Use of the Comet test and micronucleus assay on human white blood cells for in vitro assessment of genotoxicity induced by different drinking water disinfection protocols

Surface water disinfection can lead to the formation of mutagenic/carcinogenic by-products derived from reactions with naturally occurring inorganic compounds. We investigated the feasibility and potential usefulness of an integrated approach to genotoxicity analysis of drinking water. The approach employed the Comet and micronucleus (MN) assays to evaluate the DNA and chromosomal damage produced by water extracts in human blood cells. Surface water samples from Lago Trasimeno (Italy) were collected in different seasons (July 2000, October 2000, February 2001, and June 2001), and samples were disinfected with sodium hypochloride (NaClO), chlorine dioxide (ClO(2)), or peracetic acid (PAA). Extracts of untreated and treated water were incubated with primary human leukocytes. The Comet assay revealed both strong seasonal variations and differences between samples processed by the three disinfection protocols. The three disinfectants increased the genotoxicity of the water collected in July 2000 and October 2000, with PAA producing the greatest amount of DNA damage. Extracts of raw water collected in February 2001 produced so much DNA damage that the relative genotoxic potentials of the three disinfectants could not be evaluated. No increase in MN frequency was detected in any of the samples. The multi-endpoint MN assay indicated, however, that our study samples (especially the sample collected in the February 2001) were cytotoxic. We conclude that this integrated approach to genotoxicity assessment may be useful both for the quality control of raw drinking water and to help compare the potential health risks associated with alternative disinfection processes.     

Genotoxicity of surface water treated with different disinfectants using in situ plant tests

Disinfection of surface drinking water, in particular water chlorination, results in many by-products with potential genotoxic and/or carcinogenic activity. In the present study, we evaluated the genotoxicity of surface water after treatment with different disinfectants by means of in situ plant genotoxicity assays (micronucleus and chromosomal aberration tests) which can detect both clastogenic and aneugenic effects. The study was carried out at a pilot plant using lake water after sedimentation and filtration. This water supplied four stainless steel basins: three basins were disinfected with sodium hypochlorite, chlorine dioxide, and peracetic acid and the fourth basin containing untreated lake water was used as a control. Plants were exposed in situ in the basins. The study was carried out using water collected in different seasons over a period of about 1 year in order to assess the treatments in different physical and chemical lake water conditions. The micronucleus test in root cells of Vicia faba (Vicia faba/MCN test) revealed genotoxicity in many samples of disinfected water. The micronucleus test in Tradescantia pollen cells and the chromosome aberration test in root cells of Allium cepa showed genotoxic effects only in some disinfected samples, but also revealed genotoxicity in raw water. The results of the study indicated that the Vicia faba/MCN test was the most sensitive plant assay for disinfected water and that peracetic acid disinfection produced similar or lower genotoxicity than sodium hypochlorite or chlorine dioxide treatment.     

Evaluation of chemicals used for drinking water disinfection for production of chromosomal damage and sperm-head abnormalities in mice

Chemical oxidants are commonly added during water treatment for disinfection purposes. These chemicals have not been tested previously for their ability to induce genetic damage in vivo. Chlorine (hypochlorite and hypochlorous acid), monochloramine, chlorine dioxide, sodium chlorite, and sodium chlorate were evaluated for induction of chromosomal aberrations and micronuclei in bone marrow of CD-1 mice, and for induction of sperm-head abnormalities in B6C3F1 mice. Oral administration of chlorine at pH 8.5 (where hypochlorite predominates) at dose levels equivalent to approximately 4 and 8 mg/kg/day induced significant increases in the level of sperm-head abnormalities. There was no evidence of other effects with any of the disinfectants. Halogenated acetonitriles, which have previously been shown to form in the stomach following oral dosing of sodium hypochlorite to rats, were also tested in the sperm-head abnormality assay but gave no indication of an effect.     

