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Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag(-/-)) or O(6)-methylguanine methyltransferase (Mgmt(-/-)), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt(-/-) neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag(-/-) neurons were for the most part significantly less sensitive than wild type or Mgmt(-/-) neurons to MAM and HN2. Aag(-/-) neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt(-/-) mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag(-/-) or MGMT-overexpressing (Mgmt(Tg+)) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt(Tg+) mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.

Drug synthesis and/or formulation can generate genotoxic impurities. For instance, strong acid/alcohol interactions during the process of drug salt formation produce alkylating agents such as alkyl halides and alkyl esters of alkyl sulfonic acids. The genotoxicity of a few classic alkylating agents such as methyl and ethyl methanesulfonate have been previously well characterized, whereas the majority of compounds from this class have only been tested in the Salmonella reversion assay. Therefore, the goal of this study was to investigate clastogenicity and DEL recombination profiles of 22 halogenated alkanes and alkylesters of sulfuric and alkane-, aryl-sulfonic acids using a battery of cellular and molecular assays. The in-vitro micronucleus assay in CHO cells was used to measure clastogenicity and the deletion recombination (DEL) assay in S. cerevisiae provided a measure of DNA deletions. We also examined the compounds' reactivity towards 4-(p-nitrobenzyl)pyridine (NBP), a surrogate molecule for biological ring nitrogens. Methylating agents were most potent in all three assays and the alkyl chlorides evaluated in our study were negative in all three assays. Also, a strong correlation was found between the MN, DEL and NBP assays. In summary, this study contributes to a better understanding of the genotoxic properties of common alkyl halides and alkyl esters with alkylating activity and might provide guidance for managing risk of genotoxic process-related impurities of drug substances and products.

The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces lung adenocarcinoma through formation of DNA adducts. Our previous research on susceptibility to tobacco-induced carcinogenesis focused on benzo[a]pyrene diol epoxide (BPDE) as the in vitro mutagen for phenotype measurements of DNA repair capacity (DRC) in mammalian cells. Here, we present a modified host-cell reactivation (HCR) assay to measure lymphocytic DRC for alkylating DNA damage as is induced by the tobacco-specific nitrosamine, NNK. We substituted dimethyl sulfate (DMS) to create alkylating damage in pCMVluc plasmid DNA and established the damage-repair dose-response curves in both normal and nucleotide excision repair-deficient lymphoblastoid cell lines and in phytohemagglutinin (PHA)-stimulated primary lymphocytes. We then successfully measured the DRC in PHA-stimulated lymphocytes from 48 patients with lung adenocarcinoma and 45 cancer-free controls and tested our hypothesis that lower DRC for alkylating damage is associated with an increased risk of lung adenocarcinoma. The cases exhibited a lower mean DRC than did the controls. A >3-fold increased risk (odds ratio = 3.21; 95% confidence interval = 1.25-8.21) was found for those with DRC levels below the control median. There was no correlation between the DRC measured with this DMS-HCR assay and that from the parallel BPDE-HCR assay. Interestingly, risk increased to >10-fold for those with sub-optimal DRC measured by both DMS- and BPDE-HCR assays. We conclude that variability in DRC is a risk factor for lung cancer and our results provide proof of principle for a new assay that can assess DRC for NNK-induced DNA damage.

The progression of a normal cell to senescence in vivo and in vitro is accompanied by a reduction in the length of the telomeres, the chromosome capping segments at the end of each linkage group. However, overexpression of the reverse transcriptase subunit (HTERT) of the ribonucleoprotein telomerase restores telomere length and delays cellular senescence. Although some data exist in the literature with respect to survival, no molecular data have shown that DNA repair in telomerase-immortalized cells is normal. Several telomerase-immortalized human skin fibroblast cell lines were constructed from a primary human fibroblast cell line. The primary line and the telomerase-immortalized cell lines were treated with either ultraviolet (UV) radiation or dimethylsulfate (DMS). UV radiation principally produces cyclobutane pyrimidine dimers that are repaired by nucleotide excision repair, whereas DMS introduces mainly N-methylpurines repaired by base excision repair. Here, we show that repair of both types of damage in the telomerase-immortalized human skin fibroblast cell lines is identical to repair observed in normal skin fibroblasts. Thus, telomerase expression and consequent immortalization of skin fibroblasts do not alter nucleotide or base excision repair in human cells.

