Structural and electronic properties of MX compounds related to TA100 mutagenicity. A semi-empirical molecular orbital QSAR study.

The structural and electronic properties of chlorofuranones including MX and its anhydride were calculated using the semi-empirical AM1 method to elucidate the key features related to the strong mutagenic activity of MX. Significant correlations were found between Ames TA100 mutagenicity and the following electronic parameters of chlorofuranones: LUMO energy (r = 0.9607, n = 17), electron affinity (r = 0.9557), LUMO electron density at the alpha-carbon (r = 0.8855) and partial charge of the alpha-carbon (r = 0.8812). Based on these results, a molecular orbital QSAR model for the mutagenic activity of 17 MX analogues is presented. The controversial role of the open-chain tautomers of MX compounds, chlorinated butenoic acids, is discussed briefly.     

Genotoxic effects of various chlorinated butenoic acids identified in chlorinated drinking water

The mutagenic activities of the chlorinated butenoic acids recently identified in chlorinated drinking waters were determined by the Salmonella microsome assay and by the SOS chromotest. The Salmonella typhimurium tester strains TA97, TA98 and TA100 were used without and with S9 mix. In the SOS chromotest Escherichia coli PQ37 was used as an indicator organism with and without metabolic activation. In addition, the extremely potent Ames test mutagen (Z)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid (MX, in the open form), was studied by the micronucleus test with mice using intraperitoneal treatment. The results of the Salmonella assay and the SOS chromotest showed that MX was by far the most potent mutagen of the compounds tested. Mutations were also induced by the reduced form of MX, (Z)-2-chloro-3-(dichloromethyl)-4-hydroxybut-2-enoic acid (red-MX), and by the geometric isomer of MX, (E)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid (EMX). However, since the solution of EMX contained approximately 5% MX, most of its activity might be attributable to MX. The oxidised form of EMX, (E)-2-chloro-3-(dichloromethyl)-butenedioic acid (ox-EMX), was marginally active in the SOS chromotest only. All these compounds were directly acting mutagens and in the presence of metabolic activation (S9 mix) they did not generate mutagenicity. The oxidised form of MX, (Z)-2-chloro-3-(dichloromethyl)-butenedioic acid (ox-MX), was not mutagenic at the dose levels tested. MX did not induce micronuclei in the bone marrow of mice.     

Induction of abasic sites by the drinking-water mutagen MX in Salmonella TA100

Mutagen X (MX) is a chlorinated furanone that accounts for more of the mutagenic activity of drinking water than any other disinfection by-product. It is one of the most potent base-substitution mutagens in the Salmonella (Ames) mutagenicity assay, producing primarily GC to TA mutations in TA100. MX does not produce stable DNA adducts in cellular or acellular DNA. However, theoretical calculations predict that it might induce abasic sites, which it does in supercoiled plasmid DNA but not in rodents. To investigate the ability of MX to induce abasic sites in cellular DNA, we used an aldehydic site assay to detect abasic sites in DNA from Salmonella TA100 cells treated for 1.5 h with MX. At 0, 2.3, and 4.6 μM, MX induced mutant frequencies (revertants/10⁶ survivors) and percent survivals of 2 (100%), 14.9 (111%), and 59.3 (45%), respectively. The frequencies of abasic sites (sites/10⁵ nucleotides) for the control and two concentrations were 5.9, 6.2, and 9.7, respectively, with the frequency at the highest concentration being significant (P < 0.001). These results provide some evidence for the ability of MX to induce abasic sites in cellular DNA. However, the lack of a dose response makes it unclear whether this DNA damage underlies the mutagenic activity of MX.     

Association between mutation spectra and stable and unstable DNA adduct profiles in Salmonella for benzo[a]pyrene and dibenzo[a,l]pyrene

Benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) are two polycyclic aromatic hydrocarbons (PAHs) that exhibit distinctly different mutagenicity and carcinogenicity profiles. Although some studies show that these PAHs produce unstable DNA adducts, conflicting data and arguments have been presented regarding the relative roles of these unstable adducts versus stable adducts, as well as oxidative damage, in the mutagenesis and tumor-mutation spectra of these PAHs. However, no study has determined the mutation spectra along with the stable and unstable DNA adducts in the same system with both PAHs. Thus, we determined the mutagenic potencies and mutation spectra of BP and DBP in strains TA98, TA100 and TA104 of Salmonella, and we also measured the levels of abasic sites (aldehydic-site assay) and characterized the stable DNA adducts ((32)P-postlabeling/HPLC) induced by these PAHs in TA104. Our results for the mutation spectra and site specificity of stable adducts were consistent with those from other systems, showing that DBP was more mutagenic than BP in TA98 and TA100. The mutation spectra of DBP and BP were significantly different in TA98 and TA104, with 24% of the mutations induced by BP in TA98 being complex frameshifts, whereas DBP produced hardly any of these mutations. In TA104, BP produced primarily GC to TA transversions, whereas DBP produced primarily AT to TA transversions. The majority (96%) of stable adducts induced by BP were at guanine, whereas the majority (80%) induced by DBP were at adenine. Although BP induced abasic sites, DBP did not. Most importantly, the proportion of mutations induced by DBP at adenine and guanine paralleled the proportion of stable DNA adducts induced by DBP at adenine and guanine; however, this was not the case for BP. Our results leave open a possible role for unstable DNA adducts in the mutational specificity of BP but not for DBP.     

Further examination of the effects of nitrosylation on Alternaria alternata mycotoxin mutagenicity in vitro

Previously, Alternaria extract and metabolite mutagenicities+/-nitrosylation were characterized using Ames Salmonella strains TA98 and TA100, which are both reverted at GC sites. To examine other targets for mutation, the metabolites Altertoxin I (ATX I), Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME), Tentoxin (TENT), Tenuazonic acid (TA) and Radicinin (RAD) were reexamined+/-nitrosylation, using Ames Salmonella strain TA97, sensitive to frameshift mutations at a run of C's, as well as strains TA102 and TA104, reverted by base pair mutations at AT sites and more sensitive to oxidative damage. ATX I was also assessed for mammalian mutagenicity at the Hprt gene locus in Chinese hamster V79 lung fibroblasts and rat hepatoma H4IIE cells. When tested from 1 to 100 microg/plate without nitrosylation, ATX I was mutagenic in TA102+/-rat liver S9 for activation and weakly mutagenic in TA104+/-S9, demonstrating direct-acting AT base pair mutagenicity. AOH was also directly mutagenic at AT sites in TA102+/-S9 while AME was weakly mutagenic in TA102+/-S9 and TA104+S9. Nitrosylation of ATX I enhanced mutagenicity at AT sites in TA104+/-S9 but produced little change in TA102+/-S9 compared to native ATX I. However, nitrosylated ATX I generated a potent direct-acting frameshift mutagen at C sites in TA97+/-S9. While ATX I was not mutagenic in either V79 cells or H4IIE cells, 5 and 10 microg/ml nitrosylated ATX I produced a doubling of 6-thioguanine resistant V79 colonies and 0.5 and 1 microg/ml were mutagenic to H4IIE cells, becoming toxic at higher concentrations. These results suggest ATX I, AME and AOH induce mutations at AT sites, possibly through oxidative damage, with nitrosylation enhancing ATX I frameshift mutagenicity at runs of C's. Nitrosylated ATX I was also directly mutagenic in mammalian test systems.     

Mutagenicity of flavones, chromones and acetophenones in Salmonella typhimurium. New structure-activity relationships

28 flavones and 11 structurally-related flavonoids, chromones, and acetophenones, were tested for mutagenicity in the Salmonella typhimurium his reversion assay. 7 flavones, all of which were hydroxy- or methoxy-substituted at position 8, were moderate to strong mutagens in strain TA100 in the presence of rat liver S9 mix. In each case, the response of strain TA98 was either not significant or was very much weaker than that observed in strain TA100. The activation by S9 is not mediated by the microsomal cytochrome P450 system, since activation was not diminished when microsomes were removed by centrifugation at 100 000 X g. The observed strain specificity and structural requirements for activity indicate a mutagenic mechanism different from that associated with previously reported mutagenic flavonols (3-hydroxy-flavones) which are most active in strain TA98. The most mutagenic flavone investigated, 5,7,8-trihydroxy-flavone (norwogonin), had a potency of 17 revertants/nmole. Simplification of the chemical structures to hydroxy-substituted chromone and acetophenone systems revealed similar strain specificity, hydroxylation requirements, and S9 dependence within these structural classes, suggesting a similar activation pathway and mutagenic mechanism. The greatest mutagenic potency was observed within the flavone series, but significant potency was retained by similarly hydroxylated chromones and acetophenones. No mutagenic activity was observed in the absence of the aryl ketone moiety.     