Genotoxicity of drinking water disinfectants in plant bioassays

The genotoxicity of two widely used drinking water disinfectants, sodium hypochlorite (NaClO) and chlorine dioxide (ClO(2)), and a new disinfectant, peracetic acid (PAA, CH(3)-CO-COOH), was evaluated in three short-term plant tests: (1) induction of anaphase chromosome aberrations in the root cells of Allium cepa, (2) micronucleus induction in the root cells of Vicia faba, and (3) micronucleus induction in Tradescantia pollen cells. The study was carried out in the laboratory by directly exposing the plants to several concentrations of the disinfectants in redistilled water at unadjusted (acid) and adjusted (neutral) pHs. Both 0.1 and 0.2 mg/l NaClO induced chromosome aberrations in the Allium cepa test at acid pH, but concentrations up to 0.5 mg/l of all the disinfectants were negative at neutral pH. Concentrations ranging from 0.1 to 0.5 mg/l NaClO, ClO(2,) and PAA induced micronuclei in Vicia faba at acid pH, while 1-2 mg/l NaClO and ClO(2) and 0.5-2 mg/l PAA gave positive responses at neutral pH. Most of concentrations of ClO(2) produced positive responses in the Tradescantia micronucleus test. In general, the highest levels of genotoxicity were observed under acid conditions; at acid pH, significant effects were induced by low concentrations of ClO(2) and PAA. Since the test concentrations of disinfectants are typical of those encountered in the biocidal treatment of tap water and similar concentrations are consumed daily by a large number of people, the genotoxicity of these compounds may constitute a significant public health concern.     

DNA adduct formation by 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000), an extremely potent mutagen

The effects on DNA, in bacteria, of 7-methoxy-2-nitronaphtho[2,1-b]furan(R7000), a very potent genotoxic product from the 2-nitronaphthofuranseries, were investigated with two different approaches: (i)measurement of the binding of the radiolabelled mutagen to DNAand (ii) detection by the ‘³²P-postlabelling’ methodof DNA adducts following treatment with unlabelled mutagens.The covalent binding of R7000 to DNA in Escherichia coli wasdemonstrated by both methods, and in the latter case it wasfound to involve the formation of nine different adducts. Formationof adducts by R7000 was shown to require metabolic activationof the compound. Deceased      

Isolation of S9 fractions from mouse and rat with increased enzyme activities after repeated administration of cytochrome P-450 and P-448 inducers

Cytochrome P-450 (cyt P-450), NADPH cytochrome P-450 reductaseand various microsomal monooxygenase activities [e.g. aminopyrineN-demethylase, p-nitroanisole O-demethyl-ase, dinemorphan N-demethylase,ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase(ERD)], were determined in hepatic post-mitochondrial supernatantfrom mice and rats. Experiments were performed on male and femaleanimals treated with a combination of sodium pheno-barbitaland ß-naphthoflavone according to the standard protocolschedule for short-term genotoxicity testing. A second inductivetreatment after 2, 3, 4 or 5 weeks was provided. The increasein cyt P-450 and in all enzymatic activities measured was enhancedin both species by a second induction treatment, particularlywhen given after 4 weeks. ERD activity was the only monooxygenaseactivity which was sex-dependent, being more active in femalethan in male animals. To extend the biochemical data, experimentswere performed with the proposed S9 fractions on styrene, whichpreviously has proved difficult to detect in short-term in vitromutagen-icity tests. Using the new induction conditions positiveresults were obtained with the D7 strain of Saccharomyces cerevisiae.It was concluded that a simple pre-induction of the animals3–4 weeks before the main induction treatment leads toa more active S9 fraction for in vitro genotoxicity studies. ^*This work was presented at the 18th Annual Meeting of the EnvironmentalMutagen Society, April 8–12, 1987, San Francisco, USA.      

