Genetic analysis of PHIP intestinal mutations in MutaTMMouse

The mutagenicity of 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) was investigated in male Muta^TMMouse mice administered20 mg/kg per os for 4 days and killed 7 days later. GenomicDNA was extracted from liver, kidney and small and large intestineand the mutation frequency (MF) at the lacZ locus was determinedusing a positive selection assay. Mutant lacZ clones from theintestine were characterized further by direct PCR amplificationand DNA sequencing. A total of 57 lacZ mutants from PhIP-treated(40) and untreated (18) mice were analysed. In mutants fromthe PhIP group, 33% were G:C     

Lack of mutagenicity of levofloxacin in lacZ transgenic mice

The mutagenic effects of levofloxacin in lacZ transgenicmice(Muta^TMMouse) have been investigated. Male Muta^TMMouse micewere administered levofloxacin i.p. at a dose of 300 or 600mg/kg. The higher dose corresponded to half the LD50 of thecompound in ddY strain mice. The mutant frequencies in the bonemarrow, liver (day 10 only), testis and sperm were examinedby the positive selection method for lacZ mutations on days1 and 10 after treatment Levofloxacin did not induce any statisticallysignificant increase in mutant frequency in any of the examinedtissues at either dose level or at either sampling time. Themutant frequency increases over the spontaneous values in thebone marrow, liver, testis and sperm were 1.0- to 1.2-fold,0.9-fold, 0.5- to 1.0-fold and 0.9- to 13-fold respectively.N-Ethyl-N-nitrosourea (100 mg/kg as a positive control), onthe other hand, induced significant increases in mutant frequenciesin both somatic cells (bone marrow and liver) and germ cells(testis and sperm) on day 10 after treatment. The mutagenicpotency for ENU was bone marrow >> liver testis sperm(50.1-fold, 3.4-fold, 2.9-fold and 23-fold respectively overthe spontaneous values). Levofloxacin was not mutagenic in thelacZ transgenic mice under the present experimental conditions. ¹To whom correspondence should be addressed. Tel: +81 3 5696 8294; Fax: +81 3 5696 8335; Email: itohsudk{at}daiichipharm.co.jp     

Mutagenicity of ethyl carbamate to lacZ- transgenic mice

The mutagenicity of the rodent carcinogen ethyl carbamate (EC,urethane) has been assessed using the lacZ^– transgenicmouse mutation assay (Muta^TMMouse). In two separate experimentsa single i.p. dose of 900 mg/kg urethane followed by a 14–16day expression period yielded statistically significant (     

Detection of gene mutation in skin, stomach and liver of MutaTMMouse following oral or topical treatment with N-methyl-N'-nitro-N-nitrosoguanidine or 1-chloromethylpyrene: some preliminary observations

Transgenic mouse assays, such as Muta^TMMouse, provide a methodto predict the potential target organ carcinogenicity of chemicalcompounds. As part of a validation study, the effects of thedirect-acting mutagens, N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) and 1-chloromethyl-pyrene (CMP), were investigated forgene mutation in the tissues of Muta^TMMice after a single oralor topical exposure. MNNG (50 or 100 mg/kg) or CMP (25 or 50mg/kg) were administered as a single oral dose and the micekilled after 3, 7 or 10 days. Mutation frequencies were determinedin stomach DNA from both MNNG and CMP-treated animals and inliver DNA from the MNNG-treated animals only. The results, althoughobtained from a limited number of animals, consistently showedthat MNNG increased the mutation frequency in stomach DNA, butnot apparently in liver DNA, at each exposure time; no clearincrease in mutation frequency was seen in the stomach DNA ofCMP treated animals. Also, MNNG (250 or 500 µg) or CMP(5 or 10 µg) in acetone were applied as a single doseto the shorn skin of mice 7, 14 or 21 days prior to death. Apositive control group was similarly given dimethyl-benz[a]anthracene(DMBA, 40 µg) and sacrificed after 14 days. Mutation frequencieswere determined in the skin DNA extracted from all animals andin the stomach DNA from MNNG-painted animals only. The results,again obtained from a limited number of animals, clearly showedthat all test compounds consistently increased the mutationfrequency of skin DNA and that these increases were far greaterin the DMBA- and MNNG-treated mice than the CMP-treated mice.No apparent increases were seen in the stomach DNA from theMNNG-painted mice. ¹To whom correspondence should be addressed     

