Toxicological Sciences 54, 52-59 (2000)
Copyright © 2000 by the Society of Toxicology
Glutathione S-Transferase in Mucus of Rat Small Intestine
,1
* Department of Biochemistry, and
Graduate Program in Pharmacology and Physiology, Emory University, Atlanta, Georgia
Received June 24, 1999; accepted November 2, 1999
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Glutathione S-transferases in the small intestine function in detoxification of electrophilic compounds ingested in foods, dietary supplements, and orally administered drug preparations. Although the required substrate glutathione (GSH) is synthesized in the intestinal enterocytes, the rate of synthesis is slow compared to both the maximal GST activity and the rate of uptake of luminal GSH. GSH is supplied to the intestinal lumen in the bile, and normal luminal concentrations in the rat are about 250 µM. The present study was designed to test the hypothesis that exogenous GSH is used for intestinal conjugation by glutathione S-transferase. The results show that 250 µM of extracellular GSH stimulated conjugation of 1-chloro-2,4-dinitrobenzene by approximately 300% in rat intestinal enterocyte preparations. However, an unexpected finding was that most of this stimulated activity did not depend upon uptake of GSH by the enterocytes but was due to glutathione S-transferase associated with mucus. Immunohistochemistry of glutathione S-transferase in the intact small intestine confirmed that a portion of the GST is present in the mucus layer. The presence of this detoxication enzyme in the extracellular mucus layer provides a novel mechanism for preventing direct contact of potentially toxic dietary electrophiles with the intestinal enterocytes.
Key Words: glutathione; 1-chloro-2,4-dinitrobenzene; conjugation; electrophiles; diet.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Glutathione (GSH) is supplied to the lumen of the small intestine from bile transported from the liver (Yi et al., 1994
). The concentration of GSH in bile is in the millimolar range (Eberle et al., 1981) and supply of GSH to bile occurs via a specific transport system, which has been extensively characterized in bile canalicular membranes (Fernandez-Checa et al., 1992, 1993; Inoue et al., 1983). A gene encoding a GSH transporter for this membrane has been cloned and sequenced (Yi et al., 1994) although this identification remains controversial (Li and Ballatori, 1997). GSH can also be provided from the diet (Wierzbicka et al., 1989), with fresh fruits and vegetables providing on average the highest concentrations (Jones et al., 1992). Direct measurements of luminal (jejeunal) GSH in rat show a concentration range of 60–200 µM in fasted animals that increases to 120–300 µM in fed animals (Hagen et al., 1990).
GSH, supplied to the lumen of the small intestine, can be transported into the intestinal cells by sodium-dependent and -independent mechanisms (Hagen and Jones, 1987; Linder et al., 1984; Vincenzini et al., 1989). The transport rate approximately equals the synthesis rate when luminal GSH is 200 µM, and exceeds the synthesis rate at higher concentrations (Hagen and Jones, 1987). Transported GSH can increase protection of the intestinal cells from oxidative damage induced by t-butyl hydroperoxide or menadione (Lash et al., 1986). Kowalski et al. (1990) showed that exogenous luminal GSH prevented the transepithelial transport of thiobarbituric acid-reactive substances in everted sacs of small intestine. In more detailed analyses of the effects of exogenous GSH on the elimination of lipid hydroperoxides, Aw et al. (1992) found that GSH serves as a reductant in this system and that decreased GSH content may compromise this function. A specific role for biliary GSH in this activity was established (Aw, 1994). Other studies have shown that in vivo depletion of GSH, through inhibitors of synthesis (L-buthionine-SR-sulfoximine), causes small intestinal cells to undergo severe degeneration (Martensson et al., 1990). This finding suggests that GSH is needed to maintain intestinal integrity and function.
The human diet contains a variety of plant and animal products which contain electrophiles that must be detoxified to prevent damage to cells (Ames, 1983; Hoensch and Schwenk, 1984; Miller and Miller, 1976). For example, quinones and their phenol precursors (Ames, 1983) and 4-hydroxyalk-2-enals, products of lipid peroxidation, are found in many food products and can be detoxified by glutathione S-transferases (GST) (Jensson et al., 1986). Measurements of GSH-reactive materials in foods have shown that many foodstuffs contain greater than 10 µM reactive compounds (Samiec et al., 1993). Many drugs are also electrophilic and when given orally, result in delivery of relatively high concentrations to the lumen of the small intestine (Hoensch and Schwenk, 1984).
