Mutagenicity of tetrachloroethene in the ames test—metabolic activation by conjugation with glutathione

The mutagenicity of tetrachloroethene (tetra) and its S conjugate, S-(1,2,2-trichlorovinyl)glutathione (TCVG) was investigated using a modified Ames preincubation assay. TCVG was a potent mutagen in presence of rat kidney particulate fractions containing high concentrations of γ-glutamyl transpeptidase (GGT) and dipeptidases. Purified tetra was not mutagenic without exogenous metabolic activation or under conditions favoring oxidative metabolism. Preincubation of tetra with purified rat liver glutathione (GSH) S-transferases in presence of GSH and rat kidney fractions resulted in a time-dependent formation of TCVG as determined by (HPLC) analysis and in an unequivocal mutagenic response in the Ames test. Experiments with tetra in the isolated perfused rat liver demonstrated TCVG formation and its excretion with the bile; bile collected after the addition of tetra to the isolated perfused liver was unequivocally mutagenic in bacteria in the presence of kidney particulate fractions. The mutagenicity was reduced in all cases by the GGT inhibitor serine borate or the β-lyase inhibitor aminooxyacetic acid. These results support the suggestion that cleavage of the GSH S conjugate formed from tetra by the enzymes of the mercapturic acid pathway and by β-lyase may be involved in the nephrocarcinogenic effects of this haloalkene in rats.     

Mutagenicity of amino acid and glutathione S-conjugates in the Ames test

The mutagenicity of the glutathione S-conjugate S-(1,2-dichlorovinyl)glutathione (DCVG), the cysteine conjugates S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2-dichlorovinyl)-DL-alpha-methylcysteine (DCVMC), and the homocysteine conjugates S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) and S-(1,2-dichlorovinyl)-DL-alpha-methylhomocysteine (DCVMHC) was investigated in Salmonella typhimurium strain TA2638 with the preincubation assay. DCVC was a strong, direct-acting mutagen; the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid decreased significantly the number of revertants induced by DCVC; rat renal mitochondria (11,000 X g pellet) and cytosol (105,000 X g supernatant) with high beta-lyase activity increased DCVC mutagenicity at high DCVC concentrations. DCVG was also mutagenic without the addition of mammalian activating enzymes; the presence of low gamma-glutamyltransferase activity in bacteria, the reduction of DCVG mutagenicity by aminooxyacetic acid, and the potentiation of DCVG mutagenicity by rat kidney mitochondria and microsomes (105,000 X g pellet) with high gamma-glutamyltransferase activity indicate that gamma-glutamyltransferase and beta-lyase participate in the metabolism of DCVG to mutagenic intermediates. The homocysteine conjugate DCVHC was only weakly mutagenic in the presence of rat renal cytosol, which exhibits considerable gamma-lyase activity, this mutagenic effect was also inhibited by aminooxyacetic acid. The conjugates DCVMC and DCVMHC, which are not metabolized to reactive intermediates, were not mutagenic at concentrations up to 1 mumole/plate. The results demonstrate that gamma-glutamyltransferase and beta-lyase are the key enzymes in the biotransformation of cysteine and glutathione conjugates to reactive intermediates that interact with DNA and thereby cause mutagenicity.     

Mutagenicity of thiol compounds in Escherichia coli WP2 tester strain IC203, deficient in OxyR: effects of S9 fractions from rat liver and kidney

Low doses of -cysteine (CYS), cysteinyl-glycine (CYSGLY) and reduced glutathione (GSH) activated by γ-glutamyl transpeptidase (GGT) were mutagenic in strain IC203 (oxyR), whereas higher doses were required to observe a weak mutagenicity in the oxyR⁺ strain WP2 uvrA/pKM101 (denoted IC188). This indicates that thiol mutagenesis is suppressed by OxyR-regulated antioxidant defenses and confirms its oxidative character. The mutagenesis by low doses of CYS, CYSGLY and GSH+GGT detected in IC203 was abolished by rat liver S9, through the activity of catalase, as well as by the metal chelator diethyldithiocarbamate (DETC), supporting the dependence of this mutagenesis on H₂O₂ production, probably in thiol autoxidation reactions in which transition metals are involved. Surprisingly, low DETC concentrations greatly potentiate the mutagenicity of low CYS doses. Mutagenesis by high doses of CYS and CYSGLY occurred in both IC203 and IC188 in the presence of liver S9, and was resistant to inhibition by catalase, although it was prevented by DETC. Mutagenesis by GSH activated by rat kidney S9, rich in GGT, was detected in IC203 and IC188 only at high doses since catalase and glutathione peroxidase, both present in kidney S9, might inhibit its induction by low GSH doses. In the presence of liver S9, almost deficient in GGT, GSH was not mutagenic. The mutagenicity of a high GSH dose occurring in the presence either of GGT plus liver S9 or of kidney S9 was weakly prevented by DETC.     