Use of transgenic mouse lacl/Z mutation assays in genetic toxicology

The Big Blue^TM (lacl) and the Muta^TMMouse (lacZ/GalE) assaysoffer a practical means to generate mutation data from a widerange of tissues of treated animals. Our experience with theseassays has so far been encouraging, but several fundamentalquestions require addressing. Chief among these is whether theseassays are to be regarded as replacements for standard rodentgenotoxicity assays, or if they are to be regarded as assaysfor the prediction of potential carcinogenic organotropy usinga multiple-dose protocol. The interplay between the independentchemical properties of mitogenesis and DNA adduction requirealso to be studied. The value and sensitivity of these assayscan only be assessed when these questions have been internationallyaddressed and concluded.     

A preliminary evaluation of the performance of the MutaTM Mouse (lacZ) and Big BlueTM (lacl) transgenic mouse mutation assays

All of the published mutagenicity data generated using the Muta^TMMouse (lacZ) and the Big Blue^TM (lacl) transgenic mouse mutationassays have been re-presented in a standard format. To date,26 chemicals have been evaluated in one or other of the assays,and eight tissues have been sampled. The present analysis shouldexpedite the further validation of these assays. In particular,it is suggested that data on the present 26 chemicals shouldbe consolidated rather than the generation of partial data setson additional chemicals. ¹To whom correspondence should be addressed     

Statistical analysis of lacZ mutant frequency data from MutaTMMouse mutagenicity assays

Transgenic mouse assays have provided an unprecedented opportunityto study mutagenesis in diverse rodent tissues. In this articledata from Muta^TMMouse mutagenicity assays based on the Escherichiacoli gene lacZ were analyzed systematically using liver andbone marrow as potential target tissues. Sources of variation,including plates (within ackaging reactions), packaging reactions(within animals) and animals, were evaluated for extrabinomialvariation. Although hardly any evidence of overdispersion wasdetected at the plate level, limited evidence of extra-binomialvariation was observed at the packaging reaction level. Therewas, however, much stronger evidence of overdispersion at theanimal level.Statistical tests for increasing trend in mutantfrequency with Increasing dose were also performed at the animallevel. A significant increasing trend following exposure to/V-nitrosodibenzylamine was detected in liver but not in bonemarrow. A logistical model was used to further describe thedose-response relationship observed hi nitrosodibenzylamine-treatedliver tissue ⁴To whom correspondence should be addressed. Tel: +1 519 253 4232; Fax: +1 519 971 3649; Email: kfung{at}uwindsor.ca     

The detection of gene mutation in transgenic mice (MutaTMMouse) following a single oral dose of 2-acetylaminofluorene

Transgenic mouse assays, such as Muta^TMMouse, provide a methodto predict the potential target organ carcinogenicity of chemicalcompounds. As part of a validation study, 2-acetylaminofluorenewas administered as a single oral dose of either 50 or 100 mg/kg.The mice were killed 3, 7, 14, 28, 56 or 112 days after thissingle treatment. Mutation frequencies were determined in liverDNA. The results showed that a mutagenic response was observedin the mice treated at the highest dose (100 mg/kg) and thisincrease was expressed from 28 up to 112 days after the singleexposure.     

