Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

Glutathione transferases (GST¹) are a multigene family of phase II drug-metabolizing enzymes that catalyze the conjugation of glutathione with a range of endogenous and exogenous substrates (Armstrong, 1997; Salinas and Wong, 1999). Soluble and membrane-bound GSTs are expressed in many tissues, and soluble GSTs constitute 1 to 5% of total cytosolic protein (Morgenstern et al., 1984; Sundberg et al., 1993; Rowe et al., 1997). Activities with model substrates and the subcellular localization of GSTA, GSTM, GSTP, GSTT, and GSTO class GSTs and of microsomal GST1 have been previously characterized (Meyer et al., 1991; Hiratsuka et al., 1994; Mannervik and Widersten, 1995; Armstrong, 1997; Otieno et al., 1997; Yin et al., 2001).

The expression of GSTs is regulated pre- and post-translationally in a tissue-specific manner resulting in protein products that differ quantitatively in different tissues (Tu et al., 1983; Rozell et al., 1993; Rowe et al., 1997). Testes express the highest amount of total GST protein per milligram cytosolic protein followed by the liver, brain, pancreas, adrenals, heart, and lung (DePierre and Morgenstern, 1983; Listowsky et al., 1998). Major GST-expressing tissues are the principal sites for drug and chemical metabolism indicative of the role of GSTs in the biotransformation of xenobiotics (Pabst et al., 1973; Armstrong, 1997). Studies on the tissue-dependent expression of GSTs have been used to infer other tissue-specific functions of the GSTs and to provide insight into tissue-selective damage by xenobiotics (Harrison et al., 1989; Sundberg et al., 1994a).

Glutathione transferase zeta (GSTZ1-1) is a recently identified, cytosolic glutathione transferase that is expressed in animals, microorganisms, and plants (Board et al., 1997). GSTZ1-1 is identical with maleylacetoacetate isomerase (Board et al., 1997; Fernández-Cañón and Peñalva, 1998), which catalyzes the penultimate step in tyrosine degradation (Knox and Edwards, 1955b). Hence, maleylacetoacetate is the endogenous substrate for GSTZ1-1 (Knox and Edwards, 1955a). GSTZ1-1 differs from GSTA-, GSTM-, and GSTP-class GSTs in that it is poorly retained on glutathione affinity columns; its substrate specificity, immunological reactivity, amino acid composition, and sequence identity also differ from other cytosolic GSTs. GSTZ1-1 shows little activity with most standard GST substrates but has low activities with tert-butyl hydroperoxide and ethacrynic acid as substrates (Board et al., 1997). GSTZ1-1 also catalyzes the biotransformation of a variety of -haloacids, including dichloroacetic acid (DCA) and chlorofluoroacetic acid (CFA) (Tong et al., 1998a,b).

Humans are exposed to DCA via environmental and medical sources. DCA is a by-product of the chlorination of drinking water, and humans may consume 0.1 to 3 µg of DCA/kg/day (Uden and Miller, 1983; Weisel et al., 1998). DCA is also a metabolite of trichloroethylene and chloral hydrate; trichloroethylene is found in industrial solvents and degreasing agents to which humans are exposed, and chloral hydrate is a sedative (Henderson et al., 1997; Merdink et al., 1998). DCA is also used in the clinical management of congenital lactic acidosis (Stacpoole et al., 1983, 1998).

DCA is teratogenic in rats and in mouse embryos (Smith et al., 1992; Hunter et al., 1996). DCA-induced toxicities, including peripheral neuropathies, testicular atrophy (Katz et al., 1981; Toth et al., 1992; Linder et al., 1994), and hepatocellular carcinomas (Bull et al., 1990; Nelson et al., 1990; DeAngelo et al., 1991; Carter et al., 1995), are observed in rats, mice, and dogs. Rats are more susceptible than mice to DCA-induced hepatocellular carcinomas, and male rats are more susceptible than female rats (Richmond et al., 1995; DeAngelo et al., 1996; Pereira, 1996).

