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. 2008 Aug 22;106(2):329–338. doi: 10.1093/toxsci/kfn179

Induction of Hepatic Glutathione S-Transferases in Male Mice by Prototypes of Various Classes of Microsomal Enzyme Inducers

Tamara R Knight 1, Supratim Choudhuri 1,1, Curtis D Klaassen 1,2
PMCID: PMC2581675  PMID: 18723825

Abstract

The underlying need for glutathione S-transferase (Gst) induction is thought to be an adaptive response to chemical stress within the cell. Classical microsomal enzyme inducers (MEIs) increase the expression of biotransformation enzymes (phase I and II) and transporters through transcription factors, such as the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator–activated receptor (PPAR) α, and nuclear factor erythroid-derived 2–related factor 2 (Nrf2). The effects of MEIs on the induction of hepatic Gsts in mice have not been comprehensively characterized. The purpose of this study was to determine the effects of 15 MEIs on the mRNA expression of 19 mouse Gsts. Male C57BL/6 mice were treated with three different activators each for AhR, CAR, PXR, PPARα, and Nrf2. In general, the Gsts are readily induced. All five transcription factors appear to play a role in Gst induction. The Nrf2 activators induced most Gsts (10), followed by the CAR, PXR, and PPARα activators (6–7), whereas the AhR ligands induced the least (1). Clofibrate, a PPARα agonist, induced most of the Gsts; however, all three PPARα agonists decreased Gstp1/2 mRNA. None of the 15 inducers was able to increase or only minimally increased eight of the Gsts (Gsta3, Gstk1, Gstm6, Gsto1, Gstp1/2, Gstt3, Gstz1, and MGst1). Thus, the protection afforded by a ligand for one of these transcription factors will depend on the activator, as well as which Gst that detoxifies the chemicals of interest.

Keywords: glutathione S-transferase, GST, induction, mRNA, bDNA

A well-studied family of phase II enzymes is the glutathione S-transferase (Gst; EC 2.5.1.18). Gsts catalyze the nucleophilic attack by reduced glutathione (GSH) on nonpolar compounds that contain an electrophilic carbon, nitrogen, or sulfur atom, resulting in the formation of (usually) less-reactive, more hydrophilic glutathione conjugates. The substrates of Gsts include halogenonitrobenzenes, arene oxides, quinones, and α,β-unsaturated carbonyls (Hayes et al., 2005). Gsts also metabolize several endogenous molecules, such as prostaglandins (Bogaards et al., 1997), steroids (Barycki and Colman, 1997), and the histidine metabolite urocanic acid (Shimizu and Kinuta, 1998).

Gsts are found in almost all organisms, 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 catalytic properties. Three major families of proteins exhibit glutathione transferase activity. Two of these, the cytosolic and mitochondrial Gsts, comprise soluble enzymes. The third family comprises microsomal Gsts and is now referred to as membrane-associated proteins in eicosanoid and glutathione metabolism. Cytosolic and mitochondrial Gsts share some structural similarities.

Cytosolic Gsts 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 mitochondria and 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 suggest that they evolved separately (Hayes and Pulford, 1995).

To date, 16 cytosolic and 6 microsomal Gsts have been identified. The alpha-class Gsts are basic proteins, class mu Gsts are neutral proteins, and members of the pi class are acidic (Hoensch et al., 2002). Less is known about the remaining classes. In humans, Gsta1, Gsta2, Gstm1, Gstp1, Gstt1, and Gstt2 appear to be the most abundant cytosolic transferases (Hoensch et al., 2002).

The underlying need for Gst induction is thought to be an adaptive response to chemical stress within the cell (Mitchell et al., 1997). Thus, understanding what controls the induction of various Gst isoforms could be very useful from a therapeutic standpoint and also as a biomarker. Modulation of Gst activity could be used to regulate the biosynthesis of arachidonic acid metabolites, such as prostaglandins and leukotrienes (van Bladeren and van Ommen, 1991), as well as to modulate the effectiveness and toxicity of chemotherapeutic drugs. In addition, it is well known that Gsts appear to be involved in drug resistance. Therefore, knowledge gained from studies on the mechanisms of induction of these enzymes can be utilized in drug development.

