Fd Chem. Toxic'. Vol. 24, No. 12, pp. 1267-1272, 1986 0278-6915/86 $3.00+0.00 Printed in Great Britain Pergamon Journals Ltd STUDIES ON BENZYL ACETATE. II. USE OF SPECIFIC METABOLIC INHIBITORS TO DEFINE THE PATHWAY LEADING TO THE FORMATION OF BENZYLMERCAPTURIC ACID IN THE RAT M. A. J. CHIDGEY, J. F. KENNEDY and J. CALDWELL Department of Pharmacology, St Mary's Hospital Medical School, London W2 1PG, England (Received 4 November 1985; revisions received 24 March 1986) Abstract--Specific metabolic inhibitors were used to define the route of metabolism of benzyl acetate leading to the formation of benzylmercapturic acid. Male Fischer 344 rats were dosed by gavage with [methylene-14C]benzyl acetate (500mg/kg) alone or together with pyrazole (200mg/kg), penta- chlorophenol (10 mg/kg) or both pentachlorophenol (10 mg/kg) and pyrazole (200 mg/kg), given in each case ip. Urine and faeces were collected and urinary metabolites were assayed by radio-TLC and HPLC. The excretion of ~4C was rapid in all cases, with most of the dose excreted in the urine within 24 hr. Co-administration of pyrazole (an inhibitor of alcohol dehydrogenase) with benzyl acetate caused an I 1-fold increase in the excretion of benzylmercapturic acid and halved the percentage of the dose excreted as benzoyl glucuronide. Pretreatment with pentachlorophenol, an inhibitor of sulphotransferase activity in vivo, abolished the excretion of benzylmercapturic acid, while excretion of the mercapturate following treatment with both pyrazole and pentachlorophenol was higher than in control or pentachlorophenol- treated rats, but much lower than in the animals given pyrazole alone. Taken together, these results suggest very strongly that the formation of benzylmercapturic acid involves the sulphate ester of benzyl alcohol as an obligatory intermediate and does not appear to involve a metabolic intermediate with sufficient reactivity to have toxicological relevance. INTRODUCTION Reaction with the nucleophilic tripeptide, gluta- thione, represents one of the cell's most effective means of dealing with biological reactive inter- mediates, both electrophiles (Chasseaud, 1979) and free radicals (Jerina & Bend, 1977). Once formed, glutathione conjugates are rapidly and extensively metabolized in the body along a variety of pathways to a plethora of products, which are all thioethers of the parent xenobiotic (Lamoureux & Bakke, 1984; Meister, 1982). Quantitatively, mercapturic acids (S- substituted N-acetylcysteines) are the most important of these products, and a wide variety of compounds that undergo glutathione conjugation are ultimately excreted in the urine as mercapturic acids (Arias & Jakoby, 1976; Jakoby, 1980). The formation of thioether conjugates is often taken as presumptive evidence of the formation of reactive intermediates, and is thus assumed to have toxicological relevance. However, it is important to appreciate that two distinct classes of xenobiotics undergo glutathione conjugation--those metabolized to electrophiles or free radicals, which are the oblig- atory reactive intermediates in the conjugation, and those with sufficient electrophilic character per se to react with the tripeptide directly, such as compounds with activated double bonds (e.g. diethyl maleate), aryl and alkyl halides and nitrocompounds, and aryl and arylalkyl esters (Caldwell, 1982). It will be appar- ent that while the formation of glutathione conju- gates of the first class of compound is directly related to the mechanisms by which they exert their toxicity, this is by no means always the case for the second group. It is thus important to define the metabolic pathway(s) leading to the formation of glutathione conjugates and related thioether metabolites before drawing conclusions about their toxicological significance in any particular case. The simple arylalkyl ester benzyl acetate is widely used as a food flavour (Fenaroli's Handbook of Flavor Ingredients, 1971) and fragrance agent in perfumes and toiletries (Opdyke, 1973). In view of the wide- spread exposure of human populations to this agent there is considerable interest in its safety evaluation. It has long been known that the metabolism of benzyl acetate proceeds by hydrolysis and oxidation to benzoic acid, which is then converted to hippuric acid (Snapper, Grunbaum & Sterkop, 1925; Williams, 1959), and this has recently been confirmed using modern techniques (Chidgey & Caldwell, 1986). In addition, a small fraction of the dose is excreted as benzylmercapturic acid (S-benzyl N-acetylcysteine; Chidgey & Caldwell, 1986; Clapp & Young, 1970). In view of the potential insight into mechanisms of toxicity that knowledge about the formation of the mercapturate might provide, it is important to define precisely the metabolic pathway leading to its for- mation. This paper gives an account of studies that use specific metabolic inhibitors to provide such information. EXPERIMENTAL Compounds. [methylene-14C]Benzyl acetate (radio- chemical purity >96%) was a custom synthesis by Amersham International plc (Amersham, Bucks.). 1267