Abstract: There have been several studies on the maternal administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and effects in the reproductive tract of male offspring, subsequent to risk assessments undertaken in 2001. This review compares the methodology and results to examine key methodological features, and consistency in reported outcomes. Maternal dosing at >0.8mug TCDD/kg causes lethality and weight loss, and it is difficult to distinguish between direct and indirect effects of TCDD at these dose levels. Statistically significant effects of maternal doses of <1mug TCDD/kg (i.e. the dose levels relevant for risk assessment) on prostate weight or epididymal sperm counts in offspring were reported in the minority of studies. The pharmacokinetics of TCDD differs considerably between acute and chronic dosing, and with dose level of TCDD. On the basis of body burden, TCDD had different potency at inducing adverse effects in the only comparison study between acute and chronic dosing. Understanding of the pharmacokinetics of TCDD and relationship to adverse effects in offspring is required. These analyses identify key features of TCDD developmental toxicity in male offspring, and identify data needs for future risk assessment.
Abstract: The Aryl Hydrocarbon Receptor (AhR) is required for the toxicity of TCDD, and so the AhR of CRL:WI and CRL:WI(Han) rats was characterised. Western blot showed AhR proteins of approximately 110 and approximately 97 kDa in individual rats from both strains. The AhR cDNA from a CRL:WI(Han) rat with the approximately 110kDa protein revealed a sequence that was identical to that of the CRL:WI and SD rat. However, cloning of the AhR from a rat with the approximately 97kDa protein revealed a point mutation, and five variants encoding two C-terminally truncated variants of the AhR protein, arising from a point mutation in the intron/exon junction and consequent differential splicing. These C-terminally truncated variants were expressed and shown to give rise to a protein of approximately 97kDa; the recombinant AhR bound TCDD with an affinity that was not statistically different from the full-length protein. A single-nucleotide polymorphism (SNP) assay was developed, and showed that both alleles were represented in a Hardy-Weinberg equilibrium in samples of CRL:WI and CRL:WI(Han) populations; both alleles are abundant. Rats from two studies of TCDD developmental toxicity were genotyped, and the association with toxicity investigated using statistical analysis. There was no plausible evidence that the AhR allele had a significant effect on the toxic endpoints examined. These data show that the two AhR alleles are common in two strains of Wistar rat, and that the AhR alleles had no effect on TCDD-induced developmental toxicity in two independent studies.
Abstract: It has been reported that fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat. We set out to replicate and extend these effects using a robust experimental design. Groups of 75 (control vehicle) or 55 (50, 200 or 1000 ng of TCDD kg(-1) bodyweight) female Wistar(Han) rats were exposed to TCDD on Gestational Day (GD) 15, then allowed to litter. The high dose group dams showed no sustained weight loss compared to control, but four animals had total litter loss. Pups in the high dose group showed reduced body weight up till day 21, and pups in the medium dose group showed reduced body weight in the first week post partum. Balano-preputial separation was significantly delayed in the high dose group male offspring. There were no significant effects of treatment when the offspring were subjected to a functional observational battery, or mated with females to assess reproductive capability. 25 males per group were killed on post natal day (PND) 70, and approximately 60 animals per group ( approximately 30 for the high dose group) on PND120 to assess seminology and other endpoints. At PND120, the two highest dose groups showed a statistically significant elevation of sperm counts, compared to control; however, this effect was small ( approximately 30%), within the normal range of sperm counts for this strain of rat, was not reflected in testicular spermatid counts nor PND70 data, and is therefore postulated to have no biological significance. Although there was an increase in the proportion of abnormal sperm at PND70, seminology parameters were otherwise unremarkable. Testis weights in the high dose group were slightly decreased at PND 70 and 120, and at PND120, brain weights were decreased in the high dose group, liver to body weight ratios were increased for all three dose groups, with an increase in inflammatory cell foci in the epididymis in the high dose group. These data show that TCDD is a potent developmental toxin after exposure of the developing fetus, but that acute developmental exposure to TCDD on GD15 caused no decrease in sperm counts.
