Elsevier

Free Radical Biology and Medicine

Volume 33, Issue 9, 1 November 2002, Pages 1268-1278
Free Radical Biology and Medicine

Original contribution
Mitochondrial reactive oxygen production is dependent on the aromatic hydrocarbon receptor

https://doi.org/10.1016/S0891-5849(02)01014-6Get rights and content

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin; TCDD) is a pervasive environmental contaminant that induces hepatic and extrahepatic oxidative stress. We have previously shown that dioxin increases mitochondrial respiration-dependent reactive oxygen production. In the present study we examined the dependence of mitochondrial reactive oxygen production on the aromatic hydrocarbon receptor (AHR), cytochrome P450 1A1 (CYP1A1), and cytochrome P450 1A2 (CYP1A2), proteins believed to be important in dioxin-induced liver toxicity. Congenic Ahr(−/−), Cyp1a1(−/−) and Cyp1a2(−/−) knockout mice, and C57BL/6J inbred mice as their Ahr/Cyp1a1/Cyp1a2(+/+) wild-type (wt) counterparts, were injected intraperitoneally with dioxin (15 μg/kg body weight) or corn-oil vehicle on 3 consecutive days. Liver mitochondria were examined 1 week following the first treatment. The level of mitochondrial H2O2 production in vehicle-treated Ahr(−/−) mice was one fifth that found in vehicle-treated wt mice. Whereas dioxin caused a rise in succinate-stimulated mitochondrial H2O2 production in the wt, Cyp1a1(−/−), and Cyp1a2(−/−) mice, this increase did not occur with the Ahr(−/−) knockout. The lack of H2O2 production in Ahr(−/−) mice was not due to low levels of Mn2+-superoxide dismutase (SOD2) as shown by Western immunoblot analysis, nor was it due to high levels of mitochondrial glutathione peroxidase (GPX1) activity. Dioxin decreased mitochondrial aconitase (an enzyme inactivated by superoxide) by 44% in wt mice, by 26% in Cyp1a2(−/−) mice, and by 24% in Cyp1a1(−/−) mice; no change was observed in Ahr(−/−) mice. Dioxin treatment increased mitochondrial glutathione levels in the wt, Cyp1a1(−/−), and Cyp1a2(−/−) mice, but not in Ahr(−/−) mice. These results suggest that both constitutive and dioxin-induced mitochondrial reactive oxygen production is associated with a function of the AHR, and these effects are independent of either CYP1A1 or CYP1A2.

Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin; TCDD) is representative of a toxicologically important class of polyhalogenated aromatic hydrocarbons (PHAHs) that produce a wide variety of adverse effects 1, 2, 3, 4, 5, 6. Dioxin and other planar PHAHs are generally poorly metabolized in mammalian cells. As a result, PHAHs are negligibly activated to form reactive, genotoxic intermediates; because PHAHs are slowly eliminated from organisms, however, they can be persistent in activating signaling pathways and receptors, including the aromatic hydrocarbon receptor (AHR). Activation of the AHR, a cytosolic ligand-activated transcription factor, modulates the expression of a diverse array of genes [7] including those of the [Ah] gene battery, and the AHR is required for the majority of dioxin’s deleterious effects 2, 5, 8, 9, 10, 11.

A growing body of evidence suggests that oxidative stress represents an important underlying cause of dioxin-induced toxicity [12], a response mediated primarily by the AHR 4, 11, 13, 14, 15, 16, 17, 18. Several laboratories have reported that dioxin increases reactive oxygen (RO) formation, lipid peroxidation, and DNA damage in various tissues of rodents 16, 18, 19. Dioxin causes sustained oxidative stress in the liver of C57BL/6 mice, characterized by increases in the ratio of oxidized to reduced glutathione (GSSG/GSH) and in urinary 8-hydroxydeoxyguanosine (8-OHdG), a by-product of oxidative DNA damage [17]. Similarly, a dioxin-induced AHR-dependent increase in 8-OHdG has been demonstrated in cultured hepatoma cells [15]. Dioxin-treated rats were recently shown to have increased serum 8-OHdG, as well as increased liver heme oxygenase and metallothionein levels [20], two classical gene markers of oxidative stress not controlled by the AHR.

We have reported that dioxin causes sustained oxidative stress in mouse liver mitochondria [21]. The response was characterized by a decrease in activity of the superoxide-sensitive enzyme mitochondrial aconitase, increases in the mitochondrial glutathione, GSR (glutathione reductase) and GPX1 (glutathione peroxidase-1) activities, and a succinate-dependent increase in mitochondrial-liberated H2O2 [21]. Similarly, dioxin-treated rats were shown to have oxidative damage to the mitochondria [12]. These data suggest that mitochondria mediate, at least in part, the dioxin-induced oxidative stress response; the mechanisms of this response, however, are unclear.

