Rat Liver S9 Mix Research Articles

Genotoxicity of 22 pesticides in microtitration SOS chromotest

AbstractThe authors adapted microtitration techniques to the SOS Chromotest, and thereby established a simple and reliable modified genotoxicity test: the Microtitration SOS Chromotest. This test employs microtitration techniques and photometric analysis with numerical readout. These data are integrated into the computerized data processing program Statistical Analysis System to obtain genotoxicity and toxicity results of a chemical or chemical mixture within 16 h.In order to test the applicability of the new system, the genotoxicity and toxicity of 22 pesticides were studied. A two‐sample t test was employed to determine the level of significance (p < 0.05). Nine of the pesticides were found to induce the SOS response in Escherichia coli cells. Their relative degrees of induction based on SOS inducing potency values are as follows: captafol > captan > folpet > dinoseb > ferbam > linuron > alachlor > phosmet > cacodylic acid. The addition of rat liver S9 mix does not transform the nongenotoxic pesticides tested into genotoxic forms but significantly decreases the inducing ability of most positive pesticides.The Microtitration SOS Chromotest is rapid and efficient in providing evidence for decision‐making related to cleanup of chemical spills and discharges where time is a critical factor. This method maintains its high accuracy and sensitivity compared with conventionally used assays. The studies of pesticide genotoxicity using this test confirm the applicability, reliability, and sensitivity of the Microtitration SOS Chromotest as a simple, efficient, and rapid genotoxicity prescreening system as well as an excellent research tool for genotoxic mechanism studies.

Studies on the metabolism of gomisin A (TJN-101). II. Structure determination of biliary and urinary metabolites in rat.

After oral administration of gomisin A (1) to rats, the bile and urine were collected and treated with beta-glucuronidase and arylsulfatase. Seven metabolites, met B (2), met A-III (3), met E (4), met D (5), met F (6), met G (7), and met H (8) were isolated from the bile treated with the enzymes. Eight metabolites 2-8, and met A-II (9) were isolated from the urine treated with the enzymes. A major metabolite 2, and two minor metabolites 3 and 9 were identified as met B, met A-III, and met A-II, respectively, which are oxidative products of 1 formed by rat liver S9 mix. The structures of five new metabolites 4-7, and 8 were determined on the basis of chemical and spectral studies.

Induction of chromosome aberrations and sister-chromatid exchanges by caprolactam in vitro

Caprolactam (CAP) induced chromosome aberrations in whole-blood cultures of human lymphocytes at 50 mM without metabolic activation (24-h treatment) and at 200 mM in the presence of rat liver S9 mix (1-h treatment). CAP also produced a dose-dependent increase in polyploid cells, the effect being statistically significant at 25 and 50 mM without S9 mix and at 100 and 200 mM with S9 mix. Without metabolic activation, there was an increase in hypodiploid cells at 50 mM and hyperdiploid cells at 12.5 mM. In Chinese hamster ovary cells, CAP produced a marginal elevation of sister-chromatid exchanges at 125 mM in the presence of S9 mix (4-h treatment). The results show that CAP is able to induce cytogenetic changes in vitro at very high toxic concentrations.

The genotoxicity of lucidin, a natural component of Rubia tinctorum L., and lucidinethylether, a component of ethanolic Rubia extracts.

The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA-protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3H/M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic.

Studies on the metabolism of gomisin A (TJN-101). I. Oxidative products of gomisin A formed by rat liver S9 mix.

Studies on the metabolism of gomisin A (TJN-101). I. Oxidative products of gomisin A formed by rat liver S9 mix.