Abstract: We compared the effects of a single acute dose, or chronic fetal exposure, to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the male reproductive system of the Wistar(Han) rat. Tissue samples were taken from dams on GD16 and GD21, and from offspring on PND70 and 120. Steady state concentration of TCDD was demonstrated in the chronic study: body burdens were comparable in both studies. Fetal TCDD concentrations were comparable after acute and chronic exposure, and demonstrate more potent toxicity after chronic versus acute dosing. In maternal liver, cytochrome P450 (CYP)1A1 and CYP1A2 RNA were induced. In fetus, there was induction of both CYP1A1 and CYP1A2 RNA at medium and high doses, but inadequate evidence for induction at low dose in either study. The low level induction of CYP1A1 RNA at low dose in fetus argues against AhR activation in fetus as a mechanism of toxicity of TCDD in causing delay in balanopreputial separation, and the greater induction of CYP1A1 RNA in PND70 offspring liver suggests that lactational transfer of TCDD is crucial to this toxicity. These data characterise the maternal and fetal disposition of TCDD, induction of CYP1A1 RNA as a measure of AhR activation, and suggest that lactational transfer of TCDD determines the difference in delay in balanopreputial separation between the two studies.
Abstract: We have investigated whether fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat, using chronically exposed rats to ensure continuous exposure of the fetus. 5-6 week old rats were exposed to control diet, or diet containing TCDD, to attain an average dose of 2.4, 8 and 46 ng TCDD kg(-1) day(-1) for twelve weeks, whereupon the rats were mated, and allowed to litter; rats were switched to control diet after parturition. Male offspring were allowed to develop until kills on PND70 (25 per group), or PND120 (all remaining animals). Offspring from the high dose group showed an increase in total litter loss, and the number of animals alive on post-natal day (PND) 4 in the high dose group was approximately 26% less than control. The high and medium dose offspring showed decreased weights at various ages. Balano-preputial separation was significantly delayed in all three dose groups, compared to control. There were no significant effects of maternal treatment when the offspring were subjected to a functional observational battery, or learning tests, with the exception that the high dose group showed a deficit in motor activity. 20 rats per group were mated to females, and there were no significant effects of maternal treatment on the fertility of these rats, nor on the F(1) or F(2) sex ratio. Sperm parameters at PND70 and 120 showed no significant effect of maternal treatment, with the exception that there was an increase in the proportion of abnormal sperm in the high dose group at PND70; this is associated with the developmental delay in puberty in this dose group. There were no remarkable findings of maternal treatment on organ weights, with the exception that testis weights were reduced by approximately 10% at PND70 (but not PND120), and although the experiment was sufficiently powered to detect small changes, ventral prostate weight was not reduced. There were no significant effects of maternal treatment upon histopathological comparison of high dose and control group organs. These data confirm that developmental exposure to TCDD shows no potent effect on adult sperm parameters or accessory sexual organs, but show that delay in BPS occurs after exposure to low doses of TCDD, and this is dependent upon whether TCDD is administered acutely or chronically.
Abstract: Physiologically-based pharmacokinetic (PBPK) models are used in mode-of-action based risk and safety assessments to estimate internal dosimetry in animals and humans. When used in risk assessment, these models can provide a basis for extrapolating between species, doses and exposure routes or for justifying non-default values for uncertainty factors. Characterization of uncertainty and variability is increasingly recognized as important for risk assessment; this represents a continuing challenge for both PBPK modelers and users. Current practices show significant progress in specifying deterministic biological models and non-deterministic (often statistical) models, estimating parameters using diverse data sets from multiple sources, using them to make predictions, and characterizing uncertainty and variability of model parameters and predictions. The International Workshop on Uncertainty and Variability in PBPK Models, held Oct 31-Nov 2, 2006, identified the state-of-the-science, needed changes in practice and implementation, and research priorities. For the short term, these include: (1) multidisciplinary teams to integrate deterministic and non-deterministic/statistical models; (2) broader use of sensitivity analyses, including for structural and global (rather than local) parameter changes; and (3) enhanced transparency and reproducibility through improved documentation of model structure(s), parameter values, sensitivity and other analyses, and supporting, discrepant, or excluded data. Longer-term needs include: (1) theoretical and practical methodological improvements for non-deterministic/statistical modeling; (2) better methods for evaluating alternative model structures; (3) peer-reviewed databases of parameters and covariates, and their distributions; (4) expanded coverage of PBPK models across chemicals with different properties; and (5) training and reference materials, such as cases studies, bibliographies/glossaries, model repositories, and enhanced software. The multi-disciplinary dialogue initiated by this Workshop will foster the collaboration, research, data collection, and training necessary to make characterizing uncertainty and variability a standard practice in PBPK modeling and risk assessment.
