Effect of hydrogen peroxide on nitric oxide (NO)-induced mutagenicity in Salmonella typhimurium.

Nitric oxide (NO) has been reported to impart, alone or in combination with reactive oxygen species (ROS), the cytotoxicity and putative genotoxicity associated with the immunological response. The present study examined the change in the mutagenic activity profile of the NO-donor spermine NONOate (SperNO) as a result of introduction of hydrogen peroxide (H(2)O(2)) to the Ames assay. The aim was to determine whether the assay could detect H(2)O(2)-induced co- or anti-mutagenic effects on NO-induced mutagenesis, and the Salmonella typhimurium base-pair substitution tester strain TA1535 provided an appropriate tool. While TA1535 was shown by the authors and others to be strongly sensitive to NO-induced mutagenesis, it has also been shown to be insensitive to H(2)O(2)-induced mutagenicity [1,2]. When H(2)O(2) (0.25-4.0 micromol/pl) was added directly to cells treated with SperNO (0.01-1.0 micromol/pl), co-mutagenicity was not detected, but a drop in reversion count and detectable toxicity was observed, especially at doses > 0.1 micromol/pl. When glucose/glucose oxidase (GOX) or reduced glutathione (GSH) were used as H(2)O(2)-generation systems the results varied. Reversion induced by SperNO (1 micromol/pl) was moderately enhanced by GOX (10-20 mUnits/pl), but the increase albeit reproducible did not reach a doubling (co-mutagenicity). GOX (40 micromol/pl) induced a reduction in reversion count, but no visible toxicity. On the other hand, GSH (20- 80 micromol/pl) gave a strong co-mutagenic effect. Co-mutagenicity was highest (> 5x) at 80 micromol/pl GSH and 0.1 micromol/pl SperNO. Based on these findings, it could be concluded that a) H(2)O(2), when steadily generated in the cell, has a modulatory effect on NO-mutagenicity, and such a conclusion is not inconsistent with the wide range of responses reported for the two chemicals, and/or b) the observed co-mutagenic effects of GSH may not be attributable solely to H(2)O(2) generation.

Rapid analysis of base-pair substitutions induced by mutagenic drugs through their oxygen radical or epoxide derivatives.

Among the drugs that induce base-pair substitution mutations in the Salmonella reversion assay are the nitric oxide (NO)-delivery drug, diethylamine NONOate (DeaNO), and the ovarian cancer chemotherapeutic drug, treosulphan (TE). The present study compared the mutation spectra generated by DeaNO and TE in the hisG46 strains, TA1535 and TA100, the hisG428 strain, TA102, and the six Ames II 7000 series strains. Using these strains, it was feasible to conduct rapid analysis of the type and magnitude of induced mutation without resorting to DNA amplification and sequencing. A putative hydrolysis product of TE, 1,2:3,4-diepoxybutane (DEB), and hydrogen peroxide (H(2)O(2)) were included in the study to allow for further comparisons between epoxide-induced damage and that induced by the hydroxyl radical. TE (0.93 micromole/pl) induced 16. 8-fold-over-background reversion or a mutagenicity ratio (MR) of 16. 8 in TA1535. The response was weaker in TA100 (MR of 3), and negative in strain TA102. Only two Ames II strains demonstrated sensitivity to TE, and they were TA7004 (CG:AT) and TA7005 (GC:AT). Like TE, DeaNO (33 micromole/pl) was mutagenic in TA1535 (MR of 24.6), TA100 (MR of 5.3), TA7004 (MR of 13.7), and TA7005 (MR of 7.7), and non-mutagenic in TA102. These results showed a preferential sensitivity to reversion of the -CCC-target in TA100 and TA1535, and a lack of sensitivity to reversion of the -TAA-target in TA102. In addition, they elucidated the selectivity of the Ames II strains, with AT targets showing little or no sensitivity to reversion. The TE-epoxide derivative DEB was mutagenic in TA1535 and TA7004, but in contrast to TE, DEB was mutagenic in TA102. Interestingly, TA102 was reverted by DEB and H(2)O(2) but not by TE or DeaNO. This study showed that analysis of mutations is achievable using the battery of strains listed above. The fact that DNA damage can be detected by reversion at specific bases offers a tool for understanding the mechanisms through which drugs may exert their DNA and cellular damage.

Spectra of spontaneous frameshift mutations at the hisD3052 allele of Salmonella typhimurium in four DNA repair backgrounds.

