ToxSci Advance Access originally published online on September 22, 2007
Toxicological Sciences 2007 100(2):513-524; doi:10.1093/toxsci/kfm233
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Constitutive mRNA Expression of Various Glutathione S-Transferase Isoforms in Different Tissues of Mice
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160
3 To whom correspondence should be addressed at Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7417. Fax: (913) 588-7501. E-mail: cklaasse{at}kumc.edu.
Received April 18, 2007; accepted August 31, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Glutathione S-transferase (Gst) enzymes are instrumental in protecting cellular macromolecules against electrophiles and products of oxidative stress. Of interest primarily to pharmacologists and toxicologists is the ability of these enzymes to metabolize cancer chemotherapeutic drugs, insecticides, herbicides, and carcinogens. Thus, constitutive expression of Gsts might determine a tissue's ability to handle certain forms of chemical stress. In the present study, the constitutive mRNA expression of 19 different Gst enzymes was investigated in 14 different tissues in mice. The information obtained from the present study could be distilled into a few generalized principles: in all tissues examined, multiple isoforms of Gst were constitutively expressed; several isoforms, such as Gstk1, Gstm1, Gstm4, Gstm6, and Gstt1, were expressed in most of the tissues studied; at least five Gst isoforms were highly expressed in the gonads, about three in heart, and at least one in brain (Gstm5). Gender differences in the expression of various Gst isoforms were pronounced. With a few exceptions, most of the Gst isoforms expressed in kidney showed higher expression in females than males; the same trend was observed for heart and gonads. At least eight Gst isoforms showed very high expression in stomach. This was a unique finding in the current study because drug-metabolizing enzymes that are highly expressed in the gastrointestinal (GI) tract tend to have the highest expression in small intestine with low or no expression in the stomach. In summary, most Gst isoforms are most highly expressed in the GI tract and liver, which strongly suggests an important role of many Gst isoforms in detoxification of ingested xenobiotics.
Key Words: glutathione transferase; Gst; tissue distribution; mRNA; bDNA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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All organisms are exposed to xenobiotics in the environment that do not provide either energy or building blocks for biological matrices. These xenobiotics gain entry into the body mainly through food but also by inhalation and through the skin. If these compounds are not properly excreted, the resulting body burden would be enormous and harmful. To combat this assault, cells have developed a number of defense mechanisms to prevent or at least minimize the accumulation of these xenobiotics and the potential harm they can cause to the organism. Membrane transporters, metabolism, and repair of the target sites make up various arms of the cellular defense systems against these xenobiotics. Glutathione S-transferase (Gst) enzymes are a part of this protective system. They conjugate substrate xenobiotics with reduced glutathione (GSH); the glutathione conjugates being more water soluble are easily eliminated from the body. Gsts are multifunctional proteins that catalyze the conjugation of GSH with a variety of endobiotics and xenobiotics. In addition to conjugation reactions, some members of the Gst superfamily can serve as peroxidases and isomerases (Mannervik and Danielson, 1988
). They have been shown to be instrumental in protecting against electrophiles and products of oxidative stress (Hayes et al., 2005).
A broad spectrum of compounds serve as substrates for Gsts such as halogenonitrobenzenes, arene oxides, quinones, and 
,ß-unsaturated carbonyls (Armstrong, 1997; Hayes and McLellan, 1999; Hayes and Pulford, 1995; Keen and Jakoby, 1978; Sheehan et al., 2001). Gsts catalyze nucleophilic attack by GSH on nonpolar compounds that contain an electrophilic carbon, nitrogen, or sulfur atom, resulting in the formation of (usually) less reactive, more hydrophilic GSH conjugates.
Gst enzymes are dimeric and are found from bacteria to humans. They exhibit a broad and overlapping substrate specificity (Mannervik and Danielson, 1988), which makes it difficult to identify and characterize individual isoforms based solely on their catalytic properties. Three major families of proteins exhibit glutathione transferase activity. Two of these, the cytosolic and mitochondrial Gst enzymes, comprise soluble enzymes that are only distantly related. The third family comprises microsomal Gst enzymes, which are now referred to as membrane-associated proteins in eicosanoid and glutathione (MAPEG) metabolism. Cytosolic (alpha, mu, omega, pi, sigma, theta, and zeta) and mitochondrial (kappa) Gsts share some structural similarities but bear no structural resemblance to the MAPEG (microsomal Gst) enzymes.