Abstract: A physiologically based pharmacokinetic (PBPK) model describing the disposition of 2-butoxyethanol (2-BE) was developed in order to predict the urinary concentration of its major metabolite, butoxyacetic acid (BAA) under a range of exposure scenarios. Based on Corley et al. [Corley, R.A., Bormett, G.A., Ghanayem, B.I., 1994. Physiologically based pharmacokinetics of 2-butoxyethanol and its major metabolite, 2-butoxyacetic acid, in rats and humans. Toxicol. Appl. Pharmacol. 129, 61-79], the model included such features as multiple entry routes into the body, varying workload conditions, metabolism in the liver and elimination of free BAA in urine by glomerular filtration and acid transport. A bladder compartment simulating the fluctuations in metabolite concentration in urine caused by micturition formed a novel aspect of the model. Good agreement between model predictions and existing experimental data of total BAA levels in the blood and urine over various exposure conditions were observed. The mechanistically based PBPK model allowed comparison of disparate studies and also enabled the prediction of urinary concentrations of BAA post-shift. By calculating the total amount of BAA, any inter-individual variability in conjugation is taken into account. This led us to conclude that a biological monitoring guidance value should be proposed for total rather than free BAA with a value of 250mmol/mol of creatinine (post-shift), based on an 8h exposure to 25ppm 2-BE at resting working conditions.
Abstract: The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.
Abstract: A physiologically based pharmacokinetic model for investigating inter-individual and inter-racial variability in ethanol pharmacokinetics is presented. The model is a substantial modification of an existing model which described some genetic polymorphisms in the hepatic alcohol dehydrogenase enzymes. The model was modified to incorporate a description of ethanol absorption from the stomach and gastro-intestinal tract and the retardation of gastric emptying due to a concentration-dependent inhibition of gastric peristalsis. In addition, intra-venous and intra-arterial routes of administration were added to investigate whether the biological structure of the model provided a core which may be easily adapted for any route of exposure. The model is proposed as suitable for the investigation of the effects of both acute and chronic ethanol exposure.
Abstract: The construction of a homology model of human cytochrome P450 2E1 (CYP2E1) is reported, based on the CYP2C5 crystallographic template. A relatively high degree of primary sequence homology (identity=59%), as expected for proteins of the same CYP family, ensured a straightforward generation of the 3-dimensional model due to relatively few deletions and insertions of amino acid residues with respect to the CYP2C5 crystal structure. Probing the CYP2E1 model with typical substrates of the enzyme showed a good agreement with experimental information in the form of positions of metabolism for substrates, and with site-directed mutagenesis data on certain residues. Furthermore, quantitative relationships between substrate binding affinity and various structural parameters associated with the substrate molecules facilitated the formulation of a procedure for estimating relative binding energy and, consequently, K(m) or K(D) values towards the CYP2E1 enzyme. This method has been based on a consideration of the active site interactions between substrates and key amino acid residues lining the haem pocket, together with compound lipophilicity data from partition coefficients.
