Elsevier

Toxicology

Volume 218, Issue 1, 20 January 2006, Pages 58-66
Toxicology

The course of CCl4 induced hepatotoxicity is altered in mGSTA4-4 null (−/−) mice

https://doi.org/10.1016/j.tox.2005.10.012Get rights and content

Abstract

Glutathione S-transferases (GSTs) play a key role in cellular detoxification of environmental toxicants through their conjugation to glutathione (GSH). Recent studies have shown that the α-class GSTs also provide protection against oxidative stress and lipid peroxidation (LPO). GSTA4-4 is a member of a sub group of the α-class GSTs. It has been shown to metabolize 4-hydroxynonenal (4-HNE) with high catalytic efficiency through its conjugation to glutathione (GSH) and has been suggested to be a major component of cellular defense against toxic electrophiles such as 4-HNE generated during LPO. Since the hepatotoxicity of carbon tetrachloride (CCl4) has been suggested to be due to the generation of free radicals leading to membrane LPO, the present studies were designed to compare hepatotoxicity of CCl4 in GSTA4-4 null (−/−) and wild type (+/+) mice. The results show that administration of a single dose of CCl4 (1 ml/kg i.p.) resulted in time dependent hepatotoxicity in both −/− and +/+ mice; the extent of cellular damage by serum enzymes suggests that progression was more rapid in −/− mice, although injury was similar by 24 h. Histopathologic examination showed similar degrees of centrilobular necrosis by 24 h but much greater surrounding degenerative change, including cellular swelling, disarray, and vacuolization, in the liver of −/− mice. As expected −/− mice did not show any expression of mGSTA4-4; after CCl4 a compensatory increase in the activities of total GST activity was noted at 24 h. Major alterations in other antioxidant enzymes was not observed. 4-HNE levels in the liver of −/− mice were about four-fold higher than in +/+ mice, suggesting a positive correlation between 4-HNE levels and the altered course of CCl4 hepatotoxicity. These studies suggest that GSTA4-4 is an important component during the early stages (1–6 h) of cellular defense against oxidative stress and LPO although, it is not effective in protecting against the ultimate degree of overall cell injury.

Introduction

The hepatotoxicity of CCl4 has been extensively studied in a variety of species (Hardin, 1954). It has been suggested that the hepatic necrosis produced by CCl4 may be due to the involvement of free radicals such as trichloromethyl radicals that are generated through bioactivation by the cytochrome P450 system in the liver (Frank and Link, 1984). These free radicals can extract hydrogen atoms from the membrane lipids of hepatic cells to produce lipid hydroperoxides (Recknagel and Ghoshal, 1966) and subsequently produce a rise in intracellular calcium, depleting GSH and resulting in dose-dependent hepatocellular damage in rats (Williams and Burk, 1990, Stoyanovsky and Cederbaum, 1996, Conner et al., 1989). Unsaturated alkenals are prominent among the end products of this lipid peroxidation process and 4-hydroxynonenal (4-HNE) is one of the most prevalent toxic metabolites generated during this process.

Glutathione (GSH) and GSH linked enzymes are a major cellular defense against the toxic effects of reactive electrophiles. Glutathione S-transferases (GSTs) are ubiquitous multifunctional enzymes which play a key role in cellular detoxification (Alin et al., 1985, Jensson et al., 1986). The isozyme GSTA4-4 is an α class GST that demonstrates substrate specificity which may be particularly important for cellular defense against oxidative stress (Singhal et al., 1994a, Singhal et al., 1994b, Singhal et al., 1995). GSTA4-4 utilizes 4-HNE as its preferred substrate, conjugating it to GSH with high affinity (Medh et al., 1991, Zimniak et al., 1992, Zimniak et al., 1994). The ability of GSTA4-4 to metabolize electrophiles generated during oxidative stress suggests that this isozyme may function as a major defense mechanism against the liver injury induced by CCl4.

Keeping in view the high catalytic efficiency of GSTA4-4 towards 4-HNE, we have utilized recently available mGSTA4-4 knock out mice to test the importance of this enzyme in various forms of toxic injury, including endothelial cell injury, and early atherosclerosis (Yang et al., 2004). The objective of the present study was to investigate and compare the toxic effects of CCl4 administration on the liver tissue of wild type and mGSTA4-4 knockout mice. We studied the extent of lipid peroxidation, the levels of various antioxidant enzymes as well as the histological progression of cellular damage following the administration of CCl4 in both wild type and mGSTA4 knock out mice. The results support the ideas that (1) in mGSTA4-4 knockout mice CCl4-mediated hepatotoxicity is exacerbated by the initiation of rapid lipid peroxidation leading to a marked increase in intracellular 4-HNE concentration and (2) GSTA4-4 plays a significant protective role only during the early stages of this toxic insult.

Section snippets

Chemicals

5,5′-Dithiobis(2-nitrobenzoic acid), 1-chloro2,4 dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, reduced glutathione and cumene hydroperoxide (CU-OOH), were obtained from Sigma Chemical Co. (St Louis, MO). 4-HNE was purchased from Cayman Chemical Co. (Ann Arbor, MI). Antiserum to (E)-4-hydroxynonenal was obtained from A.G. Scientific Inc. H2O2 was purchased from Fisher Chemicals (Fair lawn, NJ). Sources for specific reagents and kits are

Results

Western blot analysis (Fig. 1) of the extracts prepared from various organs (liver, testis, and kidney) showed expression of GSTA4-4 as expected in wild type mouse tissues (but not in spleen, which does not express GSTA4-4); no expression was found in tissues of mGST A4 knockout (−/−) mice.

Significant rises were found in the serum ALT levels of both wild type (+/+) and mGSTA4 knock out (−/−) mice following CCl4 administration when compared to control groups; these rises were nearly three-fold 24

Discussion

The present studies utilized a knock out mouse model which has been recently described. In this mouse, the 4-HNE conjugating activity is significantly lowered, though not totally obviated (Engle et al., 2004). Our data in this in vivo model indicates that GSTA4-4 is a major regulator of the baseline intracellular concentration of 4-HNE, as has been suggested previously by in vitro studies in cell lines (Cheng et al., 1999, Cheng et al., 2001). This fact was corroborated in vivo by the finding

Acknowledgements

Supported in part by NIH grants HL 65416 (PJB), ES 012171 (YCA), ES 07804 (PZ), and Center Grant ES 00676 to the University of Texas Medical Branch.

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