studies in the rat, we have demonstrated the greater acute pulmonary toxicity of a WC-Co mixture compared to Co or WC alone. The present study was undertaken to compare in the same animal model the delayed lung response after intratracheal administration of Co or WC-Co particles (cobalt particle 6.3 wt%). The responses were also compared with those obtained after treatment with arsenic trioxide and crystalline silica used a reference materials producing an acute toxic insult and a progressive fibrogenic response, respectively. Cellular (total and differential counts) and biochemical parameters (LDH, N-acetyl-beta-D-glucosaminidase, total protein, albumin, fibronectin, and hyaluronic acid) were measured in bronchoalveolar lavage fluid following single and repeated intratracheal instillations. The results indicate that the delayed lung response observed after WC-Co is different from that after cobalt metal alone. A single intratracheal dose of WC-Co (1, 5, or 10 mg/100 g body wt) induced an acute alveolitis which persisted for at least 1 month. Four months after a single instillation of WC-Co, no clear histological lung fibrosis could however be evidenced, indicating a reversibility of the lesions. The effects of cobalt (0.06, 0.3, or 0.6 mg/100 g body wt) or tungsten carbide alone (1, 5, 10 mg/ 100 g body wt) were very modest, if any. Following repeated intratracheal instillations (four administrations at 1-month interval), increased lung hydroxyproline content and histopathological evidence of interstitial fibrosis were observed after WC-Co (4 x 1 mg/100 g body wt), but not after administration of each component separately, i.e., Co (4 x 0.06 mg/100 g body wt) or WC (4 x 1 mg/100 g body wt). The mechanism of the fibrotic reaction induced by WC-Co seems different from the progressive inflammatory reaction induced by crystalline silica. We suggest that it might result from a scarring reaction elicited by repeated acute insults as observed after repeated administration of arsenic trioxide.

Abstract: Hard metal alloys (WC-Co) are made of a mixture of cobalt (Co; 6%) and tungsten carbide (WC; 94%) particles. Chronic inhalation of hard metal dust can lead to the development of a fibrosing alveolitis, the pathogenesis of which is still undefined. The present investigation was undertaken to assess the effect of Co, WC, and WC-Co particles on the release by lung phagocytes of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), fibronectin, and cystatin-c. The responses were compared with those induced by two other lung toxicants, i.e., crystalline silica (DQ12) and arsenic trioxide (As2O3). IL-1 and TNF-alpha activities produced in the presence and absence of LPS stimulation were measured with the aid of bioassays while fibronectin and cystatin-c were determined by latex immunoassays. In vitro, maximal noncytotoxic doses of As2O3, Co, WC, or WC-Co did not significantly affect the production of these mediators by rat alveolar macrophages. In contrast, DQ12 enhanced the production of TNF-alpha (with and without LPS stimulation) and IL-1 (after LPS stimulation) and decreased cystatin-c release (in the absence of LPS). Following a single intratracheal instillation of the different test preparations in the rat, the response of the lung phagocytes obtained by bronchoalveolar lavage (BAL) 24 hr later was examined. We were unable to detect any consistent effect of Co (0.06 mg/100 g body wt), WC (1 mg/100 g body wt), or WC-Co treatment (1 mg/100 g body wt) on the production of the above mediators. In contrast, after LPS stimulation, As2O3 (0.5 mg/100 g body wt) and DQ12 (1 mg/100 g body wt) stimulated the production of TNF-alpha and IL-1. In the absence of LPS, As2O3 stimulated fibronectin and cystatin-c production and DQ12 stimulated cystatin-c release. Since the dose of WC-Co used in vivo (1 mg/100 g body wt) caused pronounced lung inflammation (increased LDH, protein, and albumin levels in BAL fluid), we conclude that the acute lung toxicity of WC-Co particles is not mediated through enhanced production of the examined mediators by lung phagocytes.

Abstract: 1. Pyridostigmine (PYR) pretreatment is used by the military to obtain 20-30% whole blood acetylcholinesterase (AChE) inhibition in order to enhance the effectiveness of the standard therapeutic regimen for poisoning by an organophosphate anticholinesterase agent. The present study was undertaken to investigate in a rat model the potential muscle damage produced by this pretreatment when given alone or combined with physical exercise. 2. Grip strength and biochemical measurements, i.e. serum creatine phosphokinase (CPK) activity and creatine urinary excretion rate, together with histological studies, were performed in resting animals during a period of 14 days of PYR administration in a dose producing 20-30% whole blood acetylcholinesterase inhibition. No evidence was found of a deleterious effect of this treatment on the skeletal muscle. 3. In contrast, following physical exercise, the same treatment significantly exacerbated the biochemical changes reflecting a loss of integrity in skeletal muscles, namely, increased CPK and urinary creatine excretion rate. The significance of this observation remains to be clarified.