Dimethyl sulfate (DMS) is an alkylating agent that is carcinogenic to the respiratory tract of rodents. DNA adducts, cell proliferation, and histopathology were assessed in rats to better understand the molecular dosimetry and tissue dynamics associated with repeated inhalation exposure to DMS. For DNA methylation, rats were exposed to DMS vapor 6 h/day for up to 10 days to 0.0, 0.1, 0.7 and 1.5 ppm. N7-Methylguanine and N3-methyladenine were detected in neutral thermal hydrolysates of DNA isolated from respiratory tract tissues by high-performance liquid chromatography (HPLC) using fluorescence and ultraviolet (UV) detection. DNA methylation was greatest in DNA isolated from nasal respiratory mucosa, less in olfactory, and little was found in lung. N7-Methylguanine levels in respiratory mucosa approached steady-state levels by day 5, and N7-methylguanine persistence following exposure for 5 consecutive days was also determined. Loss of N7-methylguanine from respiratory and olfactory mucosa appeared to follow first-order kinetics. N3-Methyladenine levels were at or below detection limits in all samples. The effect of DMS on histopathology and cell proliferation in the nasal epithelium was also investigated. Rats were exposed nose-only for 2 wk to DMS vapor at concentrations of 0, 0.1, 0.7, or 1.5 ppm. Inhalation exposure to DMS induced degenerative and inflammatory changes in nasal epithelium at >or=0.7 ppm. Cell proliferation evaluations showed a trend towards an increased response at 1.5 ppm. These experiments demonstrate that DMS can induce cytotoxic and proliferative effects and is a potent methylating agent of the nasal mucosa in vivo. These experiments will provide data for the development of dosimetry models useful for risk extrapolation.

Dimethyl sulfate (DMS) is a volatile sulfuric acid ester used principally as a methylating agent in a wide variety of industrial applications. DMS reacts with organic macromolecules by a SN2 mechanism. The weight of experimental evidence suggests that DMS possesses genotoxic and carcinogenic potential. Inhalation studies have shown that repeated exposure to DMS leads to tumors in the nasal cavity and lower respiratory tract in both rats and mice. Here we present a quantitative assessment for cross-species dose extrapolation for inhaled DMS using a physiologically based pharmacokinetic (PBPK) model. The model is designed to simulate N7-methylguanine (N7 mG) DNA adduct levels in the nasal mucosa following DMS exposure in rats and humans. This model was parameterized and predictions were tested by comparison against experimentally measured N7 mG DNA adduct levels in rat nasal mucosa following inhalation exposure to DMS. The model-based interspecies dose comparison, using N7 mG adduct levels in the nasal respiratory tissue as the appropriate dose metrics, predicts a dose rate seven times higher in rats compared to humans.

It has been shown that the ethylating agent diethylsulphate (DES) induces centromere-containing micronuclei with kinetics suggesting that molecules other than DNA could be targets. In quiescent Chinese hamster fibroblasts CHEF/18, O6-alkylated bases inhibit ribosomal protein S6 kinase (S6K1), the terminal member of a kinase cascade responsible for an increased rate of protein synthesis, but not extracellular signal-activated kinases (ERK1/2) or terminal kinases of a second cascade which activates transcription. The inhibition correlates with the appearance of abnormal metaphases at the following mitosis, suggesting that alkylation of the nucleotide pool and inhibition of S6K1 could be one of the mechanisms leading to chromosome loss by alkylating agents. To clarify the role of protein kinases in chromosome loss induced by alkylating agents, we have studied the effects of DES and methylnitrosourea (MNU) on S6K1 and ERK1/2 activation by growth factors. The alkylating agents were studied in a battery of Chinese hamster fibroblasts (CHEF/18, CHO and ClB) with normal and mutated p53 to control for DNA damage-induced activation of p53, which could indirectly inhibit protein kinases. The role of repair in induction of micronuclei was studied in mismatch repair-proficient CHO and repair-deficient ClB cells. Our results indicate that DES induced micronuclei in a mismatch repair-independent manner, within 8 h of treatment, in agreement with a role for S6K1 inhibition in micronucleus formation. MNU induced centromere-containing micronuclei only in CHO cells, one cell cycle after treatment, without any detectable influences on either kinase cascade, suggesting a role for mismatch repair in chromosome loss.

