Microsomal Metabolism of the Hepatotoxin a -Naphthylisothiocyanate (ANIT) Following Phenobarbital,Chlorpromazine or Actinomycin D Treatment 1

Abstract

Previous investigations of the mechanism for a-naphthylisothiocyanate (ANIT)-induced hyperbilirubinemia have suggested that micro-somal biotransformation of ANIT must precede the onset of hyperbilirubinemia (1). Direct evidence for ANIT biotransformation as well as a similarity between the disposition of (¹⁴C) ANIT and/or its metabolites and temporal aspects of ANIT-induced hyper-bilirubinemia has previously been reported from this laboratory (2). Furthermore, a positive correlation has been found in vivo between the quantitative aspects of pheno-barbital and chlorpromazine potentiation of ANIT-induced hyperbilirubinemia and their effect on the rate of formation of ANIT metabolites (3). Actinomycin D, on the other hand, inhibits ANIT-induced hyperbilirubinemia, inhibits phenobarbital and chlorpromazine potentiation of ANIT-induced hyperbilirubinemia, and alters certain aspects of ANIT metabolism in vivo (1, 3). The present study correlates previously reported in vivo alteration of ANIT disposition and ANIT-induced hyperbilirubinemia (2, 3) by phenobarbital, chlorpromazine and actinomycin D with the effect of these agents on in vitro microsomal metabolism of ANIT.

Methods. Livers were obtained from four separate groups of pretreated male Sprague-Dawley rats (150-200 g). The rats were pretreated with either chlorpromazine (20 mg/kg, ip) or phenobarbital (60 mg/kg, ip) daily for 3 days prior to sacrifice, or with actinomycin D (100μg/kg, ip) or actinomycin D plus phenobarbital (60 mg/kg, ip) 24 and 12 hr prior to sacrifice. Control rats were treated with saline. After sacrifice, livers were immediately excised, blotted dry, weighed, placed in cold 1.15% KCl, minced with scissors and the KCl was decanted. Each liver was individually homogenized in 2 vol of 1.15% KCl with a Potter-Elvehjem homogenizer (Teflon pestle). Cell debris, cell nuclei, and mitochondria were removed by centrifugation at 9000g for 20 min. The microsomal fraction was obtained by centri-fugation at 100,000g for 60 min.