Glutathione-S-transferase-omega [MMA(V) reductase] knockout mice: Enzyme and arsenic species concentrations in tissues after arsenate administrationā˜†

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Abstract

Inorganic arsenic is a human carcinogen to which millions of people are exposed via their naturally contaminated drinking water. Its molecular mechanisms of carcinogenicity have remained an enigma, perhaps because arsenate is biochemically transformed to at least five other arsenic-containing metabolites. In the biotransformation of inorganic arsenic, GSTO1 catalyzes the reduction of arsenate, MMA(V), and DMA(V) to the more toxic +Ā 3 arsenic species. MMA(V) reductase and human (hGSTO1-1) are identical proteins. The hypothesis that GST-Omega knockout mice biotransformed inorganic arsenic differently than wild-type mice has been tested.

The livers of male knockout (KO) mice, in which 222Ā bp of Exon 3 of the GSTO1 gene were eliminated, were analyzed by PCR for mRNA. The level of transcripts of the GSTO1 gene in KO mice was 3.3-fold less than in DBA/1lacJ wild-type (WT) mice. The GSTO2 transcripts were about two-fold less in the KO mouse. When KO and WT mice were injected intramuscularly with Na arsenate (4.16Ā mg As/kg body weight); tissues removed at 0.5, 1, 2, 4, 8, and 12Ā h after arsenate injection; and the arsenic species measured by HPLC-ICP-MS, the results indicated that the highest concentration of the recently discovered and very toxic MMA(III), a key biotransformant, was in the kidneys of both KO and WT mice. The highest concentration of DMA(III) was in the urinary bladder tissue for both the KO and WT mice. The MMA(V) reducing activity of the liver cytosol of KO mice was only 20% of that found in wild-type mice. There appears to be another enzyme(s) other than GST-O able to reduce arsenic(V) species but to a lesser extent. This and other studies suggest that each step of the biotransformation of inorganic arsenic has an alternative enzyme to biotransform the arsenic substrate.

Introduction

The mechanisms of the toxicity and carcinogenicity of inorganic arsenic at the molecular level in humans remain an enigma (Abernathy et al., 1999, Aposhian and Aposhian, 2006, Goering et al., 1999, Liu et al., 2002, Kitchin, 2001, Rossman et al., 2004, NRC, 2001, IARC, 1987) even though there are millions of people in the world drinking water containing carcinogenic concentrations of inorganic arsenic. For example, more than 25 million people in Bangladesh and 6 million people in West Bengal, India, are drinking water containing arsenic concentrations above 50Ā Ī¼g/L (Chakraborti et al., 2002) even though the WHO recommends that arsenic in drinking water not exceed 10Ā Ī¼g/L. An estimated 36 million people in the Bengal Delta also are at risk for arsenic-caused cancer (Nordstrom, 2002). Chronic exposure to inorganic arsenic has led to cancer of the skin, lungs, urinary bladder tissue, kidneys, and liver (Hopenhayn-Rich et al., 1996, Chiou et al., 1995, Chen et al., 1992, Smith et al., 1992).

Human MMA(V) reductase (Zakharyan and Aposhian, 1999) and human GSTO1 (Board et al., 2000) are identical proteins (Zakharyan et al., 2001). Because the reductions of arsenate to arsenite, MMA(V) to MMA(III), and DMA(V) to DMA(III) are catalyzed in vitro by GSTO1, this enzyme is crucial in the pathway for the methylation of inorganic As since only arsenic species having an oxidation state of +Ā 3 can be methylated (Zakharyan and Aposhian, 1999, Cullen and Reimer, 1989) (Fig. 1). Thus, we hypothesized that a deletion in the GSTO1 gene might seriously impair arsenic metabolism.

GSTO1 is a member of the glutathione-S-transferase superfamily. There are seven major types of human cytosolic GSTs: alpha, mu, pi, sigma, theta, zeta, and omega. These enzymes detoxify xenobiotics usually by the catalysis of the nucleophilic attack by reduced glutathione on an electrophilic compound. GSTO1 is a dimer of identical subunits. It has the characteristic GST fold of an N-terminal GSH-binding domain and a C-terminal domain made up of Ī±-helices (Board et al., 2000).