Cytosolic Gst enzymes represent the largest family. Mammalian cytosolic Gsts are all dimeric with subunits of 199–244 amino acids in length; seven classes of cytosolic Gsts are recognized in mammalian species, designated alpha (a), mu (m), omega (o), pi (p), sigma (s), theta (t), and zeta (z). Other classes of cytosolic Gsts, namely, beta, delta, epsilon, lambda, phi, tau, and the "U" class, have been identified in nonmammalian species. In rodents and humans, cytosolic Gsts within a class typically share > 40% identity and those between classes share < 25% identity. The mammalian mitochondrial class kappa (k) Gsts are dimeric with subunits of 226 amino acids. Mouse, rat, and human possess only a single kappa Gst (Hayes and Pulford, 1995; Hayes et al., 2005). Mouse and human alpha class Gsta4 and mu class Gstm1 can also associate with mitochondrial membranes (Gardner and Gallagher, 2001; Raza et al., 2002; Robin et al., 2003). The fact that microsomal Gsts do not share any sequence identity with the cytosolic enzymes suggests that they evolved separately (Hayes and Pulford, 1995).
The alpha class enzymes are basic proteins, class mu enzymes are neutral proteins, and members of the pi class are acidic (Hoensch et al., 2002). Less is known about the remaining classes. In humans, Gst alpha1, alpha2, mu1, pi1, theta1, and theta2 appear to be the most abundant cytosolic transferases (Hoensch et al., 2002).
Of interest primarily to pharmacologists and toxicologists is the ability of these enzymes to metabolize cancer therapeutic agents, insecticides, herbicides, and carcinogens (Hayes and Pulford, 1995). Expression of various Gst isoforms increases during the development of cancer. Resistance to various anticancer drugs has been linked to overexpression of Gst enzymes in mammalian tumor cells. Likewise, individuals with lower expression of certain Gst isoforms are more susceptible to asthma (Ketterer, 2001). The expression of Gsts can influence the efficacy of drugs, detoxification capacity, as well as an individual's susceptibility to cancer (Board et al., 1997; Hayes and Pulford, 1995).
In humans, constitutive expression of the multiple isoforms of Gsts is tissue specific, suggesting that some isoforms may have specialized functions (Gupta et al., 1990). In animal models as well as cell lines, the expression of certain Gst isoforms is altered during carcinogenesis and in response to antineoplastic and chemotherapeutic drugs (Gupta et al., 1990). Gst pi expression has been shown to be increase in gastric carcinomas in humans, as well as in colon, cervical, and bladder tumors compared with levels in adjacent normal tissues (Hoensch et al., 2002).
The majority of studies examining Gst expression and activity have been performed using rat and human Gsts. In recent years, the expression of mouse Gst enzymes has also been studied, but tissue distribution and constitutive expression of all mouse Gst isoforms have not been fully characterized. Therefore, the aims of the present study were to determine tissue-specific expression of all known mouse Gst isoforms and determine whether or not the constitutive expression of various Gst isoforms demonstrate a gender difference.
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MATERIALS AND METHODS |
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Animals.
Male and female C57BL/6 mice (Charles River Laboratories, Inc., Wilmington, MA) were housed in an environmentally controlled room with a 12-h light/dark cycle and allowed free access to food and water. At approximately 8 weeks of age, after cervical dislocation, the following tissues were taken: liver, kidney, lung, stomach, duodenum, jejunum, ileum, large intestine, brain, heart, testis, ovary, placenta, and uterus (n = 5, 10 pairs of ovary per sample). The placenta was excised from 18-day-old pregnant mice. All tissues were snap frozen in liquid nitrogen and stored at – 80°C until use.
RNA extraction.
Total RNA was extracted using RNA-Bee reagent (Tel-Test Inc., Friendswood, TX) as per the manufacturer's instructions. RNA concentrations were determined spectrophotometrically at A260, and the integrity of RNA was determined by gel electrophoresis.
Branched DNA signal amplification assay.
Mouse Gst gene sequences were obtained from GenBank. Oligonucleotide probe sets were designed using Probe Designer software, version 1.0 (Bayer Diagnostics, East Walpole, MA). Due to > 90% similarity, one probe set was designed to recognize both Gsta1 and Gsta2 isoforms and another to recognize both Gstp1 and Gstp2 isoforms. Probe sequences are presented in Supplementary Data 1. Each probe was designed with a Tm of approximately 63°C to ensure optimal hybridization conditions. Probe sets were submitted to the National Center for Biotechnology Information for nucleotide comparison by the basic logarithmic alignment search tool to ensure minimal cross-reactivity with mouse genomic sequences and expressed sequence tags.
