Induction of cytochrome P450 3A and heat shock protein by tributyltin in blue crab, Callinectes sapidus

Tributyltin (TBT), a toxic contaminant in aquatic environments, breaks down cytochrome P450 enzymes (P450s) in vitro. To determine in vivo effects of long-term TBT-exposure in aquatic invertebrates, respiration rates, heat shock protein induction, microsomal cytochrome P450 levels and metabolism of [ 14C]testosterone were examined in the hepatopancreas of blue crabs fed TBT. Four groups of crabs were fed fish injected with ethanol or 2, 4 or 8 μg TBT Cl dissolved in ethanol/g meat; doses of 0, 125, 250, or 500 μg kg −1 respectively. Crabs were fed TBT-treated meat every other day for 16 days. Respiration rates were significantly decreased after TBT exposure. Hepatopancreas microsomes and cytosol were prepared 24 h after the last feeding. Heat shock protein was induced at the two highest TBT exposure concentrations. Total P450 levels were similar in all samples; however, protein cross-reacting with anti-scup CYP 3A, an isozyme responsible for 6 β-hydroxylation of testosterone in vertebrates, increased at the 250 μg and 500 μg TBT Cl kg −1 dose. EROD activity showed that CYP 1A was not elevated. Hydroxylation of [ 14C]testosterone at the 6 β, 6 α, 7 α, and 16 α positions by hepatopancreas microsomes increased significantly in crabs exposed to 250 μg TBT Cl kg −1. The lack of increase in [ 14C]testosterone hydroxylation at the 500 μg kg −1 dosage may result from TBT Cl's disruption of P450 activity at high concentrations. Incubation of control microsomes with TBT significantly reduced spectrally quantified P450 as well as P450 activity after 2 h, showing that TBT Cl interferes with crab microsomal P450 proteins. Immunoinhibition of testosterone 6 β-hydroxylation was accomplished by using scup CYP 3A antibody, showing that the antibody specifically binds to a protein with 6 β-hydroxylase activity. These results indicate that blue crabs are stressed by TBT-exposure, and upregulate P450 isozymes possibly to aid in metabolism and elimination of TBT.