The Glutathione S-Transferases of the Small Intestine in the Rau

[CANCER RESEARCH 37, 788-791 , March 1977) The Glutathione S-Transferases of the Small Intestine in the RaU

G. Clifton2 and N. Kaplowitz3

Clinical Investigation Center, Naval Regional Medical Center, Oakland, California 94627 (G. CI, and the Gastroenterology Division, Medical and Research Services, Veterans Administration, Wadsorth Hospital Center, Los Angleles, California 90073 (N. K.J

SUMMARY been identified in the small intestine and may have an important role in the metabolism of absorbed drugs and Glutathione S-transfenase activities have been identified carcinogens (12). Anyl hydrocarbon hydnoxylase activity has in the small intestine of the rat. Thmree activities obtained been induced in the small intestine of animals (7, 9), and with p-nitrobenzyl chloride (aralkyl), 1,2-epoxy-3-(p-nitro levels of cytochnome P-4S0 and hydnoxylase activity appear phenoxy)propane (epoxide), and ethacrynic acid (alkene) to be under dietary control (14, 27, 29). Thus, it seemed of as substrates were present in significant amounts. Gel filtra interest and importance to consider the possibility of the tion indicated an elution volume for the intestinal transfer parallel occurrence and distribution of the glutathione 5- ase activities that was similar to those activities in the liver transfenases in the small intestine as well as the effect of and kidney. The induction of the intestinal transferases by fasting and the possibility of drug induction in this organ. polycyclic aromatic hydrocarbons and phenobarbital is sim ilar to those effects observed previously for the hepatic and renal enzymes. The highest concentration of transferase MATERIALS AND METHODS activities occurs in the proximal small intestine; these activ ities are reduced upon fasting. Parallel observations have Animals, Treatment, and Preparation of Intestinal Cyto been reported for aryl hydrocarbon hydroxylases. Because sol. Three groups, each containing 8 male 270- to 330-g only low or negligible levels of epoxide hydrases have been Sprague-Dawley rats, were given phenobanbital (Sigma reported in the small intestine, the glutathione S-transferases Chemical Co., St. Louis, Mo.), 8 mg/100 g i.p. in 0.9% NaCI may be the primary epoxide-detoxifying system in that on solution daily for 10 days; 3,4-benzo(a)pyrene (Sigma), 1 mg gan. i.p. in corn oil twice daily for 10 days; or 3-methylcholan threne (Calbiochem, San Diego, Calif.), 1 mg i.p. in corn oil twice daily for 10 days. Two 6-animal groups were deprived INTRODUCTION of food but not water for 24 and 48 hr, respectively. A con trol group of 8 rats was given i.p. 0.9% NaCI solution. Ani The glutathione S-transfemases are detoxifying enzymes mals were killed with ether anesthesia 24 hr after the last that exist in the cytosol of liver cells. These enzymes cata treatment. Small intestines were removed; washed with lyze the conjugation of glutathione with a variety of exoge 0.01 M sodium phosphate buffer, pH 7.4; and tnisected into nous substrates (2â€”4,8,15) including the products of mi proximal, middle, and distal segments. For each weighed crosomal mixed-function oxygenases (13, 22, 23). These 2 segment, 20% (w/v) homogenates (0.01 M sodium phos enzyme systems may be considered complementary; thus, phate-0.25 M sucrose buffer, pH 7.4) were prepared and in the biotransformation of foreign compounds, 1 means of centrifuged at 105,000 x g for 60 mm in a Beckman Model detoxification of microsomally activated metabolites is in L2-65B ultracentrifuge (Beckman Instruments, Inc., Fuller teraction with glutathione catalyzed by the transfemases. ton, Calif.). Lipid layer was removed by suction. Cytosol Aside from this biochemical association of toxication-de fractions were decanted and stored at â€”15Â°. toxication, both enzyme systems are responsive to the 2 Gel Filtration. Gel filtration at 4Â°wasdone with Sephadex major kinds of enzyme inducers, phenobambital and poly G-100 (Pharmacia, Uppsala, Sweden) columns, 35 x 2.5 cyclic aromatic hydrocarbons (6, 9, 18). Furthermore, in cm, with 0.01 M sodium phosphate buffer, pH 7.4, as the addition to the liven, the occurrence and inducibility of both mobile phase. Flow rate was 30 mI/hr; 10 fractions/hr were enzyme systems have been reported for the kidney (5, 10). collected. For gel filtration experiments, concentrated in The microsomal mixed-function oxygenases also have testinal cytosol, 20% original volume, was prepared by ul trafiltration at 4Â°withDiaflo Model PM10 ultrafilters (Amicon