The mechanism by which DCA exerts it toxicity has not been elucidated. Studies on the biotransformation of DCA by GSTZ1-1 show that DCA is a mechanism-based inactivator of GSTZ1-1 (Tzeng et al., 2000). Moreover, GSTZ1-1 activities and immunoreactive GSTZ1-1 protein concentrations are reduced in hepatic cytosolic fractions from rats given DCA for 5 days (Anderson et al., 1999). The DCA-induced inactivation of GSTZ1-1 perturbs tyrosine metabolism in rats (Cornett et al., 1999), and these perturbations may be associated with the multiorgan toxicity of DCA. The multiorgan toxicity of DCA also indicates that GSTZ1-1 may be expressed in different organs. Northern blot analyses of mouse and human tissue samples show that GSTZ1-1 is expressed in multiple tissues (Board et al., 1997; Fernández-Cañón et al., 1999). Protein expression and activities of GSTZ1-1 in rat tissues have not been determined.

The objective of this study was to determine the subcellular localization of GSTZ1-1 by immunohistochemistry and to quantify the activities of GSTZ1-1 in different rat tissues with MA and CFA as substrates. The patterns of expression of GSTZ1-1 were also compared with those of other GSTs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

Immunohistochemistry. Male, Fischer 344 rats (175-200 g; Charles River Laboratories, Inc., Wilmington, MA) were anesthetized with ether and then sacrificed. Organs were removed, fixed in 10% (v/v) neutralized formalin (J. T. Baker, Phillipsburg, NJ), and embedded in paraffin. Two 5-µm serial sections were cut for each tissue and mounted on Superfrost Plus slides (VWR Scientific Products, West Chester, PA) to provide a negative control adjacent to each antibody-treated section.

GSTZ1-1 Activity Assays and Immunoblotting Studies. Male, Fischer 344 rats (175-200 g; Charles River Laboratories, Inc.) were housed in metabolic cages and provided with double-distilled water and Purina rodent chow ad libitum (Purina, St. Louis, MO). The rats were injected i.p. once daily for 5 days with normal saline or with 1.2 mmol/kg DCA (Aldrich Chemical Co., Milwaukee, WI) or CFA, prepared as described previously (Tong et al., 1998b), dissolved in normal saline, and brought to pH 7.4 with NaOH. Twenty-four hours after giving the last dose of DCA or CFA, the rats were anesthetized with ether and then sacrificed; organs were removed and placed in ice-cold 20 mM potassium phosphate buffer (pH 7.4) containing 2 mM EDTA, 2 mM DTT, 100 µM phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO), and 1.15% KCl. The tissues were homogenized, and the homogenates were centrifuged at 3,000 rpm (700g) for 15 min. The supernatant fractions were dialyzed overnight in 30 volumes of the homogenization buffer that lacked KCl and were stored at 80°C until used.