Abstract: Decreased interleukin-1 (IL-1) production by mononuclear phagocytes has been shown to contribute to benzene myelotoxicity in animals. The study presented here was designed to examine the relevance of this mechanism in humans. Fresh human blood monocytes were exposed to 0.001-10 microM hydroquinone (HQ) and assessed for their ability to release IL-1 alpha and IL-1 beta in response to a stimulation with endotoxin. Both cytokines were measured by specific ELISA. Exposure of human monocytes to micromolar concentrations of HQ for 2 hr resulted in a dose-dependent reduction of IL-1 secretion. For both IL-1 alpha and IL-1 beta, the decreases were statistically significant at concentrations of 5 microM and above. HQ also inhibited RNA and protein synthesis in a dose-dependent manner, with 50% inhibitory concentrations of 21 +/- 11 and 10 +/- 9 microM, respectively. Furthermore, monocytes treated with 5 microM HQ also displayed a reduced total protein content when compared with control cells. These data suggest that the reduction of IL-1 production caused by HQ results from a global impairment of monocyte essential functions such as transcription or translation. Taken as a whole, our results support a mechanism whereby HQ may contribute to the myelotoxicity of benzene in humans by inhibiting the production by mononuclear phagocytes of cytokines involved in the regulation of hematopoiesis.

Abstract: 1. This in vitro study was undertaken as a preliminary approach before assessing whether the alkaline elution assay can be applied to peripheral blood lymphocytes (PBL) for the monitoring of humans exposed to genotoxic agents such as polycyclic aromatic hydrocarbons (PAH). We have compared in vitro, with the aid of the alkaline elution assay, the formation and the repair of DNA single-strand breaks (ssb) induced by different genotoxic agents [gamma-irradiation, ethyl methanesulfonate (EMS), benzo<a>pyrene diol epoxide (BPDE)] on quiescent and PHA-stimulated human lymphocytes and on a fibroblast cell line. 2. Gamma-irradiation (4 Gy) induced an equivalent amount of DNA ssb in the three cell types. On the other hand, after treatment with EMS (10 mM) and BPDE (50 microM), a higher production of DNA ssb was observed in replicating cells (PHA-stimulated lymphocytes and fibroblasts) when compared with quiescent lymphocytes. 3. After gamma-irradiation, all cell types repaired more than 65% of ssb within 1 h. After treatment with EMS, we noted a deficient DNA repair capacity in quiescent lymphocytes in comparison with replicating cells. In all cell types treated with BPDE, more breaks were observed after a 2 h repair period than immediately after treatment, demonstrating the involvement of a slow repair mechanism after BPDE treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Hard metal alloys (or cemented carbides) are made of a mixture of tungsten carbide particles (WC, more than 80%) cemented in cobalt metal powder (Co, 5-10%). The inhalation of hard metal particles may cause an interstitial pulmonary disease, the mechanism of which involves an interaction between Co and WC particles. Some epidemiological data also suggest that hard metal dust can induce lung cancer in workers. In a macrophage culture model, butylated hydroxytoluene (1 mM) protected from the cytotoxicity of hard metal particles, suggesting a possible involvement of lipid peroxidation in the toxicity of these powders. In a biochemical system, a mixture of Co and WC particles, but not Co or WC alone, stimulated the production of thiobarbituric acid-reactive substances from arachidonic acid. Using a spin trapping system applied to aqueous particulate suspensions and electrochemical techniques, we present experimental evidence that the association of Co and carbide particles represents a specific toxic entity producing large amounts of activated oxygen species. The mechanism of this interaction proceeds through the oxidation of cobalt metal catalyzed at the surface of carbide particles and resulting in the reduction of dissolved oxygen. This physicochemical property of hard metal particles provides a new basis for interpreting their inflammatory action and their possible carcinogenic effect on the lung.

