Measurement of drug-metabolizing systems in Salmonella typhimurium strains G46, TA1535, TA100, TA1538 and TA98

Salmonella typhimurium strains which are commonly used in the Ames test for screening potential carcinogens were examined for a number of drug-metabolizing systems. Neither cytochrome P-450 itself nor two activities catalyzed by the cytochrome P-450 system in mammalian cells, i.e., benzpyrene monooxygenase and ethoxycoumarin O-deethylation, could be detected. Nor do these bacterial strains demonstrate any ability to detoxify epoxides by hydrating them or to conjugate p-nitrophenol with glucuronic acid.On the other hand, S. typhimurium strains G46, TA1535, TA100, TA1538 and TA98 contain considerable amounts of acid-soluble thiols, approx. 5–10% of which is glutathione. These bacteria can also enzymatically conjugate glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) and can reduce oxidized glutathione using NADPH as cofactor.Thus, enzymatic and non-enzymatic reaction of immediate carcinogens with thiol groups in S. typhimurium may have a significant effect on the outcome of the Ames test in certain cases.     

Glutathione and glutathione S-transferases in the salmonella mammalian-microsome mutagenicity test

Levels of the tripeptide glutathione (GSH) and the activity of glutathione S-transferases were investigated in S9 fractions of rats and mice and in Salmonella typhymurium tester strains TA1535, TA100, TA1538 and TA98. The S9 and Salmonella typhimurium tester strains had high levels of glutathione. Compared with S9, the activity of GSH S-transferases was lower in the bacteria. However, electrophiles such as 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and styrene oxide were effectively bound to bacterial GSH.

The mutagenicity of the direct mutagen CDNB was drastically lowered in presence of S9 fractions but not in presence of microsomes. A comparable decrease was obtained when microsomal supernatant, which contains GSH and GSH S-transferases, was added to the microsomes. Addition of GSH in excess completely abolished mutagenicity of CDNB. These results demonstrate that the conjugation of electrophiles with GSH mediated by the S9 fraction or the bacterial tester strains represents an important detoxication mechanism which may influence the results obtained with the Salmonella typhimurium mammalian-microsome mutagenicity test.     

Mutagenic activity of the glutathione S-transferase substrate 1-chloro-2,4-dinitrobenzene (CDNB) in the Salmonella mutagenicity assay

The mutagenicity of the commonly used glutathione S-transferase substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) was investigated in the Salmonella mutagenicity assay. CDNB induced a concentration-dependent mutagenic response in Salmonella typhimurium strain TA98. Incorporation of an activation system derived from Aroclor 1254-induced rats did not influence mutagenic response. Under the same conditions DCNB failed to display mutagenic activity. The mutagenic activity of CDNB was attenuated in bacterial strains under-expressing nitroreductase or O-acetylase activity but, in contrast, it was exaggerated in an O-acetylase over-expressing strain. It is inferred that CDNB exhibits a mutagenic response following reduction of the nitro-group to the hydroxylamine, which is further acetylated to form the acetoxy derivative that presumably breaks down spontaneously to generate the nitrenium ion, the likely ultimate mutagen.     

The protective action of glutathione on the microbial mutagenicity of the Z- and E-isomers of 1,3-dichloropropene

The Z(cis)- and E(trans)-isomers of 1,3-dichloropropene (DCP), in confirmation of previous reports, caused dose-dependent increases in the numbers of reverse mutations in Salmonella typhimurium TA100 in the presence and absence of a 9000 X g supernatant fraction (S9) from the livers of Aroclor-treated rats. The relevance of these findings to mammals is uncertain, not least because of major differences in the metabolism of the DCPs in the microbial assay systems and in vivo. For example, (Z)-DCP is efficiently detoxified in mammals by the operation of a glutathione (GSH)-dependent S-alkyl transferase. It is possible that such detoxification could proceed only very slowly in the microbial assays because the concentrations of GSH could be severely rate-limiting even in those assays fortified by the addition of S9. The results obtained in the current study demonstrate a dramatic reduction in the microbial mutagenicity of both (Z)- and (E)-DCP when the concentration of GSH in the microbial assays was adjusted to a normal physiological concentration (5 mM). However, this protective action of GSH was at least as effective in the absence of S9 as in its presence, suggesting that it was not mediated by mammalian GSH transferase. There appears to be little or no GSH alkyl or aryl transferase in the cytosol of S. typhimurium TA100, but intracellular GSH is present at a concentration similar to that found in mammalian cells. Since the uncatalysed reaction between the DCPs and glutathione is relatively slow, the effect is not due simply to their destruction by GSH. It is possible that a physiological concentration of extracellular GSH maintains the intracellular GSH in a reduced form in which its nucleophilic thiol group competes effectively with the nucleophilic centres in the bacterial DNA for the haloalkenes. The current results highlight the efficiency of GSH-linked systems in affording protection against the genotoxic action of the DCPs. It may be presumed that their operation would exert a major limiting effect on the genotoxicity of (Z)- and (E)-DCP in mammals.     