Dimethyl sulfate (DMS; CAS No. 77-78) is a colorless, oily liquid which is used as a chemical intermediate and as a reactant in producing polyurethane resins. In this study, groups of pregnant Crl:CD BR rats were exposed, nose-only, to either 0.1, 0.7 or 1.5 ppm DMS by inhalation for 6 hr/day from Days 7 through 16 of gestation (day in which copulation plug was detected was designated Day 1G). A control group of pregnant rats was exposed simultaneously to air only. All female rats were euthanized on Day 22G and the fetuses were examined. A suppression of both food consumption and the rate of body weight gain was seen in the 0.7 and 1.5 ppm groups. No unusual clinical signs were seen in rats exposed to DMS. None of the reproductive parameters was altered in any of the groups and no statistically significant fetal effects were detected. DMS is not a developmental toxin in the rat following inhalation exposures up to 1.5 ppm during the period of major organogenesis.

The activities and the expression of 3-methyladenine glycosylase (3-meAde gly) and O6-alkylguanine-DNA-alkyltransferase (O6 ATase) were investigated in ten human cancer cell lines. Both 3-meAde gly and O6 ATase activities were variable among different cell lines. mRNA levels of the O6 ATase gene, appeared to be related to the content of O6 ATase in different cell lines, whereas no apparent correlation was found between mRNA of 3-meAde gly and the enzyme activity. No correlation was found between the activity of the two enzymes and the sensitivity to alkylating agents of different structures such as CC-1065, tallimustine, dimethylsulphate (DMSO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cis-diamminedichloroplatinum (cDDP) and melphalan (L-PAM). The most striking finding of this study is that a correlation exists between the activity of O6 ATase and 3-meAde gly in the various cell lines investigated (P<0.01), suggesting a common mechanism of regulation of two DNA repair enzymes.

In a previous study we showed that the formation of O6-ethylguanine (O6-EtGua) in the DNA of CHO cells in culture correlated with mutations induced by ethylnitrosourea (ENU) and diethylsulfate (DES) at the hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus but not at the Na, K-ATPase locus. This study was extended to another ethylating agent, ethyl methanesulfonate (EMS). DNA adduct formation and induction of mutation at the two gene loci were determined simultaneously in CHO cells after EMS exposure. The extent of ethylation at the N7 and O6 positions of guanine and at the N3 site of adenine were measured and the possible correlations with 6-thioguanine resistance (6-TGr) and ouabain resistance (ouar) mutations were investigated. A good correlation between the levels of ethylation at O6 guanine and mutation frequency at hprt gene by all three ethylating agents was observed. In the case of the ouar locus, the frequency of O6-EtGua adducts correlated with mutation induction by EMS and ENU but not by DES. Although both EMS and DES have similar reaction mechanisms, these results highlight differences in their mutational specificity. The comparison of this type of analysis with mutational spectra revealed that correlation studies may be inadequate to analyse multicomponent phenomena like mutation formation.

