Biomedical subjects

J B Guttenplan

Publications and source records attributed to J B Guttenplan.

At least 19 recordsLinked to original sources

PubMed2001

Effects of a lycopene-rich diet on spontaneous and benzo[a]pyrene-induced mutagenesis in prostate, colon and lungs of the lacZ mouse.

Consumption of lycopene has been associated with reduced risk of prostate cancer. We have investigated the effects of lycopene, fed as a lycopene-rich tomato oleoresin (LTO) at two doses, on in vivo mutagenesis in prostate, colon, and lungs of lacZ mice. Both short-term benzo[a]pyrene (BaP)- induced and long-term spontaneous mutagenesis were monitored. Non-significant inhibition of spontaneous mutagenesis in prostate and colon was observed at the higher dose of LTO, and the observation of inhibition in colon was facilitated by an unusually high spontaneous mutagenesis rate. BaP-induced mutagenesis was slightly inhibited by LTO in prostate. However, enhancement of BaP-induced-mutagenesis was observed in colon and lung. These results indicate that any antimutagenic effects of LTO may be organospecific.

Animals↗

PubMed2001

Mutagenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine in lacZ upper aerodigestive tissue and liver and inhibition by green tea.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and nitrosonornicotine (NNN) were administered to lacZ mice (MutaMouse) at equal concentrations in drinking water (2 weeks at 0.1 followed by 2 weeks at 0.2 mg/ml) over a 4 week period, for a total estimated dose of 615 mg/kg) and mutagenesis in a number of organs was measured. For mutagenesis induced by NNK the potency order was: liver > lung> pooled oral tissues kidney > esophagus > tongue. The mutant fraction varied from approximately 6 to 40 mutants per 10(-5) plaque forming units This corresponds to approximately 2-13 times the background levels. A somewhat different pattern was observed with NNN, where the order was liver > esophagus oral tissue approximately tongue > lung > kidney. The potency of NNK was about twice that of NNN in liver and lung, but somewhat less in aerodigestive tract tissue. When compared with results previously obtained for a similar administered dose of benzo[a]pyrene, NNK was approximately 10-100% as mutagenic in the corresponding organs. Reported target organs for carcinogenesis by NNN and NNK in rodents were targets for mutagenesis, but mutagenesis was also observed at other sites, suggesting that these sites are initiated. The effect of green tea consumption on mutagenesis by NNK was also investigated. Green tea reduced mutagenesis by approximately 15-50% in liver, lung, pooled oral tissue and esophagus.

Animals↗

PubMed2000

Mutagenic activity of 4-nitroquinoline-N-oxide in upper aerodigestive tissue in lacZ mice (MutaMouse) and the effects of 1, 4-phenylenebis(methylene)selenocyanate.

4-Nitroquinoline-N-oxide (4-NQO) was administered to lacZ mice at a concentration of 20 microg/ml in drinking water for 2 weeks, and the mutagenic fractions in a number of organs were assayed. The mutant fractions in tongue, esophagus and other pooled oral tissues were, respectively, 117+/-26, 73+/-15, and 48+/-15 mutants/10(5) plaque-forming units (pfu) (ca. 15-40xbackground). 4-NQO was not mutagenic in lung, liver or colon at conditions used here. We had previously demonstrated that the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC), an established chemopreventive agent, greatly reduced carcinogenicity in 4-NQO in rat tongue, and we observed here that administration of p-XSC (10 ppm se) in the diet for 6 weeks (2 weeks before, during, and 2 weeks after 4-NQO) resulted in a 33% decrease in mutagenesis in oral tissue, a 17% decrease in esophagus, and a slight increase in tongue. Only the decrease in oral tissue reached statistical significance (p<0.04). The results reported here demonstrate that 4-NQO was extremely mutagenic in lacZ mouse tongue, with lower, but highly significant activities in esophagus and other pooled oral tissues. The high activity of 4-NQO in lacZ mouse tongue is consistent with the organ specificity of 4-NQO in the rat. Inhibition of 4-NQO-induced mutagenesis by p-XSC was observed mainly in pooled oral tissues, other than tongue. Possible reasons for the difference between inhibition of mutagenesis and carcinogenesis in tongue are discussed, as well as advantages and disadvantages of in vivo mutagenesis assays as surrogates for carcinogenicity assays in chemoprevention studies.