The lyophilized oligonucleotide probe sets were reconstituted in Tris-ethylenediaminetetraacetic acid buffer, pH 8.0, as per the manufacturer's instructions (Quantigene bDNA Signal Amplification Kit, Panomics Inc., Fremont, CA). Total RNA (1 µg/µl; 10 µl = 10 µg) was added to each well of a 96-well plate containing 50 µl capture hybridization buffer and 50 µl of each diluted probe set. Total RNA was allowed to hybridize overnight at 53°C in a hybridization oven. Hybridization and subsequent wash steps were carried out according to the manufacturer's protocol. Luminescence was measured using Quantiplex 320 bDNA luminometer, interfaced with Quantiplex Data Management Software Version 5.02.
The branched DNA (bDNA) signal amplification assay is a non-PCR and nonradioactive-based method of RNA analysis. It was initially developed and has been used extensively in clinical settings as a tool to monitor HIV and hepatitis (B and C) viral load in patients (see Hartley and Klaassen, 2000, and references therein). The suitability of the bDNA assay in determining transcript abundance for various xenobiotic-metabolizing enzymes was first studied by Hartley and Klaassen (2000). They examined the linearity, sensitivity, and reproducibility of the bDNA assay by measuring the transcript abundance of various inducible cytochrome P450 isoforms (CYP2B1/2 and CYP1A1/2) in rat livers. The results obtained by the bDNA assay were compared with the enormous wealth of information on the expression of various CYP isoforms that already exist in the literature.
The authors found that the CYP2B1/2 signal from total RNA isolated from control animals was linear from 0.1 to 100 µg of total RNA (range, 0.27–20 Relative Luminescence Unit (RLU)). However, for the controls, the response obtained from low levels of total RNA (less than 5 µg) was less reliable. In contrast, a much more robust signal was observed using 0.1 to 100 µg of total RNA isolated from phenobarbital-treated rats (range, 13–2500 RLU). Very similar results were obtained with the CYP1A1 probe set for control or 3-methylcholanthrene-treated rats. Analysis of the sensitivity of the assay using CYP2B1/2 transcript abundance demonstrated that signals were detected reliably above background when the amount of total RNA used was 5 µg or more for controls and 0.1 µg or more for phenobarbital-treated rat livers. Furthermore, the level of background noise in the assay was very low in which relative luminescence was consistently < 0.2 RLU. The assay was also found to be reproducible within an experiment on the same plate or on different plates on different days. Replicates within an experiment had a coefficient of variation of 8–15%.
Based on the findings of Hartley and Klaassen 2000, we routinely use 10 µg of total RNA from control and treated rats to ensure linearity, sensitivity, and reproducibility of the bDNA assays.
Statistical analysis.
Statistical differences were determined using Student's t-test with significance set at p 
0.05. A total of five different mice (n = 5) were used for each experiment. When the bDNA assay was performed, each sample was analyzed once. The error bars in all figures represent the mean (n = 5) ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The constitutive mRNA expression of mouse Gst isoforms was quantified in 13 different tissues. Whereas the mRNA expression of certain isoforms showed tissue-predominant expression, others were expressed in many tissues; still others showed a distinct gender difference in expression.
Mouse Gsta1/2 expression was highest in stomach, followed by kidney and intestine. The expression in stomach and intestine was similar in males and females, but in kidney, females had about a fourfold higher Gsta1/2 expression than males (Fig. 1). Mouse Gsta3 was most highly expressed in liver, with appreciable levels in kidney and lung (Fig. 1). Expression of Gsta4 mRNA was by far the highest in stomach (Fig. 1). Gender differences in Gsta4 expression were observed in two tissues, a 12-fold higher expression in female kidney and 
2.5-fold higher expression in female heart.
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In mice, Gstk1 appeared to be ubiquitously expressed in all tissues (Fig. 1). However, there was a decrease in its expression from the stomach through the small intestine, such that Gstk1 expression in stomach was about twice that in duodenum, the expression in duodenum was about twice that in jejunum, and the expression in jejunum was approximately twice that in ileum. Surprisingly, the expression of Gstk1 was higher in the large intestine. Gstk1 expression in heart showed a very distinct gender difference; the expression in female hearts being more than twice that in males (Fig. 1).