Mutagenicity of the glutathione and cysteine S-conjugates of the haloalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene and trichlorofluoroethene in the Ames test in comparison with the tetrachloroethene-analogues

The nephrotoxic and nephrocarcinogenic potential of the haloalkenes is associated with the conjugation of the chemicals to L-glutathione. Subsequent processing of the haloalkene glutathione S-conjugates via the cysteine conjugate beta-lyase pathway in the mammalian kidney yields nephrotoxic and mutagenic species. To investigate whether S-conjugates of the model chlorofluoroalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene (CAS # 431-52-7) and trichlorofluoroethene (CAS # 359-29-5) show comparable effects, we have synthesised the respective cysteine and glutathione S-conjugates and subjected them to the Ames test. The cysteine and glutathione S-conjugates of tetrachloroethene (CAS # 127-18-4), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) and S-(1,2,2-trichlorovinyl)glutathione (TCVG) were used as positive controls and reference substances. S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)-L-cysteine (DCTFPC) and S-(2,2-dichloro-1-fluorovinyl)-L-cysteine (DCFVC) showed clear dose-dependent mutagenic effects with the Salmonella typhimurium tester strains TA100 and TA98. Using TCVC as a reference substance the following ranking in mutagenic response was established: TCVC>DCTFPC>DCFVC. S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)glutathione (DCTFPG) and S-(2,2-dichloro-1-fluorovinyl)glutathione (DCFVG) showed potent dose-dependent mutagenic effects with the S. typhimurium tester strain TA100 in the presence of a rat kidney S9-protein fraction; tests carried out in the absence of the bioactivation system resulted only in background rates of revertants. Using TCVG as a reference substance the following ranking in mutagenic response was established: TCVG=DCTFPG>DCFVG.The data obtained provide a basis for further studies on the mutagenic and presumable carcinogenic potential of the substances.     

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. Activation by the isolated perfused rat liver.

In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.     

Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus.

To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified beta-lyase from Peptostreptococcus magnus GAI0663, since P. magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys). The activity of purified beta-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55 degrees C for 5 min. The molecular weight of beta-lyase was 150,000, as determined by fast protein liquid chromatography. S-Arylcysteine conjugates were good substrates for this enzyme. As determined by the Salmonella mutagenicity test, 5 ng of beta-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100. However, beta-lyase had little effect on the cysteine conjugate of 1-NP 4,5-oxide (10 nmol per plate). Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR. In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified beta-lyase or xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterial cysteine conjugate beta-lyase and the metabolism of cysteine S-conjugates: structural requirements for the cleavage of S-conjugates and the formation of reactive intermediates

The cysteine conjugate beta-lyase mediated metabolism and the mutagenicity of the synthetic cysteine conjugates S-(2-chloroethyl)-L-cysteine (CEC), S-(2-chlorovinyl)-L-cysteine (CVC), S-(1,2,3,3,3-pentachloroprop-1-enyl)-L-cysteine (PCPC), S-(pentachlorophenyl)-L-cysteine (PCPhC), S-(chloro-1,2,2-trifluoroethyl)-L-cysteine (CTFEC), S-benzyl-L-cysteine (SBC) and S-methyl-L-cysteine (SMC) were investigated in Salmonella typhimurium strains TA100, TA2638, TA102 and TA98 to establish structure/activity relationships. Bacterial 100,000 X g supernatants cleaved CTFEC, PCPC, CVC, PCPhC and SBC to pyruvate; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA) in all cases. Of the compounds tested, CEC, PCPC and CVC were mutagenic in the Ames-test. CTFEC, PCPhC and SBC failed to increase the number of revertants above control levels. The mutagenicity of PCPC and CVC could be inhibited by AOAA. CEC exerted a potent mutagenic effect in the Ames-test which was not affected by AOAA; CEC was not transformed to pyruvate by bacterial beta-lyase. Neither pyruvate formation nor mutagenicity were observed with SMC. These results indicate that the structure of the substituent on the sulfur atom is an important determinant for the biological activity of cysteine S-conjugates. Electronegative and/or unsaturated substituents are required for beta-lyase catalysed beta-elimination reactions. The formation of chemically unstable thiols, which may be converted to thioacylating intermediates, seems to be a prerequisite for beta-lyase dependent mutagenicity of S-conjugates.     