Six model ethylating agents were tested for clastogenic potency by means of a new technique of the micronucleus assay with mouse peripheral blood cells using acridine orange (AO)-coated slides, to evaluate the test. The alkylating agents were: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), diethylsulfate (DES), ethyl methanesulfonate (EMS), epichlorohydrin (ECH) and ethylene dibromide (EDB). The animals were given a single intraperitoneal injection of the following doses of the chemicals: ENNG and ENU, 25, 50 and 100 mg/kg; EMS and DES, 100, 200 and 400 mg/kg body weight. For EDB and ECH, the doses were 50, 100 and 200 mg/kg, given twice, 24 h apart. Before and after the injection, blood samples were taken from the tails at 24-h intervals up to 72 h and preparations were made on AO-coated slides. For each dose group, 4 animals were used and 1000 reticulocytes were examined per slide for the presence of micronuclei. At the optimum induction time of 48 h, ENU induced micronucleated reticulocytes (MNRETs) at all 3 doses. ENNG and EMS induced MNRETs significantly at 2 dose levels each and DES only at the highest dose. ECH and EDB failed to induce MNRETs. On the basis of the dose of chemical needed to double the spontaneous frequency, the order of clastogenic potency was ENU greater than ENNG greater than EMS greater than DES. The results obtained compared favorably with those from other in vivo methods. The present technique proves to be simple, flexible and relatively sensitive. Shifts in the optimum induction peak in individual animals and by some chemicals can be picked up easily which is important when testing weak mutagens and chemicals with an unknown mechanism of action.

DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the O6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if O6-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 O6-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.

The covalent binding of alkylating agents with DNA is considered a critical event in the mutagenic and carcinogenic effect of these chemicals. Several studies have indicated that only certain DNA alkylation products contribute to the mutagenic or carcinogenic activity of these agents. In this paper we summarize data obtained in Chinese hamster ovary (CHO) cells after treatment with two ethylating agents, N-ethylnitrosourea and diethylsulfate, which are known to alkylate DNA sites with different efficiency. A correlation study between DNA adduct formation and induction of mutations at two gene loci, i.e. hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, has been performed. The influence of DNA repair processes on the final yield of mutation is specifically discussed.

The induction by diethylsulphate of micronuclei derived from acentric fragments or from whole chromosomes was studied in Chinese hamster V79 cells using autoantibodies from the serum of a scleroderma patient (CREST-syndrome) to detect centromere--kinetochore structures. Centromere-containing micronuclei appeared early after treatment and plateaued both earlier and at lower level than centromere-lacking micronuclei. The frequency of centromere-containing micronuclei was in good agreement with that of mitotic chromosome displacement, suggesting that a high proportion of displaced chromosomes were transmitted to the cytoplasm of one of the two daughter cells, where they gave rise to micronuclei. On the contrary, centromere-lacking micronuclei were more frequent than what could be expected from chromosome fragments observed in mitotic stages.

The mutational specificity of the monofunctional alkylating agent dimethylsulphate has been determined through the DNA sequence characterization of 121 lacI-d mutations of Escherichia coli. The predominant mutation induced was the G:C----A:T transition (75%). Transversions constituted 20% of all mutation with the greatest contribution being that of G:C----T:A events (12%). Runs of G:C base pairs were the preferred sites of frameshift mutation. One 6-bp sequence (5'-CCCGCG-3') appeared to be highly susceptible to all classes of mutation and events within this sequence accounted for 33% of all mutations characterized. Although the distribution of G:C----A:T mutations appeared non-random, the site-specificity observed was quite different from that reported for SN1 alkylating agents. The results of this study highlight the differences between the consequences of SN1 and SN2 alkylation pathways.

Six monofunctional alkylating agents, trenimon, cyclophosphamide, and isoniazid were proven for transplacental cytogenetic activity in mouse embryos at day 10 of gestational age under the same conditions as used in the mammalian spot test. With the exception of isoniazid, all compounds led to an increase in the aberration frequencies in embryonal cells. The results were statistically not significant in the case of EMS, while all other chemicals showed a dose-dependent clastogenic activity. After treatment with monofunctional alkylants, chromatid breaks were dominating, while polyfunctional compounds also produced chromatid exchanges, especially in the case of trenimon. ENU and DMS showed a very early aberration maximum 6 hr after injection. For both compounds, very similar dose-response curves were found for induction of chromatid breaks in the dose range 10-75 mg/kg. There is no correlation between the Swain-Scott factors of monofunctional alkylants and their ability to induce chromosomal damage when compared in terms of pharmacological doses. A quantitative comparison of data found in the cytogenetic test in embryonal cells with those obtained in the mammalian spot test led to the conclusion that chromosomal mutations are of minor relevancy for the expression of recessive alleles in heterozygous mouse embryos. With this respect, the mammalian spot test must be considered as an in vivo test for the detection of gene mutations in somatic cells of the mouse.