Biological mechanisms responsible for controlling the expression and regulation of various Gst isoforms are complex. A number of well-known responsive elements, such as glucocorticoid response element, xenobiotic response element, and antioxidant responsive element or electrophile responsive element (ARE/EpRE), are thought to mediate transcriptional regulation of Gsts (van Bladeren, 2000). However, little is known about which Gst isoforms are induced by the various factors that are known to induce phase I enzymes. Recent studies have shown that the physiological regulation of biotransformation enzymes may be under the control of transcription factors that are responsive to an assortment of endogenous and exogenous activators (Savas et al., 1999; Staudinger et al., 2001; Xie and Evans, 2001). Classical microsomal enzyme inducers (MEIs) increase the expression of some biotransformation enzymes (phase I and II) and transporters through transcription factors, such as the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator–activated receptor (PPAR) α, and nuclear factor erythroid-derived 2–related factor 2 (Nrf2).

Rats and mice are common models used to study the pharmacology and toxicology of chemicals. However, little is known about individual Gst isoenzyme induction patterns in mice. Therefore, the purpose of this study was to investigate the effects of 15 MEIs on the expression of mouse Gst mRNAs. These inducers are prototypical activators of the five transcription factors important in the induction of phase I drug-metabolizing enzymes.

MATERIALS AND METHODS

Animals.

Male 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 Teklad 8064 rodent chow and water. At ∼8 weeks of age, mice (n = 5 per treatment group) were administered each treatment once daily for four consecutive days. On day 5, animals were euthanized by cervical dislocation. All animals were sacrificed at the same time of the day. Livers were removed, flash-frozen in liquid nitrogen, and stored at −80°C until use.

Chemicals.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; 34 μg/kg ip in corn oil) was a generous gift from Dr Karl Rozman (University of Kansas Medical Center, KS). Oltipraz (OPZ; 150 mg/kg po in corn oil) was a gift from Dr Ronald Lubet (National Cancer Institute, Bethesda, MD). Polychlorinated biphenyl 126 (PCB126; 300 μg/kg ip in corn oil) was obtained from AccuStandard (New Haven, CT). β-Naphthoflavone (BNF; 200 mg/kg ip in corn oil), diallyl sulfide (DAS; 200 mg/kg ip in corn oil), clofibrate acid (CLOF; 500 mg/kg ip in corn oil), di-(2-ethylhexyl)phthalate (DEHP; 1000 mg/kg po in corn oil), ethoxyquin (EXQ; 250 mg/kg po in corn oil), dexamethasone (DEX; 75 mg/kg ip in corn oil), pregnenolone-16α-carbonitrile (PCN; 200 mg/kg ip in corn oil), ciprofibrate (CIPRO; 40 mg/kg ip in corn oil), butylated hydroxyanisole (BHA; 350 mg/kg po in corn oil), spironolactone (SPR; 200 mg/kg ip in corn oil), phenobarbital (PB; 100 mg/kg ip in saline), and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP; 0.3 mg/kg ip in corn oil) were purchased from Sigma-Aldrich Co. (St Louis, MO).

Controls were administered vehicle only, and the route of administration in controls and the corresponding treatment group was the same.

RNA extraction.

Total RNA was isolated using RNAzol B 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 Gst1 gene sequences were obtained from GenBank. Oligonucleotide probe sets were designed using Probe Designer software, version 1.0 (Panomics, Inc., Freemont, CA). Due to > 90% similarity, one probe set was designed to recognize both Gsta1 and Gsta2 isoforms; for the same reason, one probe set was designed to recognize both Gstp1 and Gstp2 isoforms. The sequences of various capture extender and label extender probes were presented previously (Knight et al., 2007).

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.). Total RNA (10 μl = 10 μg) was added to each well in a 96-well plate containing 50 μl capture hybridization buffer and 50 μl of each diluted probe set. Hybridization was carried out at 53°C according to the manufacturer's protocol. Unhybridized probes were washed, and luminescence was measured using Quantiplex 320 bDNA Luminometer, interfaced with Quantiplex Data Management Software, version 5.02.