An insoluble hGSTO2 is also known. GSTO1 and GSTO2 are two functional class glutathione transferase (GST) genes in humans that are separated by 7.5Ā kb on chromosome 10q24.3 (Whitbread et al., 2003). Recently, Schmuck et al. (2005) solubilized hGSTO2 and found it had MMA(V) and DMA(V) reducing activity. In addition, there has been the suggestion that GSTO1 may have a role as a nuclear antioxidant system (Yin et al., 2001). An excellent review of these important enzymes has appeared recently (Hayes et al., 2005).

A new pathway for inorganic arsenic biotransformation has been suggested by Hayakawa et al. (2005). It proposes that arsenic triglutathione (ATG) and monomethylarsonic diglutathione [MA(SG)2] are substrates of CYT 19. CYT 19 may be one possible methylating enzyme that forms MADG and DMAG in the presence of S-adenosyl-methionine (SAM). As yet, this has not been tested experimentally. The methylated glutathionalated compounds are then oxidized to MMA(V) and DMA(V). The most original part of this new proposal is that +Ā 3 arsenic species are formed before the +Ā 5 analogous species. The latter are proposed end products of arsenic metabolism. But CYT 19 has not been purified and isolated from human tissues. A critical review of these enzymes for inorganic arsenic biotransformation has appeared recently (Aposhian and Aposhian, 2006).

The chemical forms of arsenic determine the toxicity and bioavailability of arsenic compounds (NRC, 2001, Cullen and Reimer, 1989). Although the tissue distributions of arsenic species with an oxidation state of +Ā 5 have been investigated extensively, this is not the case for the more reactive MMA(III) and DMA(III) recently found in human urine (Aposhian et al., 2000). Information about the concentration of these +Ā 3 arsenic species in specific tissues might be of help in elucidating their role in arsenic toxicity and carcinogenicity as well as being of value for human risk assessment.

It is ironic that inorganic arsenic, an accepted human carcinogen, has no generally accepted animal model for its carcinogenesis although the mouse model proposed by Waalkes et al. (2004) is promising. Paradoxically, arsenic trioxide is a potent chemotherapeutic agent for obtaining complete clinical remission of acute promyelocytic leukemia (Sun et al., 1992, Soignet et al., 2001). Unfortunately, however, at least five patients so treated died due to its suspected toxicity (Westervelt et al., 2001).

When inorganic arsenic is ingested, it is absorbed well, distributed throughout the body, metabolized and then excreted primarily in the urine (Hughes, 2002, Hughes et al., 1994, Vahter and Norin, 1980, Odanaka et al., 1980). It appears that many, but not all, mammals methylate inorganic arsenic to MMA(V) and DMA(V) and excrete them in the urine (Vahter, 1999). However, in some species, e.g., marmoset monkey and chimpanzee, methylated arsenicals were not found in the urine (Vahter et al., 1995, Vahter and Marafante, 1985). Zakharyan et al. (1996) and Wildfang et al. (2001) found that marmoset and tamarin monkeys lacked the enzyme activities for inorganic arsenic methylation in the liver. The guinea pig also lacked the activity to methylate inorganic arsenic (Healy et al., 1997).

Recent studies have demonstrated that when humans have been chronically exposed to inorganic arsenic, they excreted in the urine arsenic biotransformants in the +Ā 3 oxidation state (Aposhian et al., 2000, Le et al., 2000). These important studies have been confirmed by others (Del Razo et al., 2001, Mandal et al., 2001). The +Ā 3 oxidation state metabolites, MMA(III) and DMA(III), are more potent cytotoxins, genotoxins, and enzyme inhibitors than are inorganic arsenate, arsenite, or the +Ā 5 oxidation state methylated arsenic metabolites MMA(V) and DMA(V) (Chen et al., 2003, Cohen et al., 2002, Petrick et al., 2000, Petrick et al., 2001, Mass et al., 2001, Styblo et al., 2000). High concentrations of MMA(III), monomethylarsine, DMA(III), or dimethylarsine damaged DNA in vitro (Andrewes et al., 2003). Exposure in vivo to arsenic compounds appeared to generate reactive oxygen species (ROS) that may contribute to cellular toxicity and/or carcinogenicity of inorganic arsenic (Kato et al., 2003, Barchowsky et al., 1996, Okada and Yamanaka, 1994).