Six members of the Gst mu class were quantified. The expression of Gstm1 was highest in liver. The expression was also high and relatively similar in kidney, lung, stomach, and gonads (Fig. 2). No significant gender difference was observed in Gstm1 expression in any of the tissues examined. The expression of Gstm2 was highest in stomach, female heart, large intestine, and kidney. Significant gender difference in Gstm2 expression was observed in kidney and heart, both being higher in females (Fig. 2). A trend that was observed in the expression of both Gstm1 and Gstm2 was that from stomach to ileum, the expression progressively declined, but the expression was high again in large intestine (Fig. 2). In contrast, both Gstm3 and Gstm4 expressions were not high in stomach but were high in duodenum and progressively declined thereafter through jejunum and ileum (Fig. 2). In general, other than various parts of the small intestine, Gstm3 expression was very low in other tissues, but significant Gstm4 expression was observed in most of the tissues studied, which was particularly high in liver, kidney, duodenum, jejunum, and large intestine (Fig. 2). High expression of Gstm5 was observed in kidney and brain with no gender difference in expression (Fig. 2). Interestingly, Gstm5 was the only Gst isoform that showed high expression in brain relative to other tissues. With the exception of testis, where Gstm6 was more highly expressed than in any other tissue, Gstm6 was expressed at relatively low levels in all other tissues (Fig. 2).
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The expression of Gsto1 was highest in stomach, followed by uterus. However, Gsto1 expression along the gastrointestinal (GI) tract showed a similar trend to Gstk1, that is, higher expression in stomach that progressively decreased from duodenum to jejunum to ileum. Although higher expression of Gsto1 was observed in liver, kidney, heart, and gonads of female than in male mice, the difference in expression in gonads was marked, the expression in ovary being about 30-fold higher than that in testis (Fig. 3).
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Mouse Gstp1/2 expression was highest in male liver, which was more than sixfold higher than that in female liver (Fig. 3). Moderate expression of Gstp1/2 in all other tissues was observed; however, a gender difference was again observed in gonads, the expression of Gstp1/2 in ovaries being more than threefold higher than that in testes (Fig. 3). Surprisingly, Gstp1/2 expression was not detected in uterus. In this context, it is worth noting that Chanas et al. (2002)
determined the expression of Gst isoenzymes and enzymes involved in GSH biosynthesis in livers of male and female mice. The authors used TaqMan Reverse transcriptase-polymerase chain reaction (RT-PCR) and designed probe sets that distinguished Gsta1 and Gsta2 as well as Gstp1 and Gstp2. Both Gstt1 and Gstt2 were expressed in most of the tissues studied; the expression of Gstt1 was high in liver, kidney, and lung, whereas that of Gstt2 was very high in kidney compared to other tissues (Fig. 3). Gstt3 was highly expressed in lung and gonads (testis and ovary) with some expression in uterus (Fig. 3). A small but statistically significant difference in Gstt3 expression in lungs was observed between males and females.
The expression of Gstz1 was high in liver, kidney, and ovary compared to all other tissues (Fig. 3). The mean expression level of Gstz1 in ovary was more than eightfold higher than that in testis; however, the expression of Gstz1 in ovary showed a large degree of variation (Fig. 3).
Microsomal Gst1 (MGst1) expression was highest in liver, followed by ovary (Fig. 4). In most other tissues, MGst1 expression was observed but not nearly as high as in liver. Small but statistically significant gender differences in MGst1 expression were observed in kidney and heart; however, the difference between ovary and testis was marked, with ovary showing about an eightfold higher MGst1 expression than testis (Fig. 4). The expression of both MGst2 and MGst3 was highest in stomach and it declined in duodenum, jejunum, and ileum; the expression was also high again in large intestine (Fig. 4). Significant gender differences were also observed; the expression of MGst2 was higher in ovary than in testis; and the expression of MGst3 was higher in hearts of female than male mice. Therefore, the gonadal expression of MGst1, MGst2, and MGst3 showed a similar trend, that is, the expression in ovary was higher than that in testis; whereas this difference was statistically significant for MGst1 and MGst2, it was not statistically significant for MGst3 (Fig. 4).
