Abstract: The accumulation of stochastic DNA damage throughout an organism's lifespan is thought to contribute to ageing. Conversely, ageing seems to be phenotypically reproducible and regulated through genetic pathways such as the insulin-like growth factor-1 (IGF-1) and growth hormone (GH) receptors, which are central mediators of the somatic growth axis. Here we report that persistent DNA damage in primary cells from mice elicits changes in global gene expression similar to those occurring in various organs of naturally aged animals. We show that, as in ageing animals, the expression of IGF-1 receptor and GH receptor is attenuated, resulting in cellular resistance to IGF-1. This cell-autonomous attenuation is specifically induced by persistent lesions leading to stalling of RNA polymerase II in proliferating, quiescent and terminally differentiated cells; it is exacerbated and prolonged in cells from progeroid mice and confers resistance to oxidative stress. Our findings suggest that the accumulation of DNA damage in transcribed genes in most if not all tissues contributes to the ageing-associated shift from growth to somatic maintenance that triggers stress resistance and is thought to promote longevity.
Abstract: Patients carrying mutations in the XPB helicase subunit of the basal transcription and nucleotide excision repair (NER) factor TFIIH display the combined cancer and developmental-progeroid disorder xeroderma pigmentosum/Cockayne syndrome (XPCS). Due to the dual transcription repair role of XPB and the absence of animal models, the underlying molecular mechanisms of XPB(XPCS) are largely uncharacterized. Here we show that severe alterations in Xpb cause embryonic lethality and that knock-in mice closely mimicking an XPCS patient-derived XPB mutation recapitulate the UV sensitivity typical for XP but fail to show overt CS features unless the DNA repair capacity is further challenged by crossings to the NER-deficient Xpa background. Interestingly, the Xpb(XPCS) Xpa double mutants display a remarkable interanimal variance, which points to stochastic DNA damage accumulation as an important determinant of clinical diversity in NER syndromes. Furthermore, mice carrying the Xpb(XPCS) mutation together with a point mutation in the second TFIIH helicase Xpd are healthy at birth but display neonatal lethality, indicating that transcription efficiency is sufficient to permit embryonal development even when both TFIIH helicases are crippled. The double-mutant cells exhibit sensitivity to oxidative stress, suggesting a role for endogenous DNA damage in the onset of XPB-associated CS.
Abstract: UV-irradiated skin and UV-induced tumors overexpress the inducible isoform of cyclooxygenase-2 (Cox-2), and Cox-2 inhibition reduces photocarcinogenesis. To evaluate photoprotective effects of Polypodium leucotomos extract (PL), hairless Xpc(+/-) mice were fed for 10 days with PL (300 mg/kg) or vehicle then UV-irradiated, once. By 24 hours, UV-induced Cox-2 levels were increased in vehicle-fed and PL-fed mice, whereas by 48 and 72 hours, Cox-2 levels were four- to fivefold lower in PL-fed mice (P < 0.05). p53 expression/activity was increased in PL-fed versus vehicle-fed then UV-irradiated mice. UV-induced inflammation was decreased in PL-fed mice, as shown by approximately 60% decrease (P < 0.001) in neutrophil infiltration at 24 hours, and macrophages by approximately 50% (<0.02) at 24 and 48 hours. By 72 hours, 54 +/- 5% cyclobutane pyrimidine dimers remained in vehicle-fed versus 31 +/- 5% in PL-fed skin (P < 0.003). The number of 8-hydroxy-2'-deoxyguanosine-positive cells were decreased before UV irradiation by approximately 36% (P < 0.01), suggesting that PL reduces constitutive oxidative DNA damage. By 6 and 24 hours, the number of 8-hydroxy-2'-deoxyguanosine-positive cells were approximately 59% (P < 0.01) and approximately 79% (P < 0.03) lower in PL-fed versus vehicle-fed mice. Finally, UV-induced mutations in PL-fed-mice were decreased by approximately 25% when assessed 2 weeks after the single UV exposure. These data demonstrate that PL extract supplementation affords the following photoprotective effects: p53 activation and reduction of acute inflammation via Cox-2 enzyme inhibition, increased cyclobutane pyrimidine dimer removal, and reduction of oxidative DNA damage.
Abstract: The clastogenic effects of MMC and BLM and the mutagenic effects of B[a]P, N-ac-AAF and ENU were studied in mouse embryonic fibroblasts derived from wild-type (WT) and Rad54/Rad54B-deficient mice. Clastogens as well as mutagens showed a statistically significant induction of mutations in the lacZ reporter gene both in a WT and Rad54/Rad54B-deficient genetic background. Rad54/Rad54B MEFs appeared equally sensitive to the clastogens compared to WT MEFs, except for MMC. The type of mutations induced by the different compounds was investigated further by hybridizing the mutant colonies with total mouse DNA. An obvious increased number of mouse DNA positive clones was observed after BLM and MMC exposure, indicating that after these treatments genome rearrangements/translocations had occurred. In this hybridization assay, Rad54/Rad54B MEFs did not show more rearrangements/translocations than WT MEFs. As expected, the mutagens used showed no increase in chromosomal rearrangements or transloctions in MEFs derived from both genotypes. These results show that WT MEFs carrying the lacZ reporter gene on a plasmid are capable to detect both clastogenic as well as mutagenic effects of compounds in vitro. Deletion of the Rad54 and Rad54B genes did not further enhance the sensitivity of MEFs towards clastogens.
Abstract: The DNA repair-deficient Xpa(-/-)p53(+/-) (Xpa/p53) mouse is a potent model for carcinogenicity testing, representing increased sensitivity toward genotoxic but surprisingly also toward true human non-genotoxic carcinogens. The mechanism of this increased sensitivity in Xpa/p53 mice toward non-genotoxic carcinogens is still unknown. Here, we investigated the mechanism of the human non-genotoxic carcinogen cyclosporine A (CsA) in the Xpa/p53 mouse model. Xpa/p53 mice exposed to CsA for 39 weeks showed a significantly increased lymphoma incidence as compared with untreated Xpa/p53 mice and CsA-treated wild-type (WT) mice. We excluded concealed genotoxicity of CsA in Xpa/p53 mice by mutant frequency analyses. As a next step, we used a genetic approach: immunodeficient DNA-PKcs mice, defective in the catalytic subunit of the DNA-dependent protein kinase, were crossed with Xpa and Xpa/p53 mice. Xpa/p53 mice had an increased lymphoma incidence with shorter latency times as compared with DNA-PKcs-deficient WT and Xpa mice. Surprisingly, also six of 15 DNA-PKcs/Xpa/p53 females had developed an adenocarcinoma of the mammary gland. Tumor responses in CsA-treated and DNA-PKcs-deficient Xpa/p53 mice were comparable as both genotypes developed mainly splenic lymphomas enriched in B lymphocytes. From our present studies, we hypothesize that levels of initiated precancerous cells are elevated in Xpa/p53 mice. These cells are insufficiently eliminated due to either suppression of the immune system by CsA or through immune-related DNA-PKcs deficiency. Based on the current studies and those conducted previously, we conclude that the Xpa/p53 model is an excellent adjunct to the current chronic rodent bioassay.
