Biochemical and Molecular Characterization of Glutathione S-Transferase Expression in English Sole and Starry Flounder.
摘要
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 which exhibit striking differences in the prevalence of contaminant-associated liver neoplasia. English sole, the species with a high prevalence of contaminant-associated liver neoplasia, exhibited greater hepatic GST-benzo[a]pyrene-trans-7,8-dihydrodiol, 9,10-epoxide-anti (BaPDE) activity than did starry flounder, regardless of site of capture. English sole and starry flounder exhibiting greater GST-AFBO activity than English sole. Starry flounder hepatic cytosolic GSTs did not display cross-reactivity to antibodies raised against rat α (Ya or Yc), μ (Yb1/2), and π class (Yf) GSTs, whereas English sole had cytosolic GST(s) that displayed limited cross-reactivity to rat μ and π class, but not α class GST(s). Alignment of cDNA sequences for plaice liver GST (PLGSTA, θ class GST) and an octopus GST was employed to identify conserved regions for oligonucleotide primer design for polymerase chain reaction (PCR)-based studies of sole and flounder. Reverse-transcription-PCR (RT-PCR) analysis of sole and flounder hepatic total mRNA yielded an approximate 300 nucleotide cDNA fragment displaying extensive sequence homology to plaice GST-A. Northern blot analysis of sole and flounder using the PCR product from English sole as a probe detected a single band of approximately 1000 nucleotides that was expressed in English sole from polluted and control sites, and in starry flounder from the polluted site only. These results are supportive of previous work (Leaver et al., Biochem. J., 292, 189-195) indicating that a θ class GST is conserved in marine flatfish. Further studies are necessary to determine if the environmental induction of this GST, or selective expression of other GST(s) with catalytic activity towards sediment-associated carcinogens, affords protection against environmental hepatocarcinogenesis in the starry flounder