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Figure 5 shows a composite of the tissue expression of all Gst isoforms in the tissues studied. From this global view, it is apparent that the majority of the Gst isoforms are expressed in liver, kidney, and GI tract. A few are highly expressed in gonads, whereas a couple of Gsts are highly expressed in heart and brain.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Gst enzymes are a family of proteins that play a critical role in cellular protection against oxidative stress and toxic foreign substances. They detoxify a variety of electrophilic compounds, including oxidized lipid, DNA, and catechol products generated by cellular damage induced by reactive oxygen species (Hayes and Strange, 2000
). Profiling metabolic enzymes, such as Gsts, within tissues allow for better predictions of potential sites of toxicity and metabolism in response to exposure to particular environmental pollutants (Mitchell et al., 1997).
An excellent example of the utility of profiling metabolic enzyme expression, such as that of the Gsts, is provided by Sherratt et al. (2002). It was already demonstrated that the carcinogenic effects of dichloromethane (DCM) in mouse are caused by the interaction of a GSH conjugate with DNA. The GSH conjugate is produced by glutathione S-transferase theta1-1 (Gstt1-1). Sherratt et al. (2002) compared the relative capacity and locality of DCM activation in mouse and human tissues. The results showed that mouse Gstt1-1 is more efficient in catalyzing the conjugation of DCM with GSH than the orthologous human enzyme. In addition, the mouse was found to express higher levels of the Gstt1-1 than humans in liver. Histochemical analysis confirmed the presence of Gstt1-1 in the nucleus of mouse liver cells, whereas in human liver it was detected in bile duct epithelial cells and hepatocyte nuclei but was also present in the cytoplasm. Taking this information into account, it is unlikely that humans have a sufficiently high capacity to activate DCM to be considered to represent a carcinogenic risk.
The present study examined the expressions of various Gst isoforms in both male and female mice in a variety of tissues in order to obtain a global constitutive expression profile of these enzymes in various tissues. The information obtained from the study could be distilled into a few generalized principles. For example, (1) in all tissues examined, multiple isoforms of Gst were constitutively expressed, but some isoforms were expressed at higher levels than others, (2) the tissue expression of different isoforms had no relationship with their class designation; members of the same class were often highly expressed in different tissues. Some members of the same class showed similar trend in tissue expression, and (3) constitutive expression of many Gst isoforms showed gender difference in a few but not all tissues; in most such cases, females had higher expression than males (Table 1).
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For many Gst isoforms that had high constitutive expression in the GI tract, a distinct gradient in expression was observed from stomach through the small intestine; thus, stomach had higher expression than duodenum, duodenum had higher expression than jejunum, and jejunum had higher expression than ileum. In most of these cases, the expression was again high in the large intestine. Higher expression of certain Gst isoforms in stomach was an interesting finding in the present study (Fig. 5) because stomach is generally not considered to be involved in xenobiotic metabolism. It has been hypothesized that in humans, Gst theta1-null genotype is associated with an increased risk of stomach cancer (Lan et al., 2001
). Recently, Boccia et al. (2006) conducted a meta-analysis of the literature data on the relationship of Gst theta1 genotype and the risk of developing gastric carcinoma in humans. The authors came to the conclusion that Gst theta1-null status has a very small effect on the risk of gastric cancer but it may modulate the tobacco-related carcinogenesis of gastric cancer and that the combination of unfavorable genotypes may result in an additional risk of gastric cancer. Thus, although stomach is not an organ involved in significant absorption or metabolism in humans, the expression levels of certain Gst isoforms in stomach nevertheless may make a difference in the risk of developing cancers triggered by exposure to xenobiotics and environmental pollutants. Similarly, the expression of specific Gst isoforms in the mouse stomach may offer protection against potentially harmful xenobiotics that mice may be exposed to through ingestion of food.
Gupta et al. (1990) studied the suitability of mice as an animal model for studying Gst-mediated detoxification mechanisms by analyzing the expression of the alpha, mu, and pi classes of Gst isoforms in brain, heart, kidney, spleen, liver, and muscle. The authors purified each isoform and probed their expression in various tissues by enzyme activity assay and Western blots using class-specific polyclonal antibodies. They concluded that Gst isoforms were variably expressed in different mouse tissues, suggesting that their expression was tissue specific. They also concluded that mice could be used as a model to analyze the Gst-mediated detoxification mechanisms in humans. In mice, the alpha and pi class Gst were found to be differentially expressed in various organs. The authors were able to detect the presence of a highly cationic alpha form in liver, kidney, and lung and also an anionic alpha form in liver, kidney, and brain. Using enzyme activity assays, the authors also found evidence of highest Gst activity in liver and kidney.
