Expanded Simple Tandem Repeat Research Articles

The effects of extremely low frequency magnetic fields on mutation induction in mice

The growing human exposure to extremely low frequency (ELF) magnetic fields has raised a considerable concern regarding their genotoxic effects. The aim of this study was to evaluate the in vivo effects of ELF magnetic fields irradiation on mutation induction in the germline and somatic tissues of male mice. Seven week old BALB/c×CBA/Ca F1 hybrid males were exposed to 10, 100 or 300μT of 50Hz magnetic fields for 2 or 15h. Using single-molecule PCR, the frequency of mutation at the mouse Expanded Simple Tandem Repeat (ESTR) locus Ms6-hm was established in sperm and blood samples of exposed and matched sham-treated males. ESTR mutation frequency was also established in sperm and blood samples taken from male mice exposed to 1Gy of acute X-rays. The frequency of ESTR mutation in DNA samples extracted from blood of mice exposed to magnetic fields did not significantly differ from that in sham-treated controls. However, there was a marginally significant increase in mutation frequency in sperm but this was not dose-dependent. In contrast, acute exposure X-rays led to significant increases in mutation frequency in sperm and blood of exposed males. The results of our study suggest that, within the range of doses analyzed here, the in vivo mutagenic effects of ELF magnetic fields are likely to be minor if not negligible.

In utero exposure to nanosized carbon black (Printex90) does not induce tandem repeat mutations in female murine germ cells

Inhalation of particles has been shown to induce mutations in the male germline in mice following both prenatal and adult exposures in several experiments. In contrast, the effects of particles on female germ cell mutagenesis are not well established. Germline mutations are induced during active cell division, which occurs during fetal development in females. We investigated the effects of prenatal exposure to carbon black nanoparticles (CB) on induction of mutations in the female mouse germline during fetal development, spanning the critical developmental stages of oogenesis. Pregnant C57BL/6J mice were exposed four times during gestation by intratracheal instillation of 67μg/animal of nanosized carbon black Printex90 or vehicle (gestation days 7, 10, 15 and 18). Female offspring were raised to maturity and mated with unexposed CBA males. Expanded simple tandem repeat (ESTR) germline mutation rates in the resulting F2 generation were determined from full pedigrees (mother, father, offspring) of F1 female mice (178 CB-exposed and 258 control F2 offspring). ESTR mutation rates in CB-exposed F2 female offspring were not statistically different from those of F2 female control offspring.

The Dose and Dose-Rate Effects of Paternal Irradiation on Transgenerational Instability in Mice: A Radiotherapy Connection

The non-targeted effects of human exposure to ionising radiation, including transgenerational instability manifesting in the children of irradiated parents, remains poorly understood. Employing a mouse model, we have analysed whether low-dose acute or low-dose-rate chronic paternal γ-irradiation can destabilise the genomes of their first-generation offspring. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm was established in DNA samples extracted from sperm of directly exposed BALB/c male mice, as well as from sperm and the brain of their first-generation offspring. For acute γ-irradiation from 10–100 cGy a linear dose-response for ESTR mutation induction was found in the germ line of directly exposed mice, with a doubling dose of 57 cGy. The mutagenicity of acute exposure to 100 cGy was more pronounced than that for chronic low-dose-rate irradiation. The analysis of transgenerational effects of paternal irradiation revealed that ESTR mutation frequencies were equally elevated in the germ line (sperm) and brain of the offspring of fathers exposed to 50 and 100 cGy of acute γ-rays. In contrast, neither paternal acute irradiation at lower doses (10–25 cGy), nor low-dose-rate exposure to 100 cGy affected stability of their offspring. Our data imply that the manifestation of transgenerational instability is triggered by a threshold dose of acute paternal irradiation. The results of our study also suggest that most doses of human exposure to ionising radiation, including radiotherapy regimens, may be unlikely to result in transgenerational instability in the offspring children of irradiated fathers.