I Supported through funds provided by the Bureau of Medicine and Sur Corp., Lexington, Mass.). In certain experiments sulfo gery, Navy Department, for 5-48-494C, and by the Veterans Administration bromophthalein (3.0 mg) was added to the concentrated Medical Research. The opinions and assertions contained herein are those of cytosol immediately before placement on the column. Sul the authors and are not to be construed as official or as reflecting the views of the Navy Department or the naval service at large. fobromophthaleinbindingtoproteinincolumn fractionwas 2 To whom requests for reprints should be addressed. determined by alkalization and measurement of absorbance 3 Present address: Gastroenterology Division , Wadsworth Veterans Ad ministration Hospital Center, Los Angeles, Calif. 90073. at 580 nm. Received July 13, 1976; accepted December 2, 1976. Determination of Enzyme Activity. Five glutathione 5-

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1977 American Association for Cancer Research. Intestinal G!utathione S-Transferases tmansfemaseactivities were determined by previously de intestinal cytosol (3.0 ml) was filtered through a Sephadex scribed methods (11, 18, 26). The following substrates were G-100 column (Chart 1). Glutathione S-transfenase activities used in 2.0-mI reaction volumes: 1.0 mM 3,4-dichlomonitro with ethacrynic acid, epoxide, and p-nitmobenzyl chloride benzene (anyl substrate) at 37Â°(Aldrich Chemical Co. , Mil had supenimposable elution peaks when the individual col waukee, Wis.); 0.5 mM p-nitrobenzyl chloride (amalkylsub umn fractions were assayed. This elution volume (90 ml) strate) at 37Â°(Aldrich); 0.5 mM 1,2-epoxy-3-(p-nitrophen corresponds to that found for the hepatic and renal trans oxy)propane (epoxide substrate) at 37Â°(Eastman Kodak femasesdetermined with the same column conditions (17, Co. , Rochester, N. Y.); 0.2 mr@iethacrynic acid (alkene sub 19). In contrast to hepatic cytosol, there was no detectable strate) at 37Â°(agift from Merck Sharp & Dohme Research binding of sulfobromophthalein to the column fractions Laboratories, Rahway, N. J.); 1.5 mr@i[14C]methyl iodide containing the intestinal transferases. This observation is (alkyl substrate) at 20Â°(New England Nuclear, Boston, consistent with our previous demonstration of the relation Mass.). Excess glutathione (10 mM; Sigma) was used for ship between sulfobromophthalein binding to column frac each reaction except for the ethacmynic acid activity for tions of liven and kidney cytosol and the presence of 3,4- which 0.25 mM glutathione was used. Reactions were initi dichlomonitrobenzene activity (aryltransferase) (16, 17). ated by the addition of 200 p1of intestinal cytosol except for Effect of Fasting on Enzymatic Activities. The transfer the alkene activity for which 50 @tlwereused. Volumes were ase activities for the 3 readily detectable substrates were selected to minimize the error involved in determination of diminished in the intestines from animals that had been raw activities and yet meet the requirement that the enzy fasted for 48 hr (Chart 2). Transferase activities determined matic reactions be linear with respect to time and protein with epoxide and p-nitrobenzyl chloride substrates were concentration. Nonenzymatic reaction rates of substrates reduced significantly in the proximal and distal intestine were subtracted from the enzymatic rates. after 48-hr starvation. Activity with epoxide substrate Protein concentrations were determined by the method of Lowry et a!. (21). Comparison of activities between groups 03 of rats was done by unpaired Student's t test (1). â€”A- Alkene l.0 â€”Câ€” Epoe'de â€”+ â€”Aralkyl RESULTS 0.2 Distribution of Enzyme Activities in the Small Intestine 4 and Comparison with Values of Hepatic and Renal Trans S A280 ferase Activities. Three glutathione S-tnansferase activities 0.5 were present in significant amounts in the small intestine of ). I the rat: epoxide,p-nitrobenzyl chloride, and ethacrynic acid (Table 1). Activity with 3,4-dichlononitmobenzene was pres ent in trace amounts. Methyl iodide activity was not detecta I ble. For the 3 measurable transfenases, the level of enzy 0 0 matic activity in the proximal intestine was significantly greater than the levels in either the middle or distal intes 40 70 100 130 tine. The levels of specific activity in the proximal intestine Elution volume in ml were significantly lower than values previously determined Chart 1. Glutathione S-transferase activities in intestinal cytosol are given for the following substrates: ethacrynic acid (alkene); 1,2-epoxy-3-(p-nitro for the hepatic and renal tnansfenases. phenoxy)propane (epoxide); and p-nitrobenzyl chloride (aralkyl). Protein Gel Filtration of Intestinal Transferases. Concentrated concentration is shown as absorbance at 280 nm.