In the present study, only Gsta3 was found to have highest expression in liver of mice compared to other organs; the other two isoforms, Gsta1/2 and Gsta4, were most highly expressed in stomach. This apparent discrepancy is because of the detection methods, the reagents used, and also the tissues examined. Gupta et al. (1990) used Western blotting and enzyme activity assays. Western blots were performed using class-specific polyclonal antibodies that could not distinguish between isoforms within a class. In contrast, in the present study isoform-specific oligonucleotide probes were used to study and distinguish between the expressions of mRNAs of different isoforms. Gsta1/2 and Gsta4 were only minimally expressed in mouse liver. Both Gsta1/2 and Gsta4 expressions were highest in stomach; the expression of Gst alpha class in stomach was not investigated in the previous study by Gupta et al. (1990) (Fig. 1).
Because the Gst alpha class is the only Gst class with selenium-dependent glutathione peroxidase activity, one might expect that the highest level of these isoforms would be in liver where most metabolism occurs. Studies investigating the expression of Gst alpha in stomach reported the expressions as that of the entire (alpha) class, rather than the individual Gst alpha isoforms. For example, Maurya and Singh (1991) investigated whether the antineoplastic effect of diallyl sulfide (DAS), an organosulfur compound in garlic, on forestomach and lung tumors in mice has any relationship with elevated Gst expression. Western blotting revealed the presence of alpha, mu, and pi class Gsts in the stomach of mice. A significant increase in all three classes of Gst enzymes was observed in the stomach of mice treated with DAS. The authors concluded that these results suggest that DAS may exert antineoplastic effects by modulating GSH-dependent detoxification enzymes. Similarly, the expression of various Gst classes was studied in stomach and other organs in relation to their relevance in protection against cancer in rats and humans (Nijhoff et al., 1995; Van Lieshout et al., 1998). Thus, the higher expression of Gsta1/2 and a4 isoforms in stomach rather than liver is an interesting finding in the present study.
All three Gst alpha isoforms showed a gender difference in expression in kidney. Female kidneys had higher Gst alpha (a1/2, a3, a4) mRNA expression than male kidneys, suggesting that kidneys of female mice may have stronger detoxifying capacity (Fig. 1).
The tissue-specific expression and subcellular distribution of Gstk1 were recently characterized by Thomson et al. (2004) in male C57BL/6 mice. They showed that Gstk1 protein expression was highest in liver and stomach, with moderate expression in kidney, heart, large intestine, testis, and lung. The present study confirms their observations for all tissues except testis (Fig. 2). In testis, we detected minor levels of expression of Gstk1. In the present study, a gender difference in Gstk1 expression in heart was also observed; the expression in female heart was almost 2.5-fold higher than that in male heart. Subcellular localization studies have shown that Gstk1 is exclusively expressed in the mitochondria of liver and kidney leading to the speculation that this isoform may have a novel antioxidant role in protecting mitochondria from the damaging effects of reactive oxygen species (Thomson et al., 2004). If the proposed antioxidant role of hepatic Gstk1 has any merit, and if Gstk1 is also localized in the mitochondria in heart, then the same antioxidant function of Gstk1 could also be expected in the heart. In such a situation, Gstk1-mediated protection against oxidative stress might benefit hearts of female mice more than that of male mice.
Coles et al. (2002) quantified Gst protein expression in GI tracts of human subjects using high-performance liquid chromatography and examined for interindividual variability/consistency of organ-specific patterns of expression. The authors found Gstp1, Gsta1, and Gsta2 as major and Gstm1 and Gstm3 as minor constituents. Gstp1 was found to be expressed throughout the GI tract and showed a decrease in expression from stomach to colon. Gsta1 and Gsta2 were expressed at high levels in duodenum, and the expression decreased from proximal to distal small intestine. In contrast, Gsta1 and Gsta2 expression in colon and stomach, particularly colon, of all subjects was much lower than in the small intestine. The authors concluded that these data suggest that compared to duodenum and small intestine, colon and to a lesser extent stomach always have low potential for Gst-dependent detoxification of chemical carcinogens and are therefore at greater risk of genotoxic effects, particularly via substrates that are conjugated by Gsta1. This may be a factor in the greater susceptibility of stomach and colon to cancers compared to duodenum/small intestine.