4-Nitroquinoline-1-oxide↗

PubMed1999

Mutagenesis induced by oral carcinogens in lacZ mouse (MutaMouse) tongue and other oral tissues.

Animal models for carcinogenesis of the oral cavity are limited, although this disease is often fatal or disfiguring and its incidence in the USA is approximately 30 000 cases/year. Short-term whole-animal models for this disease should prove valuable in the investigation of factors affecting oral carcinogenesis. In this study we observed that a group of oral carcinogens are clearly mutagenic in the lacZ transgenic mouse oral cavity. The carcinogens 4-nitroquinoline-N-oxide (4-NQO), benzo[a]pyrene (B[a]P), N-nitroso-N-methylurea (NMU), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), nitrosonornicotine (NNN) and 7,12-dimethylbenzanthracene (DMBA) were all mutagenic in a mixture of pooled oral tissues (gingival, buccal, pharyngeal and sublingual) and in the tongue. All agents except DMBA (which was swabbed in the oral cavity) and B[a]P (by gavage) were given in drinking water for 2-4 weeks followed by a 2 week expression period before killing. With one exception, groups of 4-5 female mice were treated. The doses and mutant fractions (MF) in DNA isolated from pooled oral tissues (in mutants/10(5) p.f.u. +/- SD) were: 4-NQO (20-80 microg/ml, over 4 weeks) 78 +/- 16; B[a]P (five doses of 125 mg/ml) 33.2 +/- 10.9; NMU (20-80 microg/ml over 4 weeks) 7.8 +/- 2.6; NNK (0.1 mg/ml, weeks 1-2, 0.2 mg/ml, weeks 3-4) 9.1 +/- 3.0; NNN (same dose as NNK) 9.2 +/- 1.6 and DMBA (0.5 mg/ml in corn oil, 3 weeks) 7.1 +/- 2.7. The corresponding value for untreated controls was 3.2 +/- 1.8. Values for induced mutagenesis in tongue from the same animals were similar except for 4-NQO which was about twice as potent in tongue. Mutagenesis by several compounds was compared in other organs. B[a]P was assayed in lung and kidney and was about twice as mutagenic in oral tissues as in lung, but several times less mutagenic in kidney. Lung, but not kidney is a target organ for B[a]P-induced carcinogenesis in the mouse. NNK was somewhat more mutagenic in lung (MF of 15.0 +/- 5.5) than in oral tissues, corresponding with previous reports on carcinogenesis by NNK. Mutagenesis induced by NNN was also assayed in esophagus, a target organ in rodents, and was similar to that in oral tissue. In all cases the MF in untreated control group was about 3-4. These results suggest that: (i) the oral cavity has a significant capacity for metabolic activation of carcinogens; (ii) DNA damage in the oral cavity can be converted to mutations; and (iii) there is significant target organ specificity. The results also tend to support the concept that the anatomical components of the upper aerodigestive tract, in general, behave similarly with respect to genotoxicity. As carcinogenesis is believed to involve mutagenesis, this study demonstrates the utility of the lacZ mouse for investigations involving initiation of carcinogenesis of the oral cavity.

Animals↗

PubMed1999

Mutagenesis induced by benzo[a]pyrene in lacZ mouse mammary and oral tissues: comparisons with mutagenesis in other organs and relationships to previous carcinogenicity assays.

Thus far, in vivo mutagenic assays have detected organ-specific effects of benzo[a]pyrene (B[a]P) in a number of organs, but not in oral tissues and breast. Previous studies have shown that the mouse tongue is a target for tumorigenesis induced by B[a]P when incorporated into feed, and polycyclic aromatic hydrocarbons are carcinogens in mouse mammary tissue. In order to evaluate the capacity of the lacZ mouse in vivo mutagenesis assay to detect mutations in these target tissues, we have measured mutagenesis induced by B[a]P in breast and oral tissues. The oral tissue consisted of either tongue or a mixture of oral tissues from several sites in the oral cavity. B[a]P was more mutagenic in breast tissue than in most other organs tested (liver, lung and kidney) when administered at relatively high dose by gavage, and more mutagenic than in liver, but not lung, at low dose. When administered in an emulsion in drinking water, B[a]P was more mutagenic in oral tissues than in liver, and somewhat less mutagenic than in lung. Regardless of dose, the mutagenic activity was greatest in colon where it was much higher than in other organs. A reasonable correlation was observed between mutagenesis observed here and carcinogenesis in previous studies although some differences were noted. To our knowledge, this represents the first report of in vivo mutagenesis in non-tumor mammary and oral tissue, and the results indicate these organs can efficiently metabolize B[a]P to genotoxic products, although some transport of active metabolites from the liver cannot be ruled out. The lacZ mouse mutagenesis assay may represent a shorter term alternative to carcinogenesis assays for investigations of factors affecting initiation of carcinogenesis in mammary and oral tissues. However, it is less predictive of actual tumor formation.