Immunoblotting Analyses. Whole tissue supernatants were diluted in phosphate-buffered saline containing 0.5% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate; Sigma-Aldrich) buffer to solubilize the membranes. Polyclonal anti-GSTZ1-1 antibodies (1:1000 dilution), prepared as previously described (Board et al., 1997; Tong et al., 1998a), were incubated with 250 µg of total protein overnight at 4°C on a shaker. Protein A-Sepharose beads (Sigma-Aldrich) were added to the mixture (10% v/v), which was incubated for 2 h at 4°C with shaking. The mixture was centrifuged at 10,000 rpm for 2 min at 4°C. The centrifugates were suspended and centrifuged twice in 200 µl of 0.5% CHAPS buffer to remove unbound protein; 60 µl of Laemli's sample buffer (Bio-Rad Laboratories, Hercules CA) containing 2% -mercaptoethanol were added to the precipitates. The samples were loaded (20-40 µl per lane) on 15% polyacrylamide gels and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mini Protean gel apparatus; Bio-Rad Laboratories). Proteins were transferred to 2-µm nitrocellulose membranes with a semidry transfer apparatus (Bio-Rad Laboratories), and the membranes were soaked in blocking solution [10% (w/v) nonfat dry milk (Bio-Rad Laboratories) in 20 mM Tris HCl, 150 mM NaCl, and 0.15% (w/v) Tween 20 (pH 7.4)] overnight and then incubated with polyclonal anti-GSTZ1-1 antibodies (diluted 1:5000) for 10 h. The membranes were rinsed with blocking solution and then incubated with horseradish peroxidase conjugated with mouse anti-rabbit IgG (Bio-Rad Laboratories) diluted 1:5000 in blocking buffer lacking milk for 2 h at room temperature. The membranes were rinsed again in blocking buffer lacking milk and incubated with the enhanced chemoluminescent substrate for detection of horseradish peroxidase (SuperSignal West Pico Chemoluminescent substrate kit; Pierce Chemical Co., Rockford, IL) following manufacturer's instructions. Kodak X-OMAT films (Eastman Kodak Co., Rochester, NY) were exposed to the membranes and developed by standard procedures.

Activities with Maleylacetone as Substrate. The isomerase activities in homogenates of the different tissues were determined by measuring the rate of formation of FA from MA (Fowler and Seltzer, 1970). Reaction mixtures contained protein (10-80 µg), MA (1 mM), and glutathione (1 mM) in a final volume of 0.5 ml of 0.01 M potassium phosphate buffer (pH 7.4). The reaction mixtures were incubated for 5 min at 37°C; the reactions were initiated by addition of MA and were quenched after 1 to 5 min by addition of 50 µl of concentrated HCl. Fifty microliters of a salicylic acid solution (1.37 mg in 1 ml methanol) was added to each sample as an internal standard. Samples (50 µl) were analyzed on a Hewlett Packard 1090 liquid chromatograph (Hewlett Packard, Mississauga, ON) equipped with a µBondapak C₁₈ column (3.9 mm × 300 mm, 10-µm particle size; Waters Corp., Milford, MA). The column was eluted with a 0 to 30% methanol gradient at a flow rate of 0.75 ml/min over 30 min; solvent A contained 0.075% acetic acid in water, and solvent B contained 0.075% acetic acid and 60% methanol in water. The absorbances of MA and FA in the eluate were monitored with a diode-array detector at 312 nm. Concentrations of FA in the reaction mixtures were quantified with a calibration curve prepared with known concentrations of FA. The retention times of MA and FA were t_R = 9.6 min and t_R = 22.3 min, respectively. The rate of nonenzymatic conversion of MA to FA (0.027 nmol/min/mg of protein) was determined by analysis of a solution containing MA (1 mM), glutathione (1 mM), and heat-inactivated homogenates in 0.01 M potassium phosphate buffer (pH 7.4) and was subtracted from each sample.

Activities with Chlorofluoroacetic Acid as Substrate. The formation of glyoxylate from CFA was measured spectrophotometrically, as previously described (Tong et al., 1998b). Reaction mixtures contained 150 to 500 µg of homogenate protein, CFA (1 mM), and 1 mM glutathione in a final volume of 1 ml of 0.1 M potassium phosphate buffer (pH 7.4) and were incubated at 37°C for 20 min. The reactions were initiated by addition of CFA and were quenched by addition of 50 µl of concentrated trifluoroacetic acid.

