Oxidative Inactivation of Cytochrome P-450 1A (CYP1A) Stimulated by 3,3',4,4'-Tetrachlorobiphenyl: Production of Reactive Oxygen by Vertebrate CYP1As JENNIFER J. SCHLEZINGER, RENEE D. WHITE, and JOHN J. STEGEMAN Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts Received for publication February 12, 1999; accepted May 18, 1999 This paper is available online at http://www.molpharm.org ABSTRACT Microsomal cytochrome P-450 1A (CYP1A) in a vertebrate model (the teleost fish scup) is inactivated by the aryl hydro- carbon 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 hu- man 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 in- activation was lessened by adding cytochrome c. CYP1A inac- tivation 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 H 2 O 2 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 for- mation of ROS; formation rates correlated with native CYP1A1 content. TCB stimulated ROS formation by baculovirus-ex- pressed 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. 2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD), non-ortho polychlorinated biphenyls (PCBs), and other planar haloge- nated aromatic hydrocarbons (pHAHs) exert systemic toxic- ity in laboratory animals, wildlife species, and, ostensibly, humans, through activation of the aryl hydrocarbon receptor (AhR), a basic helix-loop-helix/Per-Arnt-Sim transcription factor (Hankinson, 1995). AhR-null mice are resistant to TCDD toxicity, indicating that toxicity of pHAHs is AhR- dependent (Fernandez-Salguero et al., 1996). AhR activation leads to isozyme-specific induction of drug-metabolizing en- zymes [cytochrome P-450 1A1 (CYP1A1), CYP1A2, CYP1B1, glutathione S-transferase, and UDP-glucuronosyltrans- ferase] and alters the expression of genes involved in cell growth control (e.g., transforming growth factor- and -, B-Jun, and Jun-D; Hankinson, 1995), yet the mechanisms of pHAH toxicity still are unclear. The occurrence of oxidative damage in animals or cells exposed to pHAHs (Stohs et al., 1990; Toborek et al., 1995; Park et al., 1996) suggests that some toxic responses to AhR agonists might involve reactive oxygen species (ROS). Oxidative damage and CYP1A1 induc- tion both occur in cells exposed to TCDD or PCB congeners, suggesting that this enzyme could be involved in that dam- age. An NADPH-dependent generation of ROS (O 2  ,H 2 O 2 , and/or OH  ) by animal liver microsomes has been known for 40 years (Gillette et al., 1957) and has been linked to CYP (Bondy and Naderi, 1994). Uncoupling of electron transfer and oxygen reduction from monooxygenation by CYP can result in the release of O 2  and H 2 O 2 ; CYP forms including CYP2E1, CYP2B4, and CYP3A have been shown to release ROS, usu- ally on substrate binding, which accelerates electron transfer 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'-dichlorodihydrofluo- rescein diacetate; EROD, ethoxyresorufin O-deethylation; HA, hydroxylamine; HBSS, Hank’s buffered salt solution; HE, dihydroethidium; MROD, methoxyresorufin O-demethylation; pHAH, planar halogenated aromatic hydrocarbon; PCB, polychlorinated biphenyl; ROS, reactive oxygen species; SOD, superoxide dismutase; TCB, 3,3',4,4'-tetrachlorobiphenyl. 0026-895X/99/030588-10$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:588 –597 (1999). 588 at ASPET Journals on June 6, 2016 molpharm.aspetjournals.org Downloaded from