Antimutagenic effects of diethyldithiocarbamate towards maleic hydrazide- and N-nitrosodiethylamine-induced mutagenicity in theTradescantia mutagenicity assay

InTradescantia, clone 4430, diethyldithiocarbamate (DEDTC) markedly decreased the frequency of somatic mutations induced by maleic hydrazide (MH) and N-nitrosodiethylamine (NDEA). In contrast, DEDTC had no such effect on N-methyl-N-nitrosourea-induced mutagenesis. The putative degradation and conversion products of MH (maleic acid diamide, succinic acid, maleic acid, lactic acid and hydrazine) exhibited no mutagenic activity in theTradescantia mutagenicity assay.     

Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.     

DNA damage induced by indirect and direct acting mutagens in catalase-deficient transgenic tobacco. Cellular and acellular Comet assays

We have measured the level of DNA damage induced by treating roots (cellular Comet assay) and isolated root nuclei (acellular Comet assay) of catalase-deficient (CAT1AS) and wild-type (SR1) tobacco with the promutagen o-phenylenediamine (o-PDA) and the direct acting genotoxic agents hydrogen peroxide and ethyl methanesulphonate (EMS). The roots of CAT1AS have about 60% less catalase activity compared to the roots of SR1. The promutagen o-PDA applied on tobacco roots induced significantly higher levels of DNA damage in the CAT1AS transgenic line than in SR1, while after application of o-PDA on isolated root nuclei, no DNA damage could be detected. In the catalase-deficient line CAT1AS about six-fold lower concentrations of H(2)O(2) are sufficient to induce the same levels of DNA damage as in SR1. By contrast, after treatment of isolated root nuclei with H(2)O(2) no difference in the induced levels of DNA damage was observed between CAT1AS and SR1. The DNA damaging effect of EMS was not affected by the presence of catalase in the tobacco roots and the levels of DNA damage measured by the cellular and acellular assay were similar.Comparing the effects of genotoxic agents in both the cellular and acellular Comet assays may help to elucidate their mechanism of action. Differences in both systems may reveal the participation of scavengers and of repair and metabolic enzymes on the activity of the genotoxic agent and the role of the cell wall in preventing the agent from reacting with nuclear DNA.     

The BRCT Domain of PARP-1 Is Required for Immunoglobulin Gene Conversion

Genetic variation at immunoglobulin (Ig) gene variable regions in B-cells is created through a multi-step process involving deamination of cytosine bases by activation-induced cytidine deaminase (AID) and their subsequent mutagenic repair. To protect the genome from dangerous, potentially oncogenic effects of off-target mutations, both AID activity and mutagenic repair are targeted specifically to the Ig genes. However, the mechanisms of targeting are unknown and recent data have highlighted the role of regulating mutagenic repair to limit the accumulation of somatic mutations resulting from the more widely distributed AID-induced lesions to the Ig genes. Here we investigated the role of the DNA damage sensor poly-(ADPribose)-polymerase-1 (PARP-1) in the repair of AID-induced DNA lesions. We show through sequencing of the diversifying Ig genes in PARP-1^−/− DT40 B-cells that PARP-1 deficiency results in a marked reduction in gene conversion events and enhanced high-fidelity repair of AID-induced lesions at both Ig heavy and light chains. To further characterize the role of PARP-1 in the mutagenic repair of AID-induced lesions, we performed functional analyses comparing the role of engineered PARP-1 variants in high-fidelity repair of DNA damage induced by methyl methane sulfonate (MMS) and the mutagenic repair of lesions at the Ig genes induced by AID. This revealed a requirement for the previously uncharacterized BRCT domain of PARP-1 to reconstitute both gene conversion and a normal rate of somatic mutation at Ig genes, while being dispensable for the high-fidelity base excision repair. From these data we conclude that the BRCT domain of PARP-1 is required to initiate a significant proportion of the mutagenic repair specific to diversifying antibody genes. This role is distinct from the known roles of PARP-1 in high-fidelity DNA repair, suggesting that the PARP-1 BRCT domain has a specialized role in assembling mutagenic DNA repair complexes involved in antibody diversification.     