Abstract: Chronic inhalation of hard metal particles can produce an interstitial lung disease (hard metal disease). Recent studies on rats and on isolated alveolar and peritoneal macrophages have demonstrated that this disorder can be explained by an interaction between cobalt metal (Co) and tungsten carbide (WC) particles, which represent the main constituents of hard metal. The exact mechanism of this interaction is still undefined. The present study was undertaken to assess in vitro whether a similar interaction also occurs between cobalt and other metallic carbide particles which may also be incorporated in hard metals depending on the desired applications. When tested separately, Co and metallic carbide particles did not affect the cell integrity. In contrast, TiC, NbC and Cr(3)C(2) exerted a synergistic effect with Co (interactive carbides) while TaC, Mo(2)C and SiC did not (non-interactive carbides). The interaction did not simply result from an increased cobalt bioavailability since cobalt uptake by the macrophages was increased 4-7-fold in the presence of interactive as well as non-interactive carbides. The interactive effect appeared dependent on the size of the carbide particles, which suggests that a physicochemical reaction taking place at the interface between certain carbides and cobalt particles may be responsible for the toxicity of the Co-carbide mixture. Other non-carbide particles (Fe, diamond, crystalline silica) did not produce a similar interaction with cobalt. This observation may contribute to the better delineation of the pathogenesis of hard metal disease.

Abstract: The oxygen dependence of ethane formation was investigated in rat lung and liver homogenates, incubated in sealed flasks, in which the peroxidation was stimulated by the addition of ferrous ions. For both tissues, the production of ethane was maximal under a 20% oxygenated gas phase, while hyperoxic conditions led to a decreased ethane in the gas phase. The formation of thiobarbituric acid-reactive substances (TBA-RS), another marker of the lipid peroxidation process, in the homogenates of lung and liver was strongly stimulated at 100% compared to 20% oxygen. Experiments were also carried out on iron-stimulated peroxidation of pure docosahexaenoic acid preparations, which under air led to a large production of ethane. As for tissue homogenates, the TBA-RS content was increased in the presence of 100% oxygen. Those conditions, however, did not induce an increase in ethane production but led to the formation of ethanol. Therefore, the quenching of ethyl radical by molecular oxygen seems to be a very attractive hypothesis to explain the lack of increased ethane production in favor of ethanol when iron-induced lipid peroxidation was stimulated by oxygen.

Abstract:

Abstract: The production of thiobarbituric acid-reactive substances (TBA-RS) and ethane, two markers of the lipid peroxidation process, was evaluated in rat lung and liver microsomal membranes incubated in the presence of either ferrous ions or a mixture of ferric ions and ascorbate. Microsomal fractions isolated from lung tissue were more resistant than those isolated from the liver. Compared to Fe2+, the association of Fe3+/ascorbate seemed to be totally ineffective in stimulating peroxidation of lung microsomes. The fatty acid profile of lung and liver microsomal membranes could not be responsible for their different susceptibility to free radical degradation. The microsomal fraction isolated from lung showed a higher vitamin E concentration than the liver. The importance of vitamin E in protecting lung membranes was assessed by using lung and liver isolated from vitamin E-deficient and vitamin E-supplemented rats. For both lung and liver microsomal fractions an inverse relationship between vitamin E concentrations and the extent of lipid peroxidation was observed. However, although the vitamin E concentrations in lung and liver microsomes isolated from rats submitted to a vitamin E-deficient diet were not different, lung microsomes still exhibited a lower production of TBA-RS and ethane than liver. In addition to vitamin E, other factors must be involved to explain the resistance of lung microsomes to lipid peroxidation.

Abstract: The intermediate syndrome in organophosphate poisoning is clinically characterized by weakness in the territory of cranial nerves, weakness of respiratory, neck and proximal limb muscles, and depressed deep tendon reflexes. It occurs between the acute cholinergic crisis and the usual onset of organophosphate-induced delayed neurotoxicity. The weakness has been ascribed to muscle fiber necrosis. Fenthion has been the most common cause. This study assesses the occurrence of the necrotizing myopathy in rats in relation to the clinical course and the acetylcholinesterase (AChE) inhibition after poisoning with organophosphates representative for each of the major types of organophosphate-related neurotoxicity. Marked differences are noted in the duration of cholinergic symptoms and of AChE inhibition after either paraoxon and mipafox, or fenthion poisoning. The necrotizing myopathy begins shortly after the initial decline in AChE activity with all organophosphates studied. Maximal muscle involvement occurs within the first 2 days of the poisoning with all organophosphates studied. The myopathy is not aggravated by a further decline in AChE activity in fenthion poisoning. Our data argues against the monophasic necrotizing myopathy being the cause of the intermediate syndrome, and is suggestive of persistent AChE inhibition being involved.