Table 1 ratSubstrate Comparison of enzyme activities in 3segments of intestine, the liver, and kidney of the protein)Smallactivity (nmoles/min/mg cytosolic intestine forglutathioneS-transferase LiverKidneyProximalEthacrynicactivityEnzyme Middle Distal 1.61 acid29.1 Â±1,9â€•'21 .1 Â±1 .6 13.1 Â±1 .7 43.5 Â±1 .947.0 Â± p

a Mean Â± S.D. 1' Spontaneous conversion of substrate: 58% of proximal value for ethacrynic acid; 1 1% for 1 ,2-epoxy-3-(p-nitrophenoxy)propane; and 19% for p-nitrobenzyl chloride.

C For statistical comparison with proximal intestine. d ND, not detectable.

MARCH 1977 789

dropped to 44% (p < 0.005) of control in the proximal Benzo(a)pymene administration caused a 49% increase of segment and to 38% (p < 0.001) of control in the distal ethacrynic acid activity in the distal intestine and 53 and segment. Activity with p-nitrobenzyl chloride was 59% (p < 23% increases of p-nitrobenzyl chloride activity in the mid 0.0025) and 55% (p < 0.025) of the respective control values. dIe and distal segments, respectively. Phenobarbital treat Activity determined with ethacrynic acid as substrate was ment resultedin26 and 33% increasesofp-nitrobenzyl affected only in the proximal segment where it was found to chloride activity in the middle and distal intestine, respec be 74% (p < 0.025) of control. None of the detectable tively; this was the only change observed with phenobarbi activities in the middle segment was significantly lower tal. There was no increase in the activities in the proximal under these experimental conditions. intestine; in fact, significant decreases were observed for Induction of Enzymatic Activities by Polycyclic Aromatic several activities. No change in the epoxide activity for any Hydrocarbons and Phenobarbital. The level of glutathione of the 3 segments of intestine was observed after drug S-tnansferase activities with ethacrynic acid and p-nitroben treatments. zyl chloride increased significantly in the middle and distal intestinal segments after treatment with benzo(a)pyrene and 3-methylcholanthrene (Table 2). 3-Methylcholanthrene DISCUSSION produced 33 and 37% increases of ethacrynic acid activity and 23 and 21% increases of p-nitrobenzyl chloride activity Several hepatic and renal glutathione S-transfenases are in the middle and distal intestine, respectively. induced by polycyclic aromatic hydrocarbons and pheno

@ Control 48-Hour Fast

Chart2.Effectof starvationonintestinal 4 glutathione S-transferase activities. Enzyme specific activities in proximal, middle, and distal small intestine from a group of 6 rats 3 fasted for 48 hr are compared to activities for a control group of 8 rats. N.S., no significant 2 difference between fasted and control groups. Bars, S.D.