To characterize the hisD3052 -1 frameshift allele of Salmonella typhimurium, we analyzed approximately 6000 spontaneous revertants (rev) for a 2-base deletion hotspot within the sequence (CG)4, and we sequenced approximately 500 nonhotspot rev. The reversion target is a minimum of 76 bases (nucleotides 843-918) that code for amino acids within a nonconserved region of the histidinol dehydrogenase protein. Only 0.4-3.9% were true rev. Of the following classes, 182 unique second-site mutations were identified: hotspot, complex frameshifts requiring DeltauvrB + pKM101 (TA98-specific) or not (concerted), 1-base insertions, duplications, and nonhotspot deletions. The percentages of hotspot mutations were 13.8% in TA1978 (wild type), 24.5% in UTH8413 (pKM101), 31.6% in TA1538 (DeltauvrB), and 41.0% in TA98 (DeltauvrB, pKM101). The DeltauvrB allele decreased by three times the mutant frequency (MF, rev/10(8) survivors) of duplications and increased by about two times the MF of deletions. Separately, the DeltauvrB allele or pKM101 plasmid increased by two to three times the MF of hotspot mutations; combined, they increased this MF by five times. The percentage of 1-base insertions was not influenced by either DeltauvrB or pKM101. Hotspot deletions and TA98-specific complex frameshifts are inducible by some mutagens; concerted complex frameshifts and 1-base insertions are not; and there is little evidence for mutagen-induced duplications and nonhotspot deletions. Except for the base substitutions in TA98-specific complex frameshifts, all spontaneous mutations of the hisD3052 allele are likely templated. The mechanisms may involve (1) the potential of direct and inverted repeats to undergo slippage and misalignment and to form quasi-palindromes and (2) the interaction of these sequences with DNA replication and repair proteins.

Formation of 8-hydroxy-2'-deoxyguanosine following treatment of 2'-deoxyguanosine or DNA by hydrogen peroxide or glutathione.

We have demonstrated that free radicals generated by hydrogen peroxide (H2O2), in the presence of divalent iron (Fe2+) and a chelator (EDTA), oxidize 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG). The 8-OHdG formed by this reaction was isolated and quantitated using reverse-phase HPLC with UV and electrochemical detection. A 1-h incubation of dG with H2O2 caused a 50% increase in 8-OHdG over background, which increased to 100% after 2 h. However, when an H2O2-generating system [glutathione (GSH), Fe2+, EDTA] was used, there was no increase in 8-OHdG yield after the 1-h incubation, but up to a 50% increase over background was observed with GSH after 2-h incubation. Attempts to detect increased levels of 8-OHdG after H2O2- or GSH-treatment of purified calf thymus or rat DNA, or purified Salmonella typhimurium DNA were not successful. This may have been because the treatment procedures used generated 8-OHdG in the control samples at sufficiently high levels to mask any H2O2-induced responses that may have been present. This artifactual production of 8-OHdG has presented a problem in all in vitro studies to date. In contrast, treatment of Salmonella cells (strain TA104) with increasing concentrations of H2O2, caused a doubling in the 8-OHdG yield. GSH-treatment of strain TA104 cells under the same conditions did not result in an increase of 8-OHdG. The study presented here shows that the ubiquitous molecule H2O2 can play a major role in DNA oxidation, mutation, and damage.

Benzo[b]fluoranthene: tumorigenicity in strain A/J mouse lungs, DNA adducts and mutations in the Ki-ras oncogene.

The polycyclic aromatic hydrocarbon benzo[b]fluoranthene (B[b]F) is a pervasive constituent of environmental combustion products. We sought to examine the lung tumorigenic activity of B[b]F in strain A/J mice, to study the relationship between formation and decay of B[b]F-DNA adducts and to examine mutations in the Ki-ras proto-oncogene in DNA from B[b]F-induced tumors. Mice were given i.p. injections of 0, 10, 50, 100 or 200 mg/kg body wt and lung adenomas were scored after 8 months. B[b]F induced significant numbers of mouse lung adenomas in a dose-related fashion, with the highest dose (200 mg/kg) yielding 6.95 adenomas/ mouse, with 100% of the mice exhibiting an adenoma. In mice given tricaprylin, the vehicle control, there were 0.60 adenomas/mouse, with 55% of the mice exhibiting an adenoma. Based on dose, B[b]F was less active than benzo[a]pyrene. DNA adducts were analyzed qualitatively and quantitatively by 32P-post-labeling in lungs of strain A/J mice 1, 3, 5, 7, 14 and 21 days after i.p. injection. Maximal levels of adduction occurred 5 days after treatment with the 200 mg/kg dose group, producing 1230 amol B[b]F-DNA adducts/microgram DNA. The major B[b]F-DNA adduct was identified by co-chromatography as trans-9, 10-dihydroxy-anti-11, 12-epoxy-5-hydroxy-9, 10, 11, 12-tetra-hydro-B[b]F-deoxyguanosine. Approximately 86% of the tumors had a mutation in codon 12 of the Ki-ras oncogene, as determined by direct DNA sequencing of PCR-amplified exon 1 and single-stranded conformation polymorphism analysis. Analysis of the Ki-ras mutation spectrum in 25 of 29 B[b]F-induced tumors revealed the predominant mutation to be a G-->T transversion in the first or second base of codon 12, congruous with the DNA adduct data. Our data are consistent with previous reports in mouse skin implicating a phenolic diol epoxide as the proximate carcinogenic form of B[b]F that binds to guanine.