The expression profiles described by Coles et al. (2002) in humans have some similarities as well as differences from that found in mice in the present study. For example, contrary to humans, highest expression of Gsta1/2 in mice was found in stomach, but in colon it was negligible as in humans. Gstp1 was found to be expressed throughout the GI tract and showed a decrease in expression from stomach to colon, both in mice and in humans. Unlike in humans where Gstm1 and Gstm3 expression were relatively low compared to that of Gsta1 or Gstp1, in mice the expressions of Gstm isoforms were high in various tissues examined.
Bammler et al. (1994) cloned Gstp1 and Gstp2 in Balb/c mouse and studied their expression in liver using RT-PCR. Both Gstp1 and Gstp2 genes were found to be expressed at higher levels in livers of male than female mice, and Gstp1 mRNA was found to be more abundant in both sexes than Gstp2. Although in the present study Gstp1 and p2 expression were not quantified separately, the expression of Gst pi class was found to be much higher in livers of male than female mice, thereby corroborating the findings of Bammler et al.. In another study, Chaubey et al. (1994) observed, using slot-blot hybridization, that basal level mRNA expression of Gst pi and mu class in mice was 18-fold higher in male than female livers, but in the lung and kidney, the expressions were similar. The authors did not observe any gender difference in the expression of Gst alpha in liver and lung, and the expression of Gst alpha in kidney was below the detection limit. The findings in the present study are consistent with that reported by Chaubey et al. (1994); however, these two studies are not fully comparable because Chaubey et al. (1994) determined the levels of total Gst alpha, Gst mu, and Gst pi mRNA, without making a distinction between the relative abundance of different isoforms. In contrast, in the present study, we examined the expression of each isoform; additionally, the bDNA assay employed in the present study is much more sensitive than slot-blot hybridization. Thus, in the present study, expression of various Gst alpha isoforms in kidney was observed. Thus, the present study yields new information that was not reported earlier.
In the present study, the majority of cytosolic Gsts had the highest constitutive expression in liver, kidney, and/or different sections of the GI tract, particularly stomach. This finding is consistent with the fact that liver and GI tract, in general, are mostly associated with detoxification of harmful endogenous and exogenous compounds (Thomson et al., 2004). However, higher constitutive expression of many Gst isoforms in the stomach is somewhat unusual because stomach is not known to be involved in xenobiotic metabolism and detoxification to any significant degree. It remains to be determined whether Gsts are expressed in all or specific cell types in the stomach, that is, mucus-secreting "mucous cells," hydrochloric acid–secreting "parietal cells," pepsin-secreting "chief cells," and gastrin-secreting "G cells." It will also be interesting to investigate whether Gsts expressed in stomach play any role in the etiology of gastric carcinomas. Also for microsomal Gsts, liver and stomach showed highest constitutive expression compared to other tissues. MGst1 had highest expression in liver, whereas MGst2 and MGst3 had the highest expressions in stomach, followed by different sections of the intestine. Such preferential expression in different sections of the GI tract and liver strongly suggests an important role of microsomal Gsts in detoxification of ingested xenobiotics. For example, rat liver microsomal Gst is involved in the detoxification of numerous carcinogenic, mutagenic, toxic, and pharmacologically active compounds (Ji et al., 1996).
Gst enzymes constitute a very ancient protein superfamily that is thought to have evolved from thioredoxin-like ancestral protein in response to development of oxidative stress. Multiple Gst classes probably arose by gene duplication, sequence divergence, and acquisition of new function. In humans, cytosolic Gst enzymes show polymorphisms, and this is likely to contribute to interindividual differences in their response to xenobiotics (Hayes et al., 2005). Another factor that can contribute to variability in response to xenobiotics is gender difference.