Evaluation of mutagenic effects of hyperbaric oxygen (HBO) in vitro. II. Induction of oxidative DNA damage and mutations in the mouse lymphoma assay

We recently showed that treatment of V79 cells with hyperbaric oxygen (HBO) efficiently induced DNA effects in the comet assay and chromosomal damage in the micronucleus test (MNT), but did not lead to gene mutations at the hprt locus. Using the comet assay in conjunction with bacterial formamidopyrimidine DNA glycosylase (FPG protein), we now provide indirect evidence that the same treatment leads to the induction of 8-oxoguanine, a premutagenic oxidative DNA base modification in V79 and mouse lymphoma (L5178Y) cells. We also demonstrate that HBO efficiently induces mutations in the mouse lymphoma assay (MLA). Exposure of L5178Y cells to HBO (98% O(2); 3bar) for 2h caused a clear mutagenic effect in the MLA, which was further enhanced after a 3h exposure. As this mutagenic effect was solely due to the strong increase of small colony (SC) mutants, we suggest that HBO causes mutations by induction of chromosomal alterations. Molecular characterization of induced SC mutants by loss of heterozygosity (LOH) analysis showed an extensive loss of functional tk sequences similar to the pattern found in spontaneous SC mutants. This finding confirmed that the majority of HBO-induced mutants is actually produced by a clastogenic mechanism. The induction of point mutations as a consequence of induced oxidative DNA base damage seems to be of minor importance.     

Somatic hypermutation of immunoglobulin genes: lessons from proliferating cell nuclear antigenK164R mutant mice

Proliferating cell nuclear antigen (PCNA) encircles DNA as a ring-shaped homotrimer and, by tethering DNA polymerases to their template, PCNA serves as a critical replication factor. In contrast to high-fidelity DNA polymerases, the activation of low-fidelity translesion synthesis (TLS) DNA polymerases seems to require damage-inducible monoubiquitylation (Ub) of PCNA at lysine residue 164 (PCNA-Ub). TLS polymerases can tolerate DNA damage, i.e. they can replicate across DNA lesions. The lack of proofreading activity, however, renders TLS highly mutagenic. The advantage is that B cells use mutagenic TLS to introduce somatic mutations in immunoglobulin (Ig) genes to generate high-affinity antibodies. Given the critical role of PCNA-Ub in activating TLS and the role of TLS in establishing somatic mutations in immunoglobulin genes, we analysed the mutation spectrum of somatically mutated immunoglobulin genes in B cells from PCNA^K164R knock-in mice. A 10-fold reduction in A/T mutations is associated with a compensatory increase in G/C mutations—a phenotype similar to Polη and mismatch repair-deficient B cells. Mismatch recognition, PCNA-Ub and Polη probably act within one pathway to establish the majority of mutations at template A/T. Equally relevant, the G/C mutator(s) seems largely independent of PCNA^K164 modification.     

Genomic damage induced in tobacco plants by chlorobenzoic acids--metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls--Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2-4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose-response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

Mutagenicity of cadmium in mammalian cells: implication of oxidative DNA damage

Cadmium and cadmium compounds are well established human carcinogens and are ubiquitously present in the environment. The carcinogenic mechanism(s) of cadmium remains largely unknown since direct mutagenic effect is weak in bacterial and in standard mammalian cell mutation assays. In this study, we show that when evaluated using the human-hamster hybrid A(L) cell mutation assay in which both intragenic and multilocus deletions can readily be detected, CdCl(2) is a strong mutagen that induces predominantly large deletion mutations. Concurrent treatment of A(L) cells with the oxyradical scavenger dimethyl sulfoxide significantly reduced the number of cadmium-induced mutations. In contrast, pre-treatment of cells with buthionine sulfoximine that depletes intracellular glutathione, increased cytotoxicity and mutagenicity of cadmium. These results demonstrate that reactive oxygen species mediate cadmium induced mutations in A(L) cells. With laser scanning confocal microscopy and the fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, we demonstrated that cadmium induced a dose and time dependent formation of intracellular oxyradicals. Using immunoperoxidase staining coupled with a monoclonal antibody-specific for 8-OHdG adducts in DNA, we demonstrated that cadmium induced a dose dependent increase of 8-OHdG adducts, which accumulated with prolonged exposure. Furthermore, we showed that at low concentration, cadmium, attenuated removal of hydrogen peroxide induced 8-OHdG adducts. Thus, the carcinogenicity of cadmium can, in part, be explained by its mutagenic activity, which is mediated by reactive oxygen species induced DNA damage and by its interference with the repair of oxidative DNA damage.     

