R.M.E. Brouns

Effect of toluene and xylenes on liver glutathione and their urinary excretion as mercapturic acids in the rat

Administration of toluene and xylenes to rats caused a decrease in liver glutathione concentration. The effect was most pronounced after the administration of o-xylene. 26% of the initial glutathione level was found three hours after treatment with o-xylene (4.0 mmoles/kg).No in vitro conjugation of o-xylene with glutathione was observed, neither spontaneously nor in the presence of 105,000 g supernatant from rat liver homogenate, containing glutathione S-transferases. Thus, a metabolite of o-xylene, which is not formed during incubation with 105,000 g supernatant, reacts with glutathione.A thioether was isolated from urine of rats given o-xylene; the compound was identified as o-methylbenzyl mercapturic acid by GC-MS and NMR. Chromatographic evidence was found for the presence of benzyl mercapturic acid in the urine of toluene-treated rats. The amounts of mercapturic acids excreted in the urine after administration of toluene, p-xylene, m-xylene, and o-xylene were 0.4–0.7, 0.6, 1.3, and 10–21% of the dose, respectively.These results demonstrate the involvement of a thusfar unknown pathway in the biotransformation of toluene and xylenes.

Non-mutagenicity of toluene, o-, m- and p-xylene, o-methylbenzylalcohol and o-methylbenzylsulfate in the Ames assay.

Abstract Toluene, o -, m - and p -xylene, o -methylbenzylalcohol and o -methylbenzylsulfate were assayed for mutagenicity in the Ames assay. These compounds were unable to revert Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100, either with or without metabolic activation by S9 mix derived from livers of rats either untreated or induced with Aroclor 1254.

Measurement of DNA-excision repair in suspensions of freshly isolated rat hepatocytes after exposure to some carcinogenic compounds: its possible use in carcinogenicity screening.

When suspensions of freshly isolated rat hepatocytes were exposed to a number of carcinogenic compounds, it was possible to measure an increased UDS by a rapid procedure via liquid-scintillation counting. For a number of carcinogenic compounds and some of their non-carcinogenic structural analogues a good correlation between the carcinogenic property and the ability to induce UDS was demonstrable. Out of 12 carcinogenic compounds, belonging to several different chemical classes, 10 gave rise to an increased UDS, whereas only 2 compounds, the polycyclic aromatic hydrocarbons benzo[alpha]pyrene and benz[alpha]anthracene, did not. All 4 noncarcinogenic compounds tested were negative. Possibly this method can be of value as a routine screening test, in combination with other short-term test systems, thus improving the predictive value of screening in vitro with respect to carcinogenicity.

Mutagenic effects of benzo[a]pyrene after metabolic activation by hepatic 9000 g supernatants or intact hepatocytes.

The activating capacities of isolated rat hepatocytes and 9000 g supernatant from these cells with respect to the mutagenic effect of benzo[a]pyrene on Salmonella typhimurium TA100 were investigated. No mutagenicity of benzo[a]pyrene was found with the cell-mediated assay, unless the hepatocytes were disrupted after pre-incubation with benzo[a]pyrene or the intracellular glutathione content was reduced. It is suggested that a retention of active metabolites and an effective detoxication may account for the absence of mutagenic response.

Metabolic activation of 2-aminofluorene by isolated rat liver cells through different pathways leading to hepatocellular DNA-repair and bacterial mutagenesis

The aromatic amine 2-aminofluorene (2-AF) is metabolised by isolated rat liver cells to reactive species, thereby causing mutagenic effects in Salmonella typhimurium TA 1538 and evoking DNA-excision repair within the liver cells. The pathway leading to the production of metabolites mutagenic in Salmonella is likely to proceed via direct N-hydroxylation of 2-AF to N-hydroxy-2-aminofluorene (N-OH-2-AF). On the other hand, the formation of intermediates giving rise to hepatocellular DNA-repair is shown to depend upon N-acetylation of 2-AF to 2-acetylaminofluorene(2-AAF), whereas a subsequent conjugation reaction, most likely to be sulfate ester formation, is also essentially involved.

Involvement of non-oxidative enzymes in mutagenic activation of urine from rats, given benzidine and some other aromatic amines.

Urinary metabolites of rats treated with benzidine and some other genotoxic aromatic amines became mutagenic in the Ames assay after activation with liver cytosol from rat, mouse and guinea pig. Most of the mutagenic metabolites appeared in urine as glucuronides. Strong evidence was found that N,O-acyltransferase is responsible for the mutagenic activation by rat liver cytosol. The inhibitory effect of paraoxon and sodium fluoride indicates that the activation by mouse liver cytosol is due to the action of deacetylase. Mutagenic activation by guinea pig liver cytosol seemed to be mediated in part by deacetylase. The metabolite activated by these enzymes most likely is a glucuronidated, N-acetylated, N-hydroxylated product.

Metabolic activation of 2-acetylaminofluorene by isolated rat liver cells

Isolated rat liver cells are able to metabolize 2-acetylaminofluorene (2-AAF) to reactive species, capable of producing mutagenic effects in Salmonella typhimurium TA 1538 and evoking unscheduled DNA synthesis within the hepatocytes. Indications are presented, that these genotoxic effects are caused by different reactive metabolites. Mutagenesis could be blocked almost completely by paraoxon, an inhibitor of the de-acetylation reaction, whereas induction of DNA excision repair was prevented by antagonizing the sulfation reaction by means of salicylamide.

Studies on the metabolic activation of benzidine by isolated rat hepatocytes

Isolated rat liver cells were shown to metabolize the aromatic amine benzidine to reactive products which are mutagenic to Salmonella typhimurium TA 1538 and which give rise to DNA excision repair within the liver cells. Intact rat liver cells are shown to be more active in the formation of mutagenic metabolites than the 9000-g supernatant from these cells. Data are presented which are in favour of the role of N-acetylation in this respect. Furthermore, indications are presented that a sulfation reaction is involved in the generation of DNA modifying metabolites, whereas formation of mutagenic products is likely to proceed via deacetylation and/or N,O-acyltransfer. Finally, data are given about the extrahepatocellular appearance of premutagenic metabolites which are more prone to metabolic activation by additional metabolic factors in the Salmonella assay than benzidine itself. The impact of these observations on the estimation of the genotoxic potential of benzidine will be discussed.