The unusual effect of pKM101 on the mutagenicity of acetaldehyde oxime in Salmonella typhimurium

Acetaldehyde oxime was found to induce more revertants in Salmonella typhimurium strain TA1535 than in TA100 in the absence of S9 metabolic activation. TA100 was originally constructed from TA1535 by the addition of the plasmid pKM101, carrying mucAB which generally enhances sensitivity to the mutagenic effects of chemicals. The role of pKM101 in lowering the sensitivity to acetaldehyde oxime was explored by: (1) increasing the incubation time of the selective agar plates from 2 to 3 days; (2) using a new strain, isogenic to TA100, constructed by introducing pKM101 into the TA1535 isolate used in these experiments; (3) by testing a strain constructed by inserting into TA1535 a plasmid carrying mucAB but otherwise unrelated to pKM101. Each of these alterations increased the number of revertants per plate in the presence of acetaldehyde oxime, indicating that the apparent nonmutagenicity of this chemical in TA100 is due to multiple factors.     

The correlation between benzo[a]pyrene-induced mutagenicity and DNA adduct formation in Salmonella typhimurium TA100

In an attempt to stabilize the dose response in the Salmonella typhimurium test (STT), the use of DNA-bound products from BP was evaluated as a measure of the biologically effective dose. In addition to the previously documented interlaboratory variation, we observed a 3-fold difference in the dose response of TA100 to BP even when the assay was repeated with the same experimental conditions. When overall BP-DNA adduct formation was related to the level of His+ revertants, a series of responses emerged with two predominating. In the first type of response around 70 revertants per plate were generated for every BP molecule bound per 10(6) nucleotides of cellular DNA. The second response gave about 1400 revertants per plate for one BP bound in every 10(6) nucleotides. Several intermediates curves were also detected. The variation in the mutational response to binding levels occurred regardless of the source of S9 or the growth stage of the cells. These experiments indicate that there was no constant level of DNA damage that would lead to a specified number of revertants of TA100 by BP and that DNA modification was not solely responsible for mutagenic potency. It is possible that an induction of an error-prone repair function of the muc gene carried by the plasmid pKM101 in TA100 may be affecting the relationship between the measured adduct level and reversion frequency.     

Mutagenicity studies of p-substituted benzyl derivatives in the Ames Salmonella plate-incorporation assay

The mutagenicity of 24 benzyl derivatives, containing a variety of substituents and leaving groups, were assayed in strain TA100 using the Ames plate-incorporation assay. p-Nitrobenzyl chloride (12 000 revertants/mumole), p-nitrobenzyl tosylate (6100 revertants/mumole), and p-acetoxybenzyl chloride (100 revertants/mumole) were mutagenic; none of the remaining 21 compounds were mutagenic. p-Nitrobenzyl chloride was also found to be mutagenic in strain TA98 (700 revertants/mumole), but not in strain TA98NR (a strain deficient in nitro reductase activity). p-Acetoxybenzyl chloride was nonenzymatically hydrolyzed to p-hydroxybenzyl alcohol and p-acetoxybenzyl alcohol. These findings suggest that nitrobenzyl derivatives were mutagenic due to nitro reductive metabolism and that p-acetoxybenzyl chloride was mutagenic due to the intermediate formation of p-hydroxybenzyl chloride during the hydrolysis of p-acetoxybenzyl chloride.     

Mutagenicity of beta-glucuronidase in the Ames test

The enzyme preparation beta-glucuronidase/arylsulphatase from Helix pomatia (Boehringer) caused base-pair substitutions in Salmonella typhimurium TA100 and TA1535 strains within the dose range of 0.50-50 microliter per plate. No effect was observed in the TA98 strain. The presence of S9 mix did not substantially affect the mutagenic potential of beta-G. The number of induced revertants decreased continually from experiment to experiment carried out in the course of 12 weeks.     

Toxicity and mutagenicity of hexavalent chromium on Salmonella typhimurium.