NanoTIO2 (UV-Titan) does not induce ESTR mutations in the germline of prenatally exposed female mice

BackgroundParticulate air pollution has been linked to an increased risk of cardiovascular disease and cancer. Animal studies have shown that inhalation of air particulates induces mutations in the male germline. Expanded simple tandem repeat (ESTR) loci in mice are sensitive markers of mutagenic effects on male germ cells resulting from environmental exposures; however, female germ cells have received little attention. Oocytes may be vulnerable during stages of active cell division (e.g., during fetal development). Accordingly, an increase in germline ESTR mutations in female mice prenatally exposed to radiation has previously been reported. Here we investigate the effects of nanoparticles on the female germline. Since pulmonary exposure to nanosized titanium dioxide (nanoTiO2) produces a long-lasting inflammatory response in mice, it was chosen for the present study.FindingsPregnant C57BL/6 mice were exposed by whole-body inhalation to the nanoTiO2 UV-Titan L181 (~42.4 mg UV-Titan/m3) or filtered clean air on gestation days (GD) 8–18. Female C57BL/6 F1 offspring were raised to maturity and mated with unexposed CBA males. The F2 descendents were collected and ESTR germline mutation rates in this generation were estimated from full pedigrees (mother, father, offspring) of F1 female mice (192 UV-Titan-exposed F2 offspring and 164 F2 controls). ESTR mutation rates of 0.029 (maternal allele) and 0.047 (paternal allele) in UV-Titan-exposed F2 offspring were not statistically different from those of F2 controls: 0.037 (maternal allele) and 0.061 (paternal allele).ConclusionsWe found no evidence for increased ESTR mutation rates in F1 females exposed in utero to UV-Titan nanoparticles from GD8-18 relative to control females.

The effects of methyl-donor deficiency on mutation induction and transgenerational instability in mice

The results of recent human and animal studies have provided strong evidence for the epigenetic effects of a dietary deficiency of methyl donors such as folate, choline and methionine on cancer risk and some other common diseases. However, the mechanisms underlying the links between epigenetic alterations and disease remain elusive. To establish whether a methyl-donor deficient diet can result in long-term changes in mutation rate in treated animals and their offspring, BALB/c male mice were maintained for 8 weeks, from 4 weeks of age, on a synthetic diet lacking in choline and folic acid. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm was established in sperm samples of treated males, as well as in sperm and brain of their first-generation offspring. ESTR mutation frequency in the germline of males sacrificed immediately after treatment or sampled 6 and 10 weeks after the end of dietary restriction did not significantly differ from that in age-matched control groups. The frequency of ESTR mutation in DNA samples extracted from sperm and brain of the first-generation offspring of treated mice was also similar to that in controls. The results of our study suggest that the effects of a methyl-donor deficient diet on mutation induction and transgenerational instability in mice are likely to be negligible.

Abstract 2550: Expanded simple tandem repeat (ESTR) mutations in offspring of benzo(a)pyrene exposed DNA repair deficient male mice

Abstract Germ line mutations can be transmitted to the offspring and may thus affect an infinite number of generations thereafter. To study germ line mutations, expanded simple tandem repeats (ESTR) can be used as a sensitive tool, because they are known to be highly unstable and therefore exhibit high mutation rates. This facilitates the assessment of germ line mutations in a relatively small number of offspring following exposures that may pose a genetic risk to the germ line. We used two ESTR loci Ms6-hm and Hm-2 in families of benzo(a)pyrene (BaP) exposed and unexposed male mice, proficient or deficient for global genome repair (Xpc-/-). Male mice (C57BL/6) were exposed to BaP during 6 weeks (13 mg/kg bw, 3 times per week) and were crossed with female mice (Balb/C) 6 weeks after the last exposure to obtain offspring and to ensure the analysis of mutations originating from spermatogonial stem cells. The overall ESTR mutation rate at both ESTR loci, irrespective of the DNA repair capacity, was higher in offspring of benzo(a)pyrene exposed mice than in control mice (9.4% vs. 4.5%, P=0.054). This effect was predominantly seen in BaP exposed DNA repair deficient Xpc-/- mice; mutation frequencies were 12.5% in the exposed group (12 out of 96 offspring carried a mutant allele) and 3.4% in the unexposed group (3 out of 87), respectively (P=0.024). In DNA repair proficient mice, BaP was unable to increase the mutation frequency transmitted to the offspring (5.8% vs. 5.5% for offspring of exposed and unexposed fathers). These results indicate that analysis of tandem repetitive sequences is a sensitive method for the detection of germline mutations transmitted to the offspring and that the environmental mutagen BaP is able to induce germ line mutations in DNA repair deficient mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2550. doi:1538-7445.AM2012-2550