Abstract: The effects of acute administration of dietary levels of ethanol and the garlic oil extract, diallyl sulphide (DAS), on cytochrome P450 2E1 (CYP2E1) activity in volunteers were studied using the selective probe substrate, chlorzoxazone (CZX). The ratio of the CZX metabolite 6- hydroxychlorzoxazone (6-OHCZX) to CZX was taken to indicate CYP2E1 activity. The mean differences between the baseline and DAS-treated (0.2 mg/kg) CYP2E1 activities were significantly different (two-tailed p value = 0.0242, n = 8). Likewise, the mean differences between the baseline and ethanol-treated (0.8 g/kg) CYP2E1 activities were also significantly different (two-tailed p value = 0.0005, n = 7). The reduction in in vivo CYP2E1 activity by DAS is consistent with reported inhibition observed in vitro. The marked reduction in CYP2E1 activity following acute ingestion of ethanol is consistent with a competitive inhibition mechanism of CZX metabolism. The inhibitory effect of DAS maybe additive with daily consumption of Allium vegetables in particular. This may explain the lower 6-OHCZX/CZX metabolic ratios measured in various European and Mexican cohorts and is consistent with the lower incidence of stomach, liver and colon cancers observed in southern Europeans.
Abstract: OBJECTIVE: To investigate whether anticardiolipin (aCL) and anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies are associated with lupus nephritis (group II patients), and whether there are differences in the prevalence of these two autoantibodies between group II patients and patients with non-nephritis SLE (group I) and primary antiphospholipid syndrome (PAPS) patients (group III). METHODS: IgG and IgM aCL were measured in 31 patients and anti-beta(2)GPI in 30 patients with systemic lupus erythematosus (SLE) nephritis and 25 without SLE nephritis and in 36 PAPS patients by validated enzyme immunoassays. Relationships of anti-double-stranded DNA (anti-dsDNA) antibodies and antibodies to the collagenous region of C1q (anti-C1q) with SLE nephritis were also examined. RESULTS: The prevalence and levels were higher for aCL, but not for anti-beta(2)GPI, antibodies in group II than in group I patients. Absolute values of aCL and anti-beta(2)GPI in all three patient groups correlated with each other. The prevalences of aCL, anti-dsDNA and anti-C1q antibodies were significantly higher in group II than in group I and group III patients. CONCLUSION: The observations in this paper suggest that raised levels of aCL antibodies are associated with lupus nephritis. We were not able to demonstrate an association between anti-beta(2)GPI antibodies and kidney disease either in patients with lupus or in patients with primary antiphospholipid syndrome. In SLE, we demonstrated that the presence of anticardiolipin antibodies in conjunction with elevated levels of anti-dsDNA and anti-C1q antibodies is highly specific for glomerulonephritis in patients with lupus.
Abstract: Occupational exposure limits (OELs) for individual substances are established on the basis of the available toxicological information at the time of their promulgation, expert interpretation of these data in light of industrial use, and the framework in which they sit. In the United Kingdom, the establishment of specific OELs includes the application of uncertainty factors to a defined starting point, usually the NOAEL from a suitable animal study. The magnitude of the uncertainty factors is generally determined through expert judgment including a knowledge of workplace conditions and management of exposure. PBPK modeling may help in this process by informing on issues relating to extrapolation between and within species. This study was therefore designed to consider how PBPK modeling could contribute to the establishment of OELs. PBPK models were developed for chloroform (mouse and human) and carbon tetrachloride (rat and human). These substances were chosen for examination because of the extent of their toxicological databases and availability of existing PBPK models. The models were exercised to predict the rate (chloroform) or extent (carbon tetrachloride) of metabolism of these substances, in both rodents and humans. Monte Carlo analysis was used to investigate the influence of variability within the human and animal model populations. The ratio of the rates/extent of metabolism predicted for humans compared to animals was compared to the uncertainty factors involved in setting the OES. Predictions obtained from the PBPK models indicated that average rat and mouse metabolism of carbon tetrachloride and chloroform, respectively, are much greater than that of the average human. Application of Monte Carlo analysis indicated that even those people who have the fastest rates or most extensive amounts of metabolism in the population are unlikely to generate the levels of metabolite of these substances necessary to produce overt toxicity in rodents. This study highlights the value that the use of PBPK modeling may add to help inform and improve toxicological aspects of a regulatory process.