Abstract: It is well established that cancer is a multi-step process which involves initiation, promotion and progression. Chemical carcinogens can alter any of these processes to induce their carcinogenic effects. The presence of multiple mutations in critical genes is a distinctive feature of cancer cells and supports the contention that cancer arises through the accumulation of irreversible DNA damage. In the majority of instances, chemical carcinogens, directly or after xenobiotic metabolism, induce DNA damage and act in a 'genotoxic' manner. There is, however, a group of carcinogens that induce cancer via non-genotoxic mechanisms. Non-genotoxic carcinogens have been shown to act as tumor promoters (1,4-dichlorobenzene), endocrine-modifiers (17beta-estradiol), receptor-mediators (2,3,7,8-tetrachlorodibenzo-p-dioxin), immunosuppressants (cyclosporine) or inducers of tissue-specific toxicity and inflammatory responses (metals such as arsenic and beryllium). The diversity of modes of action of non-genotoxic carcinogens, the tissue and species specificity, and the absence of genotoxicity makes predicting their carcinogenic potential extremely challenging. In order to better understand the mechanisms of known human non-genotoxic carcinogens and to illustrate the importance of a weight of evidence approach when evaluating their carcinogenic potential, we will (1) evaluate the proportion of non-genotoxic carcinogens among known, probable and possible human carcinogens classified by the International Agency for Research on Cancer (IARC), (2) estimate the risk of exposure of human non-genotoxic carcinogens through margin of exposure (MOE) evaluation, and (3) discuss potential alternative methods for their detection. Our analysis demonstrated that human non-genotoxic carcinogens were present in 12% (45/371) of IARC's Groups 1, 2A and 2B carcinogens and that a potential hazard was associated with 27% (12/45) of them. Consequently, it is suggested that for all genotoxic chemicals, the mode of action is investigated for hazard and risk evaluation. Further, if negative genotoxic compounds have putative non-genotoxic modes of action, appropriate risk measures should be implemented.
Abstract: The carcinogenic potential of chemicals and pharmaceuticals is traditionally tested in the chronic, 2 year rodent bioassay. This assay is not only time consuming, expensive and often with a limited sensitivity and specificity but it also causes major distress to the experimental animals. A major improvement in carcinogenicity testing, especially regarding reduction and refinement of animal experimentation, could be the application of toxicogenomics. The ultimate aim of this study is to demonstrate a proof-of-principle for transcriptomics biomarkers in various tissues for identification of (subclasses of) carcinogenic compounds after short-term in vivo exposure studies. Both wild-type and DNA repair-deficient Xpa(-/-)/p53(+/-) (Xpa/p53) mice were exposed up to 14 days to compounds of three distinct classes: genotoxic carcinogens (GTXC), non-genotoxic carcinogens (NGTXC) and non-carcinogens. Subsequently, extensive transcriptomics analyses were performed on several tissues, and transcriptomics data were screened for potential biomarkers using advanced statistical learning techniques. For all tissues analyzed, we identified multigene gene-expression signatures that are, with a high confidence, predictive for GTXC and NGTXC exposures in both mouse genotypes. Xpa/p53 mice did not perform better in the short-term bioassay. We were able to achieve a proof-of-principle for the identification and use of transcriptomics biomarkers for GTXC or NGTXC. This supports the view that toxicogenomics with short-term in vivo exposure provides a viable tool for classifying (geno)toxic compounds.
Abstract: The CHEK2 kinase (Chk2 in mouse) is a member of a DNA damage response pathway that regulates cell cycle arrest at cell cycle checkpoints and facilitates the repair of dsDNA breaks by a recombination-mediated mechanism. There are numerous variants of the CHEK2 gene, at least one of which, CHEK2*1100delC (SNP), associates with breast cancer. A mouse model in which the wild-type Chk2 has been replaced by a Chk2*1100delC allele was tested for elevated risk of spontaneous cancer and increased sensitivity to challenge by a carcinogenic compound. Mice homozygous for Chk2*1100delC produced more tumors than wild-type mice, whereas heterozygous mice were not statistically different. When fractionated by gender, however, homozygous and heterozygous mice developed spontaneous tumors more rapidly and to a far greater extent than wild-type mice, indicative of a marked gender bias in mice harboring the variant allele. Consistent with our previous data showing elevated genomic instability in mouse embryonic fibroblasts (MEFs) derived from mice homozygous for Chk2*1100delC, the level of Cdc25A was elevated in heterozygous and homozygous MEFs and tumors. When challenged with the carcinogen 7,12-dimethylbenz[a]anthracene, all mice, regardless of genotype, had a reduced lifespan. Latency for mammary tumorigenesis was reduced significantly in mice homozygous for Chk2*1100delC but unexpectedly increased for the development of lymphomas. An implication from this study is that individuals who harbor the variant CHEK2*1100delC allele not only are at an elevated risk for the development of cancer but also that this risk can be further increased as a result of environmental exposure.
Abstract: Benzo(a)pyrene (B[a]P) can induce somatic mutations, whereas its potential to induce germ cell mutations is unclear. There is circumstantial evidence that paternal exposure to B[a]P can result in germ cell mutations. Since DNA adducts are thought to be a prerequisite for B[a]P induced mutations, we studied DNA adduct kinetics by (32)P-postlabeling in sperm, testes and lung tissues of male mice after a single exposure to B[a]P (13 mg/kg bw, by gavage). To investigate DNA adduct formation at different stages of spermatogenesis, mice were sacrificed at Day 1, 4, 7, 10, 14, 21, 32, and 42 after exposure. In addition, DNA repair deficient (Xpc(-/-)) mice were used to study the contribution of nucleotide excision repair in DNA damage removal. DNA adducts were detectable with highest levels in lung followed by sperm and testis. Maximum adduct levels in the lung and testis were observed at Day 1 after exposure, while adduct levels in sperm reached maximum levels at approximately 1 week after exposure. Lung tissue and testis of Xpc(-/-) mice contained significantly higher DNA adduct levels compared to wild type (Wt) mice over the entire 42 day observation period (P < 0.05). Differences in adduct half-life between Xpc(-/-) and Wt mice were only observed in testis. In sperm, DNA adduct levels were significantly higher in Xpc(-/-) mice than in Wt mice only at Day 42 after exposure (P = 0.01). These results indicate that spermatogonia and testes are susceptible for the induction of DNA damage and rely on nucleotide excision repair for maintaining their genetic integrity. Environ. Mol. Mutagen. 2009. (c) 2009 Wiley-Liss, Inc.
Abstract: The accumulation of DNA damage is a slow but hazardous phenomenon that may lead to cell death, accelerated aging, and cancer. One of the most versatile defense mechanisms against the accumulation of DNA damage is nucleotide excision repair, in which, among others, the Xeroderma pigmentosum group C (XPC) and group A (XPA) proteins are involved. To elucidate differences in the functions of these two proteins, comprehensive survival studies with Xpa(-/-), Xpc(-/-) and wild-type control female mice in a pure C57BL/6J background were done. The median survival of Xpc(-/-) mice showed a significant decrease, whereas the median survival of Xpa(-/-) mice did not. Strikingly, Xpa(-/-) and Xpc(-/-) mice also showed a phenotypical difference in terms of tumor spectrum. Xpc(-/-) mice displayed a significant increase in lung tumors and a trend toward increased liver tumors compared with Xpa-deficient or wild-type mice. Xpa(-/-) mice showed a significant elevation in liver tumors. Additionally, Xpc-deficient mice exhibited a strong increase in mutant frequency in lung compared with Xpa(-/-) mice, whereas in both models mutant frequency is increased in liver. Our in vitro data displayed an elevated sensitivity to oxygen in Xpc(-/-) in mouse embryonic fibroblasts (MEF) when compared with Xpa(-/-) and wild-type fibroblasts. We believe that XPC plays a role in the removal of oxidative DNA damage and that, therefore, Xpc(-/-) mice display a significant increase in lung tumors and a significant elevation in mutant frequency in lung, and Xpc-deficient MEFs show greater sensitivity to oxygen when compared with Xpa(-/-) and wild-type mice.