Statistical Analysis. The activity data (Tables 2 and 3) were analyzed by two-way analysis of variance with Bonferroni's post-test. A level of p < 0.05 was chosen for acceptance or rejection of the null hypothesis. Activities with MA and CFA as substrate were compared with the two-tailed Pearson correlation analysis.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

Analysis of GSTZ1-1 Expression in Rat Tissues. Polyclonal rabbit anti-hGSTZ1-1 antibodies raised against hGSTZ1-1 recognize the rat ortholog (Board et al., 1997; Tong et al., 1998a). Immunoblot blot analyses of purified GSTs and 100 µg whole-liver homogenates showed that these polyclonal anti-GSTZ1-1 antibodies do not cross-react with other GSTs (Board et al., 1997; Tong et al., 1998a). Immunoblot blot analyses of 100 µg of total protein of whole homogenates from rat tissues revealed a faint band in the lane loaded with liver homogenate, and no bands were visible in lanes that contained homogenates of heart, testis, brain, kidney, and skeletal muscle tissue (data not shown). To increase the sensitivity of the analysis, GSTZ1-1 was immunoprecipitated from homogenates from liver, heart, testis, brain, kidney, and skeletal muscle to concentrate the antigen and determine the expression of GSTZ1-1 in about 250 µg of total protein from these tissues. This analysis also permitted the examination of the cross reactivity of polyclonal anti-GSTZ1-1 antibodies with other proteins in extrahepatic tissues.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Immunoblot analysis of GSTZ1-1 proteins immunoprecipitated from rat tissues with rabbit polyclonal anti-hGSTZ1-1 antibodies. Whole homogenates from heart, testis, brain, liver, muscle, and kidney (lane 1 to 6, respectively) were prepared from untreated rats as described under Materials and Methods. Immunoprecipitates from 250 µg of total protein from each tissue were loaded in each lane. The seventh lane was loaded with about 250 ng of hGSTZ1c-1c immunoprecipitated in parallel with the tissue samples as described under Materials and Methods. The blot shown is representative of blots obtained from tissues of three rats. The ~50 kDa bands each lane correspond to fragments of the anti-GSTZ1 antibody in the immunoprecipitates.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Immunoblot analysis of immunoprecipitated GSTZ1-1 proteins from rats given CFA. Whole homogenates from heart, testis, brain, liver, muscle, and kidney (lane 1 to 6, respectively) were prepared from rats given CFA (1.2 mmol/kg), as described under Materials and Methods. Immunoprecipitates from 250 µg total protein from each tissue were loaded in each lane. The seventh lane was loaded with about 250 ng of hGSTZ1c-1c immunoprecipitated in parallel with the tissue samples, as described under Materials and Methods. The blot shown is representative of blots obtained from tissues of three rats. The ~50 kDa bands each lane correspond to fragments of the anti-GSTZ1 antibody in the immunoprecipitates.

Immunostaining of GSTZ1-1 in Different Tissues. Immunohistochemical analyses of 16 tissues showed that GSTZ1-1 protein was detected in several rat tissues, but with differing intensities.

Liver. Hepatocytes were intensely stained; the staining was condensed and appeared as intracytoplasmic masses that were localized mostly around the perinuclear membrane (Fig. 3). The central, midzonal, and periportal regions of the hepatic lobules were uniformly stained. The bile duct epithelium and the vascular endothelium were not stained. Nonparenchymal cells that were visible in some fields were also not stained. This pattern of hepatic staining indicates that GSTZ1-1 is expressed only in hepatocytes and is not expressed in other cells present in liver lobules. The intense staining of GSTZ1-1 in the liver reflects the high activities seen in the liver (see below).

View larger version (72K): [in this window] [in a new window]   Fig. 3.   Immunohistochemical detection of GSTZ1-1 in rat liver. Liver sections were incubated with (panel A) or without (panel B) polyclonal anti-GSTZ1-1 antibodies and stained by the avidin-biotin complex procedure, as described under Materials and Methods. Panel A, hepatocytes show intense punctate intracytoplasmic staining (arrowhead) that is predominant in the perinuclear regions of the cells. Bile-duct epithelium (*) and vascular epithelium (V) were sparsely stained. Original magnification was 1000×. Panel B, no staining was observed in the absence of polyclonal anti-GSTZ1-1 antibodies (200×).