Intestinal epithelial cells contain members of the GST family that catalyze the reaction of GSH with electrophilic compounds (Clifton and Kaplowitz, 1977; Ogasawara et al., 1989; and Siegers et al., 1988). When GST activity is greater than the GSH synthesis rate, such as when cells are challenged by electrophiles, GSH depletion can result in cell death (i.e., acetaminophen toxicity). GST activity in small intestinal enterocytes is about 1.65 µmol/106 cells per h (calculated using data from Lash et al., 1986 and Tahir et al., 1988), a value that is more than 100-fold greater than the rate of GSH synthesis (1.32 ± 0.20 nmol/106 enterocytes per h; Bai, 1992) or GSH uptake (8.4 nmol/106 enterocytes per h, calculated from data in Lash et al., 1986). Because of these different rates, conjugation of electrophiles in the small intestine is likely to be limited by GSH supply. The rates further suggest that uptake of exogenous GSH may provide a stimulation of conjugation activity over that provided by endogenous GSH synthesis.
The purpose of the present study was to determine whether exogenous glutathione increased the rate of the conjugation of an electrophilic compound by rat small intestinal enterocytes. For this purpose, we used rat freshly isolated intestinal cells and examined the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB), a general substrate for major isoforms of GST (Ketterer, 1988). The results showed that transported GSH stimulated intracellular metabolism, but also revealed that a substantial conjugation rate occurred extracellularly. The study of the distribution of the activity and immunohistochemistry showed that this extracellular activity was due to GST associated with the layer of mucus lining the intestinal epithelium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
GSH, GSSG, acivicin, iodoacetic acid, 1-chloro-2,4-dinitrobenzene, 1-fluoro-2,4-dinitrobenzene (Sanger's reagent), heparin, and trichloroacetic acid were obtained from Sigma, St. Louis, MO. Dithiothreitol (DTT) and glacial acetic acid were purchased from Baxter, McGaw Park, IL. Collagenase D was purchased from GIBCO BRL, Gaithersburg, MD. The methanol was HPLC-grade and was purchased from Curtin Matheson Scientific, Inc., Houston, TX. Pentobarbitol sodium (Wyeth Laboratories) was obtained from the Emory University Hospital. SolvableTM was purchased from Dupont NEN Research Products, Boston, MA. Primary antibody (raised in rabbit) that reacted with µ and
forms of GST (Biotrin International, Dublin, Ireland) was a generous gift from Dr. Thomas Boyer, Emory University, Atlanta, GA. Secondary antibody (anti-rabbit IgG with a fluorescein isothiocyanate label, raised in goat) was purchased from Sigma, St. Louis, MO. All other chemicals were reagent grade or better.
Animals.
Male Sprague-Dawley rats (VAF/1; Sasco, Omaha, NE), weighing approximately 250–400 g, were housed in the animal care facility at Emory University under conditions of controlled temperature and humidity. A 12-h light (7:00 AM to 7:00 PM)-12-h dark cycle was maintained, and rats were allowed free access to tap water and Rat Chow (Purina 5001, St. Louis, MO).
Small intestinal cell isolation.
Cells were prepared from the first 50 cm of jejunum as described by Henninger et al., (1995). These cells have been shown to synthesize and transport GSH (Aw et al., 1993) by a Na+-dependent mechanism and this transport of GSH is inhibited by probenicid and 
-glutamyl compounds such as -glutamylglutamate. These cells consume O2 at a rate of 9.5 nmol/106 cells/min. Cells were counted and the viability was measured using trypan blue dye exclusion (0.2% wt/vol). Mean viability for all experiments reported was 93.2 ± 0.7%. Cells showed a minimal loss of viability over a 1-h period; original viability 93.7 ± 0.9% compared to 91.3 ± 2.5% after 1 h (n = 7), not statistically different, p = 0.28. Experiments were performed in media containing (in mM): NaCl (118), NaHCO3 (25), HEPES (17), KCl (4.7), KH2PO4 (1.18), and glucose (5), with a pH of 7.25–7.27 and the addition of Streptomycin (0.25 µg/ml)/Penicillin G (100 µg/ml).
Isolation of intestinal plasma membranes.
The brush-border and basolateral regions of the cell membrane were prepared according to the method of Scalera et al. (1980). The regions of the cell membrane were identified and contamination with other cellular organelles was assessed by use of marker enzymes, as described by Lash and Jones (1983). GST activity was measured according to the method of Habig et al. (1974).
HPLC methodology.
Samples were analyzed for GSH content by high performance liquid chromatography (HPLC), utilizing either an Ultrasil-NH2 column (Beckman, San Ramon, CA) or an aminopropyl column (Custom-LC, Houston) according to the method of Fariss and Reed (1987). GSH was identified by retention time of standards and concentrations were calculated relative to standards by integration.