Mutation spectra in Salmonella TA98, TA100, and TA104 of two phenylbenzotriazole mutagens (PBTA-1 and PBTA-2) detected in the Nishitakase River in Kyoto, Japan.

Previous studies have identified two potent aromatic amine mutagens in the Nishitakase River, a tributary of the Yodo River, which serves as the main drinking water supply for the Osaka area in Japan. The two potent mutagens are 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). PBTA-1 and PBTA-2 are presumed to be formed from azo dyes discharged in a reduced form from dye factories to sewage treatment plants where they become chlorinated and are then discharged into the river. PBTA-1 and PBTA-2 account for 21% and 17% of the mutagenic activity of the Nishitakase River, respectively. Here we determined the mutation spectra induced by these two mutagens in TA98, TA100, and TA104 at 30-35, 8-10, and 2x, respectively, above the background. In TA98, the PBTA compounds produced identical mutation spectra, with 100% of the revertants containing the hotspot 2-base deletion of CG within the (CG)(4) sequence. In TA100, 73% of the revertants were GC-->TA transversions, with most of the remaining being GC-->AT transitions; the spectra produced by the two compounds in TA100 were not significantly different (p=0.8). In TA104, as in TA100, the majority (83%-87%) of the revertants were GC-->TA transversions, with most of the remaining revertants (11%-13%) being AT-->TA transversions. Thus, 83%-87% of the mutations induced by the PBTA compounds in TA104 were at G/C sites. The mutation spectra produced by the two compounds in TA104 were not significantly different (p0.08). PBTA-1 and PBTA-2 are structurally similar and have similar mutagenic potencies and mutation spectra in the respective strains. The mutation spectra produced by the PBTA compounds (100% hotspot deletion in TA98 and primarily GC-->TA transversions in TA100 and TA104) are similar to those produced by other potent aromatic amines, which is the class of compounds from which the PBTA mutagens derive.     

Mutation spectra of smoky coal combustion emissions in Salmonella reflect the TP53 and KRAS mutations in lung tumors from smoky coal-exposed individuals

Nonsmoking women in Xuan Wei County, Yunnan Province, China who use smoky coal for cooking and heating in poorly ventilated homes have the highest lung cancer mortality rate in China, and their lung cancer is linked epidemiologically to their use of smoky coal. The emissions contain 81% organic matter, of which 43% is polycyclic aromatic hydrocarbons (PAHs). Exposure assessment and molecular analysis of the lung tumors from nonsmoking women who use smoky coal strongly indicate that PAHs in the emissions are a primary cause of the elevated lung cancer in this population. Here we have determined the mutation spectra of an extract of smoky coal emissions in Salmonella TA98 and TA100; the extract was not mutagenic in TA104. The extract was 8.7 x more mutagenic in TA100 with S9 than without (8.7 rev/microg versus 1.0 rev/microg) and was >3 x more mutagenic in TA100 than in TA98--consistent with a prominent role for PAHs in the mutagenicity of the extract because PAHs are generally more mutagenic in the base-substitution strain TA100 than in the frameshift strain TA98. The extract induced only a hotspot mutation in TA98; another combustion emission, cigarette smoke condensate (CSC), also induces this single class of mutation. In TA100, the mutation spectra of the extract were not significantly different in the presence or absence of S9 and were primarily (78-86%) GC --> TA transversions. This mutation is induced to a similar extent by CSC (78%) and the PAH benzo[a]pyrene (B[a]P) (77%). The frequency of GC --> TA transversions induced in Salmonella by the extract (78-86%) is similar to the frequency of this mutation in the TP53 (76%) and KRAS (86%) genes of lung tumors from nonsmoking women exposed to smoky coal emissions. The mutation spectra of the extract reflect the presence of PAHs in the mixture and support a role for PAHs in the induction of the mutations and tumors due to exposure to smoky coal emissions.     