Abstract: Ozone is a well-known oxidant air pollutant, inhalation of which can result in oxidative stress, and lead to pulmonary inflammation. The aim of this study was to evaluate the time-course events after a single ozone exposure in transcription-coupled repair defective Csb and wild type mice. Mice were exposed for 3 h to 2 ppm ozone and biological parameters related to oxidative stress and inflammation were examined in the lungs at 0, 4, 9, 24 and 48 h after exposure. In addition the procoagulant and thrombomodulin activities were explored by a combination of assays for tissue factor and thrombin generation. This study revealed a significant biological response to ozone, for both Csb and wild type mice. The onset of inflammation in Csb mice, as indicated by an increase in interleukin-6, tumor necrosis factor-alpha and total cell influx, occurred earlier compared with those seen in wild type mice. On the other hand, Csb mice showed a delayed antioxidant reaction compared with wild type mice. Both genotypes developed a procoagulant reaction characterized by a stably increased tissue factor activity and a progressive increase in thrombin generation after 2 days. These experiments have shown that ozone, a well-known toxic substance from the environment, induces not only inflammation, but also procoagulant reactions in the lungs of mice. These results have implications for understanding the systemic effects induced by oxidant air pollutants.
Abstract: Estimates of genotoxic effects of mutagens at low and protracted doses are often based on linear extrapolation of data obtained at relatively high doses. To test the validity of such an approach, a comparison was made between the mutagenicity of N-methyl-N-nitrosourea (MNU) in T-lymphocytes of the rat following two treatment protocols, i.e. sub-chronic exposure to a low dose (15-45 repeated exposures to 1mg/kg of MNU) or acute exposure to a single high dose (15, 30 or 45 mg/kg of MNU). Mutation induction appeared dramatically lower following sub-chronic treatment compared to treatment with a single high exposure. Furthermore, DNA sequence analysis of the coding region of the hprt gene in MNU-induced mutants showed that acute high dose treatment causes mainly GC-->AT base pair changes, whereas sub-chronic treatment results in a significant contribution of AT base pair changes to mutation induction. We hypothesize that O(6)-methylguanine-DNA methyltransferase is saturated after acute treatments, while after sub-chronic treatment most O(6)-methylguanine is efficiently repaired. These data suggest (i) that risk estimations at low and protracted doses of MNU on the basis of linear extrapolation of effects measured at high dose are too high and (ii) that the protective effects of DNA repair processes are relatively strong at low sub-chronic exposure.
Abstract: In order to investigate immunotoxic effects of a set of model compounds in mice, a toxicogenomics approach was combined with information on macroscopical and histopathological effects on spleens and on modulation of immune function. Bis(tri-n-butyltin)oxide (TBTO), cyclosporin A (CsA), and benzo[a]pyrene (B[a]P) were administered to C57BL/6 mice at immunosuppressive dose levels. Acetaminophen (APAP) was included in the study since indications of immunomodulating properties of this compound have appeared in the literature. TBTO exposure caused the most pronounced effect on gene expression and also resulted in the most severe reduction of body weight gain and induction of splenic irregularities. All compounds caused inhibition of cell division in the spleen as shown by microarray analysis as well as by suppression of lymphocyte proliferation after application of a contact sensitizer as demonstrated in an immune function assay that was adapted from the local lymph node assay. The immunotoxicogenomics approach applied in this study thus pointed to immunosuppression through cell cycle arrest as a common mechanism of action of immunotoxicants, including APAP. Genes related to cell division such as Ccna2, Brca1, Birc5, Incenp, and Cdkn1a (p21) were identified as candidate genes to indicate anti-proliferative effects of xenobiotics in immune cells for future screening assays. The results of our experiments also show the value of group wise pathway analysis for detection of more subtle transcriptional effects and the potency of evaluation of effects in the spleen to demonstrate immunotoxicity.
Abstract: We investigated whether inhaling peak concentrations of aldehydes several times daily is more damaging than semi-continuously inhaling low-dose aldehydes. We exposed Xpa-/-p53+/- knock-out mice either intermittently or semi-continuously to mixed acetaldehyde, formaldehyde, and acrolein. The intermittent regimen entailed exposure to the aldehydes 7 min every 45 min, 12 times/day, 5 days/week, corresponding to concentrations inhaled by smokers. Semi-continuously exposed animals received half the dose of aldehydes in 8h/day, 5 days/week. Some mice in each group were sacrificed after 13 weeks of exposure; the rest breathed clean air until the end of 1 year. Mice injected intratracheally with benzo[a]pyrene formed a positive control group. The nasal cavity, lungs, and any macroscopically abnormal organs of all mice were analysed histopathologically. After 13 weeks of exposure, the subacute, overall, histopathological changes induced by the inhalation differed noticeably between the intermittently and semi-continuously treated Xpa-/-p53+/- knock-out mice. After 13 weeks of mixed aldehyde exposure, atrophy of the olfactory epithelium generally appeared, but disappeared after 1 year (adaptation and/or recovery). Respiratory epithelial metaplasia of the olfactory epithelium occurred at a higher incidence at 1 year. Except for a significantly greater number of tumours observed in knock-out mice compared to wild mice (semi-continuous aldehyde exposure and controls), no differences between the semi-continuous and intermittent exposure groups were observed.
Abstract: Several studies suggest that MutaMouse is insensitive to clastogens, including the accompanying paper by Mahabir et al., which describes a study with bleomycin, camptothecin, m-AMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidide) and its ortho-analogue, o-AMSA (4'-(9-acridinylamino)-methanesulfon-o-anisidide). Only camptothecin was clastogenic in MutaMouse and none of these four compounds induced mutations at the lacZ locus. However, to improve exposure, dose range-finding studies were performed in CD2F1 mice, the parental strain of MutaMouse. Male CD2F1 mice (n=3) were treated with bleomycin (25-100 mg/kg bw, p.o. and i.p.), camptothecin (1-10 mg/kg bw p.o.) and m-AMSA (10-50mg/kg bw p.o. and 1-5 mg/kg bw i.p.) for 5 days and blood was sampled on day 3 and/or day 6 for analysis by flow cytometry to determine % MN-RETs. Camptothecin (1 mg/kg bw, day 6) induced a 3.6-fold increase in % MN-RET (P<0.05) but was toxic at higher doses. All day-3 camptothecin samples were positive (P<0.05). Bleomycin was negative when administered p.o. but positive at all doses on both days when given i.p. (P<0.05) whereas m-AMSA was negative when given i.p. or orally. Based on these results, male MutaMouse mice (5 per group) were dosed daily with bleomycin (50 mg/kg bw) for 5 days or with camptothecin (5 mg/kg bw) for 2 days. Peripheral blood was sampled 24 h after the final dose in each group and tissues were sampled 37 days later. Both compounds induced significant increases in % MN-RET, but only bleomycin induced a significant increase in MF (6-fold in liver, 4.5-fold in kidney and 2-fold in lung) compared with the untreated control. These studies support the view that MutaMouse is insensitive to compounds where the genotoxic mechanism of action is predominantly clastogenesis, but demonstrates that the peripheral blood micronucleus test is a useful adjunct to the transgenic gene-mutation assay.
Abstract: Unrepaired or misrepaired DNA damage has been implicated as a causal factor in cancer and aging. Xpd(TTD) mice, harboring defects in nucleotide excision repair and transcription due to a mutation in the Xpd gene (R722W), display severe symptoms of premature aging but have a reduced incidence of cancer. To gain further insight into the molecular basis of the mutant-specific manifestation of age-related phenotypes, we used comparative microarray analysis of young and old female livers to discover gene expression signatures distinguishing Xpd(TTD) mice from their age-matched wild type controls. We found a transcription signature of increased apoptosis in the Xpd(TTD) mice, which was confirmed by in situ immunohistochemical analysis and found to be accompanied by increased proliferation. However, apoptosis rate exceeded the rate of proliferation, resulting in homeostatic imbalance. Interestingly, a metabolic response signature was observed involving decreased energy metabolism and reduced IGF-1 signaling, a major modulator of life span. We conclude that while the increased apoptotic response to endogenous DNA damage contributes to the accelerated aging phenotypes and the reduced cancer incidence observed in the Xpd(TTD) mice, the signature of reduced energy metabolism is likely to reflect a compensatory adjustment to limit the increased genotoxic stress in these mutants. These results support a general model for premature aging in DNA repair deficient mice based on cellular responses to DNA damage that impair normal tissue homeostasis.