The affinity of 2 methylating agents, N-methyl-N-nitrosourea (MNU), a potent carcinogen, and dimethyl sulfate (DMS), a very weak or non-carcinogen, for specific structural or functional regions of DNA has been studied in an in vitro system using rat liver nuclei. The release of alkylated nucleotides from nuclei following limited nuclease digestion was measured. Under restrictive conditions, pancreatic DNase I preferentially digests DNA sequences active in RNA transcription while micrococcal nuclease digests spacer DNA between nucleosome cores. Nucleotides methylated by methylnitrosourea were preferentially released early during the digestions, suggesting a localization in both actively transcribing regions and spacer DNA. DMS alkylation, on the other hand, showed a random distribution in chromosomal DNA as measured by micrococcal nuclease and only limited accumulation in transcribing DNA.

DNA-adduct formation and induction of gene mutations were determined simultaneously after treatment with the four ethylating agents, ethyl methanesulfonate (EMS), ethylnitrosourea (ENU), diethyl sulfate (DES), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Both, in E. coli K-12 (NAL-resistance) and in V79 Chinese hamster cells in culture (HPRT-deficiency), the frequencies of mutation induction by all chemicals were the same when plotted against the amount of O6-ethylguanine formed in DNA, suggesting that this DNA adduct can be used as a common dosimeter for the comparisons of the frequencies of gene mutations induced by ethylating agents in various mutagenicity assay systems. Using ENU, such a comparison was performed between mutation induction in V79 cells in vitro and in the specific-locus assay in the mouse. The data indicate that at equal levels of O6-ethylguanine in the DNA of V79 cells and in testicular DNA from male mice treated with ENU, the frequencies of induced mutants in both assay systems were quite similar. These results support the concept that the determination of premutagenic DNA adducts in vivo can be used to monitor exposure to chemical mutagens and that genetic risk estimations may ultimately be performed on the basis of such measurements and of comparative mutagenesis in vitro and in vivo.

Several monofunctional alkylating agents (AA) were compared for their ability to induce chromosomal aberrations, cell killing, sister-chromatid exchanges (SCE) and point mutations in Chinese hamster cells (CHO and V79 cells). The AAs chosen varied in their reaction kinetics as well as their affinity to nucleophilic sites (different s values). AAs with low s values were more mutagenic in comparison to those with high s values, whereas the reverse was true for induction of cytotoxic effects. Neither SCEs nor chromosomal aberrations correlated with the induction of point mutations, indicating that different primary DNA lesions and repair pathways are involved in these biological processes. Molecular dosimetric studies indicate that O6 alkylation of guanine is the most probable cause of lesions in DNA leading to point mutations following treatment with ethyl methanesulphonate and ethyl nitrosourea.

Chinese hamster ovary (CHO) cells were treated with two ethylating agents, N-ethyl-N-nitrosourea (ENU) and diethylsulfate (DES), and the kinetics of DNA single strand break (ssb) induction and rejoining were determined in parallel with DNA adduct formation and removal. In the case of DES, DNA ssb as determined by alkaline elution (AE) were repaired very slowly with more than 50% of the lesions still present on DNA 3 h after treatment. In contrast, 45% of ENU-induced ssb were repaired within 10 min. From the relative concentration of the different ethylated products and their repair rates as measured by high performance liquid chromatography (HPLC) analysis of the ethylated DNA, a theoretical function was constructed that describes the number of ssb expected at each time point after exposure to the mutagen. DES-induced ssb are explained by excision repair processes active on the ethylated purines, mainly 3-ethyladenine (3-EtAde) and 7-ethylguanine (7-EtGua). On the same basis, the rapidly repaired ENU-induced ssb remain unexplained. These results are also discussed in relation to the sensitivity of the two techniques, AE and HPLC, for detecting DNA damage.