The effects of maternal irradiation during adulthood on mutation induction and transgenerational instability in mice

The long-term genetic effects of maternal irradiation remain poorly understood. To establish the effects of radiation exposure on mutation induction in the germline of directly exposed females and the possibility of transgenerational effects in their non-exposed offspring, adult female BALB/c and CBA/Ca mice were given 1Gy of acute X-rays and mated with control males. The frequency of mutation at expanded simple tandem repeat (ESTR) loci in the germline of directly exposed females did not differ from that of controls. Using a single-molecule PCR approach, ESTR mutation frequency was also established for both germline and somatic tissues in the first-generation offspring of irradiated parents. While the frequency of ESTR mutation in the offspring of irradiated males was significantly elevated, maternal irradiation did not affect stability in their F1 offspring. Considering these data and the results of our previous study, we propose that, in sharp contrast to paternal exposure to ionising radiation, the transgenerational effects of maternal high-dose acute irradiation are likely to be negligible.

Germline mutation rates in mice following in utero exposure to diesel exhaust particles by maternal inhalation

The induction of inherited DNA sequence mutations arising in the germline (i.e., sperm or egg) of mice exposed in utero to diesel exhaust particles (DEPs) via maternal inhalation compared to unexposed controls was investigated in this study. Previous work has shown that particulate air pollutants (PAPs) from industrial environments cause DNA damage and mutations in the sperm of adult male mice. Effects on the female and male germline during critical stages of development ( in utero) are unknown. In mice, previous studies have shown that expanded simple tandem repeat (ESTR) loci exhibit high rates of spontaneous mutation, making this endpoint a valuable tool for studying inherited mutation and genomic instability. In the present study, pregnant C57Bl/6 mice were exposed to 19 mg/m 3 DEP from gestational day 7 through 19, alongside air exposed controls. Male and female F1 offspring were raised to maturity and mated with control CBA mice. The F2 descendents were collected and ESTR germline mutation rates were derived from full pedigrees (mother, father, offspring) of F1 male and female mice. We found no evidence for increased ESTR mutation rates in females exposed in utero to DEP relative to control females. In contrast, a statistically significant increase in the mutation frequency of male mice exposed in utero to DEP was observed (2-fold; Fisher's exact p < 0.05). Thus, maternal exposure to DEP results in increased mutation in sperm during development.

Exposure to Residential Indoor Air Induces Heritable DNA Mutations in Mice

Exposure to the mixed indoor air pollutants released from synthetic materials and chemical products poses a serious public health problem, but little evidence has been provided to clarify whether such pollutants at environmentally relevant concentrations produce inheritable germline mutations. In the present study, mice were exposed to samples of indoor air from a newly decorated apartment bedroom. Results showed expanded simple tandem repeat (ESTR) mutations occurring in the germline of control and exposed parents, which were also detected in their offspring using three probes, Ms6-hm, Hm-2, and MMS10. Data indicated that mice being exposed to indoor air triggered a significant increase in frequency of ESTR mutations, which may be due primarily to a rise in mutations inherited through the paternal germline. These results suggest that exposure to a mixture of pollutants in indoor air obtained from an apartment in China induced ESTR mutations. Thus, humans exposed to polluted indoor apartment air in China may be at risk for developing germline mutations.

Age-Related Accumulation of Mutations Supports a Replication-Dependent Mechanism of Spontaneous Mutation at Tandem Repeat DNA Loci in Mice

