Curcumin attenuates dimethylnitrosamine-induced liver injury in rats through Nrf2-mediated induction of heme oxygenase-1

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Abstract

Curcumin (diferuloymethane), a yellow colouring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), has been reported to possess anti-inflammatory, antioxidant, antimutagenic and anticarcinogenic activities. Curcumin exerts its chemoprotective and chemopreventive effects via multiple mechanisms. It has been reported to induce expression of the antioxidant enzymes in various cell lines. Heme oxygenase-1 (HO-1) is an important antioxidant enzyme that plays a pivotal role in cytoprotection against noxious stimuli of both endogenous and exogenous origin. In the present study, we found that oral administration of curcumin at 200 mg/kg dose for four consecutive days not only protected against dimethylnitrosamine (DMN)-induced hepatic injury, but also resulted in more than three-fold induction of HO-1 protein expression as well as activity in rat liver. Inhibition of HO-1 activity by zinc protoporphyrin-IX abrogated the hepatoprotective effect of curcumin against DMN toxicity. NF-E2-related factor 2 (Nrf2) plays a role in the cellular protection against oxidative stress through antioxidant response element (ARE)-directed induction of several phase-2 detoxifying and antioxidant enzymes including HO-1. Curcumin administration resulted in enhanced nuclear translocation and ARE-binding of Nrf2. Taken together, these findings suggest that curcumin protects against DMN-induced hepatotoxicity, at least in part, through ARE-driven induction of HO-1 expression.

Introduction

Curcumin (deferuloymethane), a yellow colouring ingredient of the spice turmeric obtained from the rhizome of Curcuma longa Linn (Zingiberacea), a perennial herb distributed mainly throughout tropical and subtropical regions of the world. It has been used in indigenous herbal medicine for the treatment of inflammatory and liver disorders (Joe et al., 2004). Curcumin has potent antioxidant, anti-inflammatory, antimutagenic and anticarcinogenic properties (Araujo and Leon, 2001, Chainani-Wu, 2003, Surh et al., 2001). The protective effects of curcumin against chemically-induced hepatotoxicity are well documented, and have been attributed to its intrinsic antioxidant properties (Nanji et al., 2003, Rukkumani et al., 2004). Thus, curcumin was shown to retard lipid peroxidation and ameliorate chemically-induced oxidative stress (Okada et al., 2001, Rukkumani et al., 2004). In addition, curcumin has been shown to increase expression of the xenobiotic detoxifying enzymes, such as glutathione S-transferase, glutathione reductase and NAD(P)H: quinone oxidoreductase in both liver and kidney of mice (Iqbal et al., 2003).

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme widely distributed in many mammalian tissues and is responsible for the breakdown of heme to biliverdin, free iron and carbon monoxide (Maines and Gibbs, 2005). HO-1 expression is induced by a wide variety of stimuli including heme, heavy metals, cytokines and chemical carcinogens (McNally et al., 2006, Prawan et al., 2005). Moreover, HO-1 has been considered to be a potential therapeutic target for a number of chemopreventive agents (Prawan et al., 2005). Curcumin has been shown to up-regulate HO-1 in endothelia cells, renal epithelia cells and astrocytes (Hill-Kapturczak et al., 2001, Motterlini et al., 2000, Scapagnini et al., 2002) as well as in rat kidney in vivo (Jones et al., 2000). Upregulation of many phase-2 detoxifying and antioxidant enzymes, including HO-1, is mediated by antioxidant response element (ARE) (Li et al., 2004). The transcription factor Nrf2 plays a pivotal role in the activation of ARE-driven antioxidant gene expression. Nrf2 is normally sequestered in the cytosol as an inactive complex with its suppressor Keap-1. Upon cell stimulation, Nrf2 dissociates from Keap-1 and translocated into the nucleus where it binds to the ARE located in the promoters of target genes (Jaiswal, 2004). Induction of HO-1 by curcumin has been shown to occur via Nrf2 in porcine renal epithelial cells (Balogun et al., 2003), human monocytes (Rushworth et al., 2006) and in wild-type mouse embryo fibroblasts (Andreadi et al., 2006).

Dimethylnitrosamine (DMN) is a potent hepatotoxin, carcinogen and mutagen (George et al., 2001). DMN exerts carcinogenic effects and induces hepatic necrosis through metabolic activation by CYP2E1 (Guengerich et al., 1991) in experimental animals. Activation of DEN by CYP2E1 in mouse liver has been shown to stimulate Kupffer cells leading to generation of superoxide and other reactive oxygen species (ROS) capable of damaging liver cells (Teufelhofer et al., 2005). It has been shown that curcumin protects against a number of hepatotoxic agents including nitrosamines (Chuang et al., 2000, Sreepriya and Bali, 2006). However, the mechanisms by which curcumin elicits hepatoprotective and other chemoprotective/chemopreventive effects in association with HO-1 induction remains poorly understood. Here, we report that curcumin attenuates DMN-induced liver injury in rats via induction of Nrf2-mediated up-regulation of HO-1.

Section snippets

Materials and methods

DMN and curcumin were purchased from Sigma Chemical Co. (St Louis, MO, USA). The antibody against Nrf-2 was the product of Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Zinc-protoporphyrin (ZnPP) was obtained from Alexis (Carlsbad, CA, USA). Antibody against HO-1 was purchased from Stessgen Biotechnology Co. (Victoria, BC, Canada). ARE oligonucleotide was provided from Bionics, Korea. All other reagents used were in the purest form available commercially.

Curcumin induces HO-1 expression and HO activity in rat liver

The effect of various doses of curcumin on rat liver HO-1 protein expression and HO activity is depicted in Fig. 1. A maximal induction of HO-1 expression was achieved with oral administration of curcumin at 200 mg/kg (Fig. 1A). The increased HO-1 protein expression directly correlated with the HO activity (Fig. 1B). As shown in Fig. 2, treatment of rats with curcumin (200 mg/kg) resulted in gradual increases in both HO activity and HO-1 protein expression up to 4 days.

Curcumin increases the Nrf2 ARE binding activity in rat liver

Several investigators have

Discussion

HO-1-mediated cytoprotection has been shown to be critical for tissues that are vulnerable to oxidative stress (Perrella and Yet, 2003). Therefore, HO-1 is recognized as an important target of a number of chemopreventive and cytoprotective agents (Prawan et al., 2005). Curcumin has been reported to attenuate liver injury induced by diverse hepatotoxicants (Kaur et al., 2006, Park et al., 2000, Reys-Gordillo et al., 2007, Shapiro et al., 2006) via multiple mechanisms. In the present study, we

Acknowledgments

This work was supported by the Grant (B050007) from the Ministry of Health and Welfare and the National Research Laboratory Grant from the Ministry of Science and Engineering, Republic of Korea.

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    On leave from Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria.

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