Cytogenetic mapping of {lambda}gt10 lacZ sequences in the transgenic mouse strain 40.6 (MutaTMMouse)

The transgenic mouse strain 40.6 (Muta^TMMouse) was developedfor the detection of gene mutations induced in vivo. Strain40.6 was constructed by microinjecting the shuttle vector     

Spontaneous mutation during fetal development and post-natal growth

Somatic mutations seem to accumulate slowly with age duringadult life in both mice and men. There is, however, a substantialmutant frequency at birth, suggesting that the rate of accumulationis much higher before birth. This suggests that DNA replicationplays an important role in the generation of spontaneous mutations.Since most cell division and accompanying DNA replication occursearly in development, more mutations would arise during growthand development. Indeed, if the mutations are genetically neutral,the mutant frequency would rise very rapidly during early fetalgrowth, more slowly during later fetal growth and developmentand still more slowly after birth. To test this hypothesis,we have assayed the mutant frequencies from before birth to28 days after birth, by which time most growth has occurred.We have used the F₁ mice generated by crossing SWR females andMuta^TMMouse males. The Muta^TMMouse has a rescuable lacZ/     

Sodium phenobarbital-enhanced mutation frequency in the liver DNM of lacz transgenic mice treated with diethylnitrosamine

To investigate how a carcinogenic promoter acts on cells mutatedby an initiator, we used as a model, lacZ transgenic mouse anda positive selection system. Preliminary data for the mutationalevents in liver DNA of the mice was generated using diethylnitrosamine(DEN) and sodium phenobarbital (S-PB) as initiator and promoter,respectively. In our first experiment, male Muta^TMice receiveda single i.p. injection of saline or 100 mg/kg DEN and werefed a normal diet for 7 days and 500 p.p.m. S-PB in the dietfor 21 days. Liver DNA was harvested after a 1 night fast ondays 7 and 28 post-DEN treatment. In our second experiment,male mice received a single i.p. injection of phosphate bufferedsaline or 50 mg/kg DEN and were fed a normal diet for 7 days,a diet with S-PB for 14 days and then a normal diet for 7 days.Liver DNA was harvested after a 1 night fast on days 7, 21 and28 post-DEN treatment. The S-PB diet enhancedabsolute and relativeliver weights in all groups. The single intraperitoneal doseof 50 or 100 mg/kg DEN induced high mutation frequencies (MF)in liver, lacZ genes on days 7, 21 and 28. There were no remarkabledifferences of the MF among any sampling days for animals receivingDEN and a normal diet. S-PB feeding at 500 p.p.m. for 21 daysfailed to affect the MF in groups given saline or 100 mg/kgDEN. On the other hand, when 50 mg/kg DEN was given, S-PB feedingat 500 p.p.m. for 14 days elevated the MF in liver DNA on days21 and 28 to –1.8 and 4.0 times the MF, respectively,ofthe mice fed the normal diet. Consequently, S-PB might preferentiallypromote certain initiated cells participating in a balance betweencell death and proliferation. ¹To whom correspondence should be addressed     

The effect of dietary restriction during development in utero on the frequency of spontaneous somatic mutations

Caloric or dietary restriction is known to be protective againstcancer in humans and in mice but the mechanism is uncertain.Given that somatic mutations are important in carcinogenesis,dietary restriction may act by changing mutation rates. Indeed,previous studies have shown that reductions in caloric intakeduring development or in adult life make mice less susceptibleto high doses of mutagens. In these studies there have beenhints that the spontaneous mutant frequency may also be reduced,but no significant decrease has been observed save in one studyof very old mice. Since the spontaneous mutant frequency isalready low, reductions from this level require the use of muchlarger sample sizes than usual and larger than those used inthe previous studies. As pre-existing mutations cannot be eliminated,it is necessary to reduce the dietary intake over a period oftime when a substantial proportion of spontaneous mutationsarise in order to see an effect. To overcome such problems,the dietary restriction in this study was applied during thetime of the highest mutation rate, early development, and manymore than the usual number of animals were studied. SWR femalemice were crossed with Muta^TMMouse males to obtain F₁ progenyfor analysis of mutant frequency. At conception, the dams wereput into two groups, one that was fed ad libitum and anotherwhich was fed 80% of the ad libitum diet. Pups were killed atbirth, DNA was extracted from the whole animal and used to measurethe mutant frequencies of the mice at the cII locus. Althoughthe weights of the pups from dams whose diet was restrictedwere significantly less than those of the ad libitum mice (P= 0.003), the litter sizes in the two groups were approximatelythe same and did not differ significantly (P = 0.13). Therewas no significant difference in the mutant frequencies in thedietarily restricted and ad libitum groups (P = 0.43). In addition,there was no significant correlation between the weights ofthe pups and their mutant frequency in either the ad libitumor dietarily restricted groups (r² = 0.14 and r² = 0.024). Nodifference was observed in mutant frequency between the ad libitumand dietarily restricted mice from litters of the same size(P = 0.61). These results indicate that the protective effectof dietary restriction on cancer rates is not mediated by analteration in the spontaneous rate of mutation but rather byanother mechanism, such as its effect on induced mutation. ¹ To whom correspondence should be addressed. Tel: +1 416 7362100; Fax: +1 416 736 5698; Email: jheddle{at}yorku.ca     