Responses of the l5178y tk+/tk− mouse lymphoma cell forward mutation assay: III. 72 Coded chemicals

Seventy-two chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay, using procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al. (Mutat Res 59:61-108, 1979). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Significant responses were obtained with allyl isothiocyanate, p-benzoquinone dioxime, benzyl acetate, 2-biphenylamine HCl, bis(2-chloro-1-methylethyl)ether, cadmium chloride, chlordane, chlorobenzene, chlorobenzilate, 2-chloroethanol, chlorothalonil, cytarabine.HCl, p,p'-DDE, diazinon, 2,6-dichloro-p-phenylenediamine, N,N-diethylthiourea, diglycidylresorcinol ether, 2,4-dimethoxy aniline.HCl, disperse yellow 3, endosulfan, 1,2-epoxyhexadecane, ethyl acrylate, ethyl benzene, ethylene thiourea, F D and C yellow Number 6, furan, heptachlor, isophorone, mercuric chloride, 4,4'-methylenedianiline.2 HCl, methyl viologen, nickel sulfate.6H2O, 4,4'-oxydianiline, pentachloroethane, piperonyl butoxide, propyl gallate, quinoline, rotenone, 2,4,5,6-tetrachloro-4-nitro-anisole, 1,1,1,2-tetrachloroethane, trichlorfon, 2,4,6-trichlorophenol, 2,4,5-trimethoxybenzaldehyde, 1,1,3-trimethyl-2-thiourea, 1-vinyl-3-cyclopetene dioxide, vinyl toluene, and ziram. Apart from 2-biphenylamine.HCl, 2-chloroethanol, disperse yellow 3, ethylene thiourea, FD and C yellow number 6, phenol, and 1,1,2-tetrachloroethane, rat liver S9 mix was not a requirement for these compounds. Chemicals not identified as mutagens were acid red, 11-aminoudecanoic acid, boric acid, 5-chloro-o-toluidine, coumaphos, cyclohexanone, decabromodiphenyl oxide, di(2-ethylhexyl)adipate, ferric chloride, fluometuron, melamine, monuron, phenesterin, phthalimide, reserpine, sodium dodecyl sulfate, 4,4-sulfonyldianiline, tetrachloroethylene, and zearalenone. The assay was incapable of providing a clear indication of whether some chemicals were mutagens; these were benzyl alcohol, 1,4-dichlorobenzene, phenol, succinic acid-2,2-dimethyl hydrazide, and toluene.

Mutagenicity of halogenated propanes and their methylated derivatives.

1,2,3-Tribromopropane, 1,2,3-trichloropropane, and 1,2-dibromo-3-chloropropane are mutagenic in strains TA1535 and TA100 of Salmonella typhimurium, but only in the presence of rat liver S9 mix. This requirement for metabolic activation was unexpected for an alkyl halide and thus suggested the metabolic formation of the 2-keto derivatives (di-haloacetone). The 2-methyl derivatives of the halopropane compounds did not induce a doubling of revertants compared to controls. It was demonstrated that none of these compounds is converted into a secondary material that could be determined as structurally different by gas chromatography. These observations suggest that lack of mutagenicity of the methylated compounds is a manifestation of a steric effect.

Investigations into the genotoxic potential of loxtidine, a long-acting H2-receptor antagonist.

Loxtidine, a potent, non-competitive histamine H2-receptor antagonist was evaluated for genotoxic potential using a range of short-term mutagenicity assays. Unequivocally negative results were obtained in a Salmonella/plate incorporation assay and a liquid pre-incubation assay (using S. typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98), a fluctuation assay [using Escherichia coli strains WP2, WP2 uvrA (R46) and 343/113 lys60 (R46)], a gene conversion assay (using Saccharomyces cerevisiae JD1) and a human peripheral lymphocyte cytogenetic assay. All of these in vitro tests were carried out in the presence and absence of rat liver S9 mix. In addition, the major metabolites of loxtidine in the rat were also negative in the same range of microbial mutagenicity assays. Loxtidine was inactive in the mouse micronucleus test after oral administration. The potential nitrosatability of loxtidine was investigated using an expanded version of the WHO Nitrosation Assay Procedure, and detectable quantities of mutagenic nitroso-species were not formed. The subsequent appearance of carcinoid tumours within the gastric fundus of rodents treated orally with loxtidine for most of their natural lifespan, led to additional assays being carried out on this compound to determine whether the tumorigenic effects were due to alternative mutagenic mechanisms. Negative results were obtained in an in vitro unscheduled DNA synthesis assay using primary rat hepatocytes, and an assay for spindle damaging agents using Muntjac skin fibroblasts. It can be concluded from these results that loxtidine is unlikely to be a genotoxic carcinogen. The increase in carcinoid tumour incidence observed in rats and mice after loxtidine treatment was probably related to the prolonged achlorhydria produced by this potent unsurmountable histamine H2-receptor antagonist.