Abstract: In the present paper the capacity of the pUR288 plasmid mouse model and the MutaMouse model to detect the clastogens bleomycin, m-AMSA, o-AMSA and camptothecin, was investigated. Ethylnitrosourea (ENU) served as a positive control, methylcellulose as a negative control. Only bleomycin induced a slight but significant increase in lacZ mutant frequency (MF) in bone marrow of pUR288 plasmid mice. Exposure to the other compounds did not result in an increase in the MF in bone marrow and liver in both mouse models. For the MutaMouse this result was expected, for the plasmid mouse an increase in MF after clastogen exposure was expected. The positive control ENU induced statistically significant increases in MF compared with the negative control in both models and in both tissues analyzed. Hybridisation of DNA of mutant colonies derived from plasmid mice with labelled total mouse DNA (Hybridisation Assay) demonstrated an increase in the percentage of colonies hybridised with total mouse DNA as compared with the negative control, which suggests that there was indeed a biological response associated with treatment. The latter results indicate that the plasmid mouse assay may still be a promising model for the detection of clastogens.
Abstract: Mutant dwarf and calorie-restricted mice benefit from healthy aging and unusually long lifespan. In contrast, mouse models for DNA repair-deficient progeroid syndromes age and die prematurely. To identify mechanisms that regulate mammalian longevity, we quantified the parallels between the genome-wide liver expression profiles of mice with those two extremes of lifespan. Contrary to expectation, we find significant, genome-wide expression associations between the progeroid and long-lived mice. Subsequent analysis of significantly over-represented biological processes revealed suppression of the endocrine and energy pathways with increased stress responses in both delayed and premature aging. To test the relevance of these processes in natural aging, we compared the transcriptomes of liver, lung, kidney, and spleen over the entire murine adult lifespan and subsequently confirmed these findings on an independent aging cohort. The majority of genes showed similar expression changes in all four organs, indicating a systemic transcriptional response with aging. This systemic response included the same biological processes that are triggered in progeroid and long-lived mice. However, on a genome-wide scale, transcriptomes of naturally aged mice showed a strong association to progeroid but not to long-lived mice. Thus, endocrine and metabolic changes are indicative of "survival" responses to genotoxic stress or starvation, whereas genome-wide associations in gene expression with natural aging are indicative of biological age, which may thus delineate pro- and anti-aging effects of treatments aimed at health-span extension.
Abstract: The sensitivity of DNA-repair-deficient Rad54/Rad54B mice for clastogens was studied and compared to that of wild-type mice. LacZ mutant frequencies (MF) in Rad54/Rad54B mice, after treatment with mitomycin C (MMC), bleomycin (BLM) and gamma-irradiation, were compared to those of the wild-type mice following the same treatments. While none of the clastogens showed an induction of the lacZ MF in the wild-type mice, there was a significant increase of the lacZ MF in the bone marrow of the Rad54/Rad54B mice after treatment with BLM and gamma-irradiation and in the spleen after MMC treatment. As expected, the positive control ENU showed a significant increase in the lacZ MF in all tested organs in wild-type mice. Mutant colonies were hybridized with total mouse DNA in order to discriminate between small gene mutations and large DNA rearrangements and translocations (size-change mutations). The hybridization studies showed a significant increase in mouse DNA positive clones 4 days after treatment with MMC and BLM in the bone marrow of the wild-type mice, which is indicative for chromosomal rearrangements and translocations to occur. An even more pronounced increase was seen 28 days after treatment with the same compounds in the Rad54/Rad54B mice.
Abstract: Germ line mutations resulting from chemical or radiation exposure are a particular problem in toxicology as they affect not only the exposed generation but also an infinite number of generations thereafter. Established methods to show that these mutations occur in an F1 or subsequent population require the use of a large number of progeny for statistical significance. Consequently, many thousands of animals have been used in the past. Such a use is no longer considered desirable and is also very expensive. Several new molecular techniques (including analysis of tandem repeats and randomly amplified polymorphic DNA) now provide alternative methods of assessment, which also allow the quantification of individual mutations in individual sperm cells. These can also be applied to human offspring, making extrapolation obsolete. The downside of these methods is that they effectively determine the mutation rate in certain regions of DNA and the relevance of these to diseases, particularly cancer, is not always apparent. Therefore, it must be assumed that an increase in mutation rates in these selected regions correlates with altered phenotype. However, disease types linked to changes in tandem repeat length indicate that these may act as relevant markers for the development of phenotypes. Further research and evaluation are required to more closely link changes in DNA with altered phenotype and validate the use of tandem repeats and randomly amplified polymorphic DNA in transgenerational genotoxicity testing. This paper introduces and compares recently developed methods to assess mutations in sperm due to stem cell damage.
Abstract: Mutations in the CSB gene cause Cockayne syndrome (CS), a DNA repair disorder characterized by UV sensitivity and severe physical and neurological impairment. CSB functions in the transcription-coupled repair subpathway of nucleotide excision repair. This function may explain the UV sensitivity but hardly clarifies the other CS symptoms. Many of these, including retinopathy, are associated with premature aging. We studied eye pathology in a mouse model for CS. Csb(m/m) mice were hypersensitive to UV light and developed epithelial hyperplasia and squamous cell carcinomas in the cornea, which underscores the importance of transcription-coupled repair of photolesions in the mouse. In addition, we observed a spontaneous loss of retinal photoreceptor cells with age in the Csb(m/m) retina, resulting in a 60% decrease in the number of rods by the age of 18 months. Importantly, when Csb(m/m) mice (as well as Csa(-/-) mice) were exposed to 10 Gy of ionizing radiation, we noticed an increase in apoptotic photoreceptor cells, which was not observed in wild-type animals. This finding, together with our observation that the expression of established oxidative stress marker genes is upregulated in the Csb(m/m) retina, suggests that (endogenous) oxidative DNA lesions play a role in this CS-specific premature-aging feature and supports the oxidative DNA damage theory of aging.
Abstract: Semantic distinctions between "normal" aging, "pathological" aging (or age-related disease) and "premature" aging (otherwise known as segmental progeria) potentially confound important insights into the nature of each of the complex processes. Here we review a recent, unexpected discovery: the presence of longevity-associated characteristics typical of long-lived endocrine-mutant and dietary-restricted animals in short-lived progeroid mice. These data suggest that a subset of symptoms observed in premature aging, and possibly normal aging as well, may be indirect manifestations of a beneficial adaptive stress response to endogenous oxidative damage, rather than a detrimental result of the damage itself.
Abstract: Several mouse models with defects in genes encoding components of the nucleotide excision repair (NER) pathway have been developed. In NER two different sub-pathways are known, i.e. transcription-coupled repair (TC-NER) and global-genome repair (GG-NER). A defect in one particular NER protein can lead to a (partial) defect in GG-NER, TC-NER or both. GG-NER defects in mice predispose to cancer, both spontaneous as well as UV-induced. As such these models (Xpa, Xpc and Xpe) recapitulate the human xeroderma pigmentosum (XP) syndrome. Defects in TC-NER in humans are associated with Cockayne syndrome (CS), a disease not linked to tumor development. Mice with TC-NER defects (Csa and Csb) are - except for the skin - not susceptible to develop (carcinogen-induced) tumors. Some NER factors, i.e. XPB, XPD, XPF, XPG and ERCC1 have functions outside NER, like transcription initiation and inter-strand crosslink repair. Deficiencies in these processes in mice lead to very severe phenotypes, like trichothiodystrophy (TTD) or a combination of XP and CS. In most cases these animals have a (very) short life span, display segmental progeria, but do not develop tumors. Here we will overview the available NER-related mouse models and will discuss their phenotypes in terms of (chemical-induced) tissue-specific tumor development, mutagenesis and premature aging features.
