INDUCTION OF CYTOCHROME-P450 3A AND HEAT-SHOCK-PROTEIN BY TRIBUTYLTININ BLUE-CRAB, CALLINECTES-SAPIDUS

Tributyltin (TBT), a toxic contaminant in aquatic environments, breaks down cytochrome P450 enzymes (P450s) in vitro. To determine in vivo e ffects of long-term TBT-exposure in aquatic invertebrates, respiration rates, heat shock protein induction, microsomal cytochrome P450 level s and metabolism of [C-14]testosterone were examined in the hepatopanc reas of blue crabs fed TBT. Four groups of crabs were fed fish injecte d with ethanol or 2, 4 or 8 mu g TBT Cl dissolved in ethanol/g meat; d oses of 0, 125, 250, or 500 mu g kg(-1) respectively. Crabs were fed T BT-treated meat every other cay fbr 16 days. Respiration rates were si gnificantly decreased after TBT exposure. Hepatopancreas microsomes an d cytosol were prepared 24 h after the last feeding. Heat shock protei n was induced at the two highest TBT exposure concentrations. Total P4 50 levels were similar in all samples; however, protein cross-reacting with anti-scup CYP 3A, an isozyme responsible for 6 beta-hydroxylatio n of testosterone in vertebrates, increased al the 250 mu g and 500 mu g TBT Cl kg(-1) dose. EROD activity showed that CYP 1A was not elevat ed. Hydroxylation of [C-14]testosterone at the 6 beta, 6 alpha, 7 alph a, and 16 alpha positions by hepatopancreas microsomes increased signi ficantly in crabs exposed to 250 mu g TBT Cl kg(-1). The lack of incre ase in [C-14]testosterone hydroxylation at the 500 mu g kg(-1) dosage may result from TBT Cl's disruption of P450 activity at high concentra tions. Incubation of control microsomes with TBT significantly reduced spectrally quantified P450 as well as P450 activity after 2 h, showin g that TBT Cl interferes with crab microsomal P450 proteins. Immunoinh ibition of testosterone 6 beta-hydroxylation was accomplished by using scup CYP 3A antibody, showing thar the antibody specifically binds to a protein with 6 beta-hydroxylase activity. These results indicate th at blue crabs are stressed by TBT-exposure, and upregulate P450 isozym es possibly to aid in metabolism and elimination of TBT. (C) 1998 Else vier Science B.V. All rights reserved.