Testis. The germ cells in the seminiferous tubules showed intense staining (Fig. 4). The staining appeared to increase in intensity from the basal layers toward the superficial layers, indicating an increased expression of GSTZ1-1 associated with maturation and differentiation of germ cells into spermatozoa. The absence of staining between the germ cells and in the interstitial areas indicated that Sertoli cells and Leydig cells, respectively, did not express GSTZ1-1.

View larger version (74K): [in this window] [in a new window]   Fig. 4.   Immunohistochemical detection of GSTZ1-1 in rat testis. Sections of rat testis were incubated with (panel A) or without (panel B) polyclonal anti-GSTZ1-1 antibodies and stained by the avidin-biotin complex procedure, as described under Materials and Methods. Panel A, germ cells of the seminiferous tubules (S) were intensely stained. The more developed superficial cells showed more staining that the basal cells. The interstitial regions (I) were not stained. Original magnification was 400×. Panel B, sections processed in the absence of anti-GSTZ1-1 antibodies were negative for GSTZ1-1 staining.

Cerebrum and cerebellum. A dotted pattern of staining was observed in cross sections of the cerebral and cerebellar cortices (Fig. 5). This staining was associated with the pyramidal cells in the cerebral cortex and the Purkinje cells in the cerebellar cortex, based on their localization and sizes. Little or no staining was observed in other tissues, such as glial cells and the cells lining the choroid plexus and ventricles. GSTZ1-1 was expressed only in some neurons of the central nervous system. This pattern of expression is unique to GSTZ1-1; GSTA and GSTM are expressed mostly in the choroid plexus, ventricular lining, and vascular epithelia; GSTP and GSTO are expressed only in astrocytes or glial cells; and microsomal GST1 is expressed in most regions in the brain (Abramovitz et al., 1988; Carder et al., 1990; Terrier et al., 1990; Campbell et al., 1991; Johnson et al., 1993; Juronen et al., 1996; Otieno et al., 1997; Sherratt et al., 1997; Yin et al., 2001) (Table 1). No staining was observed in a peripheral nerve embedded in skeletal muscle, indicating that GSTZ1-1 may not be expressed in peripheral nerves (Fig. 8B). Only GSTP has been described in Schwann cells of peripheral nerves (Terrier et al., 1990).

View larger version (63K): [in this window] [in a new window]   Fig. 5.   Immunohistochemical detection of GSTZ1-1 in rat brain. Sections of rat (A) cerebrum and (B) cerebellum were incubated with polyclonal anti-GSTZ1-1 antibodies and stained by the avidin-biotin complex procedure, as described under Materials and Methods. Only neurons (arrowheads), especially the pyramidal cells of the cortex and the Purkinje cells of the cerebellum, stained positive. Original magnification was 400×.

Gastrointestinal tract. The epithelial lining of the esophagus, stomach, small intestine, and colon was stained intensely, more so in the superficial layers than in the basal layers (Fig. 6). In the stomach, the parietal cells showed more staining than the chief cells. In the intestines, the cuboidal cells of the villi were stained; sloughed-off cells in the lumen were also stained, whereas mucous-secreting cells were not stained. The interstitial tissues underlying the epithelia were not stained. GSTZ1-1 was, therefore, uniformly expressed in the epithelial lining of the gastrointestinal tract.

View larger version (119K): [in this window] [in a new window]   Fig. 6.   Immunohistochemical detection of GSTZ1-1 protein in the digestive tract. Sections of (panel A) the esophagus (100×), (panel B) stomach (400×), (panel C) duodenum (100×), and (panel D) colon (100×) were incubated with polyclonal anti-GSTZ1-1 antibodies and stained by the avidin-biotin complex procedure, as described under Materials and Methods. The epithelial lining (arrowheads) of the digestive tract was stained intensely, more so in the superficial layers than in the basal layers. The mucous lining of the lumen (L) containing sloughed cells that also stained strongly. Moderately intense staining was also observed with the smooth muscle layers (*) underlying the epithelia. The parietal cells (P) of the stomach lining stained more intensely than the chief cells (C). The interstitial regions (I) in all segments were negative for GSTZ1-1.