Localization of the amino phospholipids in sarcoplasmic reticulum membranes revealed by trinitrobenzene-sulfonate and fluorodinitrobenzene

The chemical probes for amino compounds 2,4,6-trinitrobenzenesulfonate (TNBS) and 1-fluoro-2,4-dinitrobenzene (FDNB) were utilized to determine the localization of the amino phospholipids in the sarcoplasmic reticulum membranes. At low concentrations (<1 mM), TNBS does not penetrate the sarcoplasmic reticulum membrane, while FDNB readily penetrates it. The results show that about 70% of the total phosphatidylethanolamine is located on the external surface of the membrane, about 20% is on the internal surface and 10% is probably strongly interacting with the proteins since it is not accessible to the probes. In contrast, most of the phosphatidylserine is located on the inner surface of the membrane. This molecular distribution of the amino phospholipids supports a structural assymmetry of the sarcoplasmic reticulum membrane.     

Xenobiotica-metabolizing enzymes in Drosophila melanogaster: activities of epoxide hydratase and glutathione S-transferase compared with similar activities in rat liver.

Activities of epoxide hydratase and glutathione (GSH) S-transferase were investigated in subcellular fractions of Drosophila melanogaster, and these activities were compared with analogous enzymic activities in extracts from rat liver. Microsomes of Drosophila were active in the hydratation of styrene oxide catalyzed by epoxide hydratase. The post-microsomal supernatant of Drosophila catalyzed the conjugation of GSH with 1-chloro-2,4-dinitrobenzene. However, GSH S-transferase activity with styrene oxide as the electrophilic substrate was not measurable. The respective specific activities of epoxide hydratase (per mg microsomal protein) and GSH S-transferase (per mg cytosolic protein) were factors of 5- and 10-fold lower than the corresponding activities in rat liver. However, when expressed per gram body weight, activities of both epoxide hydratase and GSH S-transferase were 3 times higher for Drosophila enzymes. The apparent Km values for the two Drosophila enzymes were higher, whereas the apparent Km values were lower, than the values found for the rat-liver enzymes. Among 3 different Drosophila strains (a wild-type, a white eye-color carrying mutant strain and a DDT-resistant strain), preliminary experiments showed no differences as far as these two enzymic activities were concerned. It is concluded that the results obtained in genetic toxicology testing with Drosophila are probably relevant to effects to be expected in mammalian systems with compounds requiring metabolic processes involving the enzymes investigated here.     

The bacterial mutagenicity of nitropolychlorinated dibenzo-p-dioxins

The bacterial mutagenicity of 2-nitrodibenzo-p-dioxin, a mixture of 2-nitro-7-chloro- and 2-nitro-8-chlorodibenzo-p-dioxin, 7-nitro-2,3-dichloro-, 8-nitro-2,3,7-trichloro-, 2-nitro-1,3,7,8-tetrachloro- and 3-nitro-1,2,4,7,8-pentachlorodibenzo-p-dioxin was determined using Salmonella typhimurium tester strains TA98 and TA100 with and without rat hepatic S9 for metabolic activation. All the nitro-PCDDs exhibited some direct-acting mutagenicity with both tester strains, however, the activity was significantly lowered in the presence of exogenous S9 and the compounds were more mutagenic to tester strain TA98. The mutagenicity of the nitro-PCDDs was also dependent on structure because there was a marked decrease in activity with increasing chlorine content. Because nitro-PCDDs have recently been identified as incomplete combustion products of municipal waste, this study confirms that this new class of compounds contains some bacterial mutagens.     

Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents.

Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The corresponding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmic and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).     

Revised methods for the Salmonella mutagenicity test

The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed.     

Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with β-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.     

Fluctuation test data on 4-chloromethylbiphenyl (4CMB), 4-hydroxymethylbiphenyl (4HMB) and Benzyl Chloride (BC) using Salmonella typhimurium TA98 and TA100

Bacterial fluctuation tests (Green et al., 1976) were performed both with and without metabolic activation using the ‘Ames’ Salmonella typhimurium strains TA98 and TA100 (Ames et al., 1975) to assay the mutagenic potential of 4CMB, 4HMB and BC.4CMB and 4HMB were tested on the same occasion. However, 4CMB was only compared to BC in one assay. The results also show an independent test of BC.     