The effect of biotransformation of 2,4-dinitrotoluene on its mutagenic potential

Because both oxidative and reductive metabolism of the hepatocarcinogen2,4-dinitrotoluene (2,4-DNT) can occur in vivo; we have examinedthe mutagenicity of compounds which can be formed from 2,4-DNTin an attempt to establish which metabolic pathways contributeto the formation of genotoxic products. A quantitative reversionassay using Salmonella typhimurium TA98 was used to evaluatethe mutagenicity of these compounds. 2,4-Dinitrobenzyl alcohol,2-amino-4-nitro-toluene and 2-nitroso-4-nitrotoluene were foundto be more mutagenic to S. typhimurium than is 2,4-DNT and didnot require metabolic activation by post-mitochondrial super-natantsof Aroclor-induced rat liver homogenates (S9) for their effect.2-Amino-4-nitrobenzoic acid was also mutagenic to S. typhimuriumTA98 in the absence of S9, but its mutagenicity was enhancedwhen S9 was included in the incubation mixture. 2,4-Diaminotoluenerequired S9 for demonstration of mutagenicity and was approximatelyas effective, on a molar basis, as 2,4-DNT in inducing reversionto histidine prototrophy. These results suggest that both oxidativeand reductive metabolism may be involved in production of mutagenicmetabolites of 2,4-DNT. ¹Present address to which correspondence should be addressed:Department of Pharmacology and Toxicology, University of MississippiMedical Center, 2500 N. State Street, Jackson, MS 39216, USA      

Calibration of the single cell gel electrophoresis assay, flow cytometry analysis and forward mutation in Chinese hamster ovary cells

The induction kinetics of genetic damage were measured in oneclone of a mammalian cell line (CHO AS52) with three genotoxicityassays, the single cell gel electrophoresis (Comet) assay, laserbeam flow cytometry and forward mutation. The first two assaysallow for the rapid analysis of genotoxic damage in individualnuclei. The alkaline Comet assay detects DNA strand breaks,alkali-labile sites and incomplete excision repair sites. Flowcytometry measures chromosome damage that results in an unequaldistribution of nuclear DNA in daughter cells. We calibratedthese assays to compare acute DNA damage and longer term clastogenicitywith forward mutation at the gpt^– locus using ethyl methanesulfonate(EMS). The EMS treatments were conducted in F12 medium for 2h. AS52 cells carry a single functional gpt gene which providesfor quantitation of gpt mutants by selecting for 6-thioguanineresistance. EMS induced a concentration-dependent response withmedian Comet tail moment values of 1.06 µm for the negativecontrol and 64.6 µm with 20 mM. The coefficient of variation(CV) of the negative control with flow cytometry was 233; theCV value increased to 4.87 in cells treated with 20 mM EMS.EMS (8 mM) induced a mutant frequency of 779.8 x10^–6 ata relative survival of 64.4%. Genetic response factors werecalculated and the data demonstrate that the induction kineticsof genetic damage as measured by the Comet assay (15.6) andflow cytometry (14.2) were more closely related than that determinedfor mutation induction (7.9). These three assays measure a widespectrum of genetic events at the level of DNA, the gene andthe chromosome and demonstrate the usefulness of the Comet assayand flow cytometry as two relatively rapid procedures to detectgenotoxic damage in mammalian cells. ³To whom correspondence should be addressed at: 364 Environmental and Agricultural Sciences Building, 1101 West Peabody Drive, Urbana, IL 61801-4178, USA. Tel: +1 217 333 3614; Fax: +1 217 333 8046; Email: m-plewa{at}uiuc.edu     

Methylglyoxal: genotoxicity studies and its effect in vivo on the hepatic microsomal mono-oxygenase system of the mouse

The genotoxic potential of methylglyoxal (MG) was studied inSaccharomyces cerevisiae D7 and in Salmonella typhimurium TA97and TA102 in the presence and in the absence of metabolic activationsystem (S9 fraction) prepared from mouse liver induced withß-naphthoflavone (ß-NF) and sodium phenobarbital(PB). The in vivo effects on the hepatic microsomal mixed functionmono-oxygenase system induced by MG were studied in untreated,ß-NF or PB pre-treated mice. MG was a direct-actingmutagen in S. typhimurium TA97 and TA102 when tested up to amaximum concentration of 0.47 mg/plate. Mitotic gene conversionwas also induced by MG in the yeast S. cerevisiae D7. A weakbut significant effect on reverse point mutation was also foundin S. cerevisiae. Genetic activity was lower in the presenceof S9 fraction in yeast test. In the in vivo studies, MG (atthe total dose of 600 mg/kg) was shown to increase the aminopyrineN-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD)activities in uninduced mice. Cytochrome P-450 content (cytP-450) and ethoxycoumarin O-deethylase activity (ECD) were alsoweakly enhanced by MG treatment. In contrast, no significantchanges in mono oxygenase activities were seen in ß-NF-or PB-treated mice after MG injection. ¹To whom correspondence should be addressed     