Abstract: Although it has been established that premature aging trichothiodystrophy (TTD) mice display typical signs of osteoporosis, exact changes in physicochemical properties of these mice have not been elucidated. We used confocal Raman microscopy and histology to study femora of TTD mice. We measured femora isolated from xeroderma pigmentosum group A (XPA)/TTD double mutant mice to establish that Raman microscopy can be applied to measure differences in bone composition. Raman data from XPA/TTD mice showed remarkable changes in bone mineral composition. Moreover, we observed a severe form of osteoporosis, with strongly reduced cortical bone thickness. We used Raman microscopy to analyze bone composition in eight wild-type and eight TTD animals, and observed decreased levels of phosphate and carbonate in the cortex of femora isolated from TTD mice. In contrast, the bands representing the bone protein matrix were not affected in these mice.
Abstract: The oxidant ozone is a well-known air pollutant, inhalation of which is associated with respiratory tract inflammation and functional alterations of the lung. It is well established as an inducer of intracellular oxidative stress. We investigated whether Cockayne syndrome B, transcription-coupled, repair-deficient mice (Csb(-/-)), known to be sensitive to oxidative stressors, respond differently to ozone than repair-proficient controls (Csb(+/-)). Mice were exposed to 0.8 parts/million ozone for 8 h, and we examined a wide range of biological parameters in the lung at the gene expression, protein, and cellular level 4 h after the ozone exposure. Relevant biological responses to ozone for both repair-deficient Csb(-/-) and repair-proficient Csb(+/-) mice, as determined by biochemical analysis of bronchoalveolar lavage fluid (e.g., increases of polymorphonuclear neutrophils, alkaline phosphatase, macrophage-inflammatory protein-2, and tumor necrosis factor-alpha), pathological examinations, and gene expression (upregulation of oxidative-stress-related genes) analyses were observed. The bronchoalveolar lavage fluid showed significantly more tumor necrosis factor-alpha in repair-deficient Csb(-/-) mice than in repair-proficient Csb(+/-) mice after ozone exposure. In addition, a clear trend was observed toward fewer differentially expressed genes with a lower fold ratio in repair-deficient Csb(-/-) mice than in repair-proficient Csb(+/-) mice. However, repair-deficient Csb(-/-) mice do not respond significantly more sensitively to ozone compared with repair-proficient Csb(+/-) mice at the level of gene expression. We conclude that, under the conditions employed here, although small differences at the transcriptional level exist between repair-proficient Csb(+/-) mice and transcription-coupled repair defective Csb(-/-) mice, these do not have a significant effect on the ozone-induced lung injury.
Abstract: Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csb(m/m)/Xpa(-/-) mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csb(m/m)/Xpa(-/-) mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1) somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csb(m/m)/Xpa(-/-) and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair-deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis.
Abstract: Mice with non-phosphorylated serine 389 in p53 are susceptible for bladder tumors induced by 2-acetylaminofluorene (2-AAF). Since p53 is a transcription factor, this might well be preceded by differences in the regulation of gene expression. Microarray analysis was used to determine early transcriptional changes that might underlie this cancer-prone phenotype. Interestingly, lack of Ser389 phosphorylation led to endogenously different gene expression levels. The number of genes affected was, however, rather small. Conversely, after short-term exposure to 2-AAF, wild-type and p53.S389A bladders demonstrated a significant number of differentially expressed genes. Differences between wild-type and p53.S389A could mainly be attributed to a delayed, rather than complete absence of, transcriptional response of a group of genes, including well-known p53 target genes involved in apoptosis and cell-cycle control like Bax, Perp and P21. An analysis of differentially expressed genes in non-tumorigenic tissue and bladder tumors of p53.S389A after long-term exposure to 2-AAF revealed 319 genes. Comparison of these with those found after short-term exposure resulted in 23 transcripts. These possible marker genes might be useful for the early prediction of bladder tumor development. In conclusion, our data indicate that lack of Ser389 phosphorylation results in aberrant expression of genes needed to execute vital responses to DNA damage. Post-translational modifications, like Ser389 phosphorylation, seem crucial for fine-tuning the transcription of a specific set of genes and do not appear to give rise to major changes in transcription patterns. As such, Ser389 phosphorylation is needed for some, but certainly not all, p53 functions.
Abstract: p53 alterations in human tumors often involve missense mutations that may confer dominant-negative or gain-of-function properties. Dominant-negative effects result in inactivation of wild-type p53 protein in heterozygous mutant cells and as such in a p53 null phenotype. Gain-of-function effects can directly promote tumor development or metastasis through antiapoptotic mechanisms or transcriptional activation of (onco)genes. Here, we show, using conditional mouse technology, that epithelium-specific heterozygous expression of mutant p53 (i.e., the p53.R270H mutation that is equivalent to the human hotspot R273H) results in an increased incidence of spontaneous and UVB-induced skin tumors. Expression of p53.R270H exerted dominant-negative effects on latency, multiplicity, and progression status of UVB-induced but not spontaneous tumors. Surprisingly, gain-of-function properties of p53.R270H were not detected in skin epithelium. Apparently, dominant-negative and gain-of-function effects of mutant p53 are highly tissue specific and become most manifest upon stabilization of p53 after DNA damage.
Abstract: BACKGROUND: Systematic, high-throughput studies of mouse phenotypes have been hampered by the inability to analyze individual animal data from a multitude of sources in an integrated manner. Studies generally make comparisons at the level of genotype or treatment thereby excluding associations that may be subtle or involve compound phenotypes. Additionally, the lack of integrated, standardized ontologies and methodologies for data exchange has inhibited scientific collaboration and discovery. RESULTS: Here we introduce a Mouse Phenotype Analysis System (MPHASYS), a platform for integrating data generated by studies of mouse models of human biology and disease such as aging and cancer. This computational platform is designed to provide a standardized methodology for working with animal data; a framework for data entry, analysis and sharing; and ontologies and methodologies for ensuring accurate data capture. We describe the tools that currently comprise MPHASYS, primarily ones related to mouse pathology, and outline its use in a study of individual animal-specific patterns of multiple pathology in mice harboring a specific germline mutation in the DNA repair and transcription-specific gene Xpd. CONCLUSION: MPHASYS is a system for analyzing multiple data types from individual animals. It provides a framework for developing data analysis applications, and tools for collecting and distributing high-quality data. The software is platform independent and freely available under an open-source license 1.
Abstract: Because thyroid nodules are frequent in areas with iodine deficiency the aim of this study was to characterise molecular events during iodine deficiency that could explain mutagenesis and nodule formation. We therefore studied gene expression of catalytic enzymes prominent for H(2)O(2) detoxification and antioxidative defence, quantified DNA oxidation and damage as well as spontaneous mutation rates (SMR) in mice and rats fed an iodine controlled diet. Antioxidative enzymes such as superoxide dismutase 3, glutathione peroxidase 4 and the peroxiredoxins 3 and 5 showed increased mRNA expression, which indicates increased radical burden that could be the cause of additional oxidized base adducts found in thyroidal genomic DNA in our experiments of iodine deficiency. Furthermore, the uracil content of thyroid DNA was significantly higher in the iodine-deficient compared to the control group. While SMR is very high in the normal thyroid gland it is not changed in experimental iodine deficiency. Our data suggest that iodine restriction causes oxidative stress and DNA modifications. A higher uracil content of the thyroid DNA could be a precondition for C-->T transitions often detected as somatic mutations in nodular thyroid tissue. However, the absence of increased SMR would argue for more efficient DNA repair in response to iodine restriction.