Four hexavalent and two trivalent chromium compounds were tested for toxicity and mutagenicity by means of the Salmonella typhimurium/mammalian-microsome test. All hexavalent compounds yielded a complete inhibition of bacterial growth at doses of 400 to 800 mug/plate, a significant increase of his(+) revertant colonies at doses ranging from 10 to 200 mug, and no effect at doses of less than 10 mug. The distinctive sensitivity of the four Salmonella strains tested (TA1535, TA1537, TA98, and TA100) suggested that hexavalent chromium directly interacts with bacterial deoxyribonucleic acid by causing both frameshift mutations and basepair substitutions. The latter mutations, which are prevalent, are amplified by an error-prone recombinational repair of the damaged deoxyribonucleic acid. On the average, 1 mumol of hexavalent chromium yielded approximately 500 revertants of the TA100 strain, irrespective of the compound tested (sodium dichromate, calcium chromate, potassium chromate, or chromic acid). The mutagenic potency of the hexavalent metal was not enhanced by adding the microsomal fraction of rat hepatocytes, induced either with sodium barbital or with Aroclor 1254. The two trivalent compounds (chromium potassium sulfate and chromic chloride), with or without the microsomal fraction, were neither toxic nor mutagenic for the bacterial tester strains.     

The mutagenic activity of the S-nitrosoglutathione/glutathione system in Salmonella typhimurium TA1535

S-nitrosoglutathine (GSNO) and reduced glutathione (GSH) were tested for mutagenicity against strain Salmonella typhimurium TA1535 in the Ames Standard plate incorporation assay. Neither GSNO not GSH were mutagenic when tested alone. In combination, the GSNO/GSH system induced a positive mutagenic response that ranged from 3 to 20 x over background at concentrations of 10 to 50 micromol (micromol)per plate, respectively. This mutagenic response can be attributable to the generation nitric oxide, among the many other reactive products generated by the reaction of GSNO with GSH.     

Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His⁺ revertants to a maximum of 4 times over background at a GTN dose of 5 μmol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 μmol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C → T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.     

Induced mutation spectra at the upp locus in Salmonella typhimurium: response of the target gene in the FU assay to mechanistically different mutagens

A novel forward mutation assay has been developed in Salmonella typhimurium based on resistance to 5-fluorouracil (FU). The mutational target in the FU assay was determined to be the uracil phosphoribosyl transferase (upp) gene. To validate the upp gene as a suitable target for monitoring a variety of induced mutations, the mutational specificity was determined for five mechanistically different mutagens. The mutagens included a polycyclic hydrocarbon (benzo[a]pyrene, B[a]P), SN1 and SN2 alkylating agents (N-nitroso-N-methylurea, MNU, and methyl methanesulfonate, MMS, respectively), a frameshift mutagen (ICR-191), and an oxidative-damaging agent (hydrogen peroxide, H2O2). Induced mutation frequencies were measured in the presence and absence of the plasmid pKM101 (strain FU100 and FU1535, respectively). pKM101 renders FU100 more susceptible to induced mutation by providing error-prone replicative bypass of DNA adducts. B[a]P, MMS, and H2O2 failed to induce the mutant frequency in FU1535, demonstrating the dependence of pKM101 on induced mutations with these agents. ICR-191 and MNU were not dependent on pKM101, and did significantly induce mutations in FU1535. In contrast to FU1535, all agents significantly induced mutations in FU100. Approximately 60 independent mutants were sequenced for each agent that significantly induced the mutant frequency above background. The resulting mutational spectra illustrated predictable molecular fingerprints based on known mutagenic mechanisms for each agent. The predominant mutations observed were G:C to T:A transversions for B[a]P, A:T to T:A and G:C to T:A transversions for MMS, G:C to T:A transversions and A:T frameshifts for H2O2, G:C frameshifts for ICR-191, and G:C to A:T transitions for MNU. It can be concluded that the upp gene in the FU assay is a sensitive and suitable target to monitor a variety of induced mutations in Salmonella.     

Platinum(II) complexes generate frame-shift mutations in test strains of Salmonella typhimurium.

cis-Diamminodichloroplatinum(II) (cis-PDD) and diaquoethylenediamineplatinum(II) induce histidine revertants in Salmonella typhimurium strains TA98 (frame-shift mutation) and TA100 (base-pair substitution mutation). A linear dose--response relationship is found with cis-PDD acting on TA98 and TA100. Salmonella typhimurium strains TA1535, TA1537 and TA1538 are not sensitive to the mutagenic action of cis-PDD. All 5 strains are sensitive to the toxic effect of cis-PDD. Platinum(II) complexes induce mutations (frame-shift or base-pair substitution) only in strains carrying the R-factor plasmid.     