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary—or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

DNA lesion and mutagenesis induced in phageM13mp2 by UVA, UVB and UVC irradiation.

Sunlight is carcinogenic and mutagenic and its genotoxic effects are believed to be the result of UV light-induced lesions in DNA. These lesions include pyrimidine dimers and (6-4) photoproducts, but it is uncertain whether the pyrimidine modifications are the sole pre-mutagenic lesions induced by UV light. Previous studies indicate that some sunlight-induced mutations in the single-stranded DNA phage M13mp2 may not be caused by these photoproducts. In this work, purified single-stranded phage DNA was exposed to UVA, UVB and UVC and the induced mutations were analyzed. All 3 types of UV light increase the mutation frequency. The mutants were sequenced and the results suggest that UVA exposure may induce formation of a non-dipyrimidine lesion in DNA.     

Induction and expression of mutations in mammalian cells in the absence of DNA synthesis and cell division

The induction of mutations by the alkylating agent ethyl methanesulfonate (EMS) was determined with Chinese hamster ovary cells maintained in serum-free medium to arrest DNA synthesis and cell division. The arrested cultures were treated with EMS and maintained in serum-free medium for various time intervals post-treatment before serum containing medium was added to initiate DNA synthesis and cell division. The concentration-dependent increase in 6-thioguanine-resistant mutants in the arrested cultures was similar to that found with exponentially dividing cultures when serum was added to the arrested cultures immediately after the EMS treatment; the time course of phenotypic expression was also similar with both cultures. In addition, maintenance of the arrested cultures in serum-free medium for up to 18 days post-treatment resulted in no change in the mutant frequency. This suggests that the mutagenic damage is not removed in these arrested cultures. Furthermore, maintenance of the arrested state for increasing time intervals before serum addition results in decreases in the time necessary for maximum phenotypic expression. Cultures maintained in serum-free medium for 16 days after mutation treatment show complete expression of the mutations with no need for subculture. This last result suggests that the mutagenic damage induced by EMS in Chinese hamster ovary cells is not removed and that this damage results in both the induction and expression of mutation in the absence of DNA replication.     

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary -- or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

Modulation of genotoxicity in Drosophila.

The extensive knowledge of the genetics of Drosophila melanogaster and the long experimental experience with this organism have made it of unique usefulness in mutation research and genetic toxicology. The development of somatic mutation and recombination tests (SMART) has provided sensitive, rapid and cheap assays for investigations of mutagenic and recombinogenic properties of chemicals. The present paper deals with the SMART wing spot assay, developed by Graf et al. (1984). The use of two genetic markers, multiple wing hair (mwh) and flare (flr) in the third chromosome, makes it possible to discern localized recombinogenic effects on the two intervals--the major, euchromatic, part of the chromosome, and the mostly heterochromatic centromere region. The distribution of induced mitotic recombination varied between test chemicals. Ethylene oxide caused a specific increase of twin spots, indicating a localized induction of somatic recombination in the centromere region. The wing spot assay has turned out to be suitable for combined treatment with chemicals in order to study antimutagenic and other modulating effects by mutagenic and recombinogenic chemicals. Examples of the use of this assay for such a purpose are presented in this paper. The inhibitor of poly ADP-ribosylation, 3-aminobenzamide (3AB), caused a pronounced increase of wing spots, induced by alkylating agents. The data indicate that this interaction between alkylating agents and 3AB is solely due to an effect on somatic recombination but not on point mutations. The inhibitor of topoisomerases, novobiocin, which presumably acts on the chromatin configuration, had different modulating effects on spots induced by methyl methanesulfonate (MMS) and ethylnitrosourea (ENU). Novobiocin essentially acted as an antirecombinogenic agent in cotreatment experiments with MMS and as antimutagenic agent with ENU. Attempts to interfere with mutagenic and recombinogenic effects of the radical-generating agents bleomycin, menadione and paraquat, by agents acting on the defence mechanisms against oxygen radicals, were essentially unsuccessful.     