Mutation spectra in salmonella of chlorinated, chloraminated, or ozonated drinking water extracts: comparison to MX.

Drinking water samples were prepared in a pilot-scale treatment plant by chlorination (Cl2), chloramination (NH2Cl), ozonation (O3), or O3 followed by Cl2 or NH2Cl; and the nonvolatile acidic organics of the raw and treated waters were extracted by XAD/ethyl acetate and evaluated for mutagenicity in Salmonella (-S9). The extracts were 2-8 times more mutagenic in TA100 than in TA98, and the mutagenic potencies of the water extracts ranked similarly in both strains: Cl2 > O3 + Cl2 > NH2Cl > O3 + NH2Cl > O3 > raw. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), which was estimated to account for approximately 20% of the mutagenic activity of the extracts, was shown to be the most potent compound tested thus far in a prophage-induction assay in Escherichia coli and a forward-mutation assay in Salmonella TM677. The mutations in approximately 2,000 revertants of TA98 and TA100 induced by MX and the water extracts were analyzed by colony probe hybridization and polymerase chain reaction/DNA sequence analysis. The water extracts and MX produced similar mutation spectra, which consisted in TA100 of predominantly of GC-->TA transversions in the second position of the CCC (or GGG) target of the hisG46 allele. This spectrum resembles that produced by large aromatic compounds and is distinct from that produced by alkylating agents and the semivolatile drinking water mutagen dichloroacetic acid. In TA98, MX and those water extracts resulting from the introduction of the chlorine atom produced 50-70% hotspot 2-base deletions and 30-50% complex frameshifts (frameshifts with an adjacent base substitution--mostly GC-->TA transversions as found in TA100). No other compound or mixture is known to induce such high frequencies of complex frameshifts. These results suggest that MX and "MX-like" compounds (possibly halogenated aromatics, such as halogenated polycyclic aromatic hydrocarbons) account for much of the mutagenic activity and specificity of the nonvolatile organics in drinking water and that these halogenated organics are especially capable of promoting misincorporation by the DNA replication complex. This study provides further evidence that the mutation spectrum of a complex mixture reflects the dominance of one or a few classes of chemical mutagens within the mixture.

Molecular analysis of mutations induced at the hisD3052 allele of Salmonella by single chemicals and complex mixtures.

More single chemicals and complex environmental mixtures have been evaluated for mutagenicity at the hisD3052 allele of Salmonella, primarily in strain TA98, than in any other mutation assay. The development of colony probe hybridization procedures and the application of the polymerase chain reaction and direct DNA sequencing has permitted rapid molecular access to this allele. We discuss these techniques and the resulting mutation spectra that have been induced by a variety of environmental mutagens and complex mixtures. A common GC or CG deletion within a hot-spot region of the sequence dominates most of the spectra. In addition to this two-base deletion, we have recovered about 200 other types of mutations within the 72-base target for reversion of the hisD3052 allele. These include a variety of deletions (as large as 35 bases), duplications (as large as 46 bases), and complex mutations involving base substitutions. The quasipalindromic nature of the target sequence and its potential to form DNA secondary structures and slippage mismatches appear to be an important basis for the mutability of this allele.

Molecular analysis of mutations induced by the intercalating agent ellipticine at the hisD3052 allele of Salmonella typhimurium TA98.