Gender differences were observed in the constitutive expression of various isoforms in the present study, and some of these differences were very dramatic. For example, Gstp1/2 expression was dramatically higher in male liver than in female liver. A possible explanation could be that Gstp1 is expressed primarily in livers of male mice, whereas both the active p1 isoform and the inactive p2 isoform are expressed in livers of female mice (Hayes and Pulford, 1995). Interestingly, the majority of the gender differences in Gst expression observed in the present study involved higher expression levels in females than in males. For example, every Gst isoform that showed gender difference in constitutive expression in kidney, lung, heart, and gonads had higher expression levels in females than in males, the only exception being Gstm6 which showed much higher expression in gonads of male than female mice (Table 1). Likewise, every Gst isoform that showed gender difference in constitutive expression in stomach had higher expression levels in males than in females (Table 1). The mechanism regulating such preferential higher constitutive expression in most tissues in female mice is yet to be determined, and it requires further investigation. Gender differences in the disposition of xenobiotics in rodents are well documented and are the result of regulation of xenobiotic-metabolizing enzymes by sex hormones and/or gender-dimorphic growth hormone (GH) secretory patterns. Interestingly, xenobiotic transporter gene expression also shows gender differences, which can also be regulated by sex hormones and/or gender-dimorphic GH secretory patterns (Cheng et al., 2006). It is tempting to contemplate that hormonally mediated gender differences in the expression of both xenobiotic transporters and xenobiotic-metabolizing enzymes could result in transport-metabolism coupling, thereby fine-tuning the gender difference in the ability to handle environmental stress.
In the context of mRNA abundance of various Gst isoforms in mouse liver, it is worth noting that recently Ruiz-Laguna et al. (2006) reported absolute transcript expression signatures of hepatic Cyp and Gst genes in Mus spretus to detect environmental pollution. In this study, mice dwelling at the Do
ana Biological Reserve site (unpolluted) were compared with those dwelling at an industrial site (polluted). Real-time PCR was used to quantitate the constitutive as well as induced expression levels of various hepatic Cyp and Gst transcripts. The results reported by Ruiz-Laguna et al. (2006) show some similarities and some differences with our results. The similarities are reflected in the finding that all Gst isoforms investigated in both studies showed higher constitutive mRNA expression levels in female livers (the only organ for which comparison could be made). The differences are reflected in the finding that in the present study, there were no statistically significant differences in the constitutive expression of various hepatic Gst isoforms in males and females, whereas in the study by Ruiz-Laguna et al. (2006), there were marked differences in the constitutive expression of various hepatic Gst isoforms between males and females, and the differences were all statistically significant. The apparent differences could be explained by the fact that M. spretus is a wild species of mice and the species used in our study is Mus musculus, strain C57BL/6 inbred mice. It is now known that even strain differences of the same species may result in differences in the expression of various enzymes (Tjalkens et al., 1998). Thus, the results of the study of Ruiz-Laguna et al. (using wild-type M. spretus) cannot be directly compared with that of the present study (using inbred M. musculus).
In conclusion, the present study examined the expression of various Gst isoforms in both male and female mice in a number of tissues. The majority of cytosolic Gsts had the highest constitutive expression in liver, kidney, and/or different sections of the GI tract. These findings are in agreement with the roles of these tissues in xenobiotic metabolism. The constitutive levels of expression of xenobiotic-metabolizing enzymes in a tissue determine its ability to handle xenobiotic load. The finding that every tissue investigated in the present study has multiple Gst isoforms expressed also supports the notion that Gst enzymes play an important role in protecting cellular macromolecules against electrophiles and products of oxidative stress (in addition to xenobiotic metabolism). Thus, the constitutive expression of Gsts could also determine a tissue's ability to handle endogenous metabolic stress. However, the specific roles of individual Gst enzymes in each tissue still remain a matter of speculation. Experiments involving isoform-specific knockout mouse models or RNAi-mediated knockdown of isoform-specific expression are required to address these issues. Such experiments will presumably also shed light on unusual findings, such as higher constitutive expression of many Gst isoforms in the stomach, as found in the present study. This is somewhat unusual, and its physiological significance remains to be determined. Gender differences were also observed in the constitutive expression of various Gst isoforms, and in most cases, Gst expression was higher in females than in males.
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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National Institutes of Health (ES-09716, ES-013714, ES-07079, and RR-021940).
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Disclaimer: The opinions expressed in this article are the authors’ personal opinions and do not necessarily reflect those of Food and Drug Administration, Department of Health and Human Services, or the Federal Government.
1 Center for Toxicology and Environmental Health, L.L.C., Little Rock, Arkansas 72201. 
2 Food and Drug Administration, Center for Food Safety and Applied Nutrition, OFAS/DBGNR, College Park, Maryland 20740.
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