The AHR is essential in the production of dioxin-induced toxicity. This is supported by the observation that disruption of the Ahr gene protects mice from the acute toxicity of dioxin [22]. C57BL/6 mice having a high-affinity AHR produce more reactive oxygen than DBA/2 mice, which have a low-affinity AHR 23, 24. In mouse hepatoma Hepa-1c1c7 cell cultures, an increase in 8-OHdG was seen following dioxin treatment of wild-type cells but was not seen in the c4 mutant line [15], which lacks the aromatic hydrocarbon receptor nuclear translocator (ARNT) protein, the dimeric partner of the AHR that is necessary for transcription. It appeared that the AHR-inducible protein mediating oxidative DNA damage was CYP1A1.

CYP1A1 has also been shown to produce elevated levels of reactive oxygen in fish and rodent microsomes when challenged with the dioxin-like compounds 3,3′,4,4′-tetrachlorobiphenyl [25] or 3,3′,4,4′,5-pentachlorobiphenyl [26]. The mechanism of RO production was speculated to be due to blockage of the normal catalytic cycle of CYP1A1 by the nonmetabolizable ligand tetrachlorobiphenyl, resulting in release of RO. A number of recent papers have established a link between CYP1A1 and oxidative stress 27, 28, 29. CYP1A2 does not appear to directly generate RO; it may, however, contribute to an oxidative stress response via a pharmacokinetic mechanism involving the binding and increased hepatic tissue concentrations of dioxin-like compounds 30, 31. CYP1A2 is also responsible for uroporphyria and liver damage resulting from dioxin exposure 32, 33, although such hepatotoxicity does not appear to involve the AHR [34].

Clearly, the AHR, CYP1A1, and CYP1A2 all appear to contribute to the dioxin-induced oxidative stress response. The goal of this study, therefore, was to understand the involvement of AHR and its downstream gene products CYP1A1 and CYP1A2 in the mechanisms underlying dioxin-induced subacute mitochondrial oxidative stress.

Section snippets

Animals and treatment

All experiments involving mice were conducted in accordance with the National Institutes of Health standards for care and use of experimental animals and the University of Cincinnati Institutional Animal Care and Use Committee (IACUC). Female mice were used for this study; they were group-housed, maintained on a 12 h light/dark cycle, and had access to standard rodent chow and water ad libitum. The C57BL/6J inbred strain was purchased from Jackson Laboratories (Bar Harbor, ME, USA). The Ahr

Superoxide production

In order to evaluate the role of dioxin in generating a mitochondrial oxidative stress response, we examined mitochondrial aconitase activity, an Fe-S cluster enzyme that is inactivated by superoxide. Aconitase inactivation has been suggested as a reliable marker for mitochondrial RO production 44, 45. Dioxin treatment decreased aconitase activity by 44% in the wt mouse, 26% in the Cyp1a2(−/−) mouse, and 24% in the Cyp1a1(−/−) mouse, while no change was observed in the Ahr(−/−) mouse (Fig. 1).

Discussion

Greater than 95% of the cellular oxygen consumed by mitochondrial respiration (Fig. 7) undergoes a tetravalent reduction to water by cytochrome c oxidase (COX); however, some oxygen may be univalently reduced to form superoxide during the course of electron transport 39, 49, 50. Depending on the cellular conditions, superoxide may, in turn, generate other more toxicologically relevant RO species, including H2O2, hydroxyl radical, and peroxynitrite 51, 52. This mitochondrial respiration-derived

Abbreviations

  • 8-OHdG—8-hydroxy-2′-deoxyguanosine

  • AHR—aromatic hydrocarbon receptor

  • Ahr(−/−)—Ahr(−/−) null mice

  • COX—cytochrome c oxidase (Complex IV)

  • Cyp1a1(−/−)—Cyp1a1(−/−) null mice

  • Cyp1a2(−/−)—Cyp1a2(−/−) null mice

  • GPX1—glutathione peroxidase-1

  • GSH—reduced glutathione

  • GSSG—oxidized glutathione

  • GSR—glutathione reductase

  • MnTMPyP—Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride

  • RCR—respiratory control ratio

  • RO—reactive oxygen

  • SOD2—Mn2+-superoxide dismutase

  • TTFA—2-thenoyltrifluoroacetone

  • wt mice—C57BL/6J inbred mice

Acknowledgements

We thank our colleagues for valuable discussions and the critical reading of this manuscript. Supported in part by NIH Grants R01 ES10133, R01 ES06321, R01 ES08147, R01 ES06273, RO1 ES08799, T32 ES07250, and Center for Environmental Genetics Grant P30 ES06096.

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