Mutagenicity in Salmonella of halonitromethanes: a recently recognized class of disinfection by-products in drinking water

Halonitromethanes (HNMs) are a recently identified class of disinfection by-products (DBPs) in drinking water. They include chloronitromethane (CHN), dichloronitromethane (DCNM), trichloronitromethane (TCNM), bromonitromethane (BNM), dibromonitromethane (DBNM), tribromonitromethane (TBNM), bromochloronitromethane (BCNM),dibromochloronitromethane (DBCNM), and bromodichloronitromethane (BDCNM). Previous studies of TCNM, DCNM, CNM, and TBNM found that all four were mutagenic in bacteria, and a recent study showed that all nine induced DNA damage in CHO cells. Here, all nine HNMs were evaluated in the Salmonella plate-incorporation assay +/- S9 using strains TA98, TA100, TA104, TPT100, and the glutathione transferase theta (GSTT1-1)-expressing strain RSJ100. All were mutagenic, most with and without S9. In the absence of S9, six were mutagenic in TA98, six in TA100, and three in TA104; in the presence of S9, these numbers were five, seven, and three, respectively. Thus, the HNMs-induced base substitutions primarily at GC sites as well as frameshifts. Although five HNMs were activated to mutagens in RSJ100 -S9, they produced < or =2-fold increases in revertants and potencies <506 rev/micromol. The rank order of the HNMs by mutagenic potency in TA100 +S9 was (BCNM DBNM) > (TBNM CNM > BNM DCNM BDCNM) > (TCNM = DBCNM). The mean rev/micromol for the three groupings, respectively, were 1423, 498, and 0, which classifies the HNMs as weak mutagens in Salmonella. Reaction of the dihalo and monohalo HNMs with GSH, possibly GSTT1-1, is a possible mechanism for formation of ultimate mutagenic products. Because the HNMs are mutagenic in Salmonella (present study) and potent clastogens in mammalian cells [Environ. Sci. Technol. 38 (2004) 62], their presence in drinking water warrants further research on their potential health effects.     

Mutagenicity of dipyridyls and their methylated derivatives in Salmonella typhimurium/rat liver microsome system

The Salmonella/microsome assay with strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 was used to examine the potential mutagenicity of 5 dipyridyls, 1 tripyridyl, 3 dipyridinium diiodides and 2 pyridinium monoiodides. The widely used herbicide paraquat (1,1-dimethyl-4,4'-dipyridinium diiodide) and its precursor 4,4'-dipyridyl gave weak and marginal mutagenic activity to Salmonella typhimurium TA1535 and TA1538 in the presence of S9-mix. Significantly high mutagenicity was obtained with 2,2'-, 3,3'-, 2,3'-, and 2,4'-dipyridyls, 2,2',2"-tripyridyl, and 5 pyridinium salts under the same conditions. The positive mutagenic response of 2,2',2"-tripyridyl suggests that higher polymers of pyridine contaminating paraquat preparations might be mutagenic. The dose-response curves of 1,1-dimethyl-3,3'-dipyridinium diiodide and 1,1'-dimethyl-2,2'-dipyridinium diiodide revealed an exponential relationship between the number of induced revertants and the compound concentrations. The results suggested that the mechanism of mutation induced by these two compounds might be attributed to the chain reactions of their free-radicals with molecular oxygen.     

Mutagenicity of alkylhydrazine oxalates in Salmonella typhimurium TA100 and TA102 demonstrated by modifying the growth conditions of the bacteria

Alkylhydrazines are important carcinogens. However, they show generally only weak mutagenicity and the activities reported from different laboratories are contradictory. We have developed a sensitive method to detect the mutagenicity of alkylhydrazines. The method is based on a modified preculturing procedures in the Ames test, the emphasis in the modification being a change in the growth period of tester strains. The optimal growth periods were found to be 11 h in Salmonella typhimurium TA100 and 5 h in Salmonella typhimurium TA102. We tested the mutagenic activity of 12 alkylhydrazines; 1,2-dimetehylhydrazine, 1,2-diethylhydrazine, 1,2-dipropylhydrazine. 1,2-dibutylhydrazine, 1,1-dimethylhydrazine, 1,1-diethylhydrazine, 1,1-dipropylhydrazine, 1,1-dibutylhydrazine, methylhydrazine, ethylhydrazine, propylhydrazine, and butylhdyrazine. All 12 alkylhydrazines were clearly mutagenic in Salmonella typhimurium TA102, and 10 hydrazines were mutagenic in Salmonella typhimurium TA100, both in the absence of S9 mix. The mutagenicity was inhibited by the addition of S9 mix or bovine serum albumin. This suggests deactivation of the mutagens by proteins.     