Reaction of chlorine dioxide with amino acids and peptides: Kinetics and mutagenicity studies

Chlorine dioxide (ClO 2) is currently being considered as an alternate to chlorine as a disinfectant for water treatment. Many organic compounds present in water and food treated with ClO 2 are subject to oxidation. 21 amino acids and 3 peptides ( l-aspartyl- l-phenylalanine methyl ester (aspartame), l-glycyl- l-tryptophan and l-tryptophylglycine) were studied for their reactivity with ClO 2. Chlorine dioxide reacted only with 6 amino acids in 0.1 M sodium phosphate buffer, pH 6.0. The reaction with cysteine, tryptophan and tyrosine was too rapid to be monitored either iodometrically or spectrophotometrically. The reaction with histidine, hydroxyproline and proline was found to be pseudo-first order. ClO 2 readily reacted with l-glycyl- l-tryptophan and l-tryptophylglycine but not with aspartame. Mutagenicity studies with the Salmonella microsome assay of the reaction mixtures of ClO 2 with those 6 reactive amino acids and the 3 peptides indicated that the reaction products of the 3 peptides, hydroxyproline, and tyrosine exerted mutagenic activity toward both tester strains of TA98 and TA100 in the presence and absence of rat-liver S9 mix.

Chromosome aberrations and sister-chromatid exchanges induced by technical grade toluene diisocyanate and methylenediphenyl diisocyanate in cultured human lymphocytes

Technical-grade toluene diisocyanate (TDI; 80% 2,4-isomer and 20% 2,6-isomer) and 4,4'-methylenediphenyl diisocyanate (MDI; about 45% MD1, 25% 4,4 ′-methylenediphenyl triisocyanate and 30% unspecified compounds of higher molecular weight), used as hardeners in the production of Polyurethane, induced chromosome aberrations after a 24 h treatment in the absence of metabolic activation in human whole-blood lymphocyte cultures, MDI at all doses tested (0.54–4.30 μl/ml) and TDI (0.019–0.150 μl/ml) at the highest two doses (0.075 and 0.150 μl/ml). In the presence of rat liver S9 mix (1.5 h treatment), both mixtures significantly increased aberrations at only one of the doses used, MDI at the highest dose (4.30 μl/ml) and TDI at the second highest dose (0.038 μl/ml). MDI also marginally increased sister-chromatid exchanges at the highest dose available (2.17 μl/ml) with and without (48 h treatment) S9 mix. On addition to culture medium, both TDI and MDI formed polymers, which were seen as small particles on the microscopic slides. At high doses the presence of these polymers made metaphase analysis impossible.

Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay to coded chemicals. I: Results for nine compounds.

Nine substances were tested for their mutagenic potential in the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay, by means of procedures based upon those described by Clive and Spector (Mutat Res 44:269-278, 1975) and Clive et al (Mutat Res 59:61-108, 1979). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The coded chemicals were tested at least twice. Significant responses were obtained with calcium chromate, 3-(chloromethyl)pyridine, 1,2-epoxybutane, monochloroacetic acid, dicyclohexylthiourea, 2,4-diaminophenol hydrochloride, and pentachloroanisole. Apart from pentachloroanisole, rat liver S9 mix was not a requirement for the clearly mutagenic activity of any of these compounds. Compounds not identified as mutagens were 3-amino-1,2,4-triazole and sucrose.