Abstract: Hard metal is an alloy of tungsten carbide (WC) in a matrix of cobalt metal (Co). The inhalation of hard metal dust can cause an alveolitis which may progress to interstitial fibrosis. This study was undertaken to compare, both in vivo and in vitro, the bioavailability of cobalt metal when mixed or not with WC and to assess whether this factor had any influence on the cellular toxicity of hard metal particles. In vivo, non-toxic doses of cobalt metal were administered intratracheally in the rat, alone (Co, 0.03 mg/100 g) or mixed with tungsten carbide (WC-Co, 0.5 mg/100 g containing 6.3% of cobalt metal particles). Sequential measurements of cobalt in the lung and in urine demonstrated that the retention time of the metal in the lung was longer in Co- than in WC-Co-treated animals. In vitro, the cellular cobalt uptake was higher when the metal was presented to the macrophages as WC-Co. However, there was no relationship between the cellular uptake of cobalt and the occurrence of toxicity, since the intracellular concentration of cobalt associated with the occurrence of a cytotoxic effect of WC-Co particles was insufficient to exert the same effect when resulting from exposure to Co alone. This clearly indicates that increased bioavailability of cobalt is not the mechanism by which hard metal particles exhibit their cellular toxicity. These observations confirm and extend our previous findings supporting the view that cobalt is not the only component responsible for the toxicity of hard metal particles which should be considered as a specific toxic entity.

Abstract: The effect of the vesicating agent sulfur mustard (SM) has been investigated in vitro using murine peritoneal macrophages. The rationale for this study was three-fold: (1) the first symptoms after exposure to SM are mucous and cutaneous erythema, itching and oedema suggesting that inflammatory cells may represent an early target of SM toxicity; (2) it has been proposed that macrophages and their secretory products may participate in the degradation of the dermal-epidermal junction; and (3) macrophages are important components of the immune system and any alteration of their metabolism may be relevant in clarifying the immune impairments caused by SM. Cell viability, measured by LDH release and lysozyme production, was reduced in a concentration-dependent manner following exposure to SM at 10 mum or higher. A reduction of superoxide anion and hydrogen peroxide production was observed on exposure to concentrations greater than 10 and 1 mum, respectively. Cell-associated plasminogen activator activity was significanty increased (130% of the control) following exposure to 10 mum and a decrease occurred with exposures to 100 mum or more. The release of arachidonic acid equivalents was not significantly affected by SM treatment. These results demonstrate the cytotoxic effects of SM towards macrophages in culture. While activated macrophages may be present in the dermis after in vivo exposure to SM, no evidence was found of a direct stimulatory effect of SM on the production of macrophage inflammatory products.

Abstract: Cobalt is an essential oligoelement which enters in the composition of vitamin B12. For the general population, food and beverages represent the main source of cobalt exposure. Traces of cobalt are also present in cement and various household products. In industry, the potential for exposure to cobalt is particularly important during the production of cobalt powder, the production, processing and use of hard metals, the polishing of diamonds with cobalt containing disks and the processing of cobalt alloys. Except in the production of cobalt powders, these activities involve exposure not only to cobalt but also to other substances such as tungsten carbide, iron and diamond which may modulate the biological reactivity of cobalt. Cobalt salts are used for the preparation of enamels and pigments. Cobalt is mainly absorbed from the pulmonary and the gastrointestinal tracts. Absorption through the skin can occur but is low. Concomitant exposure to tungsten carbide increases the pulmonary absorption rate of cobalt metal. Cobalt is not a cumulative toxin and is mainly excreted in urine and to a lesser extent via faeces. Cobalt in blood and urine mainly reflects recent exposure. In the past, outbreaks of cardiomyopathy occurred among heavy consumers of cobalt fortified beer. It is likely that poor nutrition and ethanol had played a synergistic role. Toxic manifestations, however, have mainly been reported following inhalation of cobalt containing dusts in industry. The two main target organs are the skin and the respiratory tract. Cobalt itself may cause allergic dermatitis, rhinitis and asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: We report a case of organophosphate poisoning with a commercial preparation of malathion (deliberate ingestion of Malathane Garden Spray: malathion 15% in isopropyl alcohol) in which the initial cholinergic crisis was followed by cardiac, pulmonary, neurological and renal manifestations. They occurred when erythrocyte and plasma cholinesterases were reactivating. A chemical analysis of the pesticide preparation revealed, apart from malathion itself, the presence of isopropylmalathion and O,O,S-trimethylphosphorothioate. Although pure malathion is regarded as one of the safest organophosphate insecticides, this observation underlines the possibility of severe complications after exposure to a preparation which has been stored for a long period of time.