Isolation of a mutant of Salmonella typhimurium strain TA1535 with decreased levels of glutathione (GSH-). Primary characterization and chemical mutagenesis studies

A mutant of Salmonella typhimurium strain TA1535 with decreased glutathione (GSH) levels was isolated after treatment with UV and selection for N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) resistance; this GSH- mutant also exhibited increased resistance to MNNG, the methyl analog of ENNG. Estimation of the cellular GSH content showed that the GSH- derivative contained about 20% of the GSH levels found in TA1535. In mutagenicity tests (hisG46 leads to His+), the GSH- strain required the presence of GSH or L-cysteine in the medium for an optimal phenotypic expression and/or growth of spontaneous and induced His+ revertants, and may, therefore, be allelic to cys mutants of Salmonella described earlier. The mutagenic activity of MNNG, ENNG and 1,2-dibromoethane (DBE), but not that of N-ethylnitrosourea (ENU), was strongly reduced in TA1535/GSH-; pretreatment of the strain with GSH restored the mutagenicity of the first 3 chemicals to levels normally found in TA1535. The results support the current view that MNNG, ENNG and DBE, but not ENU, can be activated via reaction with GSH to species of higher reactivity and mutagenicity. It is concluded that the present GSH- strain can be used to study more systematically the role of GSH in the bioactivation and -deactivation of xenobiotics to mutagenic factors.     

A comparative analysis of mutagenic and SOS-induced activity in three classes of chemical compounds

Mutagenic and SOS-inducing potential of 23 derivatives of fluorenone, phenanthrenequinone and biphenyl have been studied in tester strains of Salmonella typhimurium and in Escherichia coli strain PQ 37. 14 of these compounds revert the mutation hisD3052 (much less than -1 much greater than type), but none of them induce mutations in the strain TA 1535. Maximal mutagenic activity has been shown in strain TA 1538 for amide of 2,7-dinitrofluorenone-4-carbonic acid (580 revertants per nmol), 2,7-dinitrophenanthrenequinone (308 revertants per nmol), 2,4,7-trinitrophenanthrenequinone (306 revertants per nmol) and 2',4,4'-trinitrobiphenyl-2-carbonic acid (251 revertants per nmol). In plasmid-containing strain TA 98 the mutagenic potential of the compounds tested is lower than in the TA 1538 strain. It has been suggested that mutagenic activity of these compounds can be attributed to their acceptor properties, namely, the ability to form charge transfer complexes with DNA. SOS-inducing activity has been shown for 5 compounds, also positive in mutation induction. Mutagenic and SOS-inducing activities positively correlate in fluorenone derivatives. Among phenanthrenequinone derivatives, compounds with high mutagenic activity only can induce SOS response. None of the biphenyls tested induce SOS functions. The compounds giving the positive result in the SOS-chromotest have rigid co-planar structure.     

Conversion of tris(8-quinolinolato-N1, O8) aluminum to 8-hydroxyquinoline and activity in bacterial reverse mutation assays

Tris(8-quinolinolato-N1, O8) aluminum (AlQ), an aluminum chelate of 8-hydroxyquinoline (8OHQ) is an important charge transfer molecule in semiconducting imaging devices. This study was conducted to evaluate AlQ and 8OHQ for the ability to induce reverse mutations, either in the presence or absence of mammalian microsomal enzymes, and to determine if AlQ decomposes or is metabolized to 8OHQ under assay conditions. The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA (pKM101). The assays were conducted in the presence and absence of S9. AlQ doses were 1-1000 microg per plate while 8OHQ doses were 0.947-947 microg per plate to maintain molar equivalency. Stability studies were carried out for 4h at 37 degrees C under conditions designed to mimic mutation assays. Samples were analyzed by HPLC and LC/MS to tentatively identify potential metabolites of AlQ and 8OHQ. The results of the bacterial mutagenicity assay indicate that in the presence of S9, both AlQ and 8OHQ, caused increases in the mean number of revertants per plate with tester strains TA100 and WP2uvrA (pKM101). No increases were observed with any of the remaining tester strain/activation condition combinations. The stability study showed that AlQ degrades readily to 8OHQ under standard mutagenicity test conditions, and the positive test result with AlQ is due to the bioactivation of 8OHQ. In the presence of S9, 8OHQ is metabolized to one detectable product with molecular weight indicative of a one-oxygen insertion. 8OHQ N-oxide and 2,8-quinolinediol were ruled out as possible metabolites; 8OHQ epoxides and other quinolinediols were neither confirmed nor ruled out. Bacterial mutagenicity tests have not been shown to predict in vivo effects of 8OHQ; these assays are similarly expected to be poorly predictive of in vivo genotoxic and carcinogenic potential of AlQ.     