Mutagenicity of 1-fluoro-2,4-dinitrobenzene is affected by bacterial glutathione

Recently we have shown that Salmonella typhimurium tester strains have high levels of the tripeptide glutathione (GSH) and activity of GSH S-transferases (Summer et al., 1979). In continuation of the GSH-dependent suppression of mutagenicity of 1-chloro-2,4-dinitrobenzene in presence of S9 fraction (Summer et al., 1979), this paper is focused on the GSH-dependent detoxifying capacity of the bacterial tester strains. 1-Fluoro-2,4-dinitrobenzene (FDNB), an electrophilic agent, which is used to identify terminal amino acids in proteins (Sanger reagent), readily reacts with GSH leading to a dose-dependent depletion of bacterial GSH. Additionally, FDNB is a strong mutagen for Salmonella typhimurium TA100, TA1538 and TA98 without metabolic activation.Presumably owing to conjugation with bacterial GSH, FDNB in concentrations which were lower or equal to those of bacterial GSH were found to be not mutagenic. Accordingly, increasing amounts of bacteria in the test system require increasing amounts of FDNB for expression of mutagenicity.     

Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro.

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02-0.8 micrograms/plate (38-1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in "classical" nitro-reductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 micrograms NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP6, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.     

Mutagens in coffee and tea.

Coffee prepared in the usual way for drinking contains a substance(s) that is mutagenic to Salmonella typhimurium TA100 without mammalian microsomal enzymes. One cup of coffee (200 ml) contains mutagen(s) inducing 1.4-4.6 X 10(5) revertants under standard conditions. Instant coffee too is mutagenic to TA100 and one cup of instant coffee prepared from 1 g of coffee powder and 200 ml of water induced 5.6-5.8 X 10(4) revertants of TA100. Caffeine-free instant coffee also has similar mutagenicity. Addition of microsomal enzymes abolished the mutagenicity. Black tea, green tea and Japanese roasted tea were also mutagenic to TA100 without S9 mix and one cup of these teas prepared in the ordinary way produced 1.7-3.8 X 10(4) revertants of TA100. Black tea and green tea were also mutagenic to TA98 in the presence of S9 mix after treatment with a glycosidase from Aspergillus niger, hesperidinase. This type of mutagen in one cup of black tea induced 2.4 X 10(5) revertants of TA98.     

Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02–0.8 μ g/plate (38–1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in “classical” nitroreductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 μg NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP₆, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.     

Evaluation of 1,3-pentadiene for mutagenicity by the Salmonella/mammalian microsome assay

1,3-Pentadiene, a food contaminant produced by some molds when they metabolize sorbic acid, was tested for mutagenicity, using variations of the Salmonella/mammalian microsome assay. The chemical was incorporated into the test system (with and without S9 mix) by 3 methods: (a) the standard plate incorporation assay, (b) a liquid preincubation procedure and (c) exposure of test bacteria in the soft agar overlay to gaseous 1,3-pentadiene. The chemical was extremely toxic to the test bacteria with amounts as low as 2.0 microgram/plate causing cell death. However, none of the nonlethal concentrations tested by any of the methods was mutagenic to Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 or TA1538.     

The Salmonella mutagenicity assay in a surface water quality monitoring program based on a 20-year survey

Since 1979, the Environmental Agency of S?o Paulo State in Brazil, CETESB, has been using the Salmonella mutagenicity assay to assess the quality of natural waters. This paper is a compilation of data obtained during the last 20 years from more than a thousand samples. Potencies up to 30,000 revertants/l were observed in 137 positive samples. The Salmonella typhimurium strain TA98 was more sensitive than TA100; 79% of the mutagenicity was detected by this strain, regardless of the presence of S9-mix. A classification of the mutagenic response was proposed to facilitate in the dissemination of the information to the public. The classification was low, moderate, high and extreme for samples with mutagenic potency (revertants/l equivalent) of < 500, 500-2500, 2500-5000 and > 5000, respectively. As a result of this effort to standardize methodologies, compile and classify the mutagenic effect of water pollution, in 1998, the Salmonella mutagenicity assay was officially and systematically included in the S?o Paulo State Water Quality Monitoring Program. This assay has proven to be a useful tool in the identification of important pollution sources. Correction and prevention actions in Water Pollution Control Programs were generated as a result.     

Mutagenic and genotoxic effects of theophylline and theobromine in Salmonella assay and in vivo sister chromatid exchanges in bone marrow cells of mice.