We have used DNA colony hybridization, the polymerase chain reaction (PCR), and direct DNA sequencing to determine the mutations induced by the intercalating agent ellipticine in Salmonella typhimurium TA98 in the presence of S9. Of 400 ellipticine-induced revertants that were selected at a mutant yield that was ninefold over the background, 85.5% contained a GC or CG deletion within a common CGCGCGCG hotspot; this deletion occurred among 47% of the spontaneous revertants. In addition to this hotspot, the ellipticine spectrum contained two deletion warmspots that reside opposite each other in looped-out regions of a possible DNA secondary structure. Ellipticine and its metabolites likely revert Salmonella strain TA98 by forming DNA adducts that promote slippage-mismatches and by stabilizing these slipped mismatched sequences via intercalation. The involvement of these mechanisms, along with a likely role for DNA secondary structures and a possible role for DNA gyrase, may account for the site specificity exhibited by ellipticine in strain TA98.

The modulatory effects of tryptamine and tyramine on the S9-mediated mutagenesis of IQ and MeIQ in Salmonella strain TA98.

The S9-mediated mutagenesis of IQ and MeIQ in Salmonella strain TA98 was modulated by introduction to the assay of tryptamine or tyramine. Both biogenic amines inhibited or enhanced the mutagenic response as a function of amine concentration, strain of rat used as the S9 source, and the IQ-type mutagen tested. Enhancement of IQ mutagenesis by tryptamine (10-80 microM) was observed in the presence of S9 preparations derived from Aroclor 1254-pretreated Fischer rats; the enhancing effect ceased at tryptamine concentrations > 160 microM. When Sprague-Dawley-S9 or Wistar-S9 were used for activation, the enhancement of IQ mutagenesis by tryptamine shifted to inhibition at tryptamine concentrations > 40 microM, with Sprague-Dawley-S9, and > 20 microM, with Wistar-S9. By contrast, MeIQ-mutagenesis was enhanced by tryptamine (10-160 microM), regardless of the rat strain used as S9 source. Tyramine was a weaker enhancer of MeIQ mutagenesis than was tryptamine and, unlike tryptamine, its inhibitory effects on IQ mutagenesis were observed only with Wistar-S9. Tryptamine (10-80 microM) inhibited cytochromes P450IA1 and P450IA2 activities, monitored by the O-deethylation of ethoxyresorufin and Glu-P-1 mutagenesis in TA98, respectively. These data suggest that the effects of biogenic amines on IQ and MeIQ bioactivation are complex. Furthermore, this study demonstrates that tryptamine and tyramine act both as enhancers (comutagens) and as inhibitors (antimutagens) of IQ and MeIQ mutagenesis, depending on the testing conditions.

Isolation of the mutagenic and DNA adduct-inducing components from a commercial preparation of HC blue 1 using Salmonella (TA98) bioassay-directed HPLC fractionation.

In the present study we report the separation of the mutagenic impurities from the nitrophenylenediamine hair dye HC Blue 1. This was accomplished by bioassay-directed HPLC fractionation, using Salmonella strain TA98 and reverse phase HPLC analysis. The mutagenic fraction eluted between 80 and 90% methanol, whereas the HPLC fraction containing the parent compound HC Blue 1 eluted with 30% methanol and was non-mutagenic. 100% of the mutagenic activity applied to the column was recovered in fractions that did not possess the blue color of HC Blue 1. Also, HPLC-purified HC Blue 1 did not form DNA adducts (32P-postlabeling) in Salmonella strain TA98. On the other hand, commercial HC Blue 1 and the mutagenic fraction derived from commercial HC Blue 1 (HPLC-isolated) gave similar DNA-adduct profiles that consisted of 7 adducts. DNA adduction was examined concomitantly with mutagenicity and toxicity studies on the HC Blue 1 samples in TA98. The data indicated that, in Salmonella, both the mutagenicity and DNA adduction of commercial HC Blue 1 are due to impurities and not the parent compound.

Effects of Salmonella genotypes and testing protocols on H2O2-induced mutation.

Hydrogen peroxide (H2O2) was shown to be mutagenic in a number of strains of Salmonella typhimurium. Strain SB1106p (hisC3108, hisO1242, pKM101), a newly-constructed strain carrying the histidine mutation at a UGA chain-terminating codon, was more responsive to H2O2 than TA104 or TA102, the two hisG428 strains originally developed for detecting oxidative mutagens. The largest proportional increase in revertants of strain TA104 was in the fraction of intragenic deletions. Three other strains (TA97, SB1111 and SB1106) gave unequivocal positive responses to H2O2 in both the liquid pre-incubation procedure and standard plate incorporation procedure. The response of TA100 varied among experiments, ranging from negative to a weak positive. Variations in the catalase content among the tester strains did not correlate with the relative responses obtained in the mutagenicity assays.

Comparative metabolism and genotoxicity of the structurally similar nitrophenylenediamine dyes, HC Blue 1 and HC Blue 2, in mouse hepatocytes.