Expanded simple tandem repeat (ESTR) loci belong to the class of highly unstable loci in the mouse genome. The mechanisms underlying the very high spontaneous instability at these loci still remain poorly understood. Using single-molecule polymerase chain reaction, here we have compared the pattern of mutation accumulation in tissues with different proliferation capacities in male mice of age 12, 26, 48, and 96 weeks. In the nonproliferating brain, we did not observe any measurable age-related accumulation of ESTR mutations. In contrast, a highly elevated frequency of ESTR mutation was detected in the sperm samples taken from old mice; similar changes were also observed in the bone marrow tissue. The spectra of ESTR mutations accumulated in all tissues of young and old mice did not significantly differ. Taken together, these data clearly imply that spontaneous ESTR mutations occur almost exclusively in replication-proficient cells. To gain further insights into the mechanisms of ESTR mutation, we developed a stochastic model of age-related mutation accumulation. The observed spectra of ESTR mutants accumulated in the brain and sperm were fairly accurately approximated assuming the values of ESTR mutation rate, ranging from 0.01 to 0.04 per cell division. As these estimates dramatically exceed those for protein-coding genes and microsatellite loci, our data therefore suggest that ESTRs represent one of the most unstable loci in the mammalian genome. The results of our study also imply that ESTR loci can be regarded as a class of expanded microsatellites, with the mechanism of spontaneous mutation most probably attributed to replication slippage.

The effects of in utero irradiation on mutation induction and transgenerational instability in mice

Epidemiological evidence suggests that the deleterious effects of prenatal irradiation can manifest during childhood, resulting in an increased risk of leukaemia and solid cancers after birth. However, the mechanisms underlying the long-term effects of foetal irradiation remain poorly understood. This study was designed to analyse the impact of in utero irradiation on mutation rates at expanded simple tandem repeat (ESTR) DNA loci in directly exposed mice and their first-generation (F 1) offspring. ESTR mutation frequencies in the germline and somatic tissues of male and female mice irradiated at 12 days of gestation remained highly elevated during adulthood, which was mainly attributed to a significant increase in the frequency of singleton mutations. The prevalence of singleton mutations in directly exposed mice suggests that foetal irradiation results in genomic instability manifested both in utero and during adulthood. The frequency of ESTR mutation in the F 1 offspring of prenatally irradiated male mice was equally elevated across all tissues, which suggests that foetal exposure results in transgenerational genomic instability. In contrast, maternal in utero exposure did not affect the F 1 stability. Our data imply that the passive erasure of epigenetic marks in the maternal genome can diminish the transgenerational effects of foetal irradiation and therefore provide important clues to the still unknown mechanisms of radiation-induced genomic instability. The results of this study offer a plausible explanation for the effects of in utero irradiation on the risk of leukaemia and solid cancers after birth.

Single-Molecule PCR Analysis of Germ Line Mutation Induction by Anticancer Drugs in Mice

Understanding and estimating the genetic hazards of exposure to chemical mutagens and anticancer drugs in humans requires the development of efficient systems for monitoring germ line mutation. The suitability of a single-molecule PCR-based approach for monitoring mutation induction at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm by chemical mutagens and anticancer drugs has been validated. The frequency of ESTR mutation was evaluated in the germ line of male mice exposed to the well-characterized alkylating agent and mutagen, ethylnitrosourea, and four widely used anticancer drugs, bleomycin, cyclophosphamide, mitomycin C, and procarbazine. The dose-response of ethylnitrosourea-induced mutation was found to be very close to that previously established using a pedigree-based approach for ESTR mutation detection. Paternal exposure to the clinically relevant doses of bleomycin (15-30 mg/kg), cyclophosphamide (40-80 mg/kg), and mitomycin C (2.5-5 mg/kg) led to statistically significant, dose-dependent increases in ESTR mutation frequencies in the germ line of treated male mice. Exposure to procarbazine led to a maximal increase in mutation frequency at 50 mg/kg, with a plateau at the higher concentrations. The results of this study show that the single-molecule PCR technique provides a new and efficient experimental system for monitoring the genetic effects of anticancer drugs, capable of detecting increases in mutation rates at clinically relevant doses of exposure. In addition, this approach dramatically reduces the number of mice needed for the measurement of germ line mutation induction.

The effects of Atm haploinsufficiency on mutation rate in the mouse germ line and somatic tissue

Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm(+/-)) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm(+/-) males did not significantly differ from that in wild-type BALB/c mice. Acute exposure to 1 Gy of gamma-rays did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm(+/-) and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analysed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice.