Abstract: Chronic use of phenacetin-containing analgesics has been associated with the development of renal cancer. To establish genotoxicity as a possible cause for the carcinogenic effect of phenacetin, we exposed wild type and DNA repair deficient Xpa-/- and Xpa-/-/Trp53+/- mice (further referred as Xpa and Xpa/p53 mice, respectively), carrying a reporter lacZ gene, to 0.75% (w/w) phenacetin mixed in feed. Xpa mice completely lack the nucleotide excision repair pathway, and as such they are sensitive to some classes of genotoxic compounds. Phenacetin exposure induced a significant increase of lacZ mutations in the kidney of both Xpa and Xpa/p53 mice. A minor response was found in liver, whereas no lacZ mutation induction was observed in the spleen of these animals. Interestingly, the observed phenacetin-induced mutant frequencies were higher in male than those found in female mice. This gender difference is probably due to a difference in metabolic rate. Phenacetin-induced mutations mainly consisted of point mutations rather than deletions. The mutational spectra in the kidney of treated WT and Xpa mice were quite similar. Taken together, these results demonstrate that the human carcinogen phenacetin acts as a weak genotoxic agent in an in vivo mouse model system.
Abstract: Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryonic stem (ES) cells leads to a mild reduction in homologous recombination efficiency, the absence of Rad54B has little effect. However, the absence of both Rad54 and Rad54B dramatically reduces homologous recombination efficiency. Furthermore, we show that Rad54B protects ES cells from ionizing radiation and the interstrand DNA cross-linking agent mitomycin C. Interestingly, at the ES cell level the paralogs do not display an additive or synergic interaction with respect to mitomycin C sensitivity, yet animals lacking both Rad54 and Rad54B are dramatically sensitized to mitomycin C compared to either single mutant. This suggests that the paralogs possibly function in a tissue-specific manner. Finally, we show that Rad54, but not Rad54B, is needed for a normal distribution of Rad51 on meiotic chromosomes. Thus, even though the paralogs have similar biochemical properties, genetic analysis in mice uncovered their nonoverlapping roles.
Abstract: UV-induced DNA damage has been recognized as the major molecular trigger for photoimmunosuppression. IL-12 prevents UV-induced immunosuppression via its recently discovered capacity to reduce DNA damage presumably via induction of DNA repair. Because IL-18 shares some biological activities with IL-12 we studied the effect of IL-18 on UV-induced DNA damage and immunosuppression. IL-18 reduced UV-induced apoptosis of keratinocytes and supported long-term cell survival on UV exposure. Injection of IL-18 into mice that were exposed to UV radiation significantly lowered the number of apoptotic keratinocytes. Accordingly, radiation immunohistochemistry revealed reduced amounts of DNA damage in epidermal cells upon injection of IL-18. These effects were not observed in DNA repair-deficient (XpaKO) mice, indicating that IL-18 like IL-12 reduces DNA damage via DNA repair. UV-mediated suppression of the induction of contact hypersensitivity, which is known to be primarily triggered by DNA damage, was prevented upon injection of IL-18 before UV exposure in wild-type but not in XpaKO mice. In contrast to IL-12, IL-18 was not able either in wild-type or in XpaKO mice to break UV-induced immunotolerance that is mediated via regulatory T cells rather than in a DNA damage-dependent fashion. This result indicates that IL-12 is still unique in its capacity to restore immune responses because of its effect on regulatory T cells. Together, these data identify IL-18 as a further cytokine that exhibits the capacity to affect DNA repair. Though being primarily a proinflammatory cytokine through this capacity, IL-18 can also foster an immune response that is suppressed by UV radiation.
Abstract: Genetic defects in nucleotide excision repair (NER) are associated with premature aging, including cancer, in both humans and mice. To investigate the possible role of increased somatic mutation accumulation in the accelerated appearance of symptoms of aging as a consequence of NER deficiency, we crossed four different mouse mutants, Xpa-/-, Ercc6(Csb)-/-, Ercc2(Xpd)m/m and Ercc1-/m, with mice harboring lacZ-reporter genes to assess mutant frequencies and spectra in different organs during aging. The results indicate an accelerated accumulation of mutations in both liver and kidney of Xpa defective mice, which correlated with a trend towards a decreased lifespan. Until 52 weeks, Xpa deficiency resulted mainly in 1-bp deletions. At old age (104 weeks), the spectrum had undergone a shift, in both organs, to G:C-->T:A transversions, a signature mutation of oxidative DNA damage. Ercc1-/m mice, with their short lifespan of 6 months and severe symptoms of premature aging, especially in liver and kidney, displayed an even faster lacZ-mutant accumulation in liver. In this case, the excess mutations were mostly genome rearrangements. Csb-/- mice, with mild premature aging features and no reduction in lifespan, and Xpdm/m mice, exhibiting prominent premature aging features and about 20% reduction in lifespan, did not have elevated lacZ-mutant frequencies. It is concluded that while increased genomic instability could play a causal role in the mildly accelerated aging phenotype in the Xpa-null mice or in the severe progeroid symptoms of the Ercc1-mutant mice, shortened lifespan in mice with defects in transcription-related repair do not depend upon increased mutation accumulation.
Abstract: Inborn defects in nucleotide excision DNA repair (NER) can paradoxically result in elevated cancer incidence (xeroderma pigmentosum [XP]) or segmental progeria without cancer predisposition (Cockayne syndrome [CS] and trichothiodystrophy [TTD]). We report generation of a knockin mouse model for the combined disorder XPCS with a G602D-encoding mutation in the Xpd helicase gene. XPCS mice are the most skin cancer-prone NER model to date, and we postulate an unusual NER dysfunction that is likely responsible for this susceptibility. XPCS mice also displayed symptoms of segmental progeria, including cachexia and progressive loss of germinal epithelium. Like CS fibroblasts, XPCS and TTD fibroblasts from human and mouse showed evidence of defective repair of oxidative DNA lesions that may underlie these segmental progeroid symptoms.
Abstract: Bis(tri-n-butyltin)oxide (TBTO) is one of the organotin compounds that have been used as biocides and occur as persistent environmental pollutants. Human exposure to these compounds occurs through consumption of meat and fish products in which they accumulate. The most sensitive endpoint of TBTO exposure is immunotoxicity. TBTO causes thymus atrophy and thereby interferes with T-lymphocyte-mediated immune responses. Tributyltin compounds have been found to adversely affect a wide range of cellular components and processes in many species, organ systems, and cell types. Both inhibition of proliferation and induction of apoptosis have been observed in thymocytes. We conducted microarray experiments in mice and rats in order to investigate if the immunosuppressive actions of TBTO could be detected by gene expression profiling, and if so, to elucidate the mechanisms of action. Gene expression changes that were detected in mouse thymuses after exposure to a maximum tolerable dose of TBTO correlated to previously observed effects. Most notably, reduction of expression of cell surface determinants and T-cell receptor chains, suppression of cell proliferation, and a possible involvement of nuclear receptors in interference with lipid metabolism by TBTO were observed. The TBTO-induced thymus involution may therefore primarily be caused by inhibition of thymocyte proliferation. In contrast, in rats only limited effects of a lower dose of TBTO were found at the gene expression level in the thymus, even though thymus involution was observed. Here, most gene expression regulation by TBTO was detected in the liver. These preliminary results indicate that gene expression analysis is able to reveal effects of TBTO and to gain insight into its molecular mechanism of action. It may even be a suitable tool to investigate immunotoxicology in general. However, dose and inter-species differences are apparently clearly reflected in the gene expression profiles.
