* 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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Key Words: glutathione; 1-chloro-2,4-dinitrobenzene; conjugation; electrophiles; diet.
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INTRODUCTION |
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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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Animals.
Male Sprague-Dawley rats (VAF/1; Sasco, Omaha, NE), weighing approximately 250400 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 7.257.27 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 mM1cm1.
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 510 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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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 (0500 µM) (Fig. 3
).
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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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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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NOTES |
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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) 7273231. E-mail: dpjones{at}emory.edu.
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