Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays.

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

Mutagenic activation of aromatic amines by a human hepatoma cell (Hep G2) supernatant tested by means of Salmonella typhimurium strains with different acetyltransferase activities.

The study was carried out to characterize hepatoma cells (Hep G2) as activation system relevant to man and to investigate which are the tester strains most suitable for the mutagenic assay of aromatic amines. A supernatant prepared from the human hepatoma cell line Hep G2 was used to activate benzidine, 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) in the Salmonella typhimurium reversion assay. Activation by Hep G2 supernatant was studied with increasing concentrations of the three compounds, in tester strains TA98, YG1024, DJ400 and DJ460. Benz[alpha]anthracene (BA) pretreatment of cells increases the mutagenicity of benzidine in strains YG1024, DJ460 and DJ400. Activation of 2-AAF and 2-AF was observed in strains YG1024, DJ400 and, at the highest tested dose, in DJ460. These results were compared with those obtained with S9 from control and Aroclor 1254 (Aro)-pretreated rat liver. With strain TA98 comparable responses were obtained except for 2-AF which was better activated using rat liver S9. The use of strain YG1024 greatly increases the sensitivity of the response. Strain DJ460 makes it possible to detect activation of 2-AF and 2-AAF by Aro-induced rat liver. These results indicate that Hep G2 supernatant is a useful metabolic activation system of human origin that can be used to replace rat liver S9. An appropriate choice of the Salmonella strain not only can increase the sensitivity of the response, but may also help to overcome certain metabolic shortcomings of the Hep G2 cell line and rat liver S9.

Role of kidney S9 in the mutagenic properties of 1,2-dibromoethane.

The mutagenic properties of 1,2-dibromoethane (DBE) were studied in the Ames Salmonella typhimurium assay using the strains TA 1535 and TA 100. Kidney S9 fraction alone did not modify the direct mutagenic activity of DBE; but an addition of kidney S9 to liver S9 fraction yielded a higher mutagenic activity of DBE than with liver S9 fraction alone. Moreover, the addition of glutathione (GSH) to kidney S9 increased the mutagenic activity of DBE. Methimazole, a competitive inhibitor of the flavin-containing monooxygenase, reduced mutagenic activity suggesting that this enzyme may contribute to renal damage from DBE. No mutagens could be detected in the urine of rats treated with DBE.

Metabolic activation by a supernatant from human hepatoma cells: a possible alternative in mutagenic tests.

The supernatant from human Hep G2 hepatoma cells was examined for typical enzymatic activities involved in the metabolism of xenobiotics. Neither cytochrome P-450 nor b5 was detectable, but associated enzymatic activities were found especially after induction with hydrocortisone (HC) and benzanthracene (BA) suggesting that this Hep G2 supernatant contains cyt P-450 IA1 and IA2. Other critical enzymes are also present, but, as expected, at lower activities than in Aroclor 1254 rat liver S9, except for NADH and NADPH cytochrome c reductase. Results of the Ames test indicate that the induced Hep G2 supernatant is a suitable activator for the evaluation of genotoxicity of indirect mutagens.

Oxidative activation of 2-aminofluorene by human red blood cell cytosol.

Purified red blood cell cytosol is able to activate 2-aminofluorene (2AF) to N-hydroxy-2-aminofluorene. Apparent kinetic parameters are determined with and without methylene blue. The latter, which maintains haemoglobin in the reduced form and stimulates NADPH production, increases the affinity of the enzyme for the 2AF. This activity is inhibited by carbon monoxide while cyanide is without effect. The involvement of a peroxidative reaction or a one-electron oxidative mechanism involving free radicals may be excluded.

Enzymic N-demethylation reaction catalysed by red blood cell cytosol.

Red blood cell cytosol promotes enzymic N-demethylation reactions which display typical Michaelis-Menten kinetics with respect to N-methylaniline as substrate. The demethylase activity is linked with hemoglobin (Hb) and is enhanced in the presence of NADH and the NADH-methemoglobin reductase system. It has been adduced that Hb in its oxygenated form is involved in the reaction.