Mutagenicity of high fat diets in the colon and small intestine of transgenic mice

Dietary fat has been implicated as a cause of colon cancer byepidemiological studies. Unfortunately, these studies are compatiblewith high fat as either initiator or promoter. Since initiatorsare normally mutagenic, we have tested the mutagenicity of highfat diets in the intestinal epithelium at two loci. The Dlb-Iassay, used in the small intestine, detects a wide spectrumof mutations. The lacI assay (Big Blue^TM Mouse assay) is notas sensitive to some types of mutation as is Dlb-I but was usedin the colonic epithelium. Mice suitable for both assays werefed isocaloric high fat diets and subsequently assayed for somaticmutation. The diets consisted of: (i) a mixture of beef tallow,butter and lard totalling to 31% w/w of the diet (AIN-76A) upto 17 weeks; and (ii) corn oil, beef tallow, lard or butterindividually, at 31% w/w of the diet for 5 and 9 weeks. Thesediets provided 50% of the calories from fat. The weights ofthe experimental and control mice were similar throughout theexperiment. No significant increases in mutant frequencies wereobserved on any high fat diet compared to controls, so we concludethat uncooked fats are not mutagenic and are not initiatorsof carcinogenesis in the intestinal epithelium. ¹To whom correspondence should be addressed     

Germ cell mutagenesis in {gamma}IacZ transgenic mice treated with ethylnitrosourea; comparison with specific-locus test

Germ cell mutagenesis was studied in male lacZ transgenic micein such a way that the data can be compared with literaturedata for germ cell mutagenesis obtained with the specific-locustest. This comparison is of interest for validation of the transgenicmouse model. We studied mutagenesis induced by ethylnitrosourea(ENU) in mature spermatozoa isolated from epididymis and vasdeferens. In order to investigate mutagenesis in different phasesof spercmatogenesis, animals were killed at various time pointsafter treatment. After an ENU dose of 150 mg/kg, an increaseof the mutant frequency (MF) occurred only in stem cells (9-fold;100 days post-treatment) but not in post-stem cells (0.1 and7 days post-treatment). Specific locus test data from literatureshowed a larger induction of mutations in stem cells (44-fold;>42 days post-treatment). Although spermatogenesis in micenormally takes {small tilde}42 days, we found only a limitedincrease of the MF at 50 days post-treatment. This may be dueto a delay of spermatogenesis, which was confirmed by the observationthat O⁶-ethylguanine levels in spermatozoan DNA remained approximatelythe same between 0.1 and 50 days and were reduced to backgroundlevels at 100 days. Dose-response studies with the lacZ miceindicated a threshold for mutation induction in stem cells atlow ENU dosages, which is in accordance with the specific-locustest data. In summary, our experiments suggest that lacZ transgenicmice are a suitable model to study germ cell mutagenesis inspermatogonial stem cells. ¹To whom correspondence should be addressed     