Animals↗

PubMed1998

1,N2-propanodeoxyguanosine adducts: potential new biomarkers of smoking-induced DNA damage in human oral tissue.

Highly DNA-reactive alpha,beta-unsaturated aldehydes such as acrolein and crotonaldehyde are common environmental pollutants present in cigarette smoke and automobile exhaust and are also released endogenously by lipid peroxidation. Acrolein- and crotonaldehyde-derived 1,N2-propanodeoxyguanosine (AdG and CdG, respectively) have been detected in the tissues of carcinogen-treated rodents and as background lesions in DNA from humans and untreated rodents. To determine whether cigarette smoking increases the levels of AdG and CdG, gingival tissue DNA from 11 smokers (4 males and 7 females; 30-58 years old) and 12 nonsmokers (8 males and 4 females; 21-66 years old) was analyzed using a previously described 32P-postlabeling high-performance liquid chromatography method. The results showed that the mean AdG levels in smokers were significantly higher than those in nonsmokers (1.36 +/- 0.90 micromol/mol guanine in smokers versus 0.46 +/- 0.26 micromol/mol guanine in nonsmokers; P = 0.003). The mean CdG 1 levels in smokers and nonsmokers were 0.53 +/- 0.44 and 0.06 +/- 0.07 micromol/mol guanine, respectively, corresponding to an 8.8-fold increase for smokers (P = 0.0015). Similar to CdG 1, levels of CdG 2 were increased 5.5-fold in smokers as compared to nonsmokers, from 0.31 +/- 0.40 to 1.72 +/- 1.26 micromol/mol guanine (P = 0.0014). Furthermore, the total levels of cyclic adduct (AdG and CdG) in smokers were 4.4-fold greater than those in nonsmokers (P = 0.0003). This study shows the detection of the potentially promutagenic 1,N2-propanoguanine adducts in human oral tissues and demonstrates for the first time an increase of structurally identified adducts in oral tissue DNA by cigarette smoking.

Adult↗

PubMed1996

Unexpected genetic toxicity to rodents of the N',N'-dimethyl analogues of MNU and ENU.

Lijinsky and his colleagues have reported that the N',N'-dimethyl analogues of ENU and MNU [N',N'-dimethyl-N-ethyl-N-nitrosourea (DMENU) and trimethylnitrosourea (TMNU), respectively] are carcinogenic to rats despite their extreme hydrolytic stability which would reduce or preclude generation of alkylating species analogous to those formed upon hydrolysis of ENU and MNU. Lijinsky and his colleagues were unable to rationalize those activities of DMENU and TMNU despite extensive experimentation. We therefore decided to study this problem further. Whichever mode is accepted for the generation of electrophilic/mutagenic/carcinogenic reactive species from ENU and MNU, blocking of the free-NH2 group with methyl groups (-NMe2) should ablate or abolish activity. Consistent with this DMENU and TMNU gave negative results in the NBP alkylation test while the parent compounds gave an instantaneous deep blue coloration. Studies of the rate of hydrolysis of these four compounds revealed ENU and MNU to have half-lives of 8 min, while the alkylated analogues (DMENU and TMNU) had half-lives of 25 and 41 days, respectively. Hydrolysis of ENU and MNU, to yield the alkylating species, proceeds either via proton abstraction from the -NH2 group or by attack by water on the carbon of the carbonyl group. Methylation will inhibit both of these pathways, the first absolutely (no -NH2 protons) and the second partially, via steric inhibition. The slow hydrolysis observed for DMENU and TMNU suggests that the latter route of hydrolysis is applicable. Studies with strain TA1535 of Salmonella typhimurium (without S9 mix) confirmed the potent mutagenic activity for ENU and MNU (approximately 300-fold increase in revertants at 2,000 micrograms/plate and approximately 180-fold increase in revertants at 150 micrograms/plate respectively). In contrast, the methylated analogues showed only weak mutagenic activity (approximately 3-fold) at approximately 100-fold higher dose-levels. Addition of S9 mix did not affect the mutagenicity of DMENU or TMNU. To this point, hypothesis and data coincide. ENU and MNU are potent micronucleus-inducing agents to the mouse bone marrow, and given the above data, it was expected that DMENU and TMNU would show weak or no activity in that assay. In fact, the methylated analogues were as effective as ENU and MNU as clastogens to the mouse bone marrow. Four possible reasons for this conflict of theory and data are explored. The speculative explanation we favour for these effects is that the net alkylation of bone marrow DNA is the same for all four chemicals. With ENU and MNU, most of the alkylating activity is dissipated by rapid hydrolysis. Thus, only a small fraction of the administered dose survives to alkylate the bone marrow. Due to the enhanced stability of the methyl analogues most of the delivered dose will reach the bone marrow. However, because of their lower intrinsic reactivity, only a small fraction of the target dose will alkylate the bone marrow DNA during the time window of the experiment. If these opposing influences happen to balance out, the essentially identical bone marrow genetic toxicity for the four chemicals could be explained.