Effects of X-rays on the induction of somatic mutations and growth in the retinal pigmented epithelium during development of the mouse eye

The coat and eye colour mutant beige (bg) leads to the productionof distinctive retinal melanocytes with abnormally large pigmentgranules. Heterozygotes for bg were given 2 Gy acute X-irradiationat various times between day 11.5 of fetal life and 3 days afterbirth, at which age whole mounts were prepared of the retinalpigmented epithelium (RPE). These were scanned for the presenceof mutant retinal melanocytes with large granules, either assingle cells or as clones. The earlier the fetal irradiation,the greater was the effect on RPE area at 3 days post-partum(p.p.), which fell to about half normal with the 11.5–dayfetal exposures. However, the ultimate size of the retinal melanocytesseemed little affected by the irradiation, although their normalsize increased {small tilde}3-fold between 12.5 days post-coitum(p.c.) and 3 days p.p. Mean numbers of mutant melanocytes pereye were markedly higher than in +/bg controls at all irradiationages other than 3 days p.p.; when allowance was made for finalsizes of irradiated RPE's mutation frequencies fell steadilyfrom 30.0 x 10^'5 at 11.5 days p.c. to 1.0 x ^10'5 at 3 daysp.p., with 0.8 x ^10'5 in +bg and 0.1 x ^10'5 in +/+ controls.The doubling dose of 0.18 Gy at 16.5 days p.c. was similar tothat found at 17.5 days p.c. in a previous somatic mutationexperiment in which follicular melanocytes were scanned formutations at different (d and In) loci. In the present experiment,numbers of mutant melanocytes per clone tended to decrease withlater irradiation, but not to the extent expected from the greatlyincreased numbers of cells at risk over the same period. Itwas concluded that the bg test system showed great promise forthe study of in vivo mutagenesis in somatic cells, especiallyas the fetal RPE is pigmented from 11.5 days p.c. in the mouse. ¹Present address: Roswell Park Memorial Institute, 666 Elm Street,Buffalo, NY 14263, USA      

Study on the resistance of severe acute respiratory syndrome-associated coronavirus

In this study, the persistence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was observed in feces, urine and water. In addition, the inactivation of SARS-CoV in wastewater with sodium hypochlorite and chlorine dioxide was also studied. In vitro experiments demonstrated that the virus could only persist for 2 days in hospital wastewater, domestic sewage and dechlorinated tap water, while 3 days in feces, 14 days in PBS and 17 days in urine at 20 degrees C. However, at 4 degrees C, the SARS-CoV could persist for 14 days in wastewater and at least 17 days in feces or urine. SARS-CoV is more susceptible to disinfectants than Escherichia coli and f2 phage. Free chlorine was found to inactivate SARS-CoV better than chlorine dioxide. Free residue chlorine over 0.5 mg/L for chlorine or 2.19 mg/L for chlorine dioxide in wastewater ensures complete inactivation of SARS-CoV while it does not inactivate completely E. coli and f2 phage.     