Mutagenic action of a series of epoxides

The mutagenicity of a series of 13 epoxide compounds was studied using a bacterial plate assay system. The histidine-dependent tester strains TA98 (for frameshift mutagens) and TA100 (for base-pair substitution mutagens) of Salmonella typhimurium were used. Mutagenicity was evaluated both with and without the addition of rat liver microsomal extract. Dieldrin, diglycidyl ether of bis phenol A and 3 of its homologues were not mutagenic. Allyl glycidyl ether, n-butyl glycidyl ether, vinyl cyclohexene diepoxide, glycidol, glycidaldehyde, diglycidyl ether, diepoxybutane and diglycidyl ether of substituted glycerine were mutagenic in the TA100 strain, causing reversion of the bacteria to histidine independence. Dose—response curves of the mutagenicity of the latter 4 compounds were obtained. On a molar basis, glycidaldehyde was about 20–50 times more potent in producing mutation that were the other 3 epoxides in the dose—response test. In general, the mutagenicity of the epoxides was not enhanced or diminished by the addition of microsomal extract.     

Dependence of the mutagenic power of heteroatomic dyes on their DNA-base-pair specificity

23 compounds structurally related to proflavin were tested with Salmonella typhimurium TA1537 for their ability to induce mutants in the quantitative mutagenicity test of Ames et al. Of these compounds, 13 were mutagenic. Their mutagenic power decreased with increasing GC specificity of the dye. Because the mutational site in the tester strain is assumed to consist of a run of 4 adjacent GC pairs of the same polarity we conclude that for mutation to occur the dye should not be bound within this run of identical base pairs.Some of the compounds showed a negative slope at higher concentrations. Because these dyes also showed a comparatively high tendency to dimerize we conclude that mutation induction requires the intercalator to be bound as a monomer.Those compounds that were not mutagenic either did not intercalate or were inactivated by reduction.     

Inhibition of polymorphonuclear leukocyte functions by fluorinated nitrobenzenes

The bifunctional fluorinated nitrobenzenes, 1,5-difluoro-2,4-dinitrobenzene (DFDNB) and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (DFDNDPS), and the monofunctional 1-fluoro-2,4-dinitrobenzene (FDNB) inhibit chemotaxis, phagocytosis, exocytosis and the respiratory burst of rabbit polymorphonuclear leukocytes. Inhibition occurs in the micromolar concentration range; the bifunctional compounds are stronger inhibitory than the monofunctional one. The inhibitory effect can be counteracted by sulfhydryl compounds and not with amino-group containing compounds. The results suggest that an interaction with vulnerable sulfhydryl groups, located in a hydrophobic surrounding, is the basis of the inhibitory effect of the fluorinated nitrobenzenes.     

Mutagenic effect of furocoumarin monoadducts and cross-links on bacteriophage lambda

The mutagenicities of 17 closely related oxiranes were determined in 4 tester strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537). The test compounds comprised all possible oxides of benzene and its partially hydrogenated congeners. In TA100 and TA1535, 12 of the tested oxiranes were weak to moderate mutagens. 4 of these were also active in TA98. No mutagenicity was observed with the remaining 5 compounds in any of the 4 strains.The presence of a double bond in formal conjugation with the epoxide ring increased the mutagenicity relative to that of the saturated oxirane. Interestingly, additional epoxide rings within the same molecule did not markedly increase the mutagenic activity, and for the oxiranes that are not activated by a double bond, the relationship between mutagenic activity and the number of epoxide rings in the molecule was even inverse.The influence of bromo and hydroxyl substitution on oxirane mutagenicity is discussed. Most notably, a compound having a 4-hydroxyl group in syn position to a 1,2-epoxide ring fused to the cyclohexane ring, a structure which has been suggested to increase the electrophilic reactivity of dihydrodiol epoxides through hydrogen bonding, was almost inactive.     