The GSH-CDNB conjugate (S-DNP-GSH) was also detected by HPLC. The mobile phase was 85% solvent A and 15% solvent B, run isocratically at 1 ml/min for 30 min with detection at 365 nm. The conjugate eluted at approximately 10 min and was identified by standards prepared according to Habig et al. (1974). Concentrations of S-DNP-GSH in samples were calculated from peak integrated areas relative to the corresponding standard.
GST activity.
GST activity was measured using the method of Habig et al. (1974). Briefly, 1 mM CDNB was added to buffer containing 1 mM GSH and an aliquot of sample to be tested. Upon addition of CDNB, the change in absorbance at 340 nm was measured as a function of time. The extinction coefficient for this reaction is 9.6 mM–1cm–1.
Slide preparation and immunohistochemical staining.
Rats were anesthetized with pentobarbital (50 mg/kg, ip), a midline laparotomy was made, and the jejunum was removed. The jejunum was either flushed with media (same as isolation media), flushed with media and incubated for 5–10 min with DTT (10 mM) to remove the mucus layer, or left with digesta intact. Sections (approximately 5 cm) of rat small intestine were cut with a scalpel and placed in an acetone/formaldehyde/citrate fixative.
Sections were washed with water, graded alcohol solutions, and xylene before being embedded in paraffin. Paraffin molds containing the intestinal sections were thinly cut with a microtome (5-micron thickness) and placed on slides. Slides were dried at room temperature overnight before staining.
Paraffin was removed by repeated washes in xylene, then graded alcohol washes. Slides were washed with distilled water, incubated with 0.1% trypsin for 20 min, then washed with phosphate-buffered saline (pH 7.4). Primary antibody to rat GST (µ and forms) was placed on the sections and incubated for 1.5 h, then washed off with Tris buffer (pH 8.2). Secondary antibody (anti-rabbit IgG with a fluorescein isothiocyanate label) was placed on the sections and incubated for 1 h. Fluorescent images were visualized using a Zeiss 10 Axiovert microscope equipped with a fluorescent filter set.
Statistics.
Comparisons between multiple groups were made by ANOVA and comparisons between 2 treatment groups were made with paired t-tests. Criterion for significance was p 
0.05 for all comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial studies showed that the rate of conjugation of CDNB by isolated enterocytes could not be measured spectrophotometrically at 340 nm because of cellular turbidity. Therefore, we used an HPLC method for measurement of S-DNP-GSH that we developed from the method of Fariss and Reed (1987) for the analysis of GSH. The S-DNP-GSH was made by addition of 1 mM GSH, various concentrations of CDNB, and purified glutathione S-transferase (GST). The samples that resulted were analyzed spectrophotometrically at 340 nm and the concentration was calculated using the extinction coefficient of this reaction (9600 M–1cm–1) according to Habig et al. (1974). The conjugate was run on the HPLC under the same conditions used for GSH determination according to Fariss and Reed (1987), and the conjugate was found to elute at approximately 10 min. The peak height and area were strictly dependent on the concentration of S-DNP-GSH in the sample. Using this approach, we were able to detect levels of conjugate that were well below the level of detection for the spectrophotometric method.
To determine whether exogenous glutathione enhanced the rate of conjugation of CDNB, freshly isolated enterocytes were incubated with 20 µM CDNB, with and without 250 µM GSH. These initial concentrations were selected because they are within the ranges of electrophiles and GSH that can occur in the intestinal lumen. Conjugation with added GSH was enhanced by approximately 3-fold over that without added GSH (Fig. 1). Variations in CDNB concentration showed that conjugation was dependent upon the concentration of CDNB supplied through the incubation medium (Fig. 2) at concentrations below the range of the Km for GSH S-transferases (0.86±0.12 mM) present in these cells. Similarly, variations in GSH concentration in the medium showed there was also a concentration dependence on GSH over the range studied (0–500 µM) (Fig. 3).
