Isolation and cloning of homologous glutathione S-transferase cDNAs from English sole and starry flounder liver

Abstract

We are currently investigating hepatic glutathione S-transferase (GST) expression in English sole ( Pleuronectes vetulus) and starry flounder ( Platichthys stellatus), two closely related benthic fish that exhibit striking differences in the prevalences of contaminant-associated liver neoplasia. Alignment of cDNA sequences encoding plaice ( Pleuronectes platessa) liver GST (PLGST-A, a theta class GST, Leaver et al., 1993) and a GST-related lens crystallins protein from octopus ( Octopus vulgaris) revealed conserved regions for oligonucleotide primer design for polymerase chain reaction (PCR)-based studies of sole and flounder GSTs. Reverse-transcriptase-PCR (RT-PCR) analysis of sole and flounder hepatic total mRNA yielded 469 nucleotide cDNA fragments that displayed extensive sequence homology (98%) to plaice GST-A. Northern blotting analysis of English sole and starry flounder liver using the PCR product from English sole as a probe detected a single band of approximately 1000 nucleotides that was highly expressed in English sole and in starry flounder liver. Biochemical and immunological studies using diagnostic GST reference substrates and class specific polyclonal antibodies suggested little homology among English sole and starry flounder GSTs, and rodent alpha, mu, and pi class GSTs. Interestingly, English sole, the species exhibiting a relatively high prevalence of contaminant-associated liver neoplasia, exhibited higher hepatic GST activity toward the environmental epoxides aflatoxin B 1-8,9-epoxide (AFBO) and benzo[ a]pyrene- trans-7,8-dihydrodiol-9,10-epoxide- anti (BPDE). BPDE, in particular, is a carcinogenic epoxide intermediate implicated as a possible causative agent in the etiology of these hepatic lesions. In summary, our studies indicate that a GST related to non-mammalian theta class GST is conserved in marine flatfish. Further mechanistic studies are necessary to determine the role of GSTs in the observed susceptibility differences among English sole and starry flounder to sediment-associated carcinogens.