0 px0025 MS NS px00025 r4S p.c0025 Proxinat Middle Distal M@dis Pro,umai M@dis Distal Ethacrvnic Acid p-Nitrobenzyt c@hioride phenoxy)propane Table 2 DrugS-transferasesSegmentEnzymatic induction of the intestinal glutathione activity (nmoles/min/mg cytosolicprotein)3-Methylcholan

reneEthacrynicSubstrateof intestineControl Phenobarbital Benzo(a)pyreneth acidProximal Â±1.9â€• 24.9 Â±1.3 25.9 Â±1.9 Â±1.3 NSb NS NS Middle 21.1 Â±1.6 23.0 Â±1.9 26.9 Â±0.3 27.8 Â±1.9 NS p

Â±0.33 2.06 Â±0.33 2.32 Â±0.40 Â±0.40 p < 0.025 p < 0.025 p < 0.01 Middle 2.52 Â±0.33 2.06 Â±0.33 2.26 Â±0.53 1.92 Â±0.33 NS NS NS Distal3.65 2.46 Â±0.13 3.38 Â±0.60 2.32 Â±0.46 1.99 Â±0.31 NSp-Nitnobenzyl NS NS2.52 chlorideProximal Â±0.45 4.16 Â±0.27 4.92 Â±0.36 Â±0.40 p<0.Oi NS NS Middle 2.95 Â±0.18 3.71 Â±0.40 4.52 Â±0.49 3.62 Â±0.18 p<0.05 p<0.005 p<0.Oi Distal5.63 2.95 Â±0.27 3.93 Â±0.27 3.62 Â±0.31 3.59 Â±0.22 p < 0.0125 P < 0.055.05 p < 0.05

a Mean Â± S.D. b NS, not significant.

C For statistical comparison of each group versus control.