Kidney. Staining was weak throughout the kidney (Fig. 7A). The juxtaglomerular regions of the renal cortex showed the most intense staining. Moderate staining of a subset of proximal tubule epithelial cells was also observed. The staining was more intense in the apical side of the cells (Fig. 7B). Staining was also observed in the lumen of the tubules but was absent in glomerular cells and in the epithelia of the distal convoluted tubules and collecting ducts. No staining was observed in the medulla and renal pelvis. GSTZ1-1 was, therefore, discretely expressed in the proximal tubular epithelial cells.

View larger version (68K): [in this window] [in a new window]   Fig. 7.   Immunohistochemical detection of GSTZ1-1 in rat kidney. Rat kidney sections were incubated with (panel A) or without (panel B) polyclonal anti-GSTZ1-1 antibodies and stained by the avidin-biotin complex procedure, as described under Materials and Methods. Panel A, tubular regions of the cortex (C), particularly the juxtamedullary region (JM), were weakly stained. The medullar (M) and pelvic (P) regions were not stained. Original magnification was 20×. Panel B, the apical side of the proximal convoluted tubules (P) was sparsely stained; perinuclear staining is visible in some cells. The glomerular cells (G) were not stained. Original magnification was 1000×.

Heart. Staining in the heart was moderate and uniformly distributed in the sarcoplasm of all cardiomyocytes (Fig. 8A). Nuclear staining was not observed. GSTP is highly expressed in both human and rat heart tissue, whereas expression of GSTA and GSTM is low (Hayes and Mantle, 1986; Terrier et al., 1990; Sundberg et al., 1993; Juronen et al., 1996; Sherratt et al., 1997). GSTO is expressed in human cardiac myocytes (Yin et al., 2001). GSTZ1-1, GSTO, and GSTP appear to be the most abundant GSTs in the heart.

View larger version (70K): [in this window] [in a new window]   Fig. 8.   Immunohistochemical detection of GSTZ1-1 in rat heart and other muscle tissue. Sections of tissue from (panel A) heart and (panel B) skeletal muscle were incubated with polyclonal anti-GSTZ1-1 antibodies and stained by the avidin-biotin complex procedure, as described under Materials and Methods. Panel A, myocardial fibers were homogenously and moderately stained regardless of their location throughout the heart (400×). Panel B, myocytes of skeletal muscle (M) were weakly stained. In the cross-section, the staining of smooth muscle myocytes (S) of the major artery and nearby arterioles were strongly stained. The vascular endothelial lining of the artery was not stained. Embedded in the interstitial region around the artery is a peripheral nerve fiber (N) that was not stained (400×).

Skeletal muscle and vascular smooth muscle. The staining in the skeletal muscle was weak and uniformly distributed in the sarcoplasm (Fig. 8B). Smooth muscle tissue in blood vessels and the intestinal muscularis were intensely stained. GSTZ1-1 was, therefore, expressed in muscle tissue, more so in smooth muscle than skeletal muscle. GSTP, GSTM, and GSTT have been identified in skeletal muscle, and GSTM is the only other GST expressed in smooth muscle (Terrier et al., 1990; Hussey and Hayes, 1993; Sundberg et al., 1993; Juronen et al., 1996; Sherratt et al., 1997).

Adrenal gland. Staining in the adrenal gland was diffuse (Fig. 9, left panel), but was most prominent in the adrenal medulla. Staining of the zona glomerulosa, zona fasciculata, and zona reticularis of the adrenal cortex was weak. In rat adrenals, microsomal GST1 has been detected in the medulla (Otieno et al., 1997). In human adrenals, moderate amounts of GSTA, GSTM, and GSTP are expressed in the cortex, and only GSTP is expressed in the medulla (Terrier et al., 1990; Campbell et al., 1991; Sundberg et al., 1993). The adrenal medulla is the body's major source of epinephrine, which is a metabolite of tyrosine. A high expression of GSTZ1-1 in the adrena medulla indicates that tyrosine metabolism to catecholamines and tyrosine degradation to fumarylacetoacetate are competing pathways for the biotransformation of tyrosine within the adrenals; hence, inactivation of GSTZ1-1 by DCA may affect catecholamine homeostasis.