Bacterial reversion assay and micronucleus test carried out on hydrogenated glucose syrups 'Malti-Towa' (powder) and maltitol crystal

Two preparations of maltitol (4-O-alpha-D-glucopyranosyl-D-sorbitol), hydrogenated glucose syrups and maltitol crystal, were examined for genotoxic potential by a battery of short-term tests. In the bacterial reversion assay, maltitol induced no detectable revertants in any of the tester strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538, or Escherichia coli WP2/pKM101 at doses of 0.5-50 mg per plate with and without rat liver S9 mix. In the micronucleus test, no significant increase in the frequency of micronucleated erythrocytes was observed in bone marrow of mice after administration of the two preparations at 3.75-30 g per kg by gastric intubation.     

Peroxyacetyl nitrate: measurement of its mutagenic activity using the Salmonella/mammalian microsome reversion assay

Exposures of Salmonella typhimurium strain TA100 with and without S9 metabolic activation to low ppm levels of pure peroxyacetyl nitrate (PAN) in the gas phase were conducted. Measurements of the gas-phase PAN exposure concentration and the concentration of its decomposition products in surrogate test media led to a measured mutagenic activity of 34 +/- 5 revertants/mumole. The data indicate that PAN is a relatively weak direct-acting mutagen with TA100.     

Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates

The mutagenic potential of three alkyl 2-cyanoacrylate adhesives, three commercial alkyl 2-cyanoacrylate adhesives and three methyl 2-cyano-3-phenylacrylates, was assessed using the Salmonella/microsome mutagenicity assay. Compounds were tested with and without Aroclor 1254-induced rat-liver homogenate (S9 mix). The methyl 2-cyanoacrylate adhesives were mutagenic in the standard plate test with S. typhimurium strain TA100 with and without S9 activation. Methyl 2-cyano-3-(2-bromophenyl)acrylate revealed a direct mutagenic action to S. typhimurium strain TA1535. The compounds most toxic towards the bacterium S. typhimurium, were the methyl 2-cyanoacrylate adhesives (greater than 500 micrograms/plate). All alkyl 2-cyanoacrylate adhesives were tested in a modified spot test for volatile compounds with tester strain TA100. Mutagenic and toxic effects were observed with the three methyl 2-cyanoacrylate adhesives. It can be concluded from the results that the bacterial toxicity and mutagenicity of methyl 2-cyanoacrylate adhesives may be due to the methyl 2-cyanoacrylate monomer.     

Mutagenicity of aryl propylene and butylene oxides with salmonella

10 aryl propylene oxides and 6 aryl butylene oxides were synthesized. Dose-mutagenicity relationships were studied for these compounds and for 1,2-epoxybutane, using both the preincubation and plate incorporation Ames tests with Salmonella typhimurium strains TA100 and TA1535. Structure-mutagenicity relationships were further examined by concurrent testing at single doses with the plate incorporation assay in strain TA100. In both series of compounds, mutagenicity showed very correlation to chemical reactivity, molar volume and partition values. However, all compounds were mutagenic in at least one system with the propylene oxides being more mutagenic than the corresponding butylene oxide derivatives. The naphthyl derivatives in each series were the most mutagenic.     

The mutagenicities of safrole, estragole, eugenol, trans-anethole, and some of their known or possible metabolites for Salmonella typhimurium mutants.