Previous studies indicated that HC Blue 1 induced heptocellular carcinomas in B6C3F1 mice whereas the structurally similar nitroaromatic amine HC Blue 2 did not. In an attempt to elucidate the biochemical mechanisms responsible for their different carcinogenic potencies, comparative metabolism and genetic toxicity studies were undertaken. Eighteen-hour urinary recovery of administered radioactivity was equivalent for both compounds following oral gavage (100 mg/kg) in female B6C3F1 mice. By HPLC analysis, HC Blue 1 yielded 3 major polar metabolite peaks, one of which was susceptible to glucuronidase. In vivo metabolism of HC Blue 2 yielded a single major metabolite peak which was not hydrolyzed by glucuronidase. Metabolism by B6C3F1 mouse hepatocytes yielded metabolite profiles which were qualitatively similar to the profiles observed after in vivo metabolism. HC Blue 1 was metabolized by hepatocytes at approximately twice the rate of HC Blue 2. Cytogenetic evaluations of mouse hepatocytes after in vitro treatment indicated HC Blue 1 was more potent than HC Blue 2 in inducing chromosomal aberrations while both chemicals showed weak activity for inducing sister-chromatid exchanges. Furthermore, in the V79 cell metabolic cooperation assay, HC Blue 1, but not HC Blue 2, inhibited cell-to-cell communication suggesting a non-genotoxic activity may be present for HC Blue 1. It is concluded that qualitative and quantitative differences exist in the metabolism of these compounds and that genotoxic as well as nongenotoxic effects may contribute to their different carcinogenic potencies.

Effect of tryptamine on the mutagenic activity of 2-amino-3-methylimidazo(4,5-f) quinoline (IQ) and related azaarenes in the Ames test.

The bioactivation of the azaarenes 2-amino-3-methylimidazo(4,5-f) quinoline (IQ), 2-amino-3,4-dimethylimidazo(4,5-f) quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo(4,5-f) quinoxaline (MeIQx) to mutagens by hepatic S9 preparations derived from Aroclor-pretreated Wistar rats was inhibited by tryptamine (2-50 microM). However, with similar preparations derived from Sprague-Dawley rats, bioactivation of IQ and MeIQx was less markedly inhibited by tryptamine while metabolic activation of MeIQ was enhanced. In the absence of cytosol, activation of IQ by microsomal preparations of both rat strains was inhibited by tryptamine. Cytosolic fractions from both rat strains were incapable of activation of IQ per se but increased the mutagenicity of the microsomal metabolite(s). This potentiation of the mutagenic activity by cytosol derived from Wistar rats was also inhibited by tryptamine whereas no significant inhibition was observed with cytosolic preparations from Sprague-Dawley rats. There appear to be two alternative pathways of microsomal metabolism of IQ: a tryptamine-sensitive pathway, probably involving the formation of the N-hydroxymetabolite; and a tryptamine-insensitive pathway producing weakly mutagenic or non-mutagenic metabolites which are activated to a potent mutagen by the cytosol. The tryptamine-insensitive pathway appears to be the major route of activation of the azaarenes in microsomal preparations from Sprague-Dawley rats and the principal activation route for MeIQ in both rat strains.

Metabolic activation of 2-amino-3-methylimidazo(4,5-f)quinoline by hepatic preparations--contribution of the cytosolic fraction and its significance to strain differences.

In accordance with previous studies the bioactivation of 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) to mutagens in the Ames test was preferentially catalysed by the 3-methyl-cholanthrene-induced cytochromes P-448, in contrast to the phenobarbital-induced forms of the cytochrome. The mutagenicity of IQ catalysed by microsomes, in the absence of cytosol, was much lower when compared with that observed with S9 fractions. Cytosol itself could not activate IQ but markedly potentiated the microsome-mediated mutagenicity of the carcinogen. The effect of the cytosol was still evident when microsomal metabolism was terminated, indicating that the cytosol contains enzyme(s) that can further convert the microsome-generated metabolites of IQ to more potent mutagens. The cytosolic enzyme(s) were inducible by pre-treatment of the rats with Aroclor 1254. The higher efficiency of activation of IQ to mutagens by Sprague-Dawley S9 mixes when compared with similar preparations from the Wistar rat could be attributed not only to differences in the rate of microsomal metabolism but also to the higher ability of the Sprague-Dawley cytosolic fraction in further metabolizing the microsome-generated metabolite(s). The present study demonstrates clearly that the mutagenic response of this compound in the Ames test may be profoundly modulated by the cytosolic fraction and its role in the metabolic activation of pre-mutagens merits further investigation.