Statistical analysis.

Data were analyzed using ANOVA followed by a Duncan's multiple range post hoc test. Asterisks (*) represent a statistically significant difference (P ≤ 0.05) from controls.

RESULTS

Mice were treated with 15 different MEIs that are prototypical activators of five different transcription factors: AhR, CAR, PXR, PPARα, and Nrf2. The inducers used in this study were also shown to induce the expression of various phase I and II enzymes (Hartley and Klaassen, 2000; Shelby and Klaassen, 2006).

Mouse hepatic Gsta1/2 mRNA expression increased between ∼800 and ∼1200% by all three Nrf2 activators. All three CAR activators and two PXR activators, PCN and SPR, also significantly increased Gsta1/2 expression. Gsta3 mRNA expression was not increased by any of the inducers, whereas Gsta4 was significantly induced by CAR and Nrf2 activators. The increase in Gsta4 expression was about 100–200% by the Nrf2 activators OPZ and EXQ and about threefold by the CAR activator DAS. Surprisingly, the PXR activator DEX significantly decreased Gsta4 mRNA expression (Fig. 1).

FIG. 1.

FIG. 1.

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Induction response of alpha and kappa classes of Gst: Gsta1/2, Gsta3, Gsta4, and Gstk1. Both Gsta1/2 and Gsta4 showed similar profiles of increase in mRNA expression by the microsomal inducers used. All three Nrf2 activators and only one PXR activator (DAS) significantly increased Gsta1/2 and Gsta4. DEX decreased the expression of Gsta4. Two of the PPARα activators, clofibrate and CIPRO, significantly increased Gstk1 mRNA.

The PPARα ligands CLOF and CIPRO increased hepatic Gstk1 expression by about 50%, and the increase was statistically significant. The activators of AhR, CAR, PXR, and Nrf2 had no effect on Gstk1 expression (Fig. 1).

Figure 2 shows the expression profiles of the mu (m) class of hepatic Gst isoforms in mice. One pattern that is clear in the current study is that the expression of Gstm1 through Gstm4 mRNA was increased by all Nrf2 ligands studied.

FIG. 2.

FIG. 2.

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Induction response of three mu class of Gst: Gstm1 through Gstm6. The mRNA expression of Gstm1 through Gstm4 is significantly increased by all three Nrf2 activators used in the study. The CAR and PXR activators also significantly used Gstm1 through Gstm3 mRNA expression. However, the effects of PB and SPR on Gstm2 were not statistically significant. Gstm5 mRNA expression was deceased by PCB126 and PCN.

The expression of hepatic Gstm1 mRNA was increased by all three Nrf2 activators by about 200%. All three CAR and PXR activators also increased Gstm1 expression by 100–200%; however, the increase by the PXR activator DEX was not statistically significant. The AhR activators PCB126 and BNF increased Gstm1 expression by about 70%. In contrast, all the PPARα ligands studied failed to increase the expression of Gstm1 mRNA.

Hepatic Gstm2 mRNA expression was increased by about 400–600% by Nrf2 activators and by about 200–300% by CAR and PXR activators. The PXR activator SPR tended to increase Gstm2 mRNA expression, but it was not statistically significant (Fig. 2).

Of all members of the Gst mu class, Gstm3 mRNA expression showed the largest increase by CAR, PXR, and Nrf2 activators; the magnitude varied between 500 and 1200% (Fig. 2).

The other mu isoforms (Gstm4, Gstm5, and Gstm6) appeared to be less responsive to induction, and the inducibility did not show any specific trend. Hepatic Gstm4 expression was increased by all three Nrf2 activators by about 200–300% as well as by the CAR activator DAS by about 200%. PCB126 and PCN significantly decreased Gstm5 expression; the decrease by PCB126 was quite dramatic, about 90%. It is interesting to note that of the three AhR activators used, PCB126 dramatically decreased Gstm5 expression whereas the other two did not have any effect. Likewise, of the three PXR activators used, PCN decreased but SPR increased Gstm5 expression, whereas DEX did not have any effect. Gstm5 expression was increased by about 200% by SPR (PXR activator) and CLOF (PPARα activator), respectively. In terms of magnitude, the inducibility of Gstm6 expression was found to be the least of all the hepatic Gst mu isoforms studied; the increase in expression was about 70% by OPZ (Nrf2 activator) and SPR (PXR ligand) (Fig. 2).