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) cells (106 cells/ml), CDNB (20 µM), and GSH (250 µM); (
) cells (106 cells/ml) and CDNB (20 µM); (
) non-enzymatic conjugation, CDNB (20 µM) and GSH (250 µM). Data are the mean of 3 trials. Error bars depict standard error (SE). *Significantly different from condition without added GSH, p
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) cells (106 cells/ml), GSH (250 µM), and CDNB (10 µM); (
) cells (106 cells/ml), GSH (250 µM), and CDNB (40 µM); and (
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) cells (106 cells/ml), CDNB (20 µM) and GSH (500 µM);. Data were corrected for non-enzymatic conjugate formation. Insert: Plot of initial rate of conjugate formation (nmol/ml/min) vs. concentration of GSH. Concentrations of GSH were below its Km for the enzyme. R2 = 0.895. Data shown are the mean of 4 trials. Error bars depict SE.To determine whether synthesis of GSH from its amino acid precursors could support conjugation at a similar rate, cells were incubated with 1 mM each of glycine, cysteine, and glutamate along with the CDNB. The results showed that the amino acid precursors did not enhance conjugation over that which occurred without the added GSH (Fig. 4
). Cells were also preincubated 20 min with 1-mM L-buthionine-SR-sulfoximine (BSO) to inhibit synthesis of GSH and then incubated, either in the presence or the absence of the amino acid precursors to GSH. BSO-treated cells had a small loss of conjugate formation (compared to the no-BSO condition) probably due to the loss of intracellular GSH (Fig. 4). To further investigate whether stimulation by GSH involved degradation and re-synthesis of GSH, cells were preincubated 30 min with 0.25-mM acivicin, an inhibitor of glutathione breakdown, in addition to BSO. Results showed no effect of acivicin pretreatment on the conjugation rate (Data not shown). These results support the interpretation that intact exogenous GSH is being used to directly increase the rate of conjugation of CDNB as opposed to involving the breakdown of GSH and its re-synthesis inside the cell.
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To determine whether transport of GSH into cells is necessary for this enhanced conjugate formation, inhibitors of GSH transport [probenecid and
-glutamyl glutamate (GG); Lash et al., 1986] were added to the incubation media at concentrations found to give 75–80% inhibition of GSH uptake. Results provided the unexpected finding of only about 20% inhibition (p > 0.05) of conjugate formation (Fig. 5). Experiments with medium containing GSH and CDNB without cells showed that the conjugate formation was almost entirely dependent upon the presence of cells and was not a result of non-enzymatic reaction. Because the transport of GSH into the small intestinal cells was previously shown to be inhibited by 75% in a Na+-free media (Hagen and Jones, 1987), cells were incubated in a Na+-free media (choline chloride was used in place of NaCl) with or without added GSH. Elimination of sodium resulted in only approximately 25% inhibition of conjugation (p > 0.05) in the presence of GSH, and similarly had little to no effect in the absence of added GSH (Fig. 6). These findings, taken together with the inhibitor experiments, lead to the conclusion that uptake of GSH into the cells cannot account for the stimulated rate of CDNB conjugation by extracellular GSH. The results suggest that increased conjugation with exogenous GSH is due to reactions outside the cell, either in the medium or associated with the plasma membrane.
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To determine whether the activity could be due to a GST associated with the cell membrane, the small intestinal mucosa was fractionated into different subcellular components and assayed for GST activity. No GST activity above that which could be accounted for by contamination with cytosol was found on either the brush-border or basolateral membranes of these cells (Fig. 7
). For brush-border membrane, cytosolic contamination accounted for 7.6 ± 2.9% and GST activity was 2.25 ± 1.32% of cytosolic activity; for basolateral membrane, cytosolic contamination accounted for 4.1 ± 1.4% and GST activity was 3.7 ± 1.8% of cytosolic activity. These results show that conjugation due to exogenously added GSH is not due to GST associated with the plasma membrane.
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Because increased conjugation was consistently observed with added GSH, and GST was not associated with the plasma membrane, we tested whether cells released a sufficient amount of GST into the incubation medium to account for the activity. Isolated, washed cells were incubated in media for 5 min at 37°C and then spun down. The supernatant was incubated with CDNB and GSH. The results showed that conjugate formation was observed at rates sufficient to account for most of the increase due to extracellular GSH (Fig. 8
). This conjugate formation was not due to non-enzymatic conjugation, as the rate in medium alone under these conditions was much lower (Fig. 8). There was no loss in cell viability during the 5 min time course of the incubation and centrifugation procedure; initial viability was 89.4 ± 2.2%, and final viability was 85.0±3.9% (not statistically different, p = 0.27). Thus, the results show that exogenously added GSH stimulates CDNB conjugation in small intestinal enterocytes, and that this stimulation is due to the release of a significant amount of GST activity into the incubation medium.
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The detection of substantial GST activity released into the medium from enterocytes, along with the evidence that GSH is continually supplied to the intestinal lumen, suggested that these two processes could provide a mechanism for extracellular detoxification in the lumen of the small intestine. In order to determine how stable GST would be if released into the luminal contents during digestion in a physiological setting, purified GST (activity of 0.0572
A/min after dilution factor, 5.50 nmol/ml/min) was incubated with partially digested ingesta flushed from the lumen. GST activity was measured at 0, 5, and 10 min after addition. Results showed that GST activity was lost immediately after addition to the luminal contents (Fig. 9). Thus, GST is not sufficiently stable in the presence of luminal digestive enzymes to permit a function in detoxification.