Mutagenicity of some heterocyclic amines in Salmonella typhimurium with metabolic activation by human red blood cell cytosol.

Purified human red blood cell cytosol was used to activate the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) into mutagenic intermediate(s) in the Salmonella test. The liquid preincubation method in the presence of strain TA98 was utilized. In order to understand the mechanism involved in this metabolic activation, some modulators were incorporated in the medium. The results suggest that an oxygenated hemoprotein, probably oxyhemoglobin, is involved in the activation into genotoxic intermediate(s).

Activation of some aromatic amines to mutagenic products by human red blood cell cytosol.

The ability of human red blood cell cytosol to activate aromatic amines was evaluated with the Ames test using Salmonella typhimurium TA98 in the liquid preincubation condition. While negative results were obtained with 4-acetylaminofluorene (4AAF) and 1-naphtylamine (1NA), a slight response was observed for 4-aminobiphenyl (4ABP) and 2-naphthylamine (2NA). Human red blood cell cytosol was able to activate 2-aminofluorene (2AF), 2-acetylaminofluorene (2AAF) and 2-aminoanthracene (2AA) to mutagenic intermediates. Extracts of human red blood cell cytosol incubated with 2AF were analyzed by gas chromatography: N-hydroxy-2-aminofluorene was identified as a metabolite.

[Comparison of the teratogenic properties manifested in BALB c mice, by diphenylhydantoin and deuterated diphenylhydantoin]. / Comparaison des propriétés tératogènes manifestées, chez les souris de race BALB-c, par la diphénylhydantoïne et la diphénylhydantoïne deutérée.

The aim of this study was to investigate the role of an arene oxide pathway in the teratogenicity displayed by DPH, a highly effective antiepileptic agent. This approach was carried out by comparing the teratogene potential of DPH and of p2-H-DPH administrated at days 8, 9 and 12 of gestation. The present findings support the hypothesis that substitution of protium by deuterium at the para position of one of the phenyl rings, which favours an arene oxide pathway, causes an increase in some to the teratogenic effects of DPH.

Detection of mutagenic activity in urine samples using a new concentration procedure.

A study performed with cyclophosphamide (CP) and nor-nitrogen mustard (NNM), one of its main urinary metabolites, has shown that separation on Polygosil C-18 resin is preferable to one on XAD-2 resin as a means of concentrating the mutagenic activity present in urine of rats given cytostatics such as CP. Mutagenic activity was detected, using Salmonella typhimurium tester strain TA1535. While NNM is irreversibly bound to XAD-2 resin, it can be recovered after elution with methanol on Polygosil C-18. The better efficacity of Polygosil C-18 in concentrating CP and its metabolite(s) was confirmed with experiments with urine of rats treated with increasing doses of CP.

Mutagenicity evaluation of azaperone in the Salmonella/microsome test.

Azaperone was evaluated for its mutagenic potential by the Salmonella/microsome test. No mutagenic activity towards six S. typhimurium strains could be evidenced with azaperone at doses up to 2,000 micrograms/plate, either without or with metabolic activation at usual test conditions. Higher concentrations of liver post-mitochondrial fraction from Aroclor 1254 (ARO)-pretreated rats did not reveal any increase in the number of revertants towards S. typhimurium strains TA1537, TA1538 and TA98. Moreover, a plate-incorporation test with liver post-mitochondrial fractions from mice pretreated with phenobarbital (PB) and a liquid preincubation test with liver post-mitochondrial fractions from rats pretreated with ARO also failed to reveal any mutagenic action of azaperone towards S. typhimurium strain TA98. Thus, none of the tests used provided any indication of azaperone having a mutagenic action.

Metabolism and mutagenicity of acrylonitrile: an in vivo study.