Gsto1 mRNA expression was increased about 50% by BHA, an activator of Nrf2 (Fig. 3). Hepatic Gstp1/2 expression was not increased by any of the inducers used in the study and was the only Gst isoform whose expression was significantly decreased by about 40% by all three PPARα activators (Fig. 3).

FIG. 3.

FIG. 3.

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Induction response of Gsto1, Gstp1/2, Gstt1, Gstt2, Gstt3, and Gstz1. All three PPARα activators significantly decreased the hepatic expression of Gstp1/2 mRNAs. Gstt1 mRNA expression was increased by CAR activators TCPO and PB, as well as by PPARα activator CLOF and Nrf2 activator EXQ. PPARα activators CLOF and CIPRO significantly increased Gstt2 mRNA, while CLOF increased Gstz1 mRNA expression.

Of the three mouse hepatic theta isoforms, the increased expression of Gstt1 and Gstt2 by some of the CAR and PPARα activators was found to be statistically significant. The expression of Gstt1 mRNA (by TCPO, PB, CLOF, and EXQ) and Gstt2 mRNA (by CLOF and CIPRO) was in the order of 100–200% (Fig. 3). In contrast, the expression of Gstt3 mRNA by any of the inducers was not statistically significant, even though TCPO (CAR activator), SPR (PXR activator), and EXQ (PPARα activator) tended to increase the mean expression levels.

The expression of Gstz1 was increased only by the PPARα activator CLOF. Surprisingly, CIPRO, a hypolipidemic agent and a CYP4A1 inducer like CLOF, did not increase the expression of Gstz1 (Fig. 3).

Figure 4 shows the expressions of hepatic microsomal GST isoforms MGst1 and MGst3 in mice. Whereas MGst1 mRNA expression was induced less than 100% by SPR and DEX (PXR activators), CLOF and CIPRO (PPARα activators), and EXQ (Nrf2 activator), MGst3 mRNA expression was increased appreciably by CLOF (PPARα activator) and all the Nrf2 activators. The increase by BHA was the highest, the mean increase being about 400% over controls. Although not statistically significant, TCDD, an AhR ligand and activator, decreased MGst1 mRNA expression by 36%, whereas PCB126 in the same group decreased MGst1 mRNA expression even more markedly, which was statistically significant. Of the three PXR activators used, SPR significantly increased MGst3 expression, whereas the other two (PCN and DEX) significantly decreased MGst3 mRNA expression. Constitutive expression of MGst2 was below detection level in male mouse liver and was not inducible (data not shown).

FIG. 4.

FIG. 4.

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Induction response of microsomal GSTs: MGst1 and MGst3. The expression of MGst1 mRNA was induced by PXR activators SPR and DEX, PPARα activators CLOF and CIPRO, and Nrf2 activator EXQ. The expression of MGst3 mRNA was induced by CLOF (PPARα activator) and all three Nrf2 activators, OPZ, EXQ, and BHA. MGst3 expression was decreased by PCB126, PCN, and DEX.

Figure 5 shows the overall induction profile of all hepatic Gst isoforms studied. From this global view, it is apparent that the AhR activators used in this study are relatively poor inducers, whereas the Nrf2 activators are relatively good inducers of the majority of Gst isoforms. Among the CAR activators, TCPOBOP and DAS were found to be better inducers than PB; among the PXR activators, PCN and SPR were found to be better inducers than DEX; and among the PPARα activators, clofibrate (CLOF) and CIPRO were found to be better inducers than DEHP.

FIG. 5.

FIG. 5.