Abstract: OBJECTIVE: The aim was to examine the relation between environmental and biological (blood and urine) indices of exposure to different chemical forms of cobalt. METHODS: A cross sectional study was undertaken in workers exposed to cobalt metal, oxides, and salts in a refinery and to a mixture of cobalt and tungsten carbide in a hard metal producing plant. RESULTS AND CONCLUSION: Although biological monitoring of workers exposed to cobalt oxides showed higher blood and urine concentrations than in non-exposed subjects, these indices poorly reflected the recent exposure level. By contrast, when exposure was to soluble cobalt compounds (metal, salts, and hard metals), the measurement of urine or blood cobalt at the end of the workweek could be recommended for the assessment of recent exposure. An eight hour exposure to 20 or 50 micrograms/m3 of a soluble form of cobalt would lead to an average concentration in a postshift urine sample collected at the end of the workweek of 18.2 or 32.4 micrograms of cobalt/g creatinine, respectively.

Abstract: The lung thiobarbituric acid-reactive substances (TBA-RS) content and the amount of ethane exhaled, two potential markers of the lipid peroxidation process, were measured in rats following intratracheal administration of chemicals stimulating the production of free radicals, i.e. paraquat, phorbol myristate acetate and ferrous ions. Five hours after treatment, autopsy revealed gross pulmonary damage but the lung TBA-RS and the ethane exhalation were not different from control animals. On the contrary, a large increase in ethane production was observed 2 hr after intraperitoneal administration of the hepatotoxic carbon tetrachloride. In vitro, incubation of lung and liver homogenates from control rats with ferrous iron led to the development of a lipid peroxidation process in both tissues but the accumulation of TBA-RS and ethane was much lower with homogenates from lung as compared to liver tissue. Those results suggest that the lung may be more resistant than the liver to the initiation and/or propagation of a lipid peroxidation process. The possibility that others markers than ethane and TBA-RS are more appropriate to detect this process in the lung must also be considered.

Abstract: The lung toxicity of a carbide-cobalt mixture is more important than that of each individual component; the mechanism of this interaction is not understood. The capacity of cobalt metal particles alone and mixed with different carbides to generate hydroxyl radicals was examined with the deoxyribose assay. In a chemical system, cobalt ions and cobalt metal particles (Co) were found to catalyse the degradation of deoxyribose in the presence of hydrogen peroxide. Carbides were able to directly oxidize deoxyribose, but their respective activities did not support such a mechanism to explain the carbide-cobalt interactive toxicity, since there was no direct relationship between deoxyribose degradation ability and cytotoxicity toward macrophages. Tungsten, niobium, titanium and chromium carbides (interactive carbides) were only weak oxidants and conversely molybdenum, vanadium and silicon carbides (non-interactive carbides) were the most potent ones. The ability of cobalt metal to produce hydroxyl radicals in the presence of hydrogen peroxide was not increased by tungsten carbide. The role of reactive radical formation in the toxicity of these particles was further assessed in a macrophage culture model. Catalase (4000 U/ml), superoxide dismutase (300 U/ml), sodium azide (1 mM), sodium benzoate, mannitol, taurine and methionine (all 20 mM) were all unable to protect against the cytotoxic effects of cobalt ions and cobalt metal alone or mixed with tungsten carbide. In conclusion, no evidence was found that production of reactive oxygen species contributes to the elective toxicity of carbide-cobalt mixtures.

Abstract: Mature spermatozoa contain a number of proteases that are supposed to contribute to their fertilizing ability. The present study was directed at plasminogen activator (PA), a protease that belongs to the group of serine proteases and converts the zymogen plasminogen to the active broad-spectrum protease plasmin. To investigate the possible role of PA in the fertilization process, we have measured sperm-bound PA activity in 63 patients included in an in-vitro fertilization (IVF) programme and assessed their relationship to standard semen parameters and the rate of fertilization. PA activity was correlated significantly with the sperm count, as well as with sperm motility and morphology. Using logistic regression analysis, specific PA (pmol pNA 10(-6) cells min-2) was found to significantly influence the probability of fertilization. Other significantly predictive factors were motility and the percentage of spermatozoa with normal morphology. The sperm concentration (10(6) cells ml-1) did not significantly affect the outcome of IVF. We suggest that sperm-bound PA is involved in the fertilization process and may represent a potential indicator of sperm fertilizing capacity.