Transgenerational effects from exposure to environmental toxic substances

Exposure of mouse germ cells to radiation and chemicals results in mutation, malformation, cancer and other adverse effects (e.g., functional disorders) in the offspring, though these findings have not been proven in human studies. Environmental toxic substances such as urethane (ethyl carbamate) which had been injected subcutaneously to 50 million people as a co-solvent of analgesics and dioxin (an endocrine disruptor) have been found to be associated with adverse effects in the progeny of mice after parental exposures. There are some reports on congenital malformations in the progeny of fathers who had been exposed to dioxin. However, these substances have not shown mutagenicity in in vitro assay systems such as bacterial systems even with S9, cell transformation assays, etc., in spite of their potent teratogenicity and carcinogenicity in in vivo systems. Urethane was negative in the mouse specific locus test for germ cell mutations, but elicited a significant response at the same loci in the offspring of mice treated during pregnancy. Further, urethane is a mutagen in Drosophila germ cell tests, specifically inducing point mutations. Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) does not induce in vivo somatic mutations in mice and rats. It does not induce chromosomal aberrations when the mouse and/or human sperm are treated, but induces mutations at ESTR (expanded simple tandem repeat) loci in mice at low frequencies and also congenital malformations. In this paper, we first present an overview of the results of our studies on transgenerational effects of these toxic substances, compare the results with those obtained after radiation exposure, and then discuss our subsequent studies to reconcile the problems underlying their mutagenicity, teratogenicity and carcinogenicity.

Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline

Mouse expanded simple tandem repeat (ESTR) loci are the most unstable loci in the mouse genome. Despite the fact that over the last decade these loci have been extensively used for studying germline mutation induction in mice, to date little is known about the mechanisms underlying spontaneous and induced ESTR mutation. Here we used flow cytometry and single-molecule PCR to compare the frequency of ESTR mutation in four flow-sorted fractions of the mouse male germ cells – spermatogonia, spermatocytes I, round and elongated spermatids. The frequency and the spectrum of ESTR mutation did not significantly differ between different stages of mouse spermatogenesis. Considering these data and the results of other publications, we propose that spontaneous ESTR mutation is mostly attributed to replication slippage in spermatogonia and these loci may be regarded as a class of expanded microsatellites.

Influence of a contaminated fish diet on germline expanded‐simple‐tandem‐repeat mutation frequency in mice

Herring gulls (Larus argentatus) in polluted areas on the North American Great Lakes were previously shown to have elevated germline mutation frequencies at minisatellite DNA loci. Airborne or dietary contaminants likely caused induced mutations, but the importance of each exposure type was unknown. Follow-up experiments with lab mice determined that air pollution significantly induced germline mutations; however, an evaluation of mutations induced by the diet of herring gulls has not yet been conducted. To address this issue, we fed mice a high-fish diet (58% wet mass) of the most common prey species for herring gulls nesting in Hamilton Harbour, a polluted industrial area on Lake Ontario. We bred the mice and screened pedigrees for germline mutations at expanded-simple-tandem-repeat (ESTR) DNA loci. Mutation frequencies were compared to those in a reference group that was fed fish from Atlantic Canada, and a control group that was fed commercial chow. Germline mutation frequencies were highest in mice fed contaminated fish, but were only marginally or not significantly affected by diet treatment. Statistical power to detect differences among treatment groups was low, and the effect of diet may have more clearly emerged if larger sample sizes were available. Levels of organic pollutants in the fish from Hamilton Harbour were higher than those from Atlantic Canada, but their ability to induce ESTR mutations is unknown. Our findings suggest that a contaminated fish diet may contribute to the elevated germline mutation frequencies observed previously in gulls at this site, but air pollution is likely a more important route of exposure.

Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location.

Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.

The effects of in utero irradiation on mutation induction and transgenerational instability in mice