Safrole, estragole, anethole, and eugenol and some of their known or possible metabolites were tested for mutagenic activity for S. typhimurium TA1535, TA100, and TA98. Highly purified 1'-hydroxyestragole and 1'-hydroxysafrole were mutagenic (approximately 15 and 10 revertants/micromole, respectively) for strain TA100 in the absence of fortified liver microsomes; trans-anethole and estragole appeared to have very weak activity. 3'-Hydroxyanethole was too toxic for an adequate test. Supplementation with NADPH-fortified rat-liver microsomes and cytosol converted 3'-hydroxyanethole to a mutagen(s) and increased the mutagenic activities for strain TA100 of 1'-hydroxyestragole, 1'-hydroxysafrole, estragole, and anethole. No mutagenicity was detected for safrole or eugenol with or without added NADPH-fortified liver preparations. The electrophilic 2',3'-oxides of safrole, 1'-hydroxysafrole, 1'-acetoxysafrole, 1'-oxosafrole, estragole, 1'-hydroxyestragole, and eugenol showed dose-dependent mutagenic activities for strain TA1535 in the absence of fortified liver microsomes. These mutagenic activities ranged from about 330 revertants/micromole for 1'-oxosafrole-2',3'-oxide to about 7000 revertants/micromole for safrole-2',3'-oxide. The arylalkenes, their hydroxylated derivatives, or their epoxides did not show mutagenic activity for strain TA98, except for 1'-oxosafrole-2',3'-oxide, which had weak activity. Since the arylalkenes are hydroxylated and/or epoxidized by hepatic microsomes, hydroxy and epoxide derivatives appear to be proximate and ultimate mutagenic metabolites, respectively, of the arylalkenes.     

Mutagenicity studies on fenitrothion in bacteria and mammalian cells

The mutagenicity of fenitrothion was determined in strains of Salmonella typhimurium and Escherichia coli. Fenitrothion was found to be non-mutagenic in Salmonella typhimurium strains of TA98, TA1535 and TA1537 and in Escherichia coli WP2uvrA both with and without S9 mix, while weak mutagenicity was observed only in Salmonella typhimurium TA100 and enhanced by the addition of S9 mix. The mutagenicity observed in the TA100 strain was not expressed in a nitroreductase-deficient strain, TA100 NR, and decreased in a transacetylase-deficient strain, TA100 1,8-DNP6. The mutagenicity of fenitrothion was also examined by a gene mutation assay using the gene for hypoxanthine-guanine phosphoribosyltransferase (hgprt) in V79 Chinese hamster lung cells. Fenitrothion did not induce any increment of 6-thioguanine-resistant mutant cells at doses ranging from 0.01 to 0.3 mM regardless of the presence or absence of S9 mix. These results suggest that reduction of fenitrothion by a bacterial nitroreductase of TA100 to an active form is essential for the expression of the mutagenicity of fenitrothion in TA100 and that a bacterial transacetylase of TA100 also has an important role in the process of mutagenic activation.     

Mutagenicity of styrene and styrene oxide

Incubation of S. typhimurium strain TA 1535 with styrene increased the number of his⁺ revertants/plate in presence of a fortified S9 rat-liver fraction. Styrene was also highly cytotoxic for Salmonella cells. Styrene oxide, the presumed first metabolite, had a mutagenic effect towards strains TA 1535 and TA 100 both with and without metabolic activation. Styrene is probably mutagenic because it is metabolized to styrene oxide.     

Mutation spectrum of a binary mixture of mutagens (methapyrilene and sodium azide) in strain TA1535 of Salmonella

Methapyrilene (MP) is a rat-liver carcinogen and cocarcinogen that exhibits a narrow spectrum of mutagenic activity in Salmonella typhimurium, inducing only a 2-fold increase in revertants only in the base-substitution strain TA1535; it also enhances the mutagenic activity of sodium azide (NaN₃) in the same strain. To examine the effects of MP at the molecular level, we used the colony probe hybridization procedure dveloped by Cebula and Koch (Mutation Res., 229 (1990) 79–87) to identify the base substitutions in ~800 background, MP-, NaN₃-, and MP + NaN₃-induced revertants of the hisG46 allele of strain Ta1535. The predominant mutation in all 4 mutation spectra was a CCC → CTC transition. the results suggest a mechanism by which MP enhances the infidelity of the DNA replication complex or inhibits a DNA reapir of proofreading function, resulting in the production of more of the same error that occurs normally and that is also induced by NaN₃. Such a mechanism might be the basis for the carcinogenic and cocarcinogenic activities of MP. To our knowledge, this is the first report of the molecular analysis of mutants produced by exposure of cells to a binary mixture of mutagens.