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Under physiological conditions, however, intestinal cells have a barrier between the cells and the luminal contents, i.e., the mucus. To determine if the GST is present in the intestinal mucus, the lumen of the small intestine (50 cm) was flushed out and filled with a solution (10 ml) containing 10 mM DTT, a condition known to release the mucus from the epithelial surface without damaging the cells. After a short incubation time (10-min) this luminal solution was collected and assayed for GST activity. Measurement of GST showed an activity of approximately 1.3 µmol/min/50 µl sample (0.013
A/min/50 µl sample) was found in this mucosal suspension. This activity represents approximately 25% of the total epithelial GST activity. Microscopic examination showed a very low contamination by cells; estimates of activity associated with these cells and due to non-enzymatic activity showed that >95% of the mucus activity was enzymatic and acellular.
To determine whether this activity could be due to GST in the mucus, we performed immunohistochemical staining of segments of the small intestine with antibody recognizing the µ and forms. Results showed that GST µ and/or GST is present in the mucus layer associated with the small intestinal villi as well as within the enterocyte (Fig. 10). Control experiments in which the primary antibodies were omitted did not result in staining. Furthermore, staining performed on intestinal segments devoid of mucus following incubation with DTT and then washing, showed GST to be present only within the enterocytes. Thus, based upon the criteria of activity and immunohistochemical staining, GST is located in the mucus layer and this activity is sufficient to allow conjugation of electrophiles in the extracellular space of the intestinal lumen.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we have found that exogenous GSH can increase the capacity of intestinal cells to conjugate a model electrophile, CDNB. This enhanced conjugation is minimally affected when inhibitors of GSH transport are present and does not involve a membrane-bound GST. The results show that this activity is due to GST that is released into the medium during incubation and that this released activity can account for a substantial fraction of the total conjugate formation. Additionally, examination of the mucus that lines villi of the small intestinal enterocyte showed the presence of GST activity, and immunohistochemistry confirmed the association of GST with the mucus. Thus, the data suggest that electrophile conjugation in the small intestine involves an extracellular mechanism catalyzed by GST(s) present in the mucus and utilizes GSH supplied by the bile and food.
This extracellular detoxification mechanism would appear to complement other known mechanisms that protect against ingested electrophilic chemicals, namely, cellular conjugation and cell turnover. However, the present studies do not address the mechanism for deposition of GST in the mucus. Presumably, the results with isolated cells were due to mucus remaining adherent to the cells, because the amount of activity released decreased with successive washes. In vivo, the deposition of GST in the mucus could be due to release along with mucus from goblet cells. Although this is not specifically excluded by the present study, no cells were visible with a fluorescent intensity similar to that of mucus. An alternative possibility is that the GST from dying enterocytes is deposited in the mucus. If this occurs, it would link the extracellular conjugation mechanism to the normal cell turnover. Rat small intestinal cells have a life span of approximately 48 h (Cornell and Meister, 1976; Henning, 1984), forming in crypts at the base of the villi and slowly migrating up to the villus tip, where they are sloughed and die. Although the process has some features of apoptosis, cell elimination is morphologically distinct (e.g., see Han et al., 1993) and may have specialized processes to allow deposition of detoxication enzymes such as GST in the mucus.
Because the intestine is one of the first sites of exposure to electrophiles found in foods, an extracellular mechanism for detoxication would appear to be very effective to protect epithelial cells. Many electrophilic compounds can freely diffuse into cells and cause damage to the cell or find access to the blood system and damage tissues at distant sites. Surfaces of oral, nasal, vaginal, and bronchiolar epithelia have mucus linings that could also contain GST. The presence of GST in mucus could explain why GSH is found in saliva and other extracellular fluids, and that presence may also provide a mechanistic basis for the epidemiological finding (Flagg et al., 1994) of decreased risk of oral and pharyngeal cancer in association with consumption of foods with high GSH contents.
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ACKNOWLEDGMENTS |
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This research was supported by NIH grant ES09047.
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NOTES |
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1 Present address: Barry University, Miami Shores, FL.
2 Present address: Pfizer, Inc., Central Research Division, Groton, CT.
3 To whom correspondence should be sent at Emory University, Rollins Research Center, 1510 Clifton Rd., Room 4172, Atlanta, GA 30322. Fax: (404) 727–3231. E-mail: dpjones{at}emory.edu.
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