It is well known that the developing embryo is especially sensitive to ionising radiation. However, to date little is known about the long-term effects of in utero exposure on mutation rates during adulthood. To evaluate the effects of in utero irradiation on mutation induction and transgenerational instability, BALB/c pregnant mice (Theiler stage 20, 12 days of gestation) were exposed to 1 Gy of acute X-rays. The in utero exposed 8-week-old males and females were mated to control partners. To evaluate the effects of in utero irradiation on mutation induction in the germline of exposed mice, all parents and offspring were profiled using two mouse-specific expanded simple tandem repeat (ESTR) probes Ms6-hm and Hm-2. The results of our study show that ESTR mutation rates in the germline of in utero irradiated male and female mice remain highly elevated during adulthood. Using single-molecule PCR, the frequency of ESTR mutation was established in DNA samples prepared from sperm, bone marrow and brain taken from the in utero irradiated animals. In all animals, a statistically significant ~2.8-3.7 fold increase in the mean mutation frequency was found in all tissues of the in utero irradiated animals. The results of our study show that the mutagenic effects of in utero irradiation in mice are well manifested during adulthood and therefore suggest that the susceptibility of early stages of mouse development to ionising radiation may be higher than previously thought. To analyse the effects of parental irradiation on transgenerational instability, the frequency of ESTR mutation was established in DNA samples prepared from sperm, bone marrow and brain taken from the first-generation offspring of in utero irradiated male and female mice. The results of our study show that in the offspring of in utero exposed males the frequency of ESTR mutation is considerably elevated across multiple tissues, whereas in the offspring of irradiated females it does not significantly differ from that in controls. A comparison with the results of our previous studies on transgenerational in stability among the offspring of BALB/c male mice irradiated during adulthood showed that that the magnitude of transgenerational effects is not affected by the stage of paternal exposure. This work has therefore established that an instability signal induced in the germline of in utero irradiated males is manifested during adulthood. The potential implications of our findings to for further understanding of the possible mechanisms of transgenerational genomic instability will be discussed.

Tandem repeat mutation, global DNA methylation, and regulation of DNA methyltransferases in cultured mouse embryonic fibroblast cells chronically exposed to chemicals with different modes of action

Mutations at expanded simple tandem repeat (ESTR) DNA sequences provide a useful tool for screening germline mutation. However, the mechanisms resulting in induced mutations are unknown and provide an impediment to the utility of the method. Induced ESTR mutations arise through a nontargeted mechanism resulting in destabilization of the repeat locus. We hypothesized that alterations in DNA methylation, or in DNA methyltransferase expression, may be associated with this indirect mechanism of mutation. DNA mutation frequency was measured in C3H/10T1/2 mouse embryonic fibroblast cells following chronic exposure to six chemicals exhibiting different modes of genotoxic action: N-nitroso-N-ethylurea (ENU); benzo(a)pyrene (BaP); etoposide (ETOP); okadaic acid (OA); cisplatin (CisPt); and 5-azacytidine (5azadC). Induced mutation ranged from 2-fold (ENU, BaP, ETOP), to 1.3-1.4 fold (OA, 5azadC), to nonresponsive (CisPt). Global DNA methylation, measured using the cytosine extension assay, revealed hypomethylation following exposure to ENU and 5azadC, hypermethylation following BaP and OA exposure, and no change following treatment with ETOP or CisPt. DNA methyltransferase transcription (Dnmt1, Dnmt3a, Dnmt3b) was significantly affected by all treatments except ETOP, with the vast majority of changes being downregulation. There was no direct correlation between ESTR mutation, global methylation, or DNA methyltransferase transcription. However, 4/5 ESTR mutagens caused changes in global methylation, while the noninducer (CisPt) did not cause changes in methylation. We hypothesize that chemicals that modify chromatin conformation through changes in methylation may compromise the ability of mismatch repair enzymes (or other enzymes) to access and repair secondary structures that may form across ESTR loci resulting in mutation.

The Combined Effects of Xeroderma Pigmentosum C Deficiency and Mutagens on Mutation Rates in the Mouse Germ Line

Spontaneous and induced mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germ line of xeroderma pigmentosum group C (Xpc) knockout mice defective in global genome nucleotide excision repair. Spontaneous and radiation-induced mutation rates in homozygous Xpc(-/-) males were significantly higher than those in isogenic wild-type (Xpc(+/+)) and heterozygous (Xpc(+/-)) mice. In contrast, exposure to the monofunctional alkylating agent ethylnitrosourea resulted in similar increases in ESTR mutation rates across all genotypes. ESTR mutation spectra in the germ line of Xpc(-/-), Xpc(+/-) and Xpc(+/+) did not differ. Considering these data and the results of other publications, we propose that the Xpc-deficient mice possess a mutator phenotype in their germ line and somatic tissues that may significantly enhance carcinogenesis across multiple tissues.