Studies of error-prone DNA repair in Escherichia coli K-12 and Ames Salmonella typhimurium strains using a model alkylating agent

4-Acetoxy-3-acetoxymethyl acetophenone (AAMAP) is muta-genicin Ames Salmonella typhimurium tester strains TA100 and TA98,which carry plasmid pKM101, but not in the isogenic plasmid-lessstrains TA1535 and TA1538. Similarly, no AAMAP-induced reversionof the his-4 allele is detectable in Escherichia coli K-12 umuCstrains in the absence of the plasmid, even when the strainsare treated with ethylene-diaminetetraacetate to increase permeability,or when the uvrB allele is introduced to increase error-proneDNA repair. AAMAP is, however, mutagenic in umuC⁺ strains orin umuC strains in which plasmid pKM101 has been introduced,suggesting that the plasmid-encoded MucAB or the chromosomallydetermined UmuDC proteins are required for mutagenesis. Mutationfrequencies are higher in E. coli umuC (pKM101) strains, whichresemble Ames tester strains of S.typhimurium, than in E.coliumuC⁺ or even umuC⁺ (pKM101) strains. Therefore, providing thatthe recommended pKM101-containing tester strains are used, theapparent absence of Umu-like protein activity in S. typhimuriummay actually increase the sensitivity of the Ames test for thedetection of mutagens that require error-prone DNA repair foractivity. ^* Parts of this paper were communicated to the Fourth InternationalConference on Environmental Mutagens, Stockholm, 1985. ²To whom correspondence should be addressed      

Biomonitoring human exposure to environmental carcinogenic chemicals

A coordinated study was carried out on the development, evaluationand application of biomonitoring procedures for populationsexposed to environmental genotoxic pollutants. The proceduresused involved both direct measurement of DNA or protein damage(adducts) and assessment of secondary biological effects (mutationand cytogenetic damage). Adduct detection at the level of DNAor protein (haemoglobin) was carried out by ³²P-postlabelling,immunochemical, HPLC or mass spectrometric methods. Urinaryexcretion products resulting from DNA damage were also estimated(immunochemical assay, mass spectrometry). The measurement ofadducts was focused on those from genotoxicants that resultfrom petrochemical combustion or processing, e.g. low-molecular-weightalkylating agents, PAHs and compounds that cause oxidative DNAdamage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei,chromosome aberrations and sister chromatid exchanges) and mutationfrequency was estimated at a number of loci including the hprtgene and genes involved in cancer development. Blood and urinesamples from individuals exposed to urban pollution were collected.Populations exposed through occupational or medical sourcesto larger amounts of some of the genotoxic compounds presentin the environmental samples were used as positive controlsfor the environmentally exposed population. Samples from ruralareas were used as negative controls. The project has led tonew, more sensitive and more selective approaches for detectingcarcinogen-induced damage to DNA and proteins, and subsequentbiological effects. These methods were validated with the occupationalexposures, which showed evidence of DNA and/or protein and/orchromosome damage in workers in a coke oven plant, garage workersexposed to diesel exhaust and workers exposed to ethylene oxidein a sterilization plant. Dose response and adduct repair werestudied for methylated adducts in patients treated with methylatingcytostatic drugs. The biomonitoring methods have also demonstratedtheir potential for detecting environmental exposure to genotoxiccompounds in nine groups of non-smoking individuals, ³²P-postlabellingof DNA adducts being shown to have the greatest sensitivity. ^*Synthesis report on E.U. STEP Contract No. EV5V-CT91-0013 ¹⁴To whom correspondence should be addressed      

In vitro reaction of hydroxyamino derivatives of MelQx, Glu-P-1 and Trp-P-1 with DNA: 32P-postlabelling analysis of DNA adducts formed in vivo by the parent amines and in vitro by their hydroxyamino derivatives

The synthetic hydroxyamino derivatives of three mutagenic andcarcinogenic heterocyclic amines present in cooked foods andamino acid pyrolysates, 2-amino-3,8-dimethylimidazo-[4,5-f)quinoxaIine(MeIQx), 2-amino-6-methyldipyrido[l,2-a:3',2'-d]imidazole (Glu-P-1)and 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), werereacted with DNA in vitro. Their reactivities were increasedby addition of 10-fold excess of acetic anhydride. ³²P-Postlabellinganalysis of the adducts formed in these in vitro reactions revealedthat almost all the adducts of the hydroxyamino derivativesof MeIQx and Glu-P-1 were the same as those formed in liverDNA of rats intragastrically treated with the parent amines.In contrast, analysis of Trp-P-1 -DNA adducts showed that theadducts formed in vitro were minor components of those formedin vivo; the two main adducts formed in vivo were not formedin vitro. Thus, MeIQx and Glu-P-1 may be metabolized in vivoto hydroxyamino derivatives and/or their esterified forms, suchas N-acetoxy derivatives that form DNA adducts. Formation ofadducts by Trp-P-1, however, may occur through more complicatedmetabolic pathways. Elucidation of the structures of DNA adductsin vivo is necessary to clarify this problem. ¹Present addresses: Department of Chemistry, Swedish Universityof Agricultural Sciences, PO Box 7015, S-750 07 Uppsala, Sweden      