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A global view of the induction profile of various Gst isoforms in male mouse liver following treatment with various MEIs that activate specific classes of transcription factors. It is apparent that the AhR activators used in this study are relatively poor inducers while the Nrf2 activators are relatively good inducers of the majority of the hepatic Gst isoforms. Among the CAR activators, TCPOBOP and DAS were found to be better inducers than PB; among the PXR activators, PCN and SPR were found to be better inducers than DEX; and among the PPARα activators, clofibrate (CLOF) and CIPRO were found to be better inducers than DEHP. The box showing modulation of Gstp1/2 (shaded) shows three bars plotting downward from the base line (control), indicating statistically significant decrease in the expression of Gstp1/2 by all three PPARα activators.

DISCUSSION

Gst induction has been correlated with lower incidences of cancer in rodents and humans (Clapper, 1998; Clapper and Szarka, 1998; Talalay, 2000). Clapper and Szarka (1998) observed that decreased Gst activity was associated with increased risk for colorectal cancer. However, increased Gst activity is not always beneficial. Overexpression of these enzymes in tumors may be detrimental to the host as it can be associated with resistance to anticancer drugs such as BCNU [1,3-bis (2-chloroethyl)-1-nitrosourea], chlorambucil, mechlorethamine, melphalan, cyclophosphamide, and thiotepa (Hayes and Pulford, 1995).

A general trend that becomes apparent is that none of the MEIs used in this study increased the expression of Gsta1, Gstk1, Gsto1, Gstp1/2, Gstt1, and Gstz1 to any great extent (although a few marginal but statistically significant increase in the expression of hepatic Gstk1, Gsto1, and Gstz1 was observed as discussed above). The decrease in Gstp1/2 mRNA expression by PPARα activators is interesting, and if many more PPARα activators produce the same results, such a decrease in Gstp1/2 expression can be used as a marker of exposure to potential PPARα-activating compounds. The expression of several other Gsts was also decreased, such as decrease of Gsta4 by DEX, decrease of Gstm5 by PCB126 and PCN, and decrease of MGst3 by PCB126, PCN, and DEX. The implications of such sporadic decrease of some of the Gst isoforms by some of these classical microsomal inducers are not clear at the moment.

It is postulated that Nrf2 controls both constitutive and inducible expression and activity of various Gst isoforms in mice (Chanas et al., 2002; Hayes et al., 2000; Ikeda et al., 2002; Owuor and Kong, 2002). Basal levels of Gst expression are diminished in Nrf2-null mice (Chanas et al., 2002; Hayes et al., 2000). Likewise, the effect of BHA, which is a known Gst inducer through ARE/EpRE–driven transcription, is lost in Nrf2-null mice (Chanas et al., 2002). However, Nrf2 does not appear to regulate the basal expression of class pi Gst isoforms (Hayes et al., 2000). Of the 18 Gst isoforms examined in the present study, seven appear to be significantly induced by at least two of the three Nrf2 activators (Table 1). Thus, the present finding identifies the specific Gst isoforms whose expressions are modulated the most by Nrf2.

TABLE 1.

Statistically Significant Changes (% change over controls) in Mouse Hepatic Gst mRNA Expression Levels by Prototypical MEIs that Act Through the Following Five Classes of Transcription Factors

MGst isoforms AhR (%) CAR (%) PXR (%) PPARα (%) Nrf2 (%)
Gsta1/2 –– 250–950 –– –– 800–1200
Gsta3 –– –– –– –– ––
Gsta4 –– 80–200 –– –– 100–200
Gstk1 –– –– –– 50 ––
Gstm1 30–80 100–200 30–150 –– 200
Gstm2 –– 100–300 200–300 –– 400–600
Gstm3 –– 400–1000 500–1200 –– 600–1000
Gstm4 –– 70–200 –– –– 300
Gstm5 90↓ (PCB126) –– 150 200 ––
Gstm6 –– –– 70 –– 70
Gsto1 –– –– –– –– 50
Gstp1/2 –– –– –– ↓40 ––
Gstt1 –– 100–150 –– 150 150
Gstt2 –– –– –– 150 ––
Gstt3 –– –– –– –– ––
Gstz1 –– –– –– 50 ––
MGst1 –– –– 40–80 40–70 ––
MGst2 –– –– –– –– ––
MGst3 –– –– –– 40–160 100–350
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Chaubey et al. (1994) reported marked induction of hepatic Gstm mRNA in both male and female mice by BHA. The authors reported more induction in females than males. They also reported marked induction of hepatic Gstp mRNA by BHA in females but marginal increase in males and some induction of Gsta mRNA in females but no induction in males. 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. determined the levels of total Gsta, Gstm, and Gstp mRNA, without making a distinction between the relative abundance of different isoforms.

