Nrf2 protects against As(III)-induced damage in mouse liver and bladder

Abstract

Arsenic compounds are classified as toxicants and human carcinogens. Environmental exposure to arsenic imposes a big health issue worldwide. Arsenic elicits its toxic efforts through many mechanisms, including generation of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls expression of a main cellular antioxidant response, which is required for neutralizing ROS and thus defending cells from exogenous insults. Previously, we demonstrated a protective role of Nrf2 against arsenic-induced toxicity using a cell culture model. In this report, we present evidence that Nrf2 protects against liver and bladder injury in response to six weeks of arsenic exposure in a mouse model. Nrf2^−/− mice displayed more severe pathological changes in the liver and bladder, compared to Nrf2^+/+ mice. Furthermore, Nrf2^−/− mice were more sensitive to arsenic-induced DNA hypomethylation, oxidative DNA damage, and apoptotic cell death. These results indicate a protective role of Nrf2 against arsenic toxicity in vivo. Hence, this work demonstrates the feasibility of using dietary compounds that target activation of the Nrf2 signaling pathway to alleviate arsenic-induced damage.

Introduction

Groundwater contaminated with arsenic is the major source of human exposure to arsenic. Chronic arsenic poisoning imposes a global health issue (Tchounwou et al., 2003, Walvekar et al., 2007). Therefore, the World Health Organization (WHO) has set the standard limit for arsenic to 10 parts per billion (ppb) (Kayajanian, 2003). However, approximately 57 million people are drinking groundwater with arsenic concentrations above 10 ppb. Arsenic has caused poisonings in Bangladesh, Bengal, Thailand, Finland, Hungary, Chile, Taiwan, Vietnam, Cambodia, Mexico, Argentina, and China, where geological environments are conducive to generate high amounts of arsenic compounds in groundwater (Smith et al., 2000, Kayajanian, 2003, Tchounwou et al., 2003). Furthermore, many states within the United States also have significant concentrations (up to 50 ppm) of arsenic in the groundwater (Tchounwou et al., 2003, Knobeloch et al., 2006).

The most common species of arsenic in groundwater are arsenate (As(V)) and arsenite (As(III)). Arsenic is able to undergo redox conversion between As(V) and As(III). Following uptake from drinking water, inorganic arsenic species are converted in the liver into methylated arsenic species that are excreted from the bladder. Thus, the bladder is the major target organ that is exposed to methylated arsenic species in populations who consume arsenic-contaminated water (Tchounwou et al., 2003, Kenyon et al., 2005). Chronic exposure to arsenic can cause skin, lung and bladder cancers (Hsueh et al., 1995, Brown et al., 1997, Cohen et al., 2000, Smith et al., 2006). A small but measurable increase in the incidence of bladder cancer was associated with exposure to concentration as low as 10 ppm of inorganic arsenic (Chu and Crawford-Brown, 2006). In addition to cancers, epidemiological studies have also established a strong correlation between chronic arsenic exposure and various non-cancer human diseases, such as hyperkeratosis, atherosclerosis, diabetes, and chronic obstructive pulmonary diseases (Tseng, 2002, Tchounwou et al., 2003, Navas-Acien et al., 2006).

The transcription factor, Nrf2, controls a cellular antioxidant response through transcriptional upregulation of an array of downstream genes, such as glutamate cysteine ligase (GCL), heme oxygenase-1 (HO-1), glutathione s-trasferase (GST), multidrug-resistance proteins (MRPs), and NAD(P)H quinone oxidoreductase-1 (NQO1) (Lau et al., 2008, Hayes and McMahon, 2009). Coordinated upregulation of the Nrf2-mediated endogenous antioxidants, phase II detoxifying enzymes, and drug transporters is essential in defending cells or animals from damage by environmental insults. This has been clearly demonstrated in many studies using Nrf2^−/− mice. Nrf2^−/− mice displayed a greater sensitivity to toxic effects induced by benzo[a]pyrene, diesel exhaust, pentachlorophenol, and bleomycin (Aoki et al., 2001, Ramos-Gomez et al., 2001, Cho et al., 2004, Umemura et al., 2006).

Arsenic exerts its toxicity in part by generation of ROS (Hei et al., 1998, Kitchin and Ahmad, 2003, Liu et al., 2003, Das et al., 2005). Consistent with the role of ROS in arsenic toxicity/carcinogenicity, endogenous sulfhydryl groups and the non-protein sulfhydryl glutathione (GSH) have been reported to play an important role in detoxification of arsenic (Duyndam et al., 2001). The exogenous antioxidant N-acetylcysteine is also able to prevent arsenic-induced toxicity (Liu et al., 2003). Arsenic itself was reported to induce the Nrf2-dependent antioxidant response, although the detailed mechanism of Nrf2 induction by arsenic remains to be explored (He et al., 2006, Wang et al., 2008). Numerous studies have been performed to elucidate events associated with arsenic-induced damage, both in animal and cell culture models. Results from these studies have revealed that arsenic induces multiple biological effects, such as DNA damage, apoptotic cell death, and global DNA hypomethylation, which represent both genotoxic and non-genotoxic mechanisms of action (Huang et al., 1999, Kirkpatrick et al., 2003, Chen et al., 2004). Methylation is a covalent modification of genomic DNA and occurs on cytosines followed by guanines (CpG). DNA methylation appears to influence gene expression through alteration in chromatin structure and in DNA protein interaction. An overall decrease in the content of 5-methylcytosine (5-mc) in DNA has been viewed as an early event in tumor formation in humans and animal models (Lapeyre et al., 1981, Jones and Buckley, 1990, Umemura et al., 1990).

Recently, by using both genetic and biochemical approaches, we demonstrated a protective role of Nrf2 against arsenic-induced toxicity in a cell culture model (Wang et al., 2007). In vivo, even though Nrf2^−/− mice have been demonstrated to display increased sensitivity to chemical toxicants and carcinogens (Chan and Kan, 1999, Aoki et al., 2001, Enomoto et al., 2001, Ramos-Gomez et al., 2001, Cho et al., 2002, Yu and Kensler, 2005), currently no study has been carried out to determine the effect of Nrf2 deficiency on arsenic-induced toxicity and carcinogenicity. In this report, we hypothesized that Nrf2^−/− mice are more vulnerable to the arsenic-induced toxicity, compared to Nrf2^+/+ mice. Our results confirmed that Nrf2^−/− mice were more sensitive to arsenic-induced DNA hypomethylation, oxidative DNA damage, and apoptotic cell death. Furthermore, more severe pathological alterations were observed in the liver and bladder of Nrf2^−/− mice, compared to Nrf2^+/+ mice.