At one time, the disposition of inorganic arsenic in laboratory animals was believed to be well characterized. Mice exposed to arsenate (po or iv), accumulated arsenic in the liver, kidneys, urinary bladder tissue, gall bladder, lungs, and blood (Hughes et al., 1994, Vahter and Marafante, 1983). Recently, when hamsters were given a single ip dose of arsenate, the new, highly toxic metabolites MMA(III) and DMA(III) were detected in the liver (Sampayo-Reyes et al., 2000). MMA(III) also has been detected in the liver and kidneys of rats after a single iv dose of arsenate or arsenite (Csanaky and Gregus, 2003). The rat, however, is not a good model of how the human processes arsenic because the rat red blood cells tightly bind DMA(III) (Lu et al., 2004). Such binding would be expected to atypically change inorganic arsenic pharmaco- and toxico-kinetics. Thus, the rat model is not appropriate as to how the human processes arsenic.

The knockout technology of molecular biology has not been applied previously to arsenic metabolism and toxicology. The purpose of the present study was to examine the metabolism and disposition of arsenic in the GSTO1 knockout (KO) and wild-type (WT, DBA/1LacJ), mice at different time intervals after a single intramuscular (im) injection of sodium arsenate. In addition, GSTO1 mRNA and enzyme activity were determined.

Section snippets

Reagents

TRIZMA (Reagent grade, minimum 99.95), bovine serum albumin, and glutathione (GSH) were purchased from Sigma Chemical Co. (St. Louis, MO). [14C]-MMA(V) (0.55Ā Ī¼Ci/nmol, 98% purity) was synthesized by Professor Eugene A. Mash, Jr., Department of Chemistry, The University of Arizona. Disodium methylarsenate was obtained from ChemService, Inc. (West Chester, PA). Monoflow-3 scintillation cocktail was from National Diagnostics (Atlanta, GA). Sodium arsenate (ACS reagent grade) was purchased from MCB

The GSTO1 gene of KO mice has a deletion in Exon 3

By RT-PCR analysis of the total RNA of KO or WT mouse liver, the transcript of the KO GSTO1-1 gene was determined to be 450ā€“500Ā bp in size as compared to the 700- to 750-bp transcript of the wild mice (Fig. 3, Fig. 4). Part of Exon 3 was deleted. Consistent with these observations were data from real-time PCR analysis of the alterations in the transcription of GSTO1-1 gene in the liver of knockout as compared to wild-type mice.

The transcripts of the GSTO1 gene in KO mice were 3.3-fold less than

Detoxication of inorganic arsenic

Methylation of arsenic species has been considered for many years to be a universal detoxication process, but the discovery that species such as chimpanzee; marmoset; and guinea pig (Vahter et al., 1995, Vahter and Marafante, 1985, Zakharyan et al., 1996, Wildfang et al., 2001, Healy et al., 1997) lack arsenic methyltransferase activity and lack the presence of methylated arsenic species in the urine has weakened the methylationā€“detoxication hypothesis. In fact, the lack of arsenic

Acknowledgment

This work was supported in part by the Superfund Basic Research Program NIEHS Grant Number ES 04940 from the National Institute of Environmental Health Sciences and the Southwest Environmental Health Sciences Center P30-ES-06694.

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    ā˜†

    This paper is dedicated to recently retired William R. Cullen, Professor of Chemistry at The University of British Columbia. His generous gifts of arsenic compounds synthesized in his laboratory and made available to all who requested them for research has aided the advancement of our knowledge about one of the oldest group of toxic compounds that humans have used for good (cancer chemotherapy) and bad (homicides). In addition, his subtle, gentle humor has enlivened a deadly subject. His research productivity and presence will be missed by all of us investigating the biochemistry and molecular biology properties of arsenic compounds.

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