Pretreatment of mice or rats with OPZ has been shown to protect against acute hepatotoxicity caused by carbon tetrachloride, acetaminophen, allyl alcohol, and also against carcinogenesis caused by benzo[a]pyrene, diethylnitrosamine, uracil mustard, and aflatoxin B1 (Davidson et al., 1990 and references therein). Cole et al. (1985) suggested that, in rats, the detoxification of aflatoxin through conjugation with glutathione is primarily catalyzed by Gst homodimer YaYa. Davidson et al. (1990) demonstrated that OPZ treatment, indeed, results in transcriptional increase in Gst Ya subunit mRNA levels. Recently, El-Sayed et al. (2006) reported the induction of only Gstm isoform by OPZ in mice, but not Gsta and Gstp. In the present study, OPZ was found to increase the expression of Gstm1-4, but not Gstm5 and Gstm6. As in the study of El-Sayed et al., no induction of Gstp was observed by OPZ in the present study. However, in the present study, OPZ was found to increase Gsta1/2 and Gsta4, but not Gsta3. The apparent discrepancy between the study by El-Sayed et al. and the present study on Gsta induction may be due to the differences in the technique used to study the mRNA abundance. First, El-Sayed et al. used northern blot, which is less sensitive than the branched DNA assay employed in the present study. Second, El-Sayed et al. used cDNA probes for Gsta, Gstm, and Gstp, and the probes were designed to capture as many members within a class as possible by utilizing the regions with highest homology within the genes of the class. Because each probe will capture multiple members of one class, this method could undermine the significant increase of one isoform because of the lack of increase of another isoform from the same class, particularly if the uninduced isoform has a high basal expression level. In contrast, in the present study, isoform-specific probes were used to capture the inducibility/lack of inducibility of each individual isoforms. Thus, the information obtained from the present study on the inducibility of each isoform is much more precise and informative.

Although Nrf2 has been shown to influence Gst expression, the involvement of other transcription factors or nuclear receptors has not been fully characterized. The present study shows that the inducible expression of various hepatic Gst isoforms is also subject to modulation by other transcription factors, such as PXR and CAR, and to a minor extent PPARα. Thus, chemicals that activate these transcription factors will also influence the expression levels of different Gst isoforms in the liver. The importance of this observation is yet to be determined.

PXR and CAR are important in regulating overlapping sets of genes that function in the hepatic clearance of toxic compounds (Sonoda et al., 2005). The role of PXR in the induction of Gst isoforms is consistent with its suggested role as a xenobiotic sensor (Kliewer et al., 2002). Several Gst knockout mice models have been generated in which one or more Gst isoforms have been inactivated. Some of these models do not show any clear-cut phenotype (such as, class mu Gst-null mice), whereas others show clear-cut phenotype (deficiency) when the appropriate selection pressure is applied in the form of specific substrates or other xenobiotics. For example, Gsta4−/− mice are more susceptible to bacterial infection and more sensitive to paraquat toxicity (Hayes et al., 2005). Overall, Gst knockout mice models emphasize Gsts’ role in xenobiotic detoxification as well as protecting against oxidative stress.

The occurrence of multiple Gst isoforms presents a big challenge in determining their role in processes other than conjugation reactions, such as drug resistance and cancer susceptibility. Deletions of the GSTM1 and GSTT1 genes in humans are common in the general population. Application of Gstm1 assays to a breast cancer case-control study showed a significantly increased risk of breast cancer for the +/+ genotype compared to the −/− genotype, suggesting that deletion/inactivation of Gstm1 has a protective effect (Roodi et al., 2004). Thus, true Gstm1 and Gstt1 genotyping of additional or previously analyzed groups with breast cancer or other malignancies should improve our understanding of human Gst isoforms in cancer development.