Bacterial ribonuclease: mutagenic effect in microbial test-systems

Pure enzyme samples of ribonuclease from Bacillus intermedius7P (known commercially as ‘binase’) were investigatedfor genotoxicity in four microbial tests: the Ames plate incorporationmethod, Ara^R-assay; the prophage induction test; and the DNA-repairtest. The weak mutagenic effect of binase at high concentrations(0.1 mg/plate, 1 mg/plate) was established by induction of forwardAra^R-mutations and histidine-reverse mutations (both frameshiftmutations and base pair substitution). Metabolic activationwith rat or chicken liver, human placenta or plant (from tulipbulbs) microsomal fractions in vitro was seen to abolish thebinase mutagenicity. Bacillus intermedius 7P ribonuclease appearsto possess DNA damaging activity in uvrA^– and polA^–mutants, but not in the recA-deficient Escherichia coli strain,and exhibits an induction of recA-dependent mutagenesis detectedby the 8-fold increase of the prophage-induction level in lysogenicBacillus subtilis culture and by the 5-fold increase of thislevel in the Streptomyces lavendulae 3 lysogenic strain. Theimportance of the roles of both of enzyme catalytic activityand native structure is emphasized. A proposed mechanism forexogenous ribonuclease action is discussed. Bacillus intermedius7P ribonuclease probably does not act as a direct genotoxicagent interacting with DNA, but could provoke nucleotide imbalancethrough its catalytic action on membrane-associated RNAs, whichresults in alteration of DNA replication and, as a consequence,in recA-dependent mutagenesis. ¹To whom correspondence should be addressed     

Genome wide spontaneous mutation in human cells determined by the spectrum of mutations in hprt cDNA genes

We have studied spontaneous mutagenesis in five hprt cDNA genesintegrated at five different genomic positions in a human lymphoblastoidcell line (TK6). The spectra of 40 mutants from each positionwere combined to obtain a mutation spectrum of the overall genome.This collection of mutants was used to assess the contributionof several mutagenic processes to spontaneous mutagenesis. Deletionsand single base pair changes account for the majority of themutants and arise in approximately equal amounts (43 and 41%,respectively).The majority of the deletions and insertions are >5 bp andare likely to be caused by template-directed misalignment (slippage)during replication. To account for frameshifts at non-iteratedsites we propose a slightly different template-directed replicationerror model. A considerable amount of the observed base pairchanges can also be explained by this last model but severalother processes leading to base pair changes such as depurination,deamination or spontaneously arising DNA damage are likely tocontribute as well. We have compared this spectrum with mutationspectra in the endogenous hprt genes using published mutationdata. It is shown that in the endogenous genes the contributionof base pair substitutions is much larger (71%) than in thehprt cDNA integrates and that deletions are less frequentlyobserved (20%) The mutation rates of the integrated hprt cDNAgenes show a mean increase of 30-foldas compared with the endogenoushprt gene. This results in a 60-fold increase of the absoluterate of deletion in the hprt cDNA genes and in a 15-fold increaseof the base pair substitution rate. Replication errors suchas slippage or the mechanism proposed in this study probablyaccount to a large extent for this increase. ²To whom correspondence should be addressed     

