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Differential induction of glutathione S-transferase subunits by spironolactone in rat liver, jejunum and colon
Life Sciences,Vol. 63, No. 26, pp. 22X.5-2293,1998 Copyright Q1998 F!lsevierScienceInc. Printed in the USA. All rights resewed 0024-3205/98 s19.00 + 80 PI1 SOO24-3205(98)00517-7
ELSEVIER
DIFFERENTLdL INDUCTION OF GLUTATHIONE S-TRANSFERASE SUBUNITS BY SPIRONOLACTONE IN BAT LIVER, JEJUNUM AND COLON Viviana A. Catania ‘, Marcel0 G. Luquita, Emique J. Sanchez Pozzi and Aldo D. Mottino Jnstituto de Fisiologia Experimental, CONJCET. Facultad de Ciencias Bioqtimicas y Farmaceuticas, U.N.R. Suipacha 570. (2000) Rosario. ARGENTINA (Received
in tinal form October
8, 1998)
Summary The effect of spironolactone pretreatment on ghrtathione S-transferase activity and on the relative content of the principal subunits (Ya, Yc, Ybr, m and Yp or 1, 2, 3, 4 and 7 respectively) was studied in rat liver, jejunum and colon. Male Wistar rats were injected with spironolactone i.p. at daily doses of 50, 100 and 200 p.mol/kg body wt for 3 consecutive days. Glutathione S-transferase activities were assayed using 1-chloro-2,4-dinitrobenzene as substrate. Changes in subunit composition were evaluated by Western blot analysis in rats treated with the highest dose of spironolactone. The results demonstrated a dose-dependent increase in enzyme activity in liver, while in jejunum the three tested doses exhibited the same magnitude of induction. No significant difference in ghrtathione S-transferase activity was observed between control and treated rats for the colon. Immunoblot analysis revealed more Ya and Yp protein in liver (140 and 118 % increase respectively) and jejunum (45 and 145 % increase respectively) from treated rata. While Ya and Yp relative contents were similar in jejumun, the latter subunit slightly contributed to total GST in liver, even in SL-treated animals. The inducer produced no change in subunit composition in colon. Jn conclusion, spironolactone was able to increase glutathione S-transferase activity mainly by induction of Ya subunit in liver and Yp subunit in jejunal mucosa, without affecting colonic enzyme. Key Words:
glutathione
S-transferase,
liver, jejunum,
colon, spironolactone
The ghrtathione S-transferases (GSTs) are a group of multifunctional enzymes that catalyze the nucleophilic attack of glutathione (GSH) on electrophilic centers in a wide variety of endogenous or exogenous organic molecules (1). In addition to their detoxifying properties, GSTs play an important role in the binding and intracellular traffic of several endogenous compounds. GSTs are primarily cytosolic and exhibit broad and overlapping substrate specificities (2-4). The isoenzymes, constituted by homo- or heterodhners, have been grouped into four speciesindependent classes: Q, u, rc and 8, based on their substrate and inhibitor binding properties, their immunological cross-reactivities and the similarity of their primary structures, as determined by ’ Correspondence
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recombinant DNA analysis (5, 6). An invariable feature of GSTs is that no heterodimers exist between subunits of the different classes. The o-class GSTs formed by the Ya and Yc subunits (or subunits 1 and 2, respectively) have a similar substrate specificities and are able to form heterodimers with each other (7, 8). The u-class formed by Ybr and Ybr subunits (or subunits 3 and 4, respectively) differs from the YaiYc isozymes in their substrate specificities and form heterodimers with each other but not the Ya/yc subunits (8). Yp subunit (or subunit 7) belongs to n-class GSTs. GST isoenzymes show unique patterns of tissue-specific distribution (2, 3) and are differentially induced by xenobiotics, carcinogens and other drugs (9). On the other hand, it is known that the administration of agents which increase the activity of the hepatic drugmetabolizing enzymes may modify pharmacological effects (10) or metabolic-mediated hepatotoxicity of drugs given therapeutically (11, 12). Thus, it appears important to establish the existence of inducing properties of commonly used therapeutic agents. Spironolactone (SL), a widely used diuretic agent (13), increases the activity of several Phase I and Phase II metabolizing systems (13- 17), and accelerates biotransformation or elimination of numerous compounds (13, 18) in experimental animals. The inducer effect was also evidenced in humans when the steroid was administered as a diuretic in patients with alcoholic cirrhosis (19). Regarding the inducer properties of SL on GST system, the data available up to now are indirect evidences about increases in the uptake and metabolism of bromosulfophthalein in rats treated with the steroid, phenomena that depend in part on the intrahepatic content of GSTs (20, 21). Based on these previous findings, we focused our attention on the evaluation of the effect of SL pretreatment on total GST activity and on the relative level of the principal subunits in rat liver. Due to the significant role of the intestine as the first place for metabolism of xenobiotics that enter the body via the gastrointestinal tract, the studies were also performed in jejunum and colon.