Seasonal variations in mutagenic activity of air pollutants at an industrial district of Silesia

Organic material from airborne particulate pollutants collected over a 7-month period at a highly industrialized region in Silesia (Poland) was tested for mutagenicity using the Ames test. Sequential elution solvent chromatography (SESC) was used for the separation of crude benzene extracts. Five out of 8 fractions showed mutagenic activity with differential direct and indirect responses. The mutagenicity of each active fraction was tested during the whole sampling period (from August to February 1984/1985) and seasonal variations were observed. All of the fractions, except fraction 3, showed only quantitative distinctions in mutagenic potential, expressed as a number of revertants per m3 of air. Over a period of 7 months, a steady increase of activity of fractions 2 and 4 was observed but the type of mutagenic response, indirect and direct respectively, remained unchanged in the summer and winter months. Fraction 3 (the most abundant component, probably containing polar derivatives of PAHs and heterocyclics) differed quantitatively and qualitatively between summer and winter time. From August to December samples showed enhanced mutagenic potency upon addition of rat liver microsomal enzymes, whereas in January a 4-5-fold increase in direct response was noted. This significant increase in direct mutagenic activity was accompanied by a considerable decrease in mean air temperature and resulted most probably from the intensive use of coal for domestic heating.     

Bacterial beta-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene

The metabolism of beta-lyase and the mutagenicity of the synthetic cysteine conjugates S-1,2-dichlorovinylcysteine (DCVC), S-1,2,2-trichlorovinylcysteine (TCVC), S-1,2,3,4,4-pentachlorobuta-1,3-dienylcysteine (PCBC) and S-3-chloropropenylcysteine (CPC) were investigated in Salmonella typhimurium strains TA100, TA2638 and TA98. The bacteria contained significantly higher concentrations of beta-lyase than mammalian subcellular fractions. Bacterial 100,000 X g supernatants cleaved benzthiazolylcysteine to equimolar amounts of mercaptobenzthiazole and pyruvate. DCVC, TCVC and PCBC produced a linear time-dependent increase in pyruvate formation when incubated with bacterial 100,000 X g supernatants; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA). CPC was not cleaved by bacterial enzymes to pyruvate. DCVC, TCVC and PCBC were mutagenic in three strains of S. typhimurium (TA100, TA2638 and TA98) in the Ames-test without addition of mammalian subcellular fractions; their mutagenicity was decreased by the addition of AOAA to the preincubation mixture. CPC was not mutagenic in any of the strains of bacteria tested. These results indicate that beta-lyase plays a key role in the metabolism and mutagenicity of haloalkenylcysteines when tested in S. typhimurium systems. The demonstrated formation in mammals of the mutagens DCVC, TCVC and PCBC during biotransformation of trichloroethylene (Tri), tetrachloroethylene (Tetra) and hexachlorobutadiene (HCBD) may provide a molecular explanation for the nephrocarcinogenicity of these compounds.     

Release of mutagenic metabolites of benzo[a]pyrene from the perfused rat liver after inhibition of glucuronidation and sulfation by salicylamide

The role of glucuronide and sulfate conjugation in presystemic inactivation of benzo[a]pyrene (BP) metabolites was investigated with rat livers perfused with BP (12 mumol). Comparisons were made between metabolite profiles and mutagenicity of medium from perfusions with and without salicylamide, a selective inhibitor of glucuronide and sulfate conjugation. After 4 h perfusion in the presence of salicylamide, certain BP metabolites (diols, quinones, phenols, and metabolites more polar than BP-9,10-diol) were significantly increased at the expense of quinones and phenols in the glucuronide fraction. Mutagenicity of medium (detected by the Ames test, using tester strains TA98 and TA100) was low in perfusion without salicylamide. Mutagenicity detected with tester strain TA98 was significantly increased in perfusions with salicylamide. Involvement of glucuronidation in BP inactivation was also observed at the subcellular level; when cofactors of glucuronidation were added to liver homogenates along with the NADPH regenerating system in the Ames test, BP mutagenicity was markedly decreased. Both the activation of BP to mutagenic metabolites and the inactivation of BP metabolites by glucuronidation was much more pronounced with liver homogenates from 3-methylcholanthrene-treated rats than with those from phenobarbital-treated animals or untreated controls. The results suggest an important role for glucuronidation and sulfation in the inactivation and elimination of polycyclic aromatic hydrocarbons.     