Abstract: Thyroid tumors are a frequent finding not only in iodine-deficient regions. They are predominantly characterized by somatic genetic changes (e.g. point mutations or rearrangements). Because slow thyroid proliferation is a apparent contradiction to a high frequency of tumor initiation, we characterized mutational events in thyroid. First we studied the frequency of certain base exchanges in somatic TSH receptor (TSHR) mutations and determined the spontaneous mutation rate in thyroid and liver. Then we applied different protocols of the comet assay to quantify genomic DNA damage and conducted immunohistochemistry for 8-oxoguanine as a molecular marker for oxidative stress. Among 184 somatic mutations of the human TSHR found in thyroid tumors, C-->T transitions had a unexpectedly high frequency (>32%). The mutation rate in thyroid is 8-10 times higher than in other organs. The comet assay detected increased levels of oxidized pyrimidine (2- to 3-fold) and purine (2- to 4-fold) in thyroid, compared with liver and lung, and a 1.6-fold increase of oxidized purine, compared with spleen. Immunohistochemistry revealed high levels of 8-oxoguanine in thyroid epithelial cells. We have shown a strikingly high mutation rate in the thyroid. Furthermore, results of the comet assay as well as immunohistochemistry suggest that oxidative DNA modifications are a likely cause of the higher mutation rate. It is possible that free radicals resulting from reactive oxygen species in the thyroid generate mutations more frequently. This is also supported by the spectrum of somatic mutations in the TSHR because more frequent base changes could stem from oxidized base adducts that we detected in the comet assay and with immunohistochemistry.
Abstract: BACKGROUND: The high and steadily increasing incidence of ultraviolet-B (UV-B)-induced skin cancer is a problem recognized worldwide. UV introduces different types of damage into the DNA, notably cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs). If unrepaired, these photolesions can give rise to cell death, mutation induction, and onset of carcinogenic events, but the relative contribution of CPDs and 6-4PPs to these biological consequences of UV exposure is hardly known. Because placental mammals have undergone an evolutionary loss of photolyases, repair enzymes that directly split CPDs and 6-4PPs into the respective monomers in a light-dependent and lesion-specific manner, they can only repair UV-induced DNA damage by the elaborate nucleotide excision repair pathway. RESULTS: To assess the relative contribution of CPDs and 6-4PPs to the detrimental effects of UV light, we generated transgenic mice that ubiquitously express CPD-photolyase, 6-4PP-photolyase, or both, thereby allowing rapid light-dependent repair of CPDs and/or 6-4PPs in the skin. We show that the vast majority of (semi)acute responses in the UV-exposed skin (i.e., sunburn, apoptosis, hyperplasia, and mutation induction) can be ascribed to CPDs. Moreover, CPD-photolyase mice, in contrast to 6-4PP-photolyase mice, exhibit superior resistance to sunlight-induced tumorigenesis. CONCLUSIONS: Our data unequivocally identify CPDs as the principal cause of nonmelanoma skin cancer and provide genetic evidence that CPD-photolyase enzymes can be employed as effective tools to combat skin cancer.
Abstract: The murine local lymph node assay (LLNA) was originally developed as a predictive test method for the identification of chemicals with sensitizing potential. In this study we demonstrated that an adapted LLNA can also be used as an immune function assay by studying the effects of orally administered immunomodulating compounds on the T-cell-dependent immune response induced by the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). C57Bl/6 mice were treated with the immunotoxic compounds cyclosporin A (CsA), bis(tri-n-butyltin)oxide (TBTO) or benzo[a]pyrene, (B[a]P). Subsequently, cell proliferation and interferon-gamma (IFN-gamma) and interleukin (IL)-4 release were determined in the auricular lymph nodes (LNs) after DNCB application on both ears. Immunosuppression induced by CsA, TBTO and B[a]P was clearly detectable in this application of the LLNA. Cytokine release measurements proved valuable to confirm the results of the cell proliferation assay and to obtain an indication of the effect on Th1/Th2 balance. We believe to have demonstrated the applicability of an adapted LLNA as an immune function assay in the mouse.
Abstract: The immunostimulatory cytokine IL-12 is able to antagonize immunosuppression induced by solar/ultraviolet (UV) radiation via yet unknown mechanisms. IL-12 was recently found to induce deoxyribonucleic acid (DNA) repair. UV-induced DNA damage is an important molecular trigger for UV-mediated immunosuppression. Thus, we initiated studies into immune restoration by IL-12 to discern whether its effects are linked to DNA repair. IL-12 prevented both UV-induced suppression of the induction of contact hypersensitivity and the depletion of Langerhans cells, the primary APC of the skin, in wild-type but not in DNA repair-deficient mice. IL-12 did not prevent the development of UV-induced regulatory T cells in DNA repair-deficient mice. In contrast, IL-12 was able to break established UV-induced tolerance and inhibited the activity of regulatory T cells independent of DNA repair. These data identify a new mechanism by which IL-12 can restore immune responses and also demonstrate a link between DNA repair and the prevention of UV-induced immunosuppression by IL-12.
Abstract: The nucleotide excision repair (NER) pathway comprises two sub-pathways, transcription coupled repair (TCR) and global genome repair (GGR). To establish the importance of these separate sub-pathways in tumor suppression, we exposed mice deficient for either TCR (Csb), GGR (Xpc) or both (Xpa) to 300 ppm 2-acetylaminofluorene (in feed, ad libitum) in a unique comparative exposure experiment. We found that cancer proneness was directly linked to a defect in the GGR pathway of NER as both Xpa and Xpc mice developed significantly more liver tumors upon 2-AAF exposure than wild type or Csb mice. In contrast, a defect in TCR appeared to act tumor suppressive, leading to a lower hepatocellular tumor response in Xpa mice (tumor incidence of 25%) as compared to Xpc mice (53% tumor-bearing mice). The link between deficient GGR and tumor proneness was most pronounced in the liver, but this phenomenon was also found in the urinary bladder. As tumor induction by 2-AAF appeared almost exclusively dependent on a defect in GGR, we examined whether gene mutation induction in the non-transcribed lacZ locus could reliably predict tumor risk. Interestingly, however, short-term 2-AAF exposure induced lacZ mutant levels in Csb mice almost as high as those found in Xpa or Xpc mice. This indicates that lacZ mutant frequencies are not correlated with a specific DNA repair defect and eventual tumor outcome, at least not in the experimental design presented here.
Abstract: Cellular activity of the tumor suppressor protein p53 is primarily regulated by posttranslational modifications. Phosphorylation of the COOH terminus, including Ser389, is thought to result in a conformational change of the p53 protein, enhancing DNA binding and transcriptional activity. In vitro studies presented here show that, in addition to UV radiation, Ser389 is phosphorylated upon exposure to 2-acetylaminofluorene (2-AAF). Both agents induce bulky DNA adducts repaired by nucleotide excision repair (NER). In contrast, ionizing radiation, known to induce DNA damage not repaired by NER, does not result in Ser389 phosphorylation. Previously, we have shown that p53.S389A mutant mice, lacking the Ser389 phosphorylation site, are sensitive to developing UV-induced skin tumors. Here, we show that p53.S389A mice are also prone to developing 2-AAF-induced urinary bladder tumors, whereas no increased tumor response was found upon ionizing irradiation. These results provide evidence for our hypothesis that phosphorylation of Ser389 is important for activation of p53 to exert its function as a tumor suppressor not exclusively upon the presence of UV-induced DNA damage, but also upon exposure to other bulky adduct-inducing agents. Analysis of 2-AAF- and UV-induced tumors from p53.S389A mice revealed the presence of additional p53 mutations, indicating that lack of Ser389 phosphorylation by itself is not sufficient to abrogate p53 function in tumor suppression. In addition, analyses of skin tumors of p53.S389A mice revealed an interesting hotspot mutation previously found exclusively in NER-deficient mice and patients.