Mutational spectrum of bleomycin in lacZ mouse kidney: a possible model for mutational spectrum of reactive oxygen species

The mutational spectrum of bleomycin was compared with the spontaneous mutational spectrum in lacZ mouse kidney. Mice were treated with four 20 mg/kg of doses of bleomycin over a two-week period, leading to a mutant fraction several times greater than that of controls. The major class of bleomycin-induced mutations consisted of small deletions, in particular -1 deletions at AT base pairs and hot spots for deletions at 5'-GTC-3' sequences. Smaller, but significant fractions of GC > AT followed by GC > TA substitutions were also observed. In untreated mice, the major class of mutations consisted of GC > AT substitutions followed by GC > TA mutations, and a much smaller fraction of deletions. Other than the specificity of bleomycin for AT base pairs and the 5'-GTC-3' hotspots, the mutational spectrum of bleomycin in mice is similar to that reported for ionizing radiation. However, bleomycin initially mediates the formation of oxidized DNA via reduction of molecular oxygen, as opposed to the radiolysis of water. In this respect mutagenesis induced by bleomycin may be more similar to that induced by endogenous reactive oxygen species (ROS) than mutagenesis induced by ionizing radiation. If bleomycin-induced mutagenesis is an appropriate model for mutagenesis induced by ROS, then, based on the difference between the mutational spectrum of bleomycin and spontaneous mutagenesis, the latter appears not to result predominantly from ROS, at least in mouse kidney.     

Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent.

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT --> TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10(-6), Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.     

Frameshift mutagenesis in Escherichia coli by reversible DNA intercalators: sequence specificity

The mutagenic potency of the simple reversible intercalators isopropyl-OPC (iPr-OPC) and 9-aminoacridine (9-AA) is assessed in E. coli using reversion assays based on plasmids derived from pBR322 carrying various frameshift mutations within the tetracycline resistance gene in repetitive sequences: +/- 2 frameshift mutations within alternating GC sequences; +/- 1 frameshift mutation at runs of guanines. The results obtained show that iPr-OPC and 9-AA have a sequence specificity for mutagenesis: they revert +1 and -1 frameshift mutations within runs of monotonous G:C base pairs. The precise determination of the size of a small restriction fragment which contains the mutation allowed us to demonstrate that reversion occurred by -1 deletions for the +1 frameshift mutations and by +1 additions for the -1 frameshift mutations. The possible relations of this specific reversion with the base sequence specificity of the mutagenesis are briefly discussed.     

The spectra of base substitutions induced by the impCAB,mucAB and umuDC error-prone DNA repair operons differ following exposure to methyl methanesulfonate

We have used the lacZ reversion assay to study the mutation spectra induced by the Escherichia coli chromosomal umuDC operon and of its two plasmid-borne analogues impCAB and mucAB following exposure of cells to UV light and methyl methane-sulfonate (MMS). We have shown that the impCAB, mucAB and umuDC operons all produce a similar response to UV light which results almost exclusively in AT → GC transitions. However, we found that the three operons produced different responses to alkylating agents. We found that with MMS the chromosomal umuDC operon produced almost exclusively AT → GC transitions, whilst both mucAB and impCAB produced predominantly transversions. In the case of the impCAB operon the mutation spectrum contained more AT → TA than GC → TA transversions; this balance was reversed with mucAB. The effect of the copy number of the error-prone DNA repair operons upon the mutagenic spectra was also studied. The results obtained suggest that the copy number of the imp operon does not greatly affect the specificity of base substitutions observed. However, an increase in the copy number of the umuDC operon greatly affected the specificity of base substitution, such that virtually no transitions were produced and the spectrum was dominated by GC/AT → TA transversions. It appears that the three error-prone DNA repair operons impCAB, mucAB and umuDC, despite showing strong structural and functional homologies, can display major differences in the spectrum of base changes induced during mutagenesis. We propose that the type of misincorporation/chain extension which DNA polymerase III is allowed to synthesize on a damaged DNA template is extremely sensitive to both the amount and type of error-prone repair proteins present. The modulation of these events by the different proteins can result in widely different mutagenic changes in the repaired DNA.     