The mutagenic and genotoxic effects of two methylxanthines, theophylline (TH) and theobromine (TB), were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice. These are the two most commonly used nervous system stimulators throughout the world. TH is used in the long-term treatment of asthma. Bacterial mutagenicity assay showed very weak mutagenic effects of both drugs in Salmonella strains TA102 and TA104 only in certain concentrations when S9 was added to it. No mutagenic effects were observed in any other strains used in this assay either with or without metabolic activation. But results of in vivo SCE assay indicate that these two drugs can induce significant SCE in bone marrow cells of mice.     

Glutathione activation of chloropicrin in the Salmonella mutagenicity test

Chloropicrin (CCl3NO2) is a major soil fumigant for control of fungi, insects and nematodes and may by formed by chlorination of drinking water. It is also a strong lacrimator and induces sister chromatid exchanges in cultured human lymphocytes. Mutagenicity assays of CCl3NO2 in Salmonella typhimurium TA100 establish that it is toxic but not mutagenic at 500 nmol/plate but becomes mutagenic but not toxic on addition of S9 (previous work) or 1-2 molar equivalents of glutathione (GSH) (this study). The preincubation assay is superior to the plate incorporation test giving 2-4-fold higher revertants/nmol. Using the preincubation assay with GSH at 5 mM (a biomimetic level) in the top agar gives linear dose-response relationships for CCl3NO2 and its dechlorination products CHCl2NO2 and CH2ClNO2 with 0.56, 0.56 and 1.8 revertants/nmol, respectively. The mutagenicity values for CHCl2NO2 and CH2ClNO2 are the same in the presence and the absence of GSH, which only improves the linearity at high levels by reducing toxicity to the bacteria. GSH activation of CCl3NO2 mutagenicity may be due to reductive dechlorination of the trichloro compound to the more active CHCl2NO2 and CH2ClNO2. Alternatively, the mutagenicity may result from an intermediate GSH conjugate such as GSCCl2NO2 or GSCHClNO2. In comparison, the mutagenicity of CH2Br2 and CH2I2 is affected little if any by addition of GSH and these dihalomethanes are much less active than the halonitromethane series. It therefore appears that CCl3NO2 is not mutagenic in the absence of activation and that the dechlorinated metabolites CHCl2NO2 and CH2ClNO2 are moderately potent bacterial mutagens, consistent with the possible genotoxicity of CCl3NO2 in mammals.     

Inhibition of dinitropyrene mutagenicity in vitro and in vivo using Salmonella typhimurium and the intrasanguinous host-mediated assay

Dinitropyrenes (DNP), present in polluted air, are potent direct-acting mutagens in Salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of rat-liver S9 or microsomes. This has now been confirmed using mouse hepatic fractions. Since most in vitro test systems do not adequately simulate conditions encountered in the intact animal, we have investigated dinitropyrene mutagenicity to Salmonella in the host-mediated assay. 1,8-Dinitropyrene (1,8-DNP) given p.o. to BALB/c mice induced a weak mutagenic effect in S. typhimurium TA98 recovered from the liver 1 h after i.v. administration (optimum time). Over the entire dose range tested no toxicity to bacterial cells was detected. Mutation induction in vivo was dose-related with maximum response at 1 mg DNP/kg body weight. This optimum dose, however, was non-mutagenic to strains TA98/1,8-DNP6 (O-transacetylase-deficient) or TA98NR/1,8-DNP6 (nitroreductase- and O-transacetylase-deficient). 1,3-Dinitropyrene and 1,6-dinitropyrene were weakly mutagenic to TA98 at doses similar to 1,8-DNP. Studies with [14C]1,8-DNP showed that 1 h after oral dosing (1 mg/kg), over 100 ng of 1,8-DNP equivalents were present in the liver (= 0.73% dose). However, only about 5.5 ng were present in the bacterial pellet, suggesting that hepatic components in vivo, as in vitro, bind to DNP, thus interfering with its interaction with Salmonella.     

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 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.     

Some chloro derivatives of polynuclear aromatic hydrocarbons are potent mutagens in Salmonella typhimurium

A series of chlorinations of some polynuclear aromatic hydrocarbons (PAH) were carried out and the products were tested for mutagenicity on Salmonella typhimurium TA98 and TA100. We conclude that the chlorination of certain PAHs with low mutagenicity, such as pyrene and benzo[e]pyrene, resulted in the formation of two types of product. The chlorination of pyrene was studied in some detail. The major products of this chlorination were chloro-substituted pyrenes. These compounds showed an S9-dependent mutagenicity and were identified as 1-chloro-, 1,6-dichloro-, 1,8-dichloro- and 1,3-dichloropyrene. On tester strain TA100 the mutagenic effect ranged from 1.4 to 14 revertants/nmol, 1,3-dichloropyrene being the most potent of the isomers. Minor products eluting from a chromatograph in a more polar fraction than the major products were also formed. These compounds were less stable than the major products and were identified as pyrene with chloro additions in the 4- and 5-positions, with various chloro substituents at other positions. These minor products showed a high mutagenic effect on Salmonella in the absence of S9. The mutagenic effect on strain TA100 ranged from 10 to 15 revertants per ng which is at least 40 and 4000 times higher than for 1-nitropyrene and pyrenequinones, respectively. These unstable chloro derivatives of pyrene are difficult to analyse chemically because they are easily degraded and give rise to the more stable 4-chloropyrene.     