Concentrated drinking water extracts, which cause bacterial mutation and chromosome damage in CHO cells, do not induce sex-linked recessive lethal mutations in Drosophila

Concentrated extracts prepared from chlorinated drinking watersamples were tested for their ability to induce sex-linked recessivelethal mutations in Drosophila melanogaster. Adult flies wereallowed to feed on sucrose solutions prepared using neat orhalf-strength water extract. The drinking water extracts usedfor this study were also tested in bacterial fluctuation assaysusing Salmonella typhimurium TA100 and TA98 and in an in vitrocytogenetic assay using CHO cells. Although the water extractsgave positive results in both of these in vitro tests, therewas no evidence of mutagenic activity in the Drosophila studies. ¹Present address: Glaxo Group Research Ltd, Park Road, Ware,Hertfordshire SG12 ODJ, UK      

Protection of Chinese hamster cells against the cytotoxic and mutagenic effects of alkylating agents by transfection of the Escherichia coli alkyltransferase gene and a truncated derivative

The cytotoxic and mutagenic effects of various monofunctionaland bifunctional alkylating agents have been assessed in V79Chinese hamster cells that express either the entire O⁶-alkylguanine(O⁶AG) and alkylphosphotriester alkyltransferase (ATase) gene(clone 8 cells) or a truncated form that codes only for O⁶AGATase activity (clone SB cells). Protection ratios, as determinedby D₃₇ values, were greater for clone 8 cells than for SB cells.Significant protection against the mutagenic effects of N-methyl-N-nitrosoureaand ethylmethanesulphonate at the hypoxanthine phosphoribosyltransferase(HPRT) locus was observed in clone 8 and SB cells. Streptozotocinand the haloethyl nitrosoureas, chlorozotocin and bis-chloroethylnitrosoureawere less efficient in inducing HPRT-deficient mutants and asmaller degree of protection was afforded by the transfectedgenes. This is possibly due to the propensity of these compoundsto induce multi-locus deletions. Southern analysis of DNA fromclone 8 and SB cells indicated the presence of multiple copiesof the plasmid integrated into clone 8 cells but few copiesin clone SB cells. The copy number did not change but ATaselevels fell when cells were grown in the absence of G418. ²Present address: Corporate Biosciences Laboratory, ICI plc,The Heath, Runcorn, Cheshire, UK      

Adaptive response to simple alkylating agents in the phototrophic bacteria Rhodobacter capsulatus and R.sphaeroides

The presence of an adaptive response to low doses of alkylatingchemicals in the phototrophic bacteria Rhodobacter sphaeroidesand R.capsulatushas been studied. Results obtained show thatboth strains display this repair response against the challengedoses of N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), whenthey are pretreated with low doses of this compound for 120min. The adaptive response of both R.sphaeroides and R.capsulatusinduced an increase of cell survival and a decrease of mutagenesisin the MNNG-pre-treated cells. Furthermore, the MNNG-inducedadaptive repair also gives protection to diethylsulphate andethylme-thanesulphonate in both phototrophic bacteria. Finally,the MNNG-promoted adaptive response is sensitive to inhibitorsof protein synthesis such as chloramphenicol, indicating thatthis DNA repair mechanism needs protein synthesis in R.sphaeroidesand R.capsulatus, in a way similar to that which occurs in Escherichiacoli. ¹Present address: NCI-Frederick Cancer Research Facility, POBox B, Frederick, MA 21701, USA ²To whom correspondence should be addressed      

