Uptake of waterborne 3,3′,4,4′-tetrachlorobiphenyl and organ and cell-specific induction of cytochrome P4501A in adult and larval fathead minnow Pimephales promelas

Abstract

Female fathead minnows ( Pimephales promelas) were exposed to water-borne [ 14C]-3,3′,4,4′-tetrachlorobiphenyl (TCB) on days 0 and 5, for 24 h each time, in water with 0, 0.29, 2.9 or 29 mg TCB/kg total fish mass. Between treatments, fish were kept in clean water with normal feeding and light conditions. Liver, ovary and skeletal muscle were collected at 2 h, on day 5 prior to the second dosing and on day 12. Initially, TCB uptake was greatest in liver, but after 5 days the content in ovaries (ca. 5 μg TCB/g wet weight) was similar to that in liver (ca. 4 μg/g wet weight). TCB concentration in muscle was 10% of that in liver or ovary. Tissue concentrations of TCB were similar at 5 days after the first dose and 6 days after the second dose. Immunoblot analysis with monoclonal antibody 1-12-3 (specific for cytochromes P4501A; CYP1A) showed that CYP1A content in liver homogenates increased with increasing content of TCB in the liver, to about 5 pmol scup CYP1A equivalents per mg protein at 1.8 μg TCB/g liver. Between 1.8 and 4 μg TCB/g liver, the CYP1A content increased another 10-fold. Ethoxyresorufin O-deethylase (EROD) activity in liver homogenates also increased with increasing content of TCB in liver. However, EROD activity was suppressed at TCB concentrations greater than 1.8 μg TCB/g liver, consistent with other results showing that 3,3′,4,4′-TCB can inhibit or inactivate CYP1A. Immunohistochemical staining with MAb 1-12-3 showed induction in liver and in multiple extrahepatic organs; analysis with a second anti-scup CYP1A1 MAb (1-71-3) confirmed the patterns of induction. A strong CYP1A staining signal was detectable only in the highest dose group, and induction in epithelial cells, including hepatocytes, was weaker than that in endothelium. This suggests that the CYP1A seen in immunoblots of liver homogenates could reflect a substantial contribution from endothelial cell P450. There was mild staining indicating CYP1A induction in endothelium of embryos hatched from eggs of exposed fish. Analysis of induction in fathead minnows is relevant to their use in evaluating chemicals that may be activated or detoxified by CYP1A enzymes. The results further illustrate the utility of immunohistochemistry to evaluate CYP1A induction as a marker of exposure.