Mutagenic activation of biliary metabolites of 1-nitropyrene by intestinal microflora

To investigate the modifying role of intestinal microflora in the metabolism of chemical carcinogens in vivo, we subjected bile from Fischer rats treated per os with chemical carcinogens and related compounds to a mutagenicity assay in the presence and absence of a cell-free extract from human feces. A mixture of the bile sample and potassium phosphate buffer was incubated in the presence or absence of human cell-free fecal extract and then further incubated with a bacterial suspension of Salmonella typhimurium tester strains TA98 or TA100. Bile from rats treated with 1-nitropyrene (1-NP) produced about 2700 and 400 revertants per plate in strain TA98 in the presence and absence of the fecal extract, respectively. There was a drug dose- and bile volume-related response. Treatment of 1-NP-bile with beta-glucuronidase, but not aryl sulfatase, enhanced its mutagenicity. Cell-free extracts of some strains of intestinal bacteria (Bacteroides fragilis ATCC 12044, B. vulgatus ATCC 8482, B. thetaiotaomicron ATCC 12290, Bacteroides sp. strain 524, Eubacterium eligens VPI C15-48, Peptostreptococcus sp. strain 204 and Escherichia coli A-5-18) also enhanced the mutagenicity of 1-NP-bile. These bacterial cell-free extracts hydrolyzed the synthetic beta-D-glucuronides of phenolphthalein and/or p-nitrophenol. These data indicate that the glucuronide(s) of 1-NP-metabolite(s) secreted into bile can be hydrolyzed in the intestine by bacterial beta-glucuronidases to potent mutagenic aglycone(s).     

Mutagenicity of aliphatic epoxides.

The mutagenicity of 17 aliphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains TA100 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most potent mutagens in both strains. 1,1-Disubstitution resulted in the complete loss or reduction of mutagenicity, trans-1,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-1,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the presence of electron-withdrawing substituents increased mutagenicity.     

Mutagenicity of the coccidiostat diaveridine in the Salmonella/mammalian microsome assay

The coccidiostat diaveridine was tested for mutagenicity in the Salmonella/microsome assay with tester strains TA100 and TA98. This compound was not mutagenic in either tester strain in the presence and absence of rat S9 mix, but was found to be mutagenic in strain TA100 after metabolic activation with hamster S9 mix.     

Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

Evaluation of secondary nitroalkanes, their nitronates, primary nitroalkanes, nitrocarbinols, and other aliphatic nitro compounds in the Ames Salmonella assay.

The secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane and nitrocyclopentane, as well as their anionic forms (nitronates); the primary nitroalkanes 1-nitropropane, 1-nitrobutane, and 1-nitropentane and their respective nitronates; the nitrocarbinols 2-nitro-1-propanol, 2-nitro-1-butanol, 3-nitro-2-butanol, and 3-nitro-2-pentanol and their respective nitronates; 2-methyl-2-nitropropane, and 2-nitroso-2-nitropropane were tested in the Ames Salmonella assay using strains TA98, TA100 and TA102. Nitronates of the secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane, and nitrocyclopentane were significantly mutagenic in Salmonella strains TA100 and TA102 at 10-80 mumoles/plate, but the parent compounds were mutagenic at only a single dose level or were not mutagenic at all in the same dose range. The primary nitroalkanes and the nitrocarbinols were not mutagenic, or only marginally so, at the concentrations tested. The nitronates of the primary nitroalkanes and the nitrocarbinols reprotonated too rapidly under the conditions of the assay for adequate evaluation of mutagenicity. 2-Methyl-2-nitropropane was not mutagenic in strains TA100 and TA102; 2-nitroso-2-nitropropane was also not mutagenic in strains TA100 and TA102, but induced an equivocal mutagenic response in TA98. The positive Salmonella mutation data for the nitronates of the secondary nitroalkanes studied correlate very well with the very slow rate of reprotonation of secondary nitroalkane nitronates at pH 7.7 (Conaway et al. (1991) Cancer Res., 51, 3143), and provide further evidence that nitronates of secondary nitroalkanes, rather than the neutral parent forms with which they may be in equilibrium, are the more proximate mutagenic species.