Alkylating Agents↗

PubMed1993

Mutational specificities of environmental carcinogens in the lac1 gene of Escherichia coli, VII: The host-mediated assay and its comparison with in vitro mutagenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

To investigate the influence of different types of metabolic activation (9,000 x g supernatant (S9) activation vs. a host-mediated approach) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mutational specificity, we determined by DNA sequencing the distribution of forward mutations recovered in the N-terminal region of the lac1 gene of Escherichia coli. After activation with the S9 liver fraction from rats treated with Aroclor 1254, a diverse spectrum of mutations was recovered, with 55% of the events being G:C-->A:T transitions. In contrast, after the host-mediated assay in mice, G:C-->A:T transitions accounted for over 94% of the mutations recovered. Generally, NNK metabolism can proceed through two distinct pathways, involving either alpha-methyl or methylene hydroxylation. These two pathways produce different distributions of DNA damage. The difference in the mutational spectra we observed thus likely reflects the difference in the contributions of each pathway under the two different treatment conditions.

Base Sequence↗

PubMed1993

2,3-epoxy-4-hydroxynonanal as a potential tumor-initiating agent of lipid peroxidation.

Trans-4-hydroxy-2-nonenal (HNE) is a product of lipid peroxidation. In the presence of t-butyl hydroperoxide the racemic HNE readily converts to its epoxide, 2,3-epoxy-4-hydroxynonanal (EH), as a pair of diastereomers. In this study, the potential roles of HNE and EH as tumor initiating agents were assessed. The mutagenicities of HNE and EH isomers in Salmonella strains TA100 and 104 were examined. In addition, the tumor initiating activities of HNE and EH were evaluated in bioassays involving either topical application in CD-1 mice or i.p. administration in newborn CD-1 mice. In the mutagenicity assays, EH isomers induced similar levels of revertants in both tester strains, although EG isomers were previously shown to react with bases in DNA with different specificity (Sodum, R.S. and Chung, F.-L., Cancer Res., 51, 137-143, 1991). The major isomer induced approximately 20,000 revertants/mumol in TA100 and 15,000 revertants/mumol in TA104, whereas, the minor isomer induced approximately 40,000 revertants/mumol in TA100 and 20,000 revertants/mumol in TA104. HNE was, however, not mutagenic under the assay conditions. In the tumor bioassays, EH was a weak tumorigen in CD-1 mice upon topical application followed by TPA promotion, yielding 0.55 tumors/mouse and 40% tumor incidence at a total dose of 128 mumol/mouse versus 0.02 tumors/mouse and 5% tumor incidence in the control group. Both HNE and EH induced liver tumors in male mice, but not in female mice. However, the incidences were not statistically significant. EH administered i.p. at a total dose of 200 nmol/mouse exacerbated the chronic spontaneous nephropathy in newborn CD-1 mice. Although the incidence of mild nephropathy was comparable in both EH-treated and control groups, the incidence of more severe lesions in mice treated with 200 nmol/mouse was 21%; while it was 0% in the control group. Furthermore, two mice at each dose level of EH showed a tubule profile with complex hyperplastic lining, suggestive of atypical hyperplasia. Again, HNE was not as active as EH in these bioassays. These results suggest a possible role of EH in tumorigenesis associated with lipid peroxidation.