Induction of various Gst isoform expression may have far-reaching consequences in cellular signaling too. As endogenous lipid mediators influence diverse signaling pathways, their metabolism by Gst has many biological consequences. For example, the prostaglandin D2 metabolite 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) can activate gene expression mediated by different transcription factors, such as PPARγ and Nrf2 while inactivating the gene expression mediated by other transcription factors, such as nuclear factor κB (NF-κB). Increased conjugation of 15d-PGJ2 with GSH is therefore expected to influence a number of different signaling pathways. For example, xenobiotics that activate Nrf2 (as seen in the present study) will induce Gst expression. Increased Gst expression, in turn, will increase the GSH conjugation of 15d-PGJ2 and inactivate the 15d-PGJ2/Nrf2 signaling pathway–mediated expression of target genes. Another example is PPARγ, which is a critical regulator of adipocyte differentiation and is also the molecular target of the thiazolidinedione class of insulin-sensitizing drugs. Overexpression/induction of Gsts increases the conjugation of 15d-PGJ2 with GSH, thereby inactivating PPARγ-mediated gene expression (Paumi et al., 2004), and Nrf2-mediated gene expression, the latter normally occurs through the ARE (Itoh et al., 2004; Jowsey et al., 2003). The effect of 15d-PGJ2 on NF-κB is somewhat different. Normally, compounds that activate the inhibitor of κB kinase (IKKβ) result in the activation of NF-κB. This is because IKKβ phosphorylates the inhibitor of NF-κB (IκB), leading to its degradation and activation of NF-κB. Therefore, inhibition of IKKβ will fail to phosphorylate IκB and prevent NF-κB activation. 15d-PGJ2 inhibits NF-κB activation by inactivating the β subunit of IKKβ (Rossi et al., 2000). The extent to which Gst-catalyzed synthesis and/or metabolism of 15d-PGJ2 impinges on these signaling pathways is an important area that warrants further study, but the available examples strongly suggest that an increase or decrease in the expression of various isoforms of Gst may have far-reaching consequences in cellular physiology. One unintended consequence of a coordinated high expression of different Gsts may be an increased cellular GSH demand due to rapid utilization of GSH for conjugation.

Gsts constitute an ancient superfamily of proteins that in most likelihood evolved from a thioredoxin-like ancestor in response to the development of oxidative stress. Other GSH and cysteine-binding proteins also share similar structure (Martin, 1995). Different classes of Gsts probably evolved through gene amplification followed by sequence divergence, resulting in novel catalytic activities (Sheehan et al., 2001). Although Gsts have been well studied, there still remains the challenge in determining their importance in various disease processes. These enzymes appear to fulfill important functions and are present in all tissues; however, humans and mice can live without them (van Bladeren, 2000). Examples from knockout mice models demonstrate that the loss of certain Gst isoforms causes an upregulation of the remaining transferases (Hayes et al., 2005). Therefore, understanding the regulation of individual Gst expression is of great importance, and the present study has, at least in part, addressed this issue. It is worth considering in this respect that polymorphism in the promoters of various Gst genes may alter their expression, and such altered expression are often associated with clinical consequences. For example, several linked single-nucleotide polymorphisms in the proximal promoter region of the human GSTA1 gene gave rise to the GSTA1*B allele, which causes reduced expression of the GSTA1 enzyme. Individuals carrying the low-expression GSTA1*B allele may have altered responses to chemotherapy (Sweeney et al., 2003). Similar promoter polymorphisms may influence the expression of various Gst genes differently in different strains of mice. In other words, the expression profile of different Gst genes observed in the present study may vary somewhat in other strains of mice due to promoter polymorphisms. In light of the role of Nrf2, PPARγ, or NF-κB in inducing different isoforms of hepatic Gst, as well as the role of different Gst isoforms in modulating gene expressions mediated by these transcription factors, an important question that remains to be explored is whether polymorphisms in human Gst genes influence the activity of these transcription factors (Hayes et al., 2005).

FUNDING

National Institutes of Health (ES-09716, ES-013714, and ES-08156 to C.D.K.).

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