Comparative studies of the induction of somatic eye-color mutations in an unstable strain of Drosophila melanogaster by MMS and X-rays at different developmental stages

An unstable white locus in Drosophila melanogaster originally described by Rasmuson and Green (1974) and further by Rasmuson et al. (1978, 1980) contains an IS element. This constellation interacts with the zeste mutation and forms a mutationally unstable system that is sensitive to a variety of mutagens. Mutational shifts between zeste and wild-type eye color as well as deletions and transpositions of the white locus are frequently occurring in the unstable X-chromosome in germ line and in somatic tissue. Germinal mutations from zeste to wild-type eye color are associated with an insertion of a piece of DNA, proximal to the wsp site, and the shifts from red to zeste are caused by an excision of the same piece (Rasmuson, in preparation). Mutations to pigmentless phenotype are interpreted as deletions of the white locus, while they always are irreversible and show non-complementation with wsp. The somatic system can be used as a screening test for potential mutagens, described by Rasmuson et al. (1984). This survey is an attempt to correlate the size of the mutated area of the eyes with the age of the larvae at mutagen treatment. X-Rays and MMS were used to give an indication of the mechanism of the instability, according to the different kinds of DNA damage induced. The results show that the mean size of red spots decreased with increasing age of larvae at treatment, while the mutation frequencies were increased because of the multiplication of the cells in the eye anlage susceptible to the mutagens. This is contradictory to the hypothesis maintained by Fahmy and Fahmy (1980) that the somatic shifts are not mutagenic but epigenetic events, due to altered regulation of the gene expression. Red spots induced with MMS are smaller in size than X-ray-induced red spots, indicating a delay in the establishment of mutations from chemically-induced lesions compared to irradiation damage. White spots on the other hand were equally large in size, irrespective of inducing agent and about twice the size of the chemically-induced red spots, implying a faster and more direct action for fixation of deletions than for the production of MMS induced shifts in eye color from zeste to red.     

DNA damage caused by ascorbate in the presence of Cu2+ induces mutations

The DNA damage induced by ascorbate in the presence of Cu2+ was analyzed by sequencing, and the mutagenic consequences of damages to plasmid pUC18 lacZ' were assayed in a forward repairing system in E. coli JM109 in vivo. Ascorbate induced two classes of DNA damage in the presence of Cu2+, one being non-base-specific direct strand cleavage, and the other being sequence-specific base modification labile to alkali treatment. Radicals generated from ascorbate hydroperoxide were involved in DNA damaging reactions. Ascorbate and Cu2+ caused mutations in pUC18 lacZ' gene. The mutation frequency by this method was about 10(-4) at 18% survivors when measured as a loss of alpha-complementation. All the mutations found were single-base substitutions that occurred in the structural part of the lacZ' gene. They were predominantly G:C----A:T transitions.     

Spermatocyte responses in vitro to induced DNA damage

Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by initiation of meiotic homologous-recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after recombination-related DSBs have been repaired. The experiments described here tested the hypothesis that, even though actively completing meiotic recombination, pachytene spermatocytes cultured in the absence of testicular somatic cells initiate appropriate chromatin remodeling and cell-cycle responses to environmentally induced DNA damage. Two DNA-damaging agents were employed for in vitro treatment of pachytene spermatocytes: gamma-irradiation and etoposide, a topoisomerase II (TOP2) inhibitor that results in persistent unligated DSBs. Chromatin modifications associated with DSBs were monitored after exposure by labeling surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and the phosphorylated variant of histone H2AFX (herein designated by its commonly used symbol, H2AX), gammaH2AX (which modifies chromatin associated with DSBs). Both gammaH2AX and RAD51 were rapidly recruited to irradiation- or etoposide-damaged chromatin. These chromatin modifications imply that spermatocytes recruit active DNA damage responses, even after recombination is substantially completed. Furthermore, irradiation-induced DNA damage inhibited okadaic acid-induced progression of spermatocytes from meiotic prophase to metaphase I (MI), implying efficacy of DNA damage checkpoint mechanisms. Apoptotic responses of spermatocytes with DNA damage differed, with an increase in frequency of early apoptotic spermatocytes after etoposide treatment, but not following irradiation. Taken together, these results demonstrate modification of pachytene spermatocyte chromatin and inhibition of meiotic progress after DNA damage by mechanisms that may ensure gametic genetic integrity.     