Aldehydes↗

PubMed1993

Effects of cytosol on mutagenesis induced by N-nitrosodimethylamine, N-nitrosomethylurea and alpha-acetoxy-N-nitrosodimethylamine in different strains of Salmonella: evidence for different ultimate mutagens from N-nitrosodimethylamine.

N-Nitrosodimethylamine (NDMA), but not N-nitroso-N-methylurea (MNU) was more mutagenic in the Salmonella hisG428 strain, TA104, than in the hisG46 strain, TA100 in the presence of rat or hamster liver S-9 mix. As both NMDA and MNU can give rise to methyldiazonium ion (MDI) it appears that NDMA can be metabolized to an additional mutagen with a higher activity in TA104. The effects of UV and error-prone repair on NDMA and MNU-induced mutagenesis in TA104 were also different. alpha-Acetoxy-NDMA, which gives rise to the NDMA metabolite, alpha-hydroxy-NDMA, was more mutagenic in TA104 than TA100, under certain conditions. Several metabolites of NDMA (formaldehyde, 1,1-dimethylhydrazine and nitrite) were not significantly mutagenic at the concentrations that could have been generated from NDMA. It was previously reported that the microsomal-mediated mutagenesis induced by NDMA is greatly increased by cytosol in TA104, but not in TA100. The current study found that when cytosol was separated into a high and a low mol. wt fraction, neither greatly enhanced microsomal-mediated mutagenesis by NDMA in TA104. Addition of NAD to the high, but not the low mol. wt fraction resulted in greatly enhanced activation of NDMA to a mutagen in TA104. The enhancement by cytosol of NDMA-induced mutagenesis in hisG428 was only observed when both microsomes and cytosol were simultaneously present. These observations indicate that (i) the precursor to the ultimate mutagen is relatively short-lived; and (ii) the metabolism of alpha-hydroxy-NDMA to a secondary mutagenic metabolite, possibly N-nitroso-N-methylformamide, by alcohol dehydrogenase may be responsible for the ultimate mutagen with relatively high activity in TA104.

Animals↗

PubMed1993

High mutagenic activity of N-nitrosobis(2-oxopropyl)amine and N-nitrosobis(2-hydroxypropyl)amine in the host-mediated assay in hamsters: evidence for premutagenic methyl and hydroxylpropyl adducts.

The carcinogenic nitrosamines N-nitrosobis(2-oxopropyl)-amine (BOP) and N-nitrosobis(2-hydroxypropyl)amine (BHP) were tested in excision-repair-deficient strains of hisG46 Salmonella mutants in the intrasanguinous host-mediated mutagenesis assay (HMA) in male Syrian hamsters. The major adducts produced by BOP in the hamster are methylguanines, while BHP leads to hydroxypropylguanines as well as methylguanines. Both nitrosamines were potent mutagens in bacteria recovered from the liver. On a comparison of administered dose, BOP was more potent, but when compared at doses producing similar levels of O6-methylguanine (O6-MeG) in host liver DNA, or at equitoxic doses in the hamster, BHP was more potent. BHP was approximately 10 times less mutagenic in an excision-repair-proficient strain of Salmonella, but the mutagenicity of BOP was not reduced. The effects of excision repair on in vitro mutagenesis induced by the direct-acting analogs N-(2-oxopropyl)-N-nitrosourea (OPNU), a methylating agent, and N-(2-hydroxypropyl)-N-nitrosourea (HPNU), a hydroxypropylating agent, were also examined. Mutagenesis by HPNU, but not OPNU was very sensitive to excision repair. Thus BOP appears to lead to mutagenesis via methylation, while mutagenesis by BHP apparently proceeds via hydroxypropylation. BOP, BHP, OPNU and HPNU were several times less mutagenic in hisG428 than hisG46 strains. In contrast to hisG46 strains, which are reverted mainly by base-pair substitutions at G:C base pairs, hisG428 strains are generally more sensitive to mutagenesis at A:T base pairs. Taken together the above results and observations that > 90% of the adducts from BOP and BHP were alkylguanines, suggest that the major premutagenic adducts produced from BOP and BHP are alkylguanines as opposed to other alkylated bases. BOP and BHP were weak mutagens in the Salmonella/S-9 mutagenesis assay using hamster liver S-9 fraction. When compared with results in the HMA, BOP and BHP were orders of magnitude less mutagenic in vitro. This observation suggests: (i) the pathways or enzymes involved in the activation of these carcinogens (although uncertain) may be different in vivo and in vitro; or (ii) the pathways for the in vitro and in vivo metabolism may be similar, but the conditions used for the in vitro activation of these nitrosamines are inadequate to generate significant levels of nitrosamine metabolites.

