© 1993 Oxford University Press
research-article |
Hepatic CYP1A1 Induction in Rainbow Trout by Continuous Flowthrough Exposure to ß-Naphthoflavone
Medical College of Wisconsin, Pharmacology and Toxicology Department Milwaukee, Wisconsin 53226 and University of Wisconsin, Milwaukee, NIEHS Marine and Freshwater Biomedical Research CORE Center, Great Lakes Research Facility Milwaukee, Wisconsin 53204
Received May 13, 1992; accepted September 21, 1992
In order to assess the usefulness of CYP1A1 mRNA measurement as an environmental biomarker it was necessary to determine if hepatic P450 CYP1A1 mRNA induction is sustained during constant exposure to hepatic monooxygenase inducers. To accomplish this, rainbow trout (Oncorhynchus mykiss) were exposed, under flowthrough conditions, to ß-naphthoflavone (ß-NF), a known CYP1A1 inducer in fish. Trout were exposed to a ß-NF concentration of 1.0 mg ß-NF/liter, using dimethylformamide as carrier, for 1, 2, 4, and 8 days, followed by depuration in clean water for 1, 8, 14, and 35 days. In a second experiment, trout were exposed to ß-NF concentrations of 0.05, 0.10, and 0.50 mg ß-NF/liter, using dimethylformamide as carrier, for 1, 3, 7, and 14 days, followed by depuration for 7 and 28 days. At the 1.0-mg ß-NF/liter concentration, ethoxyresorufin-O-deethylase (EROD) activity was significantly decreased by 4 days of exposure when compared to controls. At ß-NF concentrations of 0.05 to 0.50 mg ß-NF/liter EROD activity was increased compared to controls but was inversely related to the ß-NF concentration. Hybridizable CYP1A1 mRNA was increased approximately 40-fold over control levels at concentrations of 0.05 to 0.50 mg ß-NF/liter for 1, 3, and 7 days of exposure. In a third experiment, trout exposed to 0.05 mg ß-NF/liter for 2, 6, 12, 24, 32, 40, and 48 hr had increased (45-to 167-fold) EROD activity by 18 and 48 hr, respectively. Immunoreactive CYP1A1 protein was increased 46-fold at 48 hr and CYP1A1 mRNA was increased 29-fold at 48 hr of continuous ß-NF exposure. This is in contrast to previous experiments using intraperitoneal injection of ß-NF in which the induced CYP1A1 mRNA decreased to near control levels by 48 hr after injection. These data indicate that both CYP1A1 catalytic activity and immunoreactive protein are decreased at high inducer concentrations while mRNA levels remain elevated and continue to increase over time during continuous exposure. In a fourth experiment trout were continuously exposed to concentrations of 0.625, 1.25, 2.5, 5.0, and 10.0µgß-NF/liter, using dimethylformamide as carrier, for 1, 3, 7, 14, and 21 days, followed by clean water depuration for 1, 3, 7, 14, and 21 days EROD activity was significantly increased in a concentration-dependent manner over control by Day 1 of exposure with concentrations of 2.5, 5.0, and 10.0 µg ß-NF/liter. EROD activity was increased approximately 3-, 5-, 16-, 21-, and 58-fold in trout exposed for 3 days to 0.625, 1.25, 2.5, 5.0, and 10.0 µgß-NF/liter, respectively. Immunoreactive CYP1A1 protein was also increased in a concentration-dependent manner approximately 1.2-, 1.9-, 3.4-, 5.2-, and 8.2-fold after 3 days of exposure, respectively, and approximately 1.2-, 1.2-, 3.4-, 5.1-, and 10.8-fold after 7 days of exposure, respectively. At Day 3, hybridizable CYP1A1 mRNA was increased over controls 1.7-, 7.0-, 33-, 44-, and 62-fold, at concentrations of 0.625, 1.25, 2.5, 5.0, and 10.0 µg ß-NF/liter, respectively. CYP1A1 mRNA levels were significantly increased over extended time periods during prolonged exposure. With the 10.0 µg ß-NF/liter exposure, CYP1A1 mRNA levels were significantly increased approximately 7-, 17-, 20-, 16-, and 8-fold over control levels at 1, 3, 7, 14, and 21 days, respectively. During a depuration period following exposure to 10.0 µg ß-NF/liter, EROD activity remained significantly increased compared to controls for 3 days and returned to control levels by 7 days. The results indicate that CYP1A1 mRNA remains elevated for at least 21 days during constant exposure to ß-NF, and the findings provide support for the feasibility of using of CYP1A1 mRNA levels in biomonitoring of water for specific chemical contamination.