Mutational biases associated with potential iron-binding DNA motifs in rodent lacI and human p53 mutational databases

The role of Fenton oxidants in DNA damage, aging, and cancer is appreciated, but not well understood. Six potential iron-binding (PIB) DNA motifs were previously identified as sites of preferential strand cleavage. Since DNA-metal binding domains are a known determinant of oxidative DNA damage, and the location of strand breaks explains where oxidant attack occurs, we sought to determine whether the likelihood of base change mutations is a function of neighboring PIB motifs. We developed a sliding window function that computes the density of PIB motifs on both strands, within 4-12bp, for each location along a target gene. This range of window sizes reflects known diffusion distances of Fenton reaction products. Using mutational databases, odds of mutation at each base were calculated relative to PIB motif density, for all PIB motif types in aggregate, or for individual PIB motifs. Using mutational data from lacI transgenic animals, we observed a non-random distribution of PIB motifs, associated with increased odds of mutation, showing a strand bias. Sensitivity analysis confirmed that the optimum association between PIB motif density and mutations occurs when a 7bp radius is used for the window size. Randomly simulated mutations showed no association with PIB motif density. When the method was applied to human TP53 mutation data, we saw similar results, but no strand bias. As PIB motif density rises, linear trends are observed for increasing odds of mutation. Sensitivity analysis revealed associations between PIB motifs and GC --> AT transitions and GC --> TA transversions-the most commonly observed types of mutations arising from oxidative DNA damage. DNA-metal binding motifs are found in a wide variety of biological contexts, including many where conformational sensitivity to redox state is important. These techniques can help elucidate how DNA-iron-binding may affect lesions and subsequent mutations from multiple agents.     

A straight correlation between mutagenic activity and beta-galactosidase activity induced by monofunctional alkylating agents.

Eight monofunctional alkylating agents were examined for their ability to induce mutation in Salmonella typhimurium. The assay was carried out in S. typhimurium TA100 with the preincubation method. The SN1-type agents were more mutagenic than the SN2-type ones; besides, methylating agents exerted more mutagenic activity than ethylating ones. Those responses in the reversion assay were quite similar to the results obtained previously with the beta-galactosidase assay in Escherichia coli CSH26/pMCP1000 (alkA'-lacZ') as to the induction of the adaptive response. A good correlation was found between mutagenic potency in the reverse mutation assay and inducing potency in the beta-galactosidase assay.     

Hycanthone and its congeners as bacterial mutagens.

Hycanthone methanesulfonate (HCT) was shown to induce "forward" and "reverse" mutations in Salmonella typhimurium and Escherichia coli. Mutational effects of HCT on S. tyhhimurium TA1532 were concentration and time dependent. A comparison of mutagenic potency for TA1532 was made between HCT and the frame-shift mutagens quinacrine and ICR-191. An investigation of structure-activity relationships revealed the substituent in the 4-position of ring A to be critical for mutagenicity. Activity was found when this group was a hydroxymethyl (i.e., HCT) or an aldehyde (Win 25,315), but the analogues having a carboxyl group (Win 25,850) or methyl group (lucanthone) in this positionwere inactive. Removal of a single ethyl group from the side chain did not affect mutagenic activity inasmuch as the potency of desethyl HCT (Win 27,262) equaled that of HCT on a molar basis. A marginal activity was found with a sample of HCT sulfoxide (win 27,266), but this sample was found to contain traces of HCT. The HCT analogue with a terminal N-oxide in the side chain (Win 29,329) was inactive at the concentration tested.     

Mutagenic fingerprint of ozone in human cells

Ozone is an important factor in urban pollution and represents a major concern for human health. The chemical reactivity of ozone toward biological targets and particularly its genotoxicity supports a possible link between exposure and cancer risk, but no molecular data exist on its mutagenic potential in human cells. Using a shuttle vector, we showed that ozone is indeed a potent mutagen and we characterized the mutation spectrum it produced in human cells. Almost all mutations are base substitutions, essentially located at G:Cs (75%), typical of reactive oxygen species (ROS), but occurring in a specific pattern, i.e. a similar extent of GC:TA (28%), GC:CG (23%) and GC:AT (23%). The targeted distribution of mutations and identification of hotspot sequences define the first molecular fingerprint of mutations induced by ozone in human cells. Possible applications derived from our results with respect to ozone genotoxicity should help determining quantifiable biomarkers of ozone exposure in human health, especially for carcinogenesis.     