Animals↗

PubMed1992

Mutational specificities of N-nitrosamines in a host-mediated assay: comparison with direct-acting N-nitroso compounds in vitro and an approach to deducing the nature of ultimate mutagens in vivo.

The mutational activities and specificities of several N-nitrosamines in Salmonella recovered from mouse liver in the host-mediated assay (HMA) were compared with the specificities of related direct-acting N-nitroso compounds in vitro. The specificities of the direct-acting methyl, ethyl, propyl, and 2-hydroxypropyl compounds were all different and presumably are attributable to the DNA adducts resulting from the corresponding alkyldiazonium or carbonium ions. Introduction of a 2-hydroxyl group greatly influenced the mutational specificity. The 2-oxopropyl compound showed the same specificity as the methyl compound. This result is consistent with one of the known breakdown pathways of the oxopropyl diazonium ion (or related reactive species), which leads to a methyl diazonium ion. The N-nitrosodialkylnitrosamines N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and N-nitrosodipropylamine (NDPA), which all require metabolic activation, showed specificities in the HMA similar to those of their direct-acting counterparts. The cyclic nitrosamine N-nitrosopyrrolidine was weakly active in the HMA, although its direct-acting derivative was a potent mutagen in vitro. The results for NDMA and NDEA were consistent with most previous studies of the metabolism of these compounds in vivo. However, NDPA can yield methylating, and probably hydroxypropylating, species in addition to propyldiazonium ion. As the specificity of NDPA was similar to that of a propylating agent, NDPA appears to lead to genotoxic products in the mouse liver mainly by direct alpha-hydroxylation. The initial results described here indicate that mutational specificity in the HMA can be used to deduce metabolic pathways leading to genotoxic products when the appropriate proximate mutagens are available as standards. Furthermore, we observed a reasonable correlation between potency in the HMA and hepatocarcinogenesis.

Animals↗

PubMed1989

An important role for cytosol in the microsomal metabolism of N-nitrosodimethylamine to a mutagen: evidence for two different mutagenic metabolites.

Microsomal-mediated mutagenesis induced by N-nitrosodimethylamine (NDMA) in Salmonella TA100 at neutral pH was only slightly affected by cytosol and was similar in its threshold type dose-response curve to mutagenesis induced by direct-acting N-nitroso-N-methyl compounds. However, mutagenesis in strain TA104 was greatly enhanced by cytosol and this mutagenesis did not exhibit a threshold. In the presence of microsomes alone NDMA was more potent in TA100 than TA104, but in the presence of microsomes plus cytosol (S-9 fraction) this order was reversed at the doses tested. A possible explanation for these results is that NDMA is metabolized by microsomes to a mutagen (presumably methyldiazonium ion; MDI) that is more potent in TA100 than in TA104, but in the presence of S-9 fraction a fraction of the NDMA is metabolized by a pathway leading to a different mutagen with a different specificity. The ratio of metabolism via these pathways appears to be dependent on pH.

Animals↗

PubMed1988

Different mutational profiles induced by N-nitroso-N-ethylurea: effects of dose and error-prone DNA repair and correlations with DNA adducts.