Abstract: 1. This study was initiated to ascertain the possibility of biochemically monitoring the rhabdomyonecrosis that occurs after organophosphate poisoning. The evolution of different parameters has been assessed in the rat 6, 16, 24 and 48 h following 0.67 x LD50 of soman. 2. Acetylcholinesterase (AChE) was inhibited to 60% of the control value in the diaphragm at 6 and 16 h and serum ChE levels inhibited to an average of 30% of the control value. At 24 h, total blood, brain and diaphragm AChE were inhibited by 40, 69 and 38%, respectively. 3. Rhabdomyonecrosis lesions occurred in the diaphragm after 24 h and were accompanied by a concurrent increase in urinary creatine excretion rate (300% of the control) and serum total creatine phosphokinase activity (280% of the control). Calcium-activated neutral protease and phosphorylase a activities were elevated in the muscle at the same time. 4. These biochemical markers will prove useful for investigating the possible relationships between the different neuromuscular syndromes occurring in the course of an OP poisoning and potential therapeutic or protective pharmacological measures.

Abstract: Plasminogen activator (PA) activity has been suggested to be an important determinant of cell migration and tissue modelling during organogenesis. We have postulated that in the developing embryo, any abnormal modulation of this enzymatic activity may lead to the production of teratogenic effects. In the present study, we investigated the effect of teratogenic doses (inducing about 50% of malformed embryos) of retinoic acid, auranofin and mercuric chloride on PA activity in post-implantation cultured mouse embryos. At the end of the culture period, PA activities of malformed and normal embryos in the same treatment group were compared. PA activities in compound-exposed embryos were also compared with those in untreated controls. The design of the present experiment allowed the identification of the effect of drugs on PA activity and its possible relation with induced malformations. An increased PA activity was observed in malformed embryos treated with mercuric chloride. PA activity was slightly increased in both groups (normal and malformed) exposed to retinoic acid. No effect of auranofin was observed on embryo PA activity. In conclusion, the data do not confirm but they also do not contradict the hypothesis that abnormal levels of PA activity lead to dysmorphogenesis.

Abstract: Several organs (lung, skin, thyroid, heart, bone marrow) are potential targets of cobalt (Co). Whereas there is no doubt that inhalation of Co alone may cause bronchial asthma, its role in the occurrence of hard metal disease is still controversial because most cases were reported in workers exposed not only to Co but also to other substances such as tungsten carbide, titanium carbide, iron, silica and diamond. To assess whether exposure to pure Co dust (metal, oxides, or salts) may lead to adverse health effects a cross sectional study was carried out among 82 workers in a Co refinery. The results were compared with those in a sex and age matched control group. The Co group had been exposed for 8.0 years on average (range 0.3-39.4). The geometric mean time weighted average exposure assessed with personal samplers (n = 82) was about 125 micrograms/m3 and 25% of the values were higher than 500 micrograms/m3. The concentrations of Co in blood and in urine after the shift were significantly correlated with those in air. Concentration of Co in urine increased during the workweek. A slight interference with thyroid metabolism (decreased T3, T4, and increased TSH), a slight reduction of some erythropoietic variables (red blood cells, haemoglobin, packed cell volume) and increased white cell count were found in the exposed workers. The exposed workers complained more often of dyspnoea and wheezing and had significantly more skin lesions (eczema, erythema) than control workers. Within the exposed group a dose-effect relation was found between the reduction of the forced expiratory volume in one second/vital capacity and the intensity of current exposure to Co assessed by the measurement of Co in air or in urine. The prevalence of dyspnoea was related to the dustiness of the workplace as reflected by statistically significant logistic regression between this symptom and the current levels of Co in air and in urine. No difference between lung volumes, ventilatory performances, carbon monoxide diffusing capacity, and serum myocardial creatine kinase and procollagen III peptide was found between the Co and control groups and no lung abnormalities were detected on the chest radiographs in both groups. The results suggest that exposure to high airborne concentrations of Co alone is not sufficient to cause pulmonary fibrosis. This finding is compatible with experimental studies indicating that interaction of other airborne pollutants with Co particles play a part in the pathogenesis of parenchymal lung lesions.