Influence of the processing of MucA protein on the reversion of the frameshift hisD3052 and the base substitution hisG46 mutations by chemical mutagens in Salmonella typhimurium

The correlation between the proficiency at promoting mutagenesis of MucA/B proteins and MucA processing has been considered to be very high (Hauser et al., 1992) on the basis of the results of UV mutagenicity (Shiba et al., 1990). Here we show that this correlation is only partial. We have assayed the mutagenicity of benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1) in Salmonella typhimurium tester strains containing plasmids which encode MucA proteins with an altered cleavage site. Reversion of the frameshift hisD3052 mutation by B[a]P or AFB1 was observed in the presence of non-cleavable MucA protein although at a lower level than that found in cells containing wild-type MucA protein. Reversion of the base substitution hisG46 mutation by AFB1 requires a significant processing of MucA, while lower levels of this processing would be enough for the hisG46 reversion by B[a]P. These results suggest that the specificity of mutations induced by mutagens forming DNA adducts is influenced by the activity of MucA protein. They also show the relevance of mutagenicity assays in the mechanistic studies of mutagenesis.     

The interactions of triethyltin with rat glutathione-S-transferases A, B and C. Enzyme-inhibition and equilibrium-dialysis studies.

Purified glutathione(GSH)-S-transferases A, B and C from rat liver are inhibited by triethyltin (SnEt3). With 1-chloro-2,4-dinitro benzene (CDNB) as the limiting substrate the inhibition is competitive in each case. At a GSH concentration of 5 . 10(-3) M the inhibition constants for transferases A and C at 25 degrees C are similar and very low, 3.2 . 10(-8) M and 5.6 . 10(-8) M respectively, whereas for transferase B the inhibition constant is 3.5 . 10(-5) M. Equilibrium-dialysis experiments carried out at 4 degrees C in the absence of GSH give apparent dissociation constants of 7.1 . 10(-4) M and 3.4 . 10(-4) M for transferases A and B respectively, but if 5 . 10(-3) M glutathione is included in the dialysis solutions these values fall to 2.0 . 10(-7) M and 2.6 . 10(-5) M, which are within an order of magnitude of the kinetic Ki-values. Chromatographic experiments with Sephadex G-10 show that GSH and SnEt3 interact in aqueous solution under the conditions of the enzyme-kinetic and equilibrium-dialysis experiments. It is suggested that the inhibited enzymes are in the form of ternary complexes, enzyme-GSH-SnEt3, in which GSH and SnEt3 may or may not interact directly; or are possibly quaternary complexes, enzyme-(GSH)2-SnEt3. SnEt3 could be valuable as a selective inhibitor of transferases A and C in mixtures of the three transferases.     

Development of an in situ microbial mutagenicity test system for airborne workplace mutagens: laboratory evaluation

A simple on-site Salmonella mutagenicity test system for the detection of airborne mutagens in the workplace is being developed. The system permits entrapment of mutagenic airborne particles and vapors by impinging unfiltered ambient air into trapping medium containing bacterial tester cells. The trapping device consists mainly of a pump, an impinger and a cyclone. The impinging air flow generated by the pump is approximately 3 1/min. New Salmonella typhimurium testers which are resistant to streptomycin (Str) and 8-azaguanine (AG) were derived from the Ames testers TA98 and TA100 and the arabinose-resistant tester SV50, and were used as mutation indicators. Microbial contamination was sufficiently controlled by addition of ampicillin, Str, AG, and cycloheximide to the trapping and plating media. New tester strains retained a high mutagenic sensitivity from their parent strains. Laboratory studies with volatile mutagens (methyl methanesulfonate, ethyl methanesulfonate, and dimethylnitrosamine) showed that the vapor trapping of this system is promising. The study with suspended silica particles coated with a known mutagen (2,4,7-trinitro-9-fluorenone) indicated that the particle trapping of the system is satisfactory. Incorporation of metabolic activation into the trapping medium by confining S9 mix and tester cells in dialysis tubing enabled this system to detect promutagens. This in situ system may be useful for mutagenic monitoring in the workplace.     

Induction of point mutations by different chemical mechanisms in the liver microsomal assay

A selection of chemical agents with different mechanisms of chemical mutability was tested with the liver microsomal assay by using different bacterial tester strains, namely: Salmonella typhimurium TA 1535, TA 1536, TA 1537, TA 1538 and G46. The tested agents had been selected according to the following criteria. They are all well-known mutagens and can be divided into alkylating agents, anti-metabolites, acridines and those that form radicals in the cell. The mutagens were: dimethylnitrosamine (DMN), diethylnitrosamine (DEN), methyl-nitro-nitrosoguanidine (MNNG), cyclophosphamide, Captan, amethopterine, azathioprine, 6-mecaptopurine, trypaflavine, isoniazide and hydrazine. All except amethopterine gave positive results, showing that this system is very sensitive.In a comparison of the different strains and mutagens we found a correlation between the diameter of the molecule and the permeability of the bacterial cell membrane.