View larger version (76K): [in this window] [in a new window]   Fig. 9.   Immunohistochemical detection of GSTZ1-1 in rat adrenal gland and pancreas. Sections of rat adrenal (left panel) and pancreatic (right panel) tissues were incubated with polyclonal anti-GSTZ1-1 antibodies and stained by the avidin-biotin complex procedure, as described under Materials and Methods. Left panel, the adrenal medulla (M) was moderately intensely stained. The staining in the adrenal cortex was sparse and more intense in the zona glomerulosa (G) than in the zona fasciculata (F) and zona reticularis (R). Original magnification was 200×. Right panel, pancreatic islets (I) showed strong staining, and the acinar cells (A) showed sparse staining that was confined to the basal side of the cells (400×).

Pancreas. The islets of Langerhans showed strong staining, and the staining appeared to be punctate within the cytoplasm and intense in the perinuclear region of cells (Fig. 9, right panel). In contrast, pancreatic acinar cells were weakly stained, and staining was most prominent in the basolateral region. The epithelial linings of the pancreatic ducts were not stained. The pattern of GSTZ1-1 expression in the pancreas differs from that of other GSTs: GSTP is expressed only in the ductal epithelium, GSTA and microsomal GST1 show moderate staining in acinar and ductal epithelial cells, and GSTM expression is very low (Campbell et al., 1991; Sundberg et al., 1993; Sherratt et al., 1997). GSTO is expressed in acinar, islet, and ductal cells of the pancreas (Yin et al., 2001).

Lung. Staining in the lung tissue was weak and limited to the cuboidal epithelial lining of bronchi and bronchioli (Fig. 10A). Sparse, spotty staining was observed in the alveoli. Other GSTs are also expressed predominantly in the bronchial cuboidal epithelium, and staining decreases progressively and in a distal direction (Terrier et al., 1990; Anttila et al., 1993; Sundberg et al., 1993; Juronen et al., 1996; Otieno et al., 1997). Little staining is observed in the alveolar Clara cells, and GSTs are not expressed in mucus-secreting goblet cells (Hayes and Mantle, 1986; Anttila et al., 1993). Alveolar macrophages also show weak staining for GSTM and GSTO (Sundberg et al., 1993; Yin et al., 2001).

View larger version (80K): [in this window] [in a new window]   Fig. 10.   Immunohistochemical detection of GSTZ1-1 in rat lung, bladder, and prostate gland. Sections of rat lung (panel A), bladder (panel B), and prostate (panel C) tissues were incubated with polyclonal anti-GSTZ1-1 antibodies and stained by the avidin-biotin complex procedure as described under Materials and Methods. Panel A, the cuboidal epithelial cells lining the bronchi (B) and bronchioles (O) showed sparsely intense staining. The epithelial cells lining the alveoli (A) showed little or no staining. The original magnification was 400×. Panel B, the superficial transitional epithelial cells of the urinary bladder showed intense staining (); staining was less intense in the deeper () layers than in the superficial layers (400×). Panel C, secretions in the prostate gland were intensely stained. The staining of the glandular epithelial lining () was faint (100×).

Bladder. The transitional epithelium was intensely stained, more so in the superficial layers than in the basal layers (Fig. 10B). Staining in the subepithelial tissue was faint. GSTP is the only other GST that has been identified in the bladder epithelium, and its expression is weak (Terrier et al., 1990; Sundberg et al., 1993).