The DNA adducts and mutational profile produced by N-nitroso-N-ethylurea (ENU) in Salmonella are examined. The adduct profile produced by ENU in isolated DNA and at two doses in Salmonella were similar, with one exception: O6-ethylguanine (O6-EtG) was not detected at the low dose in Salmonella. This adduct was presumably repaired by a constitutive repair system. The premutagenic adducts, O2-ethyl-thymidine (O2-EtdT) and O4-ethylthymidine (O4-EtdT) were detected, with the former adduct present at higher levels. The mutational profile was also determined at the same doses by utilizing a system involving a series of histidine auxotrophs of Salmonella with differing mutagenic specificities and a further subclassification of the revertants. Four different patterns of mutagenesis were observed; these were dependent on dose and on the presence or absence of the plasmid pKM101. The mutational spectrum produced at the higher dose in strains without the plasmid consisted mainly of GC----AT transitions. At the high dose, in strains harboring pKM101, three base changes contributed importantly to the mutational spectrum: GC----AT, AT----GC, and AT----CG. At the low dose in the strains without pKM101, little mutagenesis was observed, and in strains containing pKM101, mutagenesis was greatly enhanced with the most frequent mutations resulting from AT----GC and AT----CG base changes. O6-EtG was presumably responsible for the bulk of the GC----AT transitions at the high dose. Calculations and evidence are presented indicating that O2-EtdT is responsible for at least some of the mutagenesis that occurs at AT base pairs. O4-EtdT and O2-EtdT are probably responsible for a major fraction of the AT----GC transitions, and we suggest that error-prone repair activity acting on O2-EtdT and/or O4-EtdT results in the AT----CG transversions.

Alkylation↗

PubMed1988

Mutagenic activity and specificity of N-nitrosomethylaniline and N-nitrosodiphenylamine in Salmonella.

The carcinogenic nitrosamines, N-nitrosomethylaniline (NMA) and N-nitrosodiphenylamine (NDphA), which have been previously reported negative or very weakly mutagenic in the Salmonella/microsome assay, were found to be mutagenic in the hisG428 Salmonella strain, TA104. NMA was moderately potent and NDphA was about 10% as potent. Mutagenesis by both compounds was dependent on the uvrB mutation and enhanced in strains harboring the plasmid, pKM101. The mutational specificities of NMA and NDphA for base-pair substitutions were determined by assaying their activities in several mutants which are reverted by a limited number, or a single type of base-pair substitution mutation, and additionally by subclassification of revertants. NMA induced predominantly AT----CG transversions and NDphA induced AT----TA transversions. The specificity of NMA and NDphA for mutagenesis at AT base pairs and the lack of sensitivity of the previously employed hisG46 strains for these base changes may be the reason for the previous reports on the lack of mutagenic activity of these compounds. This specificity is quite unusual for nitrosamines and is consistent with the hypothesis that NMA and NDphA lead to DNA damage of different nature than that produced by other nitrosamines.

Base Sequence↗

PubMed1988

DNA binding by [2,5-14C]N-nitrosopyrrolidine in excision-repair proficient and deficient strains of Salmonella. Evidence for a major premutagenic adduct.

Little is known about the nature and possible genotoxic effects of the DNA adducts formed by N-nitrosopyrrolidine (NPYR) in whole animals. DNA binding in DNA isolated from [2,5-14C]NPYR-treated Salmonella was studied and attempts were made to monitor DNA adducts and correlate DNA binding with mutagenesis. NPYR was metabolized by hamster liver S-9 fraction in the presence of S.typhimurium TA1535 (uvrB-) or TA1975(uvrB+). DNA isolated from TA1535 contained about three times as much radioactivity as that isolated from TA1975, and NPYR-induced mutagenesis was several-fold higher in TA1535. The fraction of radioactivity incorporated into TA1535 was approximately 10(-5). Thermal hydrolysis of the 14C-containing DNA at neutral pH, followed by precipitation, released approximately 2/3 of the radioactivity into the supernatant. HPLC analysis of the supernatant revealed one major peak. This peak was absent in DNA from TA1975. Acid hydrolysis of the DNA precipitate after neutral hydrolysis released most of the residual radioactivity. Several small peaks were observed after HPLC analysis of the TA1535 acid hydrolysate or the TA1975 acid hydrolysate. These results demonstrate that NPYR is capable of binding to Salmonella DNA yielding one major product after hydrolysis and this DNA binding product appears to be repaired by the excision repair system. The fact that the major peak of radioactivity released from Salmonella is only found in the strain which is efficiently reverted by NPYR suggests that mutagenesis is dependent on the DNA modification leading to this peak.

Animals↗

PubMed1987

Effects of UV repair, error-prone repair and critical site of mutation on mutagenesis induced by N-nitrosamines.