Test of models for the sequence specificity of UV-induced mutations in mammalian cells

We have used mathematical modeling and statistical analysis to examine the correlation between UV-induced DNA damage and resulting base-substitution mutations in mammalian cells. The frequency and site specificity of UV-induced photoproducts in the supF gene of the pZ189 shuttle vector plasmid were compared with the frequency and site specificity of base-substitution mutations induced upon passage of the UV-irradiated vector in monkey cells. The hypothesis that the observed mutational spectrum is due to a preferential insertion of adenosine opposite UV photoproducts in the DNA template was found to best explain the mutational data. Models in which it was postulated that only (6-4) photoproducts, and not cyclobutane dimers, are mutagenic, or that the relative frequency of photoproduct formation does not influence mutation frequencies, fit the data much less well. This analysis demonstrates that molecular mechanisms of mutagenesis in mammalian cells can be deduced from mutational data obtained with a shuttle vector system.     

In vivo repair of ENU-induced oxygen alkylation damage by the nucleotide excision repair mechanism in Drosophila melanogaster

DNA damage caused by oxygen alkylation of bases (mainly at O6-G, O4-T and O2-T positions in DNA) has been correlated with the mutagenic and carcinogenic potency of monofunctional alkylating agents. In all kinds of organisms, repair of O6-alkylG is carried out mainly by the enzyme O6-methyl guanine-DNA methyltransferase (MGMT). However, little is known about the repair of the O-alkylT adducts or about the contribution of nucleotide excision repair (NER) to this process, especially in higher eukaryotes. To study the influence of the NER system on the repair of O-alkylation damage, the molecular mutation spectrum induced by N-ethyl-N-nitrosourea (ENU) in an NER-deficient Drosophila strain, carrying a mutation at the mus201 locus, was obtained and compared with a previously published spectrum for NER-proficient conditions. This comparison reveals a clear increase in the frequency of base pair changes, including GC --> AT and AT --> GC transitions and AT --> TA transversions. In addition, one deletion and two frameshift mutations, not found under NER-proficient conditions, were isolated in the NER-deficient mutant. The results demonstrate that: (1) N-alkylation damage contributes considerably (more than 20%) to the mutagenic activity of ENU under NER-deficient conditions, confirming that the NER system repairs this kind of damage; and (2) that in germ cells of Drosophila in vivo, NER seems to repair O6-ethylguanine and/or O2-ethylcytosine, O4-ethylthymine, and possibly also O2-ethylthymine.     

Studies on the potent bacterial mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone: aqueous stability, XAD recovery and analytical determination in drinking water and in chlorinated humic acid solutions

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was detected by gas chromatography/mass spectrometry in drinking water samples from 3 locations in the U.S.A., and also in a chlorinated humic acid solution. MX appears to account for a significant proportion of the mutagenicity of these samples, as measured in the Ames test using strain TA100 without metabolic activation. Studies on recovery of MX from spiked water samples by XAD-2/8 resin adsorption/acetone elution indicated that sample acidification prior to resin adsorption was essential to the effective recovery of MX. The stability of MX in aqueous solution was pH and temperature dependent. At 23 degrees C the order of stability, based on persistence of mutagenic activity was found to be: pH 2 greater than pH 4 greater than pH 8 greater than pH 6. The half-life at pH 8 and 23 degrees C was 4.6 days. One of the degradation products has been tentatively identified as 2-chloro-3-(dichloromethyl)-4-oxo-2-butenoic acid, an open form of MX which appears to be in the "E" configuration. Overall, these results suggest that MX is formed during water chlorination as a result of reaction of chlorine with humic substances, and that a substantial fraction of the MX formed is likely to persist throughout the distribution system.     

Comparative genotoxic effect of vincristine,vinblastine, and vinorelbine in somatic cells of Drosophila melanogaster

In this study, the vinca alkaloids vincristine (VCR), vinblastine (VBL) and vinorelbine (VNR) were investigated for genotoxicity in the wing Somatic Mutation and Recombination Test (SMART) of Drosophila. Our in vivo experiments demonstrated that all drugs assessed induced genetic toxicity, causing increments in the incidence of mutational events, as well as in somatic recombination. Another point to be considered is the fact that VNR was able to induce, respectively, approximately 13.0 and 1.7 times more mutant clones per millimolar exposure unit as their analogues VCR and VBL. The replacement of a CH(3) attached to vindoline group in VBL by a CHO in VCR seems to be responsible for the approximately seven times higher potency of the former. In contrast, the structural modifications on VNR's catharantine group could be related to its higher genotoxic potency, as well as its similar mutagenic and recombinagenic action.