The action of anticlastogens in human lymphocyte cultures and their modification by rat-liver S9 mix: II. Studies with vitamins C and E

The action of vitamins C (VC) and E (VE) on the clatogenic activity of trenimon (TR), cyclophosphamide (CP) and bleomycin (BM) was tested on cultures of human peripheral blood lymphocytes with and without addition of rat-liver S9 mix. In addition, the influence of both anticlastogens on the SCE-inducing activity of TR and CP was examined under the same conditions. A distinct dose-dependent anticlastogenic effect of VC was detected in the action of long-term treatment (24 h) with TR, if the vitamin was added to the cultures simulatneously with or before the clastogen. In the short-term tests (2 or 3 h clastogen treatment ending 23 h or 21 h before harvesting) simultaneous addition of both vitamins did reduce the chromosome-damaging action of TR whether S9 mix was present or absent. While VC also decreased the frequency of chromosome damage induced by S9-mix-activated CP, VE was inactive under the same conditions. Neither vitamin significantly affected the chromosome-breaking activity of BM if S9 mix was absent, but they increased the clastogenicity of BM metabolized by S9 mix. In contrast to their anticlastogenic efficacy neither of the vitamins displayed any significant anti-SCE effect, nor were they active in affecting the inhibition of cell proliferation caused by TR or CP.

Mutagenicity of 4,4'-methylene-bis-(2-chloroaniline) "MOCA" and its N-acetyl derivatives in S. typhimurium.

4,4'-methylene-bis-(2-chloroaniline) ("MOCA") and two identified urinary N-acetyl and N,N'-diacetyl derivatives were tested in a Salmonella/mammalian microsome assay. No mutagenic activity was observed without rat liver S9 mix activation. In the presence of rat liver S9 mix, the chemicals were mutagens, but the mutagenicity of N-acetyl derivatives to strain TA100 was reduced when compared to that of "MOCA", and a greater amount of S9 was required to exhibit the mutagenicity of the N,N'-diacetyl-"MOCA". These data suggest that N-acetylation does not account for the mutagenic effectiveness of "MOCA".

Clastogenicity of a male contraceptive, gossypol, in mammalian cell cultures with and without the metabolic activation by S9 mix

The clastogenicity of a potential male contraceptive, gossypol, was examined in cultured Chinese hamster cells with and without the presence of a metabolic activation system (rat liver S9 mix). Gossypol at concentrations of 1, 5, and 10 μg/ml did not induce chromosome breakage either in the presence or absence of the S9 mix. The ability of this compound to induce chromosome breakage and polyploidy was further examined in human lymphocyte cultures. Neither increased frequencies of chromosome breakage nor polyploidy was found in lymphocyte cultures from two healthy donors. The present study indicates that gossypol does not cause genetic damage at the chromosomal level. This is consistent with previously reported findings that it does not produce mutations in the Ames test. Gossypol has been under clinical trial in China for years and shown to be effective in 99.9% of over 10,000 men tested with no or mild side effects. If this compound can be further proven to be “safe” and approved for world-wide use as a male contraceptive, it would be for the benefit of all mankind.

The action of anticlastogens in human lymphocyte cultures and its modification by rat liver S9 mix I. Studies with AET and sodium fluoride

The action of β-aminoethylisothiouronium bromide hydrobromide (AET) and sodiuim fluoride (NaF) on the clastogenic activity of Trenimon, cyclophosphamide, and bleomycin was tested on cultures of human peripheral lymphocytes with and without the addition of rat liver S9 mix. In addition, the influence of both anticlastogens on the SCE-inducing activity of Trenimon and cyclosphosphamide was examined under the same conditions. In the absence of S9 mix both substances displayed the known anticlastogenic action when TR was the standard clastogen but acted coclastogenically in the experiments with BM. Under the influence of rat-liver S9 mix this action on TR-induced chromosome damage was decreased and only a slight anticlastogenic effect was observed in the experiments with activated cyclophosphamide. S9-activated BM lost some of its strong chromosome-damaging effect and AET proved clearly anticlastogenic under these test conditions. AET displayed a slight decreasing effect on SCE induced by TR, but had no effect on CP-induced SCE. No anti-SCE effect at all was found in the experiments with NaF. Detailed analyses revealed different actions of both anticlastogens on the different types of structural chromosome damage.

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

Two preparations of maltitol (4- O-α- 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.