Nitrogen-substitution effects on the mutagenicity and cytochrome P450 isoform-selectivity of chrysene analogs

Nitrogen-containing analogs of chrysene, 1,10-diazachrysene (1,10-DAC) and 4,10-DAC, were tested for mutagenicity in Salmonella typhimurium TA100 in the presence of rat liver S9 and human liver microsomes to investigate the effect of nitrogen-substitution. Although these DACs could not be converted to the bay-region diol epoxide because of their nitrogen atoms in the bay-region epoxide or diol moiety, DACs were mutagenic in the Ames test with rat liver S9. Both DACs also showed mutagenicity in the Ames test using pooled human liver microsomes, although chrysene itself was not mutagenic in the presence of pooled human liver microsomes. The mutagenicity of DACs (50nmol/plate) in Ames tests using human liver microsome preparations from 10 individuals was compared with cytochrome P450 (CYP) activity in each microsome preparation to investigate the CYP isoform involved in the activation of DACs to the genotoxic forms. The numbers of induced revertants obtained by 1,10-DAC varied 6.2-folds (109-680) and those by 4,10-DAC 4.8-folds (155-751) among the 10 individuals. The number of induced revertants obtained by 1,10-DAC significantly correlated with the CYP1A2-selective catalytic activity (r=0.84, P<0.01) in each microsome preparation. On the other hand, the number of induced revertants obtained by 4,10-DAC significantly correlated with the combined activity of CYP2A6 and 1A2 (CYP2A6+0.51xCYP1A2; r=0.75, P<0.01). However, in Ames tests using microsomes from insect cells expressing various human CYP isoforms, the mutagenicity of 1,10-DAC was induced only by recombinant human CYP1A2, whereas both recombinant human CYP2A6 and 1A2 contributed to the mutagenicity of 4,10-DAC. These results suggest that 1,10-DAC shows the mutagenicity through involvement of CYP1A2 in human liver, and 4,10-DAC does so through both CYP2A6 and 1A2. In conclusion, our results suggested that the difference in the nitrogen-substituted position in the chrysene molecule might affect the mutagenic activity through influencing the ratio of participation of the metabolic activation enzyme isoforms of CYP.     

Effect of 10-aza-substitution on benzo[a]pyrene mutagenicity in vivo and in vitro

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (MutaMouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.     

Mutagenicity of several derivatives of dipyrido[1,2-a:2'',3''-d]imidazoles

The mutagenicity of 2-aminofluorene, 4-aminobiphenyl and 3,2'-dimethylaminobiphenyl towards Salmonella typhimurium was studied in the presence of microsomes from liver, kidney and small intestine of untreated and pretreated rats. The aim was to study a possible correlation between the organotropism of these amines and their activation into mutagenic intermediates by these three tissues. Pretreatment of the rats with phenobarbital, Aroclor 1254 and 3-methylcholanthrene injected intraperitoneally increased the liver microsomal-mediated mutagenic activity of the three amines but remained without effect on the activating capacity of microsomes from the kidney and small intestine. However, pretreatment with 3-methylcholanthrene administered intragastrically increased the small-intestine microsomal-mediated mutagenicity of 2-aminofluorene almost 3-fold but remained without effect on the mutagenicity of 4-aminobiphenyl and 3,2'-dimethylaminobiphenyl. No mutagenic effect was observed with 4-aminobiphenyl in the presence of kidney microsomes or with 4-aminobiphenyl and 3,2'-dimethylaminobiphenyl in the presence of small-intestine microsomes, obtained from either untreated or pretreated animals. It is concluded that no relationship exists between the mutagenic activities of the three amines, as detected in the Ames test, and their carcinogenic organotropisms.     