The mutagenic activity present in urine of animals treated with acrylonitrile (ACN) is tentatively related to the excretion of three urinary metabolites: thiocyanate (SCN-), hydroxyethylmercapturic acid (OH-MA), and cyanoethylmercapturic acid (CN-MA). It is shown that the route of administration and animal species affect SCN- excretion but not the excretion of hydroxyethyl- and cyanoethylmercapturic acids or the mutagenicity of urine from ACN-treated animals. Various pretreatments (phenobarbital, CoCl2, diethylmaleate, trichloroacetonitrile) decrease the mutagenicity of urine from ACN-treated animals and decrease the excretion of SCN- and OH-MA. None of the quantified urinary metabolites is responsible for urinary mutagenicity.

In vitro covalent binding of acrylonitrile to rat liver proteins.

The irreversible protein binding of [3-14C]acrylonitrile (ACN) was evaluated in the presence of microsomes, cytosolic and post-mitochondrial fractions of rat liver. Spontaneous binding was measured after incubation of ACN in the presence of the various liver fractions at 4 degrees C and 37 degrees C respectively and in a comparative study performed at 37 degrees C in the presence of S9 or bovine serum albumin (BSA). The effect of various inducers and inhibitors was investigated. A spontaneous binding resulting from a direct alkylation by ACN seemed to play an important role; however, binding resulting from the microsomal activation of ACN into reactive intermediate(s) was also detected.

Role of glutathione in liver-mediated mutagenicity of acrylonitrile.

The mutagenic activity of acrylonitrile (ACN) towards the Salmonella typhimurium strain TA1530 was evaluated after a short preincubation time in liquid medium in the presence of microsomes, cytosolic fractions and post-mitochondrial fractions of liver from untreated and phenobarbitone (PB)-pretreated rats and mice. The effect of the presence of glutathione (GSH) was also examined. GSH enhanced the microsomal-mediated mutagenicity of ACN; that effect was abolished in the presence of CO. The effect of GSH was usually greater after pretreatment by phenobarbitone. Other sulfhydryl compounds induce a weaker mutagenic activity than GSH. These observations support the hypothesis of a mediated formation of a mutagen involving GSH, but the adduct between ACN an GSH was not mutagenic.

Mutagenicity of styrene in the Salmonella typhimurium test system.

The mutagenic activity of styrene was investigated by incubating the strains of Salmonella typhimurium in gaseous atmospheres. The sensitive strains were TA1530, TA1535 and TA100. In that method, styrene regularly showed a mutagenic activity in the presence of a fortified liver post-mitochondrial fraction. The mutagenic activity remained weak. Moreover, it seemed that a volatile mutagenic intermediate was formed metabolically during the assays; its identity remains unknown.

Effect of several factors on the liver extract mediated mutagenicity of acrylonitrile and identification of four new in vitro metabolites.

The mutagenicity of acrylonitrile (ACN) was tested with Salmonella typhimurium TA1530 after a preincubation period of the chemical with a rat liver post-mitochondrial fraction in liquid medium. Several pretreatments were applied to the animals before the preparation of the liver fractions and different compounds added to the incubation mixture, which were shown to modify the liver mediated mutagenic activity of ACN. Four metabolites: cyanoacetic acid, cyanoethanol, acetic acid and glycolaldehyde were identified after incubation of ACN with the rat liver homogenate. From both sets of results, an in vitro metabolic scheme is proposed to ACN, which postulates the intermediate formation of a radical species and an epoxide.

Identification of two urinary metabolites of rats treated with acrylonitrile; influence of several inhibitors on the mutagenicity of those urines.

Urines collected from rats injected with acrylonitrile (ACN) were mutagenic towards Salmonella typhimurium TA1530; this activity was reduced when the animals were pretreated by pyrazole (inhibitor of alcohol dehydrogenase) and suppressed after pretreatment either by CoCl2 and SKF 525-A (inhibitors of the mixed-function oxidases system) or by trichloroacetonitrile (radical trapping agent). On the other hand, two urinary metabolites (cyanoethanol and cyanoacetic acid) have been detected by gas chromatography. One possible scheme for the in vivo metabolism of ACN is presented which postulates the intermediate formation of a radical species and of an epoxide.