Increase of 2,3-epoxybutane mutagenicity by glutathione- S-transferases

The mutagenicity of 2,3-epoxybutane (2,3-EB) towards Salmonella typhimurium TA1530 is increased by rat liver S9 mix. This enhancement can be induced by Aroclor 1254 (ARO) or phenobarbitone (PB) pretreatment. Comparative assays with purified subcellular liver fractions demonstrated that cytosol is the most active in increasing the mutagenic effect of 2,3-EB. Addition of reduced glutathione (GSH) to metabolic activating mixtures containing S9 or cytosol, strengthened this effect. From these results it is suggested that 2,3-EB is stabilized through enzymic conjugation with GSH.

Mutagenicity of flavones, chromones and acetophenones in Salmonella typhimurium: New structure—activity relationships

28 flavones and 11 structurally-related flavonoids, chromones, and acetophenones, were tested for mutagenicity in the Salmonella typhimurium his reversion assay. 7 flavones, all of which were hydroxy- or methoxy-substituted at position 8, were moderate to strong mutagens in strain TA100 in the presence of rat liver S9 mix. In each case, the response of strain TA98 was either not significant or was very much weaker than that observed in strain TA100. The activation by S9 is not mediated by the microsomal cytochrome P 450 system, since activation was not diminished when microsomes were removed by centrifugation at 100000 × g. The observed strain specificity and structural requirements for activity indicate a mutagenic mechanism different from that associated with previously reported mutagenic flavonols (3-hydroxy-flavones) which are most active in strain TA98. The most mutagenic flavone investigated, 5,7,8-trihydroxy-flavone (norwogonin), had a potency of 17 revertants/nmole. Simplification of the chemical structures to hydroxy-substituted chromone and acetophenone systems revealed similar strain specificity, hydroxylation requirements, and S9 dependence within these structural classes, suggesting a similar activation pathway and mutagenic mechanism. The greatest mutagenic potency was observed within the flavone series, but significant potency was retained by similarly hydroxylated chromones and acetophenones. No mutagenic activity was observed in the absence of the aryl ketone moiety.

Bacterial mutagenicity investigation of epoxides: drugs, drug metabolites, steroids and pesticides

Although it has been observed that many epoxides are ultimate mutagens, surprisingly little is known about epoxides to which man may be extensively exposed, e.g., physiological compounds, drugs, drug metabolites and pesticides. We have now investigated 35 such and related epoxides for mutagenicity, using reversion of his − Salmonella typhimurium TA98 and TA100 as biological end-point. None of the tested steroids (12 compounds), vitamin K epoxides (3 compounds) and pesticides (dieldrin, endrin, HEOM (1,2,3,4,9,9-hexachloro-6,7-epoxy-1,4,4a5,6,7,8,8 a-octahydro-1,4-methanonaphthalene), heptachlor epoxide) showed any mutagenic activity. Negative results were also obtained with the antibiotics oleandomycin, anti-capsin and asperlin, the cardiotonic drug resibufogenin, the widely used parasympatholytic drugs butylscopolamine and scopolamine, the sedatives valtratum, didovaltratum and acevaltratum, the tranquilizer oxanamide as well as with the drug metabolites carbamazepine 10,11-oxide and diethylstilbestrol α,β-oxide. Three barbiturate epoxides, formed by metabolism of allobarbital, alphenal and secobarbital, caused weak but reproducible mutagenic effects at high concentrations. The cytostatic agent ethoglucide was the only drug having substantial mutagenic activity. Its mutagenic potency was similar to those of the control epoxides styrene 7,8-oxide, p-bromostyrene 7,8-oxide and m-bromostyrene 7,8-oxide, but much lower than those of benzo[ a]pyrene 4,5-oxide, benzo[ e]pyrene 4,5-oxide and 7,12-dimethylbenz[ a]-anthracene 5,6-oxide. Some epoxides were also tested in other Salmonella typhimurium strains or in the presence of rat-liver S9 mix. Positive results were only obtained with compounds that had already been detected as mutagens in the direct test with strain TA100.

Non-mutagenicity of 3-dimethylamino-ψ-saccharin to Salmonella typhimurium

3-Dimethylamino-ψ-saccharin has been confirmed as non-mutagenic to Salmonella typhimurium when evaluated in vitro in the presence of induced rat liver S9 mix.