Prostate. The glandular epithelium of the prostate was weakly stained, although intense staining was observed in secretory regions of the prostate gland (Fig. 10C). GSTM, GSTP, and GSTO have also been identified in the prostate, and their expression is observed in the basal layer of the epithelium (Terrier et al., 1990; Sundberg et al., 1993; Sherratt et al., 1997). GSTZ1-1, unlike other GSTs, appears be secreted by the prostate gland and concentrated in prostatic fluid; hence, its expression in prostatic fluid may be unique among the GSTs.

Effect of DCA Treatment on GSTZ1-1 Expression. To determine the effects of DCA treatment on the expression of GSTZ1-1 in different tissues, rats were given DCA (1.2 mmol/kg i.p. for 5 days prior to sacrifice) or CFA (1.2 mmol/kg i.p. for 5 days prior to sacrifice), and tissue sections were examined by immunohistochemistry. Sections from different tissues, notably liver, brain, and testis, of rats given DCA showed sparse staining for GSTZ1-1, whereas the staining on sections from rats given CFA was similar to that of untreated rats (data not shown). These data indicate that DCA reduced the amount of immunoreactive GSTZ1-1 in hepatic and extra-hepatic tissues and corroborate the observations showing that DCA-inactivated GSTZ1-1 is degraded (Anderson et al., 1999) and that CFA is a poor inactivator of GSTZ1-1 (Tzeng et al., 2000).

GSTZ1-1 Activities with MA and CFA as Substrates in Rat Tissues. GSTZ1-1 activities with MA and CFA as substrates were determined in whole homogenates of six different tissues. Activities with MA as substrate were much higher than with CFA as substrate, and with both substrates, GSTZ1-1 activities in the liver were much higher than activities in extrahepatic tissues (Tables 2 and 3). Two-tailed Pearson correlation analysis showed a significant correlation between the activities in the different tissues with MA as the substrate compared to those with CFA as the substrate (Pearson r = 0.9931; p = 0.0007). The tissue-dependent differences in activities with both substrates reflected the pattern of expression of GSTZ1-1 observed by immunohistochemistry.

Tissue-Dependent Differences in Expression and Activities of GSTZ1-1. Several mechanisms have been proposed to explain the tissue-dependent expression of GSTs. GSTZ1-1 may be regulated by its substrates in different tissues. High hepatic expression and activities of GSTZ1-1 is consistent with its role in tyrosine degradation. The abundant expression of GSTZ1-1 in the gastrointestinal epithelium indicates that GSTZ1-1 may affect the bioavailability of tyrosine and -haloacids. No evidence for induction of GSTZ1-1 expression has been reported. Hormones (Hatayama et al., 1986), such as testosterone that regulates the expression of some GSTs (Catania et al., 2000), may also regulate the expression of GSTZ1-1 in different tissues. This is consistent with the observation that many endocrine tissues, such as the pancreas, adrenals, and testis expressed high amount of GSTZ1-1.

Relationship between DCA-induced Toxicity and GSTZ1-1 Expression. As indicated in the Introduction, DCA-induced toxicity is observed in several organs and tissues in rats and dogs given DCA. These effects range from cardiac malformations in rats (Smith et al., 1992) to hepatocellular carcinomas in rats and mice (Bhat et al., 1991; DeAngelo et al., 1991, 1996; Daniel et al., 1992). In addition, DCA is spermatotoxic in rats (Bhat et al., 1991; Linder et al., 1997); causes central and peripheral nervous system disorders in humans (Stacpoole et al., 1998), dogs (Katz et al., 1981; Cicmanec et al., 1991), and rats (Moser et al., 1999); causes diffuse degeneration of the tubular epithelium and glomerular cells in rats (Mather et al., 1990); and causes bronchial toxicity and prostatic glandular atrophy in dogs (Cicmanec et al., 1991). A time- and dose-dependent decrease in plasma insulin concentrations are observed in B6C3F1 mice given DCA, and this effect has been associated with DCA-induced inactivation of GSTZ1-1 (Lingohr et al., 2001).