Assay of ptaquiloside, the carcinogenic principle of bracken, Pteridium aquilinum, by mutagenicity testing in Salmonella typhimurium

The mutagenicity of ptaquiloside, the carcinogenic principleof Pteridium aquilinum, was tested in Salmonella typhimuriumTA100 and TA98. Under weakly basic conditions (pH 8.5), ptaquilosidedecomposed into a conjugated dienone (considered to be the ultimateform), which was mutagenic in both strains. A novel bioassay,using the pre-incubation method at pH 8.5 with S. typhimuriumtester strains was developed for the assay of ptaquiloside extractedfrom plants. By this bioassay the ptaquiloside content of fernscollected at different localities during various seasons, andin various parts of the plant was determined. The ubiquitouspresence of ptaquiloside in fresh plant materials was confirmed.Bracken processed in alkali was found not to contain the carcinogen. ¹Present address: Wako Pure Chemical Industries Ltd, Kawagoe-shi,Saitama 328, Japan. ²Present address: Tochigi Health Centre, Tochigi-shi, Japan. ³To whom correspondence should be addressed      

Elevated intracellular dCTP levels reduce the induction of GC -> AT transitions in yeast by ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine but increase alkylation-induced GC -> CG transversions

The effect of an increased intracellular dCTP:dTTP ratio onthe specificities of ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) mutagenesis was examined in the yeast Saccharomyces cerevisiae.To do so, we used a dCMP deaminase-deficient (ded1) strain havinga dCTP:dTTP ratio >77-fold larger than its isogenic wild-typeparent under the treatment conditions employed. This DNA precursorimbalance lowered the frequencies of EMS- or MNNG-induced SUP4-omutations by 75 or 45% respectively, relative to the correspondingvalues for the wild-type strain. A total of 405 SUP4-o mutationsproduced by the alkylating agents in the dcd1 background werecharacterized by DNA sequencing and the mutational spectra werecompared to those for 399 mutations induced in the wild-typeparent and 207 mutations that arose spontaneously in the dcd1strain. Unexpectedly, the frequencies of EMS- and MNNG-inducedGC AT transitions in the dcd1 strain were found to be reducedby 93 and 68%, respectively, considerably more than the decreasesfor the overall SUP4-o mutation frequencies. The differenceswere due mainly to substantial increases in the frequenciesof GC CG transversions. Although these events were the predominanttype of spontaneous substitution in the dcd1 strain, they weremore frequent after alkylation treatment and were distributeddifferently than the spontaneous GC CG transversions. Preferencesfor the EMS- or MNNG-induced GC AT transitions to occur atGC sites having the guanine located on the transcribed strandor preceded by a 5' purine, respectively, also were diminishedin the dcd1 strain. Together, these findings support mispairingof O⁶-alkylguanine with thymine as the cause of most EMS andMNNG mutagenesis in wild-type yeast. However, the data alsolead us to suggest that the normally error-free repair of apurinicsites resulting from the lability or removal of EMS- or MNNG-inducedN⁷-alkylguanine can be rendered errorprone by increasing thedCTP pool. ¹Present address: Department of Biology, The University of Saskatchewan,Saskatoon, Saskatchewan S7N 0W0, Canada ²To whom correspondence should be addressed      

Tannic acid is not mutagenic in germ cells but weakly genotoxic in somatic cells of Drosophila melanogaster

Tannic acid (TA) was tested for genotoxic activity in threedifferent assays (1–3) in Drosophila melanogaster by feedingof larvae or adult flies. TA did not induce sex-linked recessivelethals (1) nor sex-chromosome loss, mosaicism or non-disjunction(2) in male germ cells. In the wing somatic mutation and recombinationtest (SMART) (3) TA was found to be toxic for larvae of thehigh bioactivation cross and produced a weak positive response.These results suggest that this compound, when administeredorally to larvae or adults of D.melanogaster, is not mutagenicand clastogenic in male germ cells, but weakly genotoxic insomatic cells of the wing imaginal disk. ³To whom correspondence should be addressed