Uncoupling of cytochrome P450 1A and stimulation of reactive oxygen species production by co-planar polychlorinated biphenyl congeners

Abstract

The non-ortho-polychlorinated biphenyl (PCB) congener 3,3′4,4′-tetrachlorobiphenyl (PCB 77) can uncouple the catalytic cycle of fish (scup) cytochrome P4501A (CYP1A) and mammalian (rat, human) CYP1A1, stimulating release of reactive oxygen species (ROS). PCB 77 also inactivates CYP1A in an NADPH-, oxygen-, and time-dependent process, linked to uncoupling. We addressed a hypothesis that planar halogenated hydrocarbons generally will uncouple CYP1A. Thus, additional PCB congeners including non-ortho-3,3′,4,4′,5′-pentachlorobiphenyl (PCB 126) and 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169), mono-ortho-2,3,3′,4,4′-pentachlorobiphenyl (PCB 105) and di-ortho-2,2′,5,5′-tetrachlorobiphenyl (PCB 52), as well as the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P), were examined for their ability to stimulate microsomal ROS production and to inactivate CYP1A. Incubated without NADPH, non-ortho-PCB 126 and -PCB 169 both inhibited microsomal CYP1A activity (ethoxyresorufin O-deethylase; EROD). When NADPH was included, these congeners caused a progressive inactivation of CYP1A, in addition to the inhibition. The determined K_Inact values for inactivation were 0.14 and 0.08 μM, respectively, for PCB 126 and PCB 169, similar to the 0.05 μM for PCB 77 previously reported. The mono-ortho-PCB 105 weakly inhibited and weakly inactivated CYP1A. The di-ortho-PCB 52 neither inhibited nor inactivated CYP1A. Alone, B[a]P strongly inhibited CYP1A, but when NADPH was added that inhibition was reversed, apparently by metabolic depletion of the substrate, and there was no inactivation. PCB 126 and PCB 169 stimulated release of ROS from induced liver microsomes, while B[a]P, PCB 52 and PCB 105 did not. ROS release and CYP1A inactivation stimulated by the non-ortho-PCB 126 and PCB 169 indicate an uncoupling of CYP1A like that previously shown with PCB 77. The uncoupling and release of ROS further suggest a participation of CYP1A in the oxidative stress associated with some planar halogenated aryl hydrocarbon receptor agonists.

Introduction

Exposure to polycyclic aromatic hydrocarbons (PAH) and planar halogenated aromatic hydrocarbons (PHAH) variously results in carcinogenesis, as well as immune system, reproductive, endocrine and developmental toxicity. PAH and PHAH, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyl (PCB) congeners bearing meta,- and para- but no ortho-chlorine substituents, activate the aryl hydrocarbon receptor (AHR) transcription factor. Much PAH toxicity depends on their metabolism by cytochromes P450 (CYP) such as CYP1As induced via the AHR. The toxicity of PHAH is also mediated by the AHR in both mammals (Fernandez-Salguero et al., 1996) and fish (Prasch et al., 2003, Dong et al., 2004). However, the mechanisms of PHAH toxicity are not clear (Safe, 1994; Tillitt et al., 1991, Newsted et al., 1995). Oxidative stress is increasingly being linked to chemical exposure in aquatic and marine systems, as well as in laboratory studies (Livingstone et al., 2000, Regoli et al., 2002, Meyer et al., 2003, Almroth et al., 2005). Consequences of oxidative stress, including oxidative damage to DNA, have also been reported in animals from locations more highly contaminated by organic chemicals (Malins and Gunselman, 1994, Malins et al., 2004). However, the mechanisms by which many chemicals, including PHAH, elicit enhanced formation of reactive oxygen species (ROS), and subsequent oxidative stress, are poorly understood.

The non-ortho-PCB 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) stimulates production of reactive oxygen species (ROS) by liver microsomal CYP1As from fish and mammals by uncoupling the catalytic cycle (Schlezinger et al., 1999). It remains to be determined if this is a generalized mechanism by which other PHAH also could stimulate oxidative stress. Here we address the hypothesis that other PHAH, particularly other non-ortho-PCBs, act similarly to uncouple CYP1A.

The oxidation of non-ortho-PCBs is slow and production of reactive metabolites is not a major avenue to toxicity (Yoshimura et al., 1987, Koga et al., 1990). Yet, PCB 77 binds tightly to scup CYP1A, initiating the catalytic cycle. Substrates (including PAH and PHAH) binding to P450s converts the heme iron to a redox potential that favors reduction and it is reduced by electron transport from NADPH-cytochrome P450 reductase. Oxygen binds and a second electron is donated to the complex forming a peroxide. A substrate radical is formed, hydroxylated and released. This process occurs rapidly with PAH substrates such as B[a]P. However, the slow oxidation of PCB77 results in uncoupling of electron transfer and oxygen reduction from substrate oxidation and in an enhanced release of ROS (White et al., 1997b). PCB 77 also stimulates ROS release from rat liver microsomal CYP1A1 and from expressed human CYP1A1 (Schlezinger et al., 1999), and PCB 77 was found to uncouple rat CYP1A1 in studies of PCB congener stimulation of bilirubin oxidation (Pons et al., 2003). Uncoupling by PCB 77 also leads to an oxidative inactivation of CYP1A (Schlezinger et al., 1999). CYP1A1 induction correlates with oxidative damage from PHAH (Toborek et al., 1995, Park et al., 1996), and in fish embryos TCDD-induced oxidative damage and apoptosis are correlated with CYP1A induction and activity (Cantrell et al., 1998). Structure–activity relationships for substrate uncoupling of CYP1As could indicate whether this process could be involved in oxidative stress induced by various CYP1A substrates. Such information could be applied in evaluating CYP1As in different vertebrate taxa for their potential roles in PHAH toxicity.

Here, we examined additional chemicals for their ability to stimulate ROS production by microsomal CYP1A and to inactivate the enzyme. The chemicals (Fig. 1) included the PCB congeners 3,3′,4,4′,5,-pentachlorobiphenyl (PCB 126) and 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169), that like PCB 77 bear mono- and para- but not ortho-chlorine substituents. PCB 77, PCB 126 and PCB 169 are potent AHR agonists and are among the most toxic PCB congeners, contributing heavily to the TCDD toxic equivalents (TEQ) in the environment (McCain et al., 1977). Of the major sources of environmental PCBs, PCB126 contributes the most to the TEQ of emissions, and PCB77 and PCB 126 contribute significantly to the TEQ of Aroclor (Alcock et al., 1998). For comparison, we also examined 2,3,3′,4,4′-pentachlorobiphenyl (PCB 105), a mono-ortho-substituted congener that is a partial AHR agonist (Hestermann et al., 2000), 2,2′,5,5′-tetrachlorobiphenyl (PCB 52), a di-ortho-substituted congener that is neither an AHR agonist nor a CYP1A substrate and the PAH B[a]P, a potent AHR agonist and a rapidly metabolized CYP1A substrate. As a model system, we employed scup liver microsomal CYP1A. CYP1A from scup is predominantly CYP1A1-like (Morrison et al., 1995), and is highly susceptible to uncoupling by PCB 77 (Schlezinger et al., 1999). The results indicate that other non-ortho-PCBs, but not PAHs or ortho-substituted PCBs, can strongly uncouple CYP1A catalytic cycles, resulting in release of ROS that could be involved in oxidative stress and toxicity of these AHR agonists.