Genotoxicity of paracetamol in mice and rats

The genotoxicity of paracetamol, including covalent bindingto DNA, induction of DNA single-strand breaks (SSBs), and inhibitionof replicative and repair synthesis of DNA, has been investigatedin rodents in vivo. In the covalent binding studies male ICRmice were fasted and pretreated with diethyl maleate to depletehepatic glutathione (GSH) and 300 mg/kg of [G-³H]paracetamolwas administered intraperitoneally (i.p.). Animals were killedat 2, 6, 24, 72 and 168 h after paracetamol and hepatic or renalDNA and protein were isolated and the extent of covalent bindingdetermined. Maximal binding to liver DNA, 8.4 ± 3.1 pmol/mgof DNA, was observed at 2 h and declined rapidly to 2.6 pmol/mgat 24 h. Measurable binding (1.4 pmol/mg of DNA) was detectedat 7 days. Protein binding in the liver in these animals peakedbetween 2 and 6 h (887 pmol/mg of protein at 2 h) and declinedmonoexponentially to 52 pmol/mg at 7 days. Although based ona limited body of data, covalent binding was also detected inDNA isolated from the kidney. DNA damage measured as SSBs byalkaline elution was induced in nuclear DNA isolated from theliver but not from the kidney, 2 h after i.p. injection of paracetamolat 600 mg/kg in male B6 mice. Only marginal DNA damage was notedat 300 mg/kg. The alkaline elution profile from damaged livernuclei was markedly biphasic, suggesting that breaks were inducedin DNA from a subpopulation of liver cells. The non-hepatotoxicparacetamol regioisomer, acetyl-m-aminophenol (600 mg/kg), whichbinds covalently to proteins, did not cause DNA SSBs. Pretreatmentof animals with diethyl maleate enhanced the paracetamol-inducedDNA SSBs, while phenobarbital and N-acetylcysteine had no markedeffects. In male Wistar rats, which are more resistant to paracetamoltoxicity, no increase in the level of DNA SSBs was seen in liveror kidney 4 h after exposure to 600 mg/kg paracetamol. Paracetamol(150 mg/kg and higher) inhibited DNA synthesis in B6 mice, asevidenced by a marked decrease in the incorporation of [³H]thymidine([³H]TdR) between 15 and 75 min in the liver, spleen, intestine,bone marrow and kidney. The decrease in DNA synthesis was transient,and between 90 and 150 min the rate of radiolabel incorporatedwas at the control level or increased in all the organs, exceptthe kidney. Paracetamol, at 300 mg/kg, increased the level ofDNA SSBs detected in the liver, spleen and kidney of both B6mice and Wistar rats 2 h after administration of 4-nitroquinolineN-oxide (NQO). Covalent binding of paracetamol metabolites toliver and renal DNA in diethyl maleate-pretreated mice and inductionof DNA SSBs in mouse liver were observed at hepatotoxic dosesof paracetamol and probably involve reactive metabolite(s) ofparacetamol. These effects could be early events in the developmentof liver necrosis. The inhibition of (³H)TdR incorporation andthe enhancement of NQO-induced DNA SSBs, on the other hand,occur at lower doses and in organs with low capacities for metabolizingparacetamol to reactive metabolite(s). These effects are mostprobably due to inhibition of ribonucleotide reductase by paracetamol,as previously demonstrated in vitro.     

Effects of alpha-naphthyl isothiocyanate and a heterocyclic amine, PhIP, on cytochrome P-450, mutagenic activation of various carcinogens and glucuronidation in rat liver

To elucidate the mechanism underlying suppression by -naphthylisothiocyanate (ANIT) of mammary carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP), we evaluated hepatic levels of cytochrome P-450 (CYP)enzymes, mutagenic activation of environmental carcinogens andUDP-glucuronyltransferase (UDPGT) activities in female Sprague–Dawleyrats fed a high fat diet. Immunoblot analyses revealed inductionof CYP1A1, newly found 51 and 53 kDa proteins and constitutiveCYP1A2 and 2B2 by intragastric treatment with 85 mg/kg PhIPeight times for 11 days. Although the extents of induction werenot as high as in the case of PhIP, 3 weeks feeding of 400 p.p.m.ANIT induced CYP1A1 and the 51 and 53 kDa proteins. CP1A2 levelwas decreased by the feeding of ANIT. The mutagenicity in strainTA98 of PhIP, four other heterocyclic amines (HCAs) and benzo[a]pyrenewas greatly enhanced in the presence of liver S9 mix preparedfrom rats pretreated with PhIP but not with ANIT. The mutagenicitiesof these five HCAs were significantly decreased in the presenceof liver S9 from rats pretreated with a combination of PhIPand ANIT as compared with that pretreated with PhIP alone. Thelevel of hepatic CYP1A2, which is known to be involved in themetabolic activation of PhIP, was consistently decreased inliver microsomes from rats administered PhIP plus ANIT as comparedwith that from rats administered PhIP alone. On the other hand,UDPGT activity towards 4-nitrophenol (4-NP) was enhanced usingliver microsomes prepared from rats pretreated with a combinationof PhIP and ANIT as compared with those pretreated with PhIPor ANIT alone. These results show that chemoprevention by ANITagainst PhIP-induced rat mammary carcinogenesis can be explainedby a dual action mechanism, i.e. a reduction in metabolic activationby hepatic CYP1A2 and an enhancement of detoxification by 4-NPUDPGT. The role of the newly found 51 and 53 kDa proteins inactivation of HCAs is also discussed. ^* To whom correspondence should be addressed. Tel: +81 58 237 3931; Fax: +81 58 237 5979; Email: ymori{at}gifu-pu.ac.jp     