Abstract: Human neuronal cells contain mutant beta-amyloid precursor protein (APP) and ubiquitin B (UBB) mRNAs, in which dinucleotide deletions ('Delta') are generated in/around GAGAG-motifs by an unknown mechanism referred to as 'Molecular Misreading.' The encoded frameshifted (+1) proteins accumulate in the neuropathological hallmarks of Alzheimer's disease (AD) and in other neurodegenerative and age-related diseases. To measure the concentration of Delta mRNAs, we developed a highly sensitive and specific assay, utilizing peptide nucleic acid-mediated PCR clamping, followed by cloning and colony hybridization with sequence-specific oligonucleotide probes. We found only a few molecules of Delta mRNA/microg of cellular RNA, at levels <10(-5) to 10(-6) x the concentration of WT mRNA, in RNA extracted from: (i) cultured human neuroblastoma cells grown under a variety of conditions, (ii) the frontal half of brains from wild type and XPA(-/-) DNA repair-deficient mice, and (iii) post-mortem temporal cortices from humans. Importantly, in RNA from the temporal cortices of AD and Down Syndrome patients that contain betaAPP+1 and UBB+1 immunoreactive cells, we found the same low levels of Delta mRNA. We infer that the accumulation of +1 proteins in neurons of these patients is not caused by an increase in the concentration of Delta mRNAs.
Abstract: This study investigates the role of cellular tyrosinase and/or peroxidase-like oxidative enzyme activity in the covalent binding of quercetin to glutathione, protein, and DNA, as well as the stability of quercetin DNA adducts in time. This was done by studying the formation of glutathionyl quercetin adducts in various in vitro models, and the covalent binding of radiolabeled quercetin to protein and DNA in cells with elevated peroxidase or tyrosinase levels and in cells devoid of nucleotide excision repair (NER). Cells with elevated tyrosinase or peroxidase levels contained approximately 2 times higher levels of covalent quercetin adducts than cells without detectable levels of these oxidative enzymes. However, this difference was smaller than expected based on the differences in tyrosinase and/or peroxidase levels, indicating that these types of oxidative enzyme activities do not play a major role in the cellular pro-oxidant activity of quercetin. Furthermore, quercetin DNA adducts were of transient nature, independent of the presence of NER, suggesting chemical instability of the adducts. Whether this transient nature reflects real reversibility or formation of genotoxic, depurinated sites remains to be investigated at the molecular level. Together, these data indicate that formation of covalent quercetin adducts can be expected in all cells, independent of their oxidative enzyme levels, whereas the transient nature of the DNA adducts formed may limit or cause their ultimate biological impact. If the transient nature represents chemical reversibility of the adduct formation, it would provide a possible explanation for the apparent lack of in vivo carcinogenicity of this in vitro mutagen. Therefore, in vitro mutagenicity studies should focus more on the transient nature of DNA adducts responsible for the mutagenicity in vitro, since this transient nature of DNA adducts may play an essential role in whether the genotoxicity observed in vitro will have any impact in vivo.
Abstract: The tumor suppressor gene p53 has an apparent role in breast tumor development in humans, as approximately 30% of sporadic tumors acquire p53 mutations and Li-Fraumeni syndrome patients carrying germ line p53 mutations frequently develop breast tumors at early age. In the present study, conditional expression of a targeted mutation is used to analyze the role of the human R273H tumor-associated hotspot mutation in p53 in mammary gland tumorigenesis. Heterozygous p53(R270H/+)WAPCre mice (with mammary gland-specific expression of the p53.R270H mutation, equivalent to human R273H, at physiologic levels) develop mammary tumors at high frequency, indicating that the R270H mutation predisposes for mammary gland tumor development and acts in a dominant-negative manner in early stages of tumorigenesis. Spontaneous tumor development in these mice is further accelerated by 7,12-dimethylbenz(a)anthracene (DMBA) treatment at young age. The majority of spontaneous and DMBA-induced carcinomas and sarcomas from p53(R270H/+)WAPCre mice is estrogen receptor alpha positive, and expression profiles of genes also implicated in human breast cancer appear similarly altered. As such, p53(R270H/+)WAPCre mice provide a well-suited model system to study the role of p53 in breast tumorigenesis and the responsiveness of mammary gland tumors to chemotherapeutics.
Abstract: Trichothiodystrophy (TTD) patients with a mutation in the XPD gene of nucleotide excision repair (NER) have a short life span and show various features of premature aging, thereby linking DNA damage to the aging process. Xpd(TTD) mutant mice share many features with TTD patients, including a shorter life span, accompanied by a segmental progeroid phenotype. Here we report new pathology features supportive to the premature aging phenotype of Xpd(TTD) mice. Strikingly, accelerated aging pathology is accompanied by signs suggestive of caloric restriction (CR), a condition usually linked to retardation of age-related pathology and life extension. Accelerated aging symptoms in Xpd(TTD) mice are most likely due to accumulation of endogenously generated DNA damage and compromised transcription leading to cell death, whereas CR symptoms may reflect the need of Xpd(TTD) mice to reduce metabolism (ROS production) in an attempt to extend their life span. Our current findings in Xpd(TTD) mice further strengthen the link between DNA damage, repair and aging.
Abstract: RAD52 and RAD54 genes from Saccharomyces cerevisiae are required for double-strand break repair through homologous recombination and show epistatic interactions i.e., single and double mutant strains are equally sensitive to DNA damaging agents. In here we combined mutations in RAD52 and RAD54 homologs in Schizosaccharomyces pombe and mice. The analysis of mutant strains in S. pombe demonstrated nearly identical sensitivities of rhp54, rad22A and rad22B double and triple mutants to X-rays, cis-diamminedichloroplatinum and hydroxyurea. In this respect, the fission yeast homologs of RAD54 and RAD52 closely resemble their counterparts in S. cerevisiae. To verify if inactivation of RAD52 affects the DNA damage sensitivities of RAD54 deficient mice, several endpoints were studied in double mutant mice and in bone marrow cells derived from these animals. Haemopoietic depression in bone marrow and the formation of micronuclei after in vivo exposure to mitomycine C (MMC) was not increased in either single or double mutant mice in comparison to wildtype animals. The induction of sister chromatid exchanges in splenocytes was slightly reduced in the RAD54 mutant. A similar reduction was detected in the double mutant. However, a deficiency of RAD52 exacerbates the MMC survival of RAD54 mutant mice and also has a distinct effect on the survival of bone marrow cells after exposure to ionizing radiation. These findings may be explained by additive defects in HR in the double mutant but may also indicate a more prominent role for single-strand annealing in the absence of Rad54.
Abstract: Cancer is caused by the loss of controlled cell growth due to mutational (in)activation of critical genes known to be involved in cell cycle regulation. Three main mechanisms are known to be involved in the prevention of cells from becoming cancerous; DNA repair and cell cycle control, important to remove DNA damage before it will be fixed into mutations and apoptosis, resulting in the elimination of cells containing severe DNA damage. Several human syndromes are known to have (partially) deficiencies in these pathways, and are therefore highly cancer prone. Examples are xeroderma pigmentosum (XP) caused by an inborn defect in the nucleotide excision repair (NER) pathway and the Li-Fraumeni syndrome, which is the result of a germ line mutation in the p53 gene. XP patients develop skin cancer on sun exposed areas at a relatively early age, whereas Li-Fraumeni patients spontaneously develop a wide variety of early onset tumors, including sarcomas, leukemia's and mammary gland carcinomas. Several mouse models have been generated to mimic these human syndromes, providing us information about the role of these particular gene defects in the tumorigenesis process. In this review, spontaneous phenotypes of mice deficient for nucleotide excision repair and/or the p53 gene will be described, together with their responses upon exposure to either chemical carcinogens or radiation. Furthermore, possible applications of these and newly generated mouse models for cancer will be given.