Renal cysteine conjugate beta-lyase. Bioactivation of nephrotoxic cysteine S-conjugates in mitochondrial outer membrane

Cysteine conjugate beta-lyase activity from rat kidney cortex was found in the cystosolic and mitochondrial fractions. With 2 mM S-(2-benzothiazolyl)-L-cysteine as the substrate, approximately two-thirds of the total beta-lyase activity was present in the cytosolic fraction. The kinetics of beta-lyase activity with three cysteine S-conjugates were different in the cytosolic and mitochondrial fractions, and the mitochondrial beta-lyase was much more sensitive to inhibition by aminooxyacetic acid than was the cytosolic activity. These results indicate that the beta-lyase activities in the two subcellular fractions are catalyzed by distinct enzymes. Nephrotoxic cysteine S-conjugates of halogenated hydrocarbons that require bioactivation by cysteine conjugate beta-lyase (S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, CTFC) were potent inhibitors of state 3 respiration in rat kidney mitochondria. Fractionation of mitochondria by digitonin treatment and comparison with marker enzyme distributions showed that the mitochondrial beta-lyase activity is localized in the outer mitochondrial membrane. Inhibition of the beta-lyase prevented the mitochondrial toxicity of DCVC and CTFC, and nonmetabolizable, alpha-methyl analogues of DCVC and CTFC were not toxic. Neither DCVC nor CTFC was toxic to mitoplasts, indicating that activation by the beta-lyase occurs on the outer membrane and may be essential for the expression of toxicity; in contrast, the direct acting nephrotoxin S-(2-chloroethyl)-DL-cysteine was toxic to both mitochondria and mitoplasts. Thus, the suborganelle localization of DCVC and CTFC bioactivation correlates with the observed pattern of toxicity.     

Effect of 10-aza-substitution on benzo[a]pyrene mutagenicity in vivo and in vitro

Benzo[a]pyrene (BaP), an environmental carcinogen, shows genotoxicity after metabolic transformation into the bay-region diol epoxide, BaP-7,8-diol 9,10-epoxide. 10-Azabenzo[a]pyrene (10-azaBaP), in which a ring nitrogen is located in the bay-region, is also a carcinogen and shows mutagenicity in the Ames test in the presence of the rat liver microsomal enzymes. In order to evaluate the effect of aza-substitution on in vivo genotoxicity, BaP and 10-azaBaP were assayed for their in vivo mutagenicity using the lacZ-transgenic mouse (Muta™Mouse). BaP was potently mutagenic in all of the organs examined (liver, lung, kidney, spleen, forestomach, stomach, colon, and bone marrow), as described in our previous report, whereas, 10-azaBaP was slightly mutagenic only in the liver and colon. The in vitro mutagenicities of BaP and 10-azaBaP were evaluated by the Ames test using liver homogenates prepared from several sources, i.e. CYP1A-inducer-treated rats, CYP1A-inducer-treated and non-treated mice, and humans. BaP showed greater mutagenicities than 10-azaBaP in the presence of a liver homogenate prepared from CYP1A-inducer-treated rodents. However, 10-azaBaP showed mutagenicities similar to or more potent than BaP in the presence of a liver homogenate or S9 from non-treated mice and humans. These results indicate that 10-aza-substitution markedly modifies the nature of mutagenicity of benzo[a]pyrene in both in vivo and in vitro mutagenesis assays.     

Dimethylnitrosamine metabolism: I. In vitro activation of dimethylnitrosamine to mutagenic substance(s) by hepatic and renal tissues from three inbred strains of mice

The potential of hepatic and renal homogenates from three inbred strains of mice (BALB/c, C57BL and DBA) to activate dimethylnitrosamine (DMN) was investigated. Microsomal enzyme (S-9) preparations of liver and kidney from mature and immature mice were used in the Ames Salmonella mutagenicity assay. No age or sex-related differences in the formation of active mutagenic DMN Metabolites by liver microsomal enzymes were observed within any of the three inbred strains. In contrast, mature male kidney S-9 fractions from all three strains had a significantly greater potential to activate DMN than mature female and immature animals. Testosterone treatment resulted in no apparent changes in the ability of hepatic tissue to biotransform DMN to its mutagenic metabolites among age and sex classes. However, after testosterone treatment, renal microsomal fractions from mature female mice of all three strains did not differ significantly from their male counterparts in their ability to transform DMN to mutagenic metabolites.     

