Hepatic CYP1A1 induction in rainbow trout by continuous flowthrough exposure to beta-naphthoflavone.
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 beta-naphthoflavone (beta-NF), a known CYP1A1 inducer in fish. Trout were exposed to a beta-NF concentration of 1.0 mg beta-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 beta-NF concentrations of 0.05, 0.10, and 0.50 mg beta-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 beta-NF/liter concentration, ethoxyresorufin-O-deethylase (EROD) activity was significantly decreased by 4 days of exposure when compared to controls. At beta-NF concentrations of 0.05 to 0.50 mg beta-NF/liter EROD activity was increased compared to controls but was inversely related to the beta-NF concentration. Hybridizable CYP1A1 mRNA was increased approximately 40-fold over control levels at concentrations of 0.05 to 0.50 mg beta-NF/liter for 1, 3, and 7 days of exposure. In a third experiment, trout exposed to 0.05 mg beta-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 beta-NF exposure. This is in contrast to previous experiments using intraperitoneal injection of beta-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 10.0 micrograms beta-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 micrograms beta-NF/liter.(ABSTRACT TRUNCATED AT 400 WORDS)
Haasch
M L
ML
Medical College of Wisconsin, Pharmacology and Toxicology Department, Milwaukee 53226.
Quardokus
E M
EM
Sutherland
L A
LA
Goodrich
M S
MS
Lech
J J
JJ
D000818:Animals; D001571:Benzoflavones / Q000633:toxicity*; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D015151:Immunoblotting; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D010088:Oxidoreductases / Q000096:biosynthesis*; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D014337:Trout / Q000378:metabolism*; D014874:Water Pollutants, Chemical / Q000633:toxicity*; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D011506:Proteins; D012333:RNA, Messenger; D014874:Water Pollutants, Chemical; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1