790 CANCER RESEARCH VOL. 37

barbital (5, 18). In this study we have reported the induction 6. Conney, A. H. Pharmacological Implications of Microsomal Enzyme Induction. Pharmacol. Rev., 19: 317-366, 1967. of glutathione S-transferase activities for ethacrynic acid 7. Dao, T. L., and Varela, A. M. On the Mechanism of Inducing Protection and p-nitrobenzyl chloride by benzo(a)pyrene and 3-methyl of the Adrenal Cortex against Injury from 7,12-Dimethyl cholanthrene in the more distal portions of the intestine. benz(a)anthracene. I. Effects of Inducers on Benzpyrene Hydroxylase Activity. Cancer Aes., 26: 1015-1021, 1966. Interestingly, the induction of microsomal benzo(a)pymene 8. Fjellstedt, T. A., Allen, A. H., Duncan, B. K., and Jakoby, W. B. Enzy hydnoxylase in the small intestine by 1,2-benzanthnacene matic Conjugation of Epoxides with Glutathione. J. Biol. Chem., 248: alsoisgreatestinthe more distalintestine(30).Fromour 3702-3707, 1973. 9. Gelboin, H. V., and Blackburn, N. A. The Stimulatory Effect of 3-Methyl observations about the distribution and inducibility of the cholanthrene on Benzpyrene Hydroxylase Activity in Several Rat Tissues: tmansfemaseactivities in small intestine, kidney, and liven as Inhibition by Actinomycin D and Puromycin. Cancer Res., 24: 356â€”360, 1964. well as the demonstration of overlapping substrate spec ific 10. Grundin, A., Jakobsson, S., and Cinti, D. L. Induction of Microsomal Aryl ities of the pure enzyme proteins from liver (11), it is appar Hydrocarbon (3,4-Benzo(alpha)pyrene) Hydroxylase and Cytochrome P ent that the regulation and effects of inducing agents on 450K in Rat Kidney Cortex. Arch. Biochem. Biophys., 158: 544-555, 1973. this system are complex. In addition, p-nitrobenzyl chloride 11. Habig, W. H., Pabst, M. J., and Jakoby, W. B. Glutathione S-Transfer activity is induced by phenobarbital in the same portions of ases. The First Step in Mercapturic Acid Formation. J. Biol. Chem., 249: 7130-7139, 1974. the small intestine. The latter activity also is induced selec 12. Hartiala, K. Metabolism of Hormones, Drugs, and Other Substances by tively by phenobambital in the nat kidney (5). Recently, gluta the Gut. Physiol. Rev., 53: 496-534, 1973. thione S-transfenase activity in intestinal cells with 1-chloro 13. Hayakawa, T., Undenfriend, S., Yagi, H., and Jerina, D. M. Substrates and Inhibitors of Hepatic Glutathione-S-Epoxide Transferase. Arch. Bio 2,4-dinitrobenzene as substrate has been shown to increase chem. Biophys., 170: 438-451, 1975. after phenobarbital administration (20). 14. Hoensch,H.,Woo, C. H.,and Schmid,R. CytochromeP-450and Drug The distribution of glutathione S-tnansfenases along the Metabolism in Intestinal Villous and Crypt Cells of Rats: Effect of Dietary Iron. Biochem. Biophys. Res. Commun., 65: 399-406, 1975. length of the small intestine is similar to that previously 15. Johnson, M. K. 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Kaplowitz, N., Kuhlenkamp, J., and Clifton, G. Drug Induction of Hepatic Glutathione 5-Transferases in Male and Female Rats. Biochem. J. , 146: ases, the effect appears to be more prominent in the proxi 351â€”356,1975. mal versus middle on distal segment. 19. Kaplowitz, N., Kuhlenkamp, J., and Clifton, G. Hepatic Glutathione 5- Transferases: Identification by Gel Filtration and in vitro Inhibition by The glutathione S-tnansfemases parallel the mixed-func Organic Anions. Proc. Soc. Exptl. Biol. Med., 149: 234-237, 1975. tion oxygenases and aryl hydrocarbon hydroxylases in var 20. Ketley,J. N., and Pincus, L. M. Glutathione 5-Transferasesas a Detoxifi ious tissues including the small intestine. Ameneoxides, the cation System of the Small Intestine. Federation Proc., 35: 1421, 1976. 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, A. J. Protein presumed carcinogenic intermediates produced by action Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265- of amylhydrocarbon hydroxylases, are enzymatically detoxi 275, 1951. fied either by conjugation with water (epoxide hydrase) or 22. Nemoto, N., and Gelboin, H. Assay and Properties of Glutathione-S Benzo(alpha)pyrene-4,5-oxide Transferase. Arch. Biochem. Biophys., glutathione (glutathione S-transferases). In the small intes 170: 739-742, 1975. tine low or undetectable levels of epoxide hydrase activity 23. Nemoto, N., Gelboin, H. V., Habig, W. H., Ketley,J. N., and Jakoby,W. B. have been reported (24, 25, 28). Therefore, in the small K Region Benzo(alpha)pyrene-4,5-oxide Is Conjugated by Homogeneous Glutathione 5-Transferases. Nature New Biol., 255: 512, 1975. intestine enzymatic detoxification of epoxide intemmediates 24. Oesch, F., Jerina, 0. M., and Daly, J. A Radiometric Assay for Hepatic under the influence of the glutathione S-transfenases may Epoxide Hydrase Activity with [7-3H]Styrene Oxide. Biochim. Biophys. Acta, 227: 685-691 , 1971. predominate. 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G. Clifton and N. Kaplowitz

Cancer Res 1977;37:788-791.

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