The spectrum of enzymes involved in activation of 2-aminoanthracene varies with the metabolic system applied

The aim of this study was to estimate the involvement of cytochrome P450s (CYPs) in the metabolic activation of 2-aminoanthracene (2AA) by use of metabolic systems such as liver S9 or hepatocytes from untreated and beta-naphthoflavone (BNF)- or phenobarbital (PB)-treated rats. Metabolic activation was determined in the Salmonella reverse mutation assay (Ames test). Unexpectedly, both enzyme inducers, BNF and PB, significantly decreased the mutagenicity of 2AA activated by S9 fractions. 2AA mutagenicity was detected in the presence of cytochrome P450 inhibitors such as alpha-naphthoflavone (ANF), clotrimazole and N-benzylimidazole to study the contribution of CYP isoenzymes to the activation process. ANF significantly decreased the activation of 2AA by S9 from untreated rats. In contrast, ANF significantly increased the metabolic activation of 2AA by S9 from BNF- and PB-treated rats. The enhanced mutagenicity was not altered by co-incubation with clotrimazole and ANF. Pre-incubation of 2AA in the presence of N-benzylimidazole significantly increased the activation of 2AA by S9 from BNF- and PB-treated rats, which suggests that CYPs play minor role in 2AA metabolic activation by rat liver S9 fractions. In contrast with the results described above, BNF treatment of rats significantly enhanced the activation of 2AA by hepatocytes. ANF attenuated the extent of this activation suggesting that different enzymes play a major role in the activation processes in these metabolic systems. Our results indicate that identification of mutagenic hazard by use of the Ames test may depend on the metabolic system applied.     

Genotoxicity of six pesticides by Salmonella mutagenicity test and SOS chromotest

Two in vitro tests (Ames test and SOS chromotest), one for bacterial mutagenicity and one for primary DNA damage, were assayed to determine the genotoxic activity of 6 pesticides (atrazine, captafol, captan, chlorpyrifosmethyl, molinate and tetrachlorvinphos). Assays were carried out both in the absence and presence of S9 fractions of liver homogenate from rat (Sprague–Dawley) pretreated with Aroclor 1254. Captan and captafol were genotoxic on both the Ames test and the SOS chromotest. Comparisons with mutagenesis data in Salmonella indicated that the SOS assay detected as genotoxic the pesticides that were mutagenic on the Salmonella test. Non-genotoxic effects were not detected in vitro either in the Salmonella/microsome assay nor in the SOS chromotest when bacterial tester strains were exposed to atrazine, molinate, chlorpyrifosmethyl and tetrachlorvinphos in the absence or presence of S9 mix.     

Glutathione-dependent dechlorination of 1,6-dichloro-1,6-dideoxyfructose.

The metabolism of 14C- and 36Cl-labelled 1,6-dichloro-1,6-dideoxyfructose (DCF) was studied in the isolated perfused rat liver system. Dechlorination of DCF occurred in the liver and erythrocytes and was GSH-dependent. The GSH conjugate formed was identified by 13C and 1H n.m.r. as the 6-chlorofructos-1-yl-SG conjugate. It is proposed that the GS- anion attacks the low steady-state concentration of the reactive keto form of DCF and that the conjugate formed cyclizes to the dominant beta-anomer. 6-Chlorofructos-1-yl-SG conjugate of hepatic origin is excreted into bile, whereas that produced in erythrocytes does not enter the liver.     

Mutagenicity and irreversible binding of the hepatocarcinogen, 2,4-diaminotoluene.

Mutagenicity of 2,4-diaminotoluene (DAT) in the Salmonella mutagenicity assay was increased with liver fractions from phenobarbital (PB) or beta-naphthoflavone (BNF) treated rats. Substitutions of the hydrogens in the methyl group of 2,4-DAT with deuterium resulted in a decrease in mutagenicity. Incubation of rat liver microsomes with tritiated 2,4-DAT in the presence of NADPH led to the formation of irreversibly bound products to microsomal protein. The rates of binding were not increased using microsomes from PB or BNF-treated rats and was not altered by deuterium substitution in the methyl group. Addition of superoxide dismutase, glutathione (GSH) or rat liver supernatant reduced 2,4-DAT irreversible binding, whereas 2,4-DAT mutagenicity was unaffected by superoxide dismutase addition. Injection of tritiated 2,4-DAT 100 mg/kg to rats lead to its irreversible binding to liver protein and ribosomal RNA and to kidney protein in vivo, again protein binding was not increased after prior treatment with PB or BNF. No irreversible interaction of tritiated 2,4-DAT with DNA either in vitro or in vivo could be demonstrated.