DNA sequence analysis of spontaneous lacl-d mutations in O6-alkylguanine-DNA alkyltransferase-proficient and -deficient Escherichia coli

Spontaneous mutagenesis in O⁶-alkylguanine-DNA alkyltransferase-proficientand -deficient (ada ogt mutants) Escherichia coli was studiedin two ways: in bacteria growing in nonselective liquid mediumand in bacteria resting on selective agar plates.ATase mutantsshowed similar spontaneous mutation rates as ATase proficientbacteria during growth phase, an excess of mutants arising innondividing cells.The resting-associated mutagenesis in ada⁺ogt⁺ uvr^– bacteria was biphasic; the high sensitive rangebeing triggered beyond the first 6 days after plating. Contrarily,spontaneous Lac^c mutants from ada^– ogt^– uvr^–cells steadily increased over the 8 day period of plate incubation.Theseresults suggested that, in the absence of nucleotide excisionrepair, the repair by both the Ada and the Ogt ATases is notsaturated until the cells have been resting for 6 days. Thespontaneous lacV* mutation spectrum of ada⁺; ogt⁺ uvr^–bacteria growing in non-selective liquid medium served as abaseline to determine the mutation events increased in the ATase–deficientderivative upon prolonged incubation on selective plates. Thepercentage of G:C     

A correlation of Salmonella mutagenicity with DNA adducts induced by the cooked-food mutagen 2-amino-1-methyl-6-6-phenylimidazo[4,5,-b]pyridine

The correlation of bacterial mutagenicity with DNA adducts fromthe heterocyclic amine cooked-food mutagen 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) was investigated in Salmonella typhimurium strains TA98(uvrB deficient) and TA1978 (uvrB proficient). Bacterial cellswere exposed to PhIP using a modification of the Ames/ Salmonellamicrosuspension assay. Half of the cells, generated from a 90min pre-incubation and washing, were plated for revertant formationwhile the remaining half was subjected to DNA adduct analysisvia ³²P-postlabeling. In TA98, DNA adducts were detected atan RAL (relative adduct labeling) of 10x10^–7 and 21x10^–7at PhIP concentrations of 5.5 and 17 µM, respectively.This corresponded to 28.8 and 20.9 adducts/revertant, respectively.These values were based on the assumption that only four repeatingGC bases within a 75 DNA base region is the gene target sitefor PhIP induced mutations. In TA1978, no revertants above backgroundwere detected at any concentration of PhIP tested. DNA adducts,however, were detected at 11x10^–7 and 21x10^–7 adductsper nucleotide at 223 and 1116 µM PhIP, respectively.The lack of detectable revertants, but the presence of DNA adducts,suggests pre-mutational lesions did occur during the 90 minpre-incubation. Presumably, when the S9 activating system andPhIP were removed (via washing with phosphate buffered saline)prior to plating, the cells containing an intact uvrB repairsystem repaired the lesions during the incubation time on theplates. In conclusion, the induction of revertants by adductsappears quite efficient, as