Many N-nitrosamines have been assayed for mutagenic activity in bacteria but few have been systematically compared in a series of strains. In this study through the use of several Salmonella tester strains, we have examined the effects of Uvr repair, error-prone repair, and the critical site for mutation (GC or AT base pair) on the mutagenic activities of a diverse group of N-nitrosamines. We have employed the histidine autotrophs, TA1975 (uvrB+), TA1535 (uvrB-) and TA100 (uvrB-/pKM101) which are hisG46 strains, sensitive mainly to G-C base damage, and TA104 (uvrB-/pKM101), a hisG428 strain, which can be reverted at the hisG428 locus by damage to A-T base-pairs, or by suppression at G-C base pairs. The N-nitrosamines studied were, N-nitroso: dimethylamine, diethylamine, dipropylamine, dibutylamine, pyrrolidine, piperidine, morpholine, methylbenzylamine, bis-(2-hydroxypropyl)amine, bis-(2-oxopropyl)amine and 3,4-dichloropyrrolidine. For all of the nitrosamines larger than diethylnitrosamine (except for methylbenzylnitrosamine) mutagenesis was greatly enhanced (3-20 X) by the lack of uvrB activity, indicating that the DNA adducts produced by these nitrosamines can be classified as "bulky adducts". For most nitrosamines the plasmid, pKM101, enhanced mutagenesis in hisG46 strains, several fold, suggesting that error-prone DNA repair plays a role in mutagenesis by these compounds. All of the compounds tested were more mutagenic in TA100 than TA104 except diethylnitrosamine and methylbenzylnitrosamine which were more potent in TA104. Revertants induced by all of the nitrosamines in TA100 were due predominantly to damage at G-C base pairs. Revertants induced by all the nitrosamines except diethylnitrosamine and dibutylnitrosamine resulted mainly from damage to G-C base pairs in TA104.

Base Sequence↗

PubMed1987

N-nitrosamines: bacterial mutagenesis and in vitro metabolism.

Many nitrosamines are potent mutagens. The rate-limiting step in their in vitro metabolism to mutagens is usually a single enzymatic reaction catalyzed by one or more of the many cytochrome P-450-dependent mixed-function oxidases present in the microsomal cell fraction. Current evidence indicates that this reaction activates nitrosamines to alpha-hydroxynitrosamines, which have half-lives on the order of seconds. This product decomposes to an aldehyde and a much shorter-lived ultimate metabolite which is probably an alkyl diazonium ion or an alkyl carbocation. This may react with DNA leading to premutagenic adducts. Such adducts represent a very small fraction of the ultimate mutagen, with the rest reacting with water to yield the corresponding alcohol. Evidence for this pathway includes (1) the observation of deuterium isotope effects in metabolism and mutagenesis, (2) products (aldehydes, alcohols, and N2) consistent with this pathway, (3) studies on metabolism of nitrosamines using purified cytochrome P-450, (4) formation of DNA adducts such as O6-alkylguanines which are consistent with those expected from the ultimate mutagen, (5) expected products and genotoxic effects of other sources of activated nitrosamines, e.g., alpha-acetoxynitrosamines, alkanediazotates and related compounds. Hydroxylation of nitrosamines at other positions also occurs in vitro (usually to a lesser extent), but these products are generally stable and must be further metabolized to exert mutagenic effects (with the exception of N-nitrosoalkyl(formylmethyl)amines, which are direct-acting mutagens). Because only low percentages of nitrosamines are metabolized in vitro, the contribution to mutagenesis by secondary metabolism is small. In this respect, in vitro metabolism can differ significantly from in vivo metabolism. Bacterial mutagenesis by nitrosamines has most often been studied in Salmonella typhimurium and to a lesser extent E. coli. Mutagenesis by nitrosamines generally requires a source of microsomes (a 9000 X g supernatant fraction is often used), and NADPH. Liver fractions from Aroclor-1254- or PB-induced rodents have been most frequently employed but liver fractions from untreated animals, and homogenates of other organs (lung, kidney, nasal mucosa, and pancreas) have also been utilized. Liver homogenates from humans are generally similar to those from untreated rats in metabolizing nitrosamines to mutagens but large interindividual variations are observed. Mutagenesis is often most effective using a liquid preincubation, a slightly acidic incubation mixture and hamster liver fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylation↗