Separation of the SOS-dependent and SOS-independent components of alkylating-agent mutagenesis.

Escherichia coli plasmids containing the rpsL+ gene (Strs phenotype) as the target for mutation were treated in vitro with N-methyl-N-nitrosourea. Following fixation of mutations in E. coli MM294A cells (recA+ Strs), an unselected population of mutant and wild-type plasmids was isolated and transferred into a second host, E. coli 6451 (recA Strr). Strains carrying plasmid-encoded forward mutations were then selected as Strr isolates, while rpsL+ plasmids conferred the dominant Strs phenotype in the second host. Mutation induction and reduced survival of N-methyl-N-nitrosourea-treated plasmids were shown to be dose dependent. Because this system permitted analysis and manipulation of the levels of certain methylated bases produced in vitro by N-methyl-N-nitrosourea, it afforded the opportunity to assess directly the relative roles of these bases and of SOS functions in mutagenesis. The methylated plasmid DNA gave a mutation frequency of 6 X 10(-5) (a 40-fold increase over background) in physiologically normal cells. When the same methylated plasmid was repaired in vitro by using purified O6-methylguanine DNA methyltransferase (to correct O6-methylguanine and O4-methylthymine), no mutations were detected above background levels. In contrast, when the methylated plasmid DNA was introduced into host cells induced by UV light for the SOS functions, rpsL mutagenesis was enhanced eightfold over the level seen without SOS induction. This enhancement of mutagenesis by SOS was unaffected by prior treatment of the DNA with O6-methylguanine DNA methyltransferase. These results demonstrate a predominant mutagenic role for alkylation lesions other than O6-methylguanine or O4-methylthymine when SOS functions are induced. The mutation spectrum of N-methyl-N-nitrosourea under conditions of induced SOS functions revealed a majority of mutagenic events at A . T base pairs.     

Radiation-induced lethality and mutation in a repair-deficient CHO cell line

A U.V.-sensitive, DNA repair-deficient mutant of Chinese hamster ovary cells was tested for its response to the lethal effects of X-irradiation and simulated solar light, and to the mutagenic actions of X-rays. A slight sensitivity to killing by X-rays and a greater sensitivity to solar light was observed relative to the wild-type CHO cells. More mutations were induced at a given dose of X-rays in the sensitive cell line than in the wild-type. These results are interpreted in terms of overlap in the repair processes which take place after U.V. damage in mammalian cells with those that take place after other types of radiation damage.     

Mitochondrial DNA exhibits resistance to induced point and deletion mutations

The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established. To test the hypothesis that mtDNA-damaging agents induce mtDNA mutations, we exposed Muta^TMMouse mice to benzo[a]pyrene (B[a]P) or N-ethyl-N-nitrosourea (ENU), daily for 28 consecutive days, and quantified mtDNA point and deletion mutations in bone marrow and liver using our newly developed Digital Random Mutation Capture (dRMC) and Digital Deletion Detection (3D) assays. Surprisingly, our results demonstrate mutagen treatment did not increase mitochondrial point or deletion mutation frequencies, despite evidence both compounds increase nuclear DNA mutations and demonstrated B[a]P adduct formation in mtDNA. These findings contradict models of mtDNA mutagenesis that assert the elevated rate of mtDNA mutation stems from damage sensitivity and abridged repair capacity. Rather, our results demonstrate induced mtDNA damage does not readily convert into mutation. These findings suggest robust mitochondrial damage responses repress induced mutations after mutagen exposure.