Oxidative Inactivation of Cytochrome P-450 1A (CYP1A) Stimulated by 3,3′,4,4′-Tetrachlorobiphenyl: Production of Reactive Oxygen by Vertebrate CYP1As
Abstract
Microsomal cytochrome P-450 1A (CYP1A) in a vertebrate model (the teleost fish scup) is inactivated by the aryl hydrocarbon receptor agonist 3,3′,4,4′-tetrachlorobiphenyl (TCB). Here, the mechanism of CYP1A inactivation and its relationship to reactive oxygen species (ROS) formation were examined by using liver microsomes from scup and rat and expressed human CYP1As. In vitro inactivation of scup CYP1A activity 7-ethoxyresorufin O-deethylation by TCB was time dependent, NADPH dependent, oxygen dependent, and irreversible. TCB increased microsomal NADPH oxidation rates, and CYP1A inactivation was lessened by adding cytochrome c. CYP1A inactivation was accompanied by loss of spectral P-450, a variable loss of heme and a variable appearance of P-420. Rates of scup liver microsomal metabolism of TCB were < 0.5 pmol/min/mg, 25-fold less than the rate of P-450 loss. Non-heme iron chelators, antioxidant enzymes, and ROS scavengers had no influence on inactivation. Inactivation was accelerated by H2O2 and azide but not by hydroxylamine or aminotriazole. TCB also inactivated rat liver microsomal CYP1A, apparently CYP1A1. Adding TCB to scup or rat liver microsomes containing induced levels of CYP1A, but not control microsomes, stimulated formation of ROS; formation rates correlated with native CYP1A1 content. TCB stimulated ROS formation by baculovirus-expressed human CYP1A1 but not CYP1A2. The results indicate that TCB uncouples the catalytic cycle of CYP1A, ostensibly CYP1A1, resulting in formation of ROS within the active site. These ROS may inactivate CYP1A or escape from the enzyme. ROS formed by CYP1A1 may contribute to the toxicity of planar halogenated aromatic hydrocarbons.
Footnotes
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Send reprint requests to: Dr. John J. Stegeman, Biology Department, Redfield 342, MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA 02543. E-mail: jstegeman{at}whoi.edu
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This research was supported in part by National Institutes of Health Grant P42-ES07381, by Environmental Protection Agency Grant R823889, by the Air Force Office of Scientific Research (AFOSR) Grant F40620-94-1039, and by a Woods Hole Oceanographic Institution-Mellon Award. This is contribution number 9661 of the Woods Hole Oceanographic Institution.
- Abbreviations:
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- AhR
- aryl hydrocarbon receptor
- ATA
- 3-amino-1,2,4-triazole
- BP
- benzo[a]pyrene
- CYP
- cytochrome P-450
- CYP-OR
- NADPH cytochrome P-450 reductase
- DCF
- 2′,7′-dichlorofluorescein
- DCDFDA
- 2′,7′-dichlorodihydrofluorescein diacetate
- EROD
- ethoxyresorufinO-deethylation
- HA
- hydroxylamine
- HBSS
- Hank’s buffered salt solution
- HE
- dihydroethidium
- MROD
- methoxyresorufinO-demethylation
- pHAH
- planar halogenated aromatic hydrocarbon
- PCB
- polychlorinated biphenyl
- ROS
- reactive oxygen species
- SOD
- superoxide dismutase
- TCB
- 3,3′,4,4′-tetrachlorobiphenyl
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- Received February 12, 1999.
- Accepted May 18, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