Material and Methods Chemicals SL and GSH were purchased from Sigma Chemical dinitrobenzene (CDNB) was from Tokyo Kasei Kogyo Co. against rat Ya, Ybr, m, Yc and Yp subunits of GST were (Dublin, Ireland). All other reagents were of the highest grade
Co (St. Louis, MO). l-chloro-2,4Ltd. (Japan). Polyclonal antibodies obtained from Biotrin International commercially available.
Animals and treatment Adult male Wistar rats (340-400 g) were used. They were maintained ad libitum on a standard laboratory pellet diet until the moment of sacrifice and were allowed free access to water and saline solution during treatment. Groups of rats were injected with SL i.p. at daily doses of 50 (SLsO), 100 (SLrss) and 200 (SL& pmovkg body wt, dissolved in propylene glycol, for 3 consecutive days prior to the experiments as was previously described (16, 17) with the last dose administered 18 h before sacrifice. Control rats (C) were injected with propylene glycol (vehicle of SL) according to the same schedule described above. All animals received humane care as outlined in the “Guide for the Care and Use of Laboratory Animals” (NH publication no. 86-23, revised 1985).
Preparation of cytosolic fractions All animals were (50 mgikg body weight) The livers were perfused were promptly removed.
killed by bleeding after cardiac puncture under per&barbital anesthesia between 10 and 11 a.m., to avoid possible effects of diurnal variations. in situ with iced 0.9 % NaCl solution through the portal vein, and then The ratio liver to body wt was calculated. Mucosa samples were obtained
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from the proximal jejunum (40 cm) and the whole colon. The segments were isolated, washed with ice-cold saline solution, opened leugthwise and then everted. The mucus was carefully removed by blotting with moist tissue paper. Mucosa was fIeed from the underlying muscular layer by scrapping with a glass slide on an ice-cold surface (22). Homogenates from the three tissues (25 % w/v) were prepared in 0.15 M Tris-HCI buffer, pH 7.40. Cytosolic fractions were obtained by ultracentrifugation at 105000 g for 1 h at 4 “C as previously described (23). Protein content was determined by the biuret reaction (24), with bovine serum albumin as standard. Enzyme assay GST activities toward CDNB were assayed by a reported procedure (25) except that GSH concentration was raised to 5 mM and CDNl3 concentration was 1.25 mM added in dimethyl sulfoxide. Assays were routinely performed at 37 ‘C, and in 0.13 M sodium phosphate buffer pH 6.50 to decrease the background due to non-enzymatic conjugation. Under these experimental conditions, enzyme activities were linear function of time and protein concentration. Western blot analysis SDS-PAGE (10 %) was performed under reducing conditions according to the method of Laemmli (26). Proteins were transferred onto nitrocellulose sheets by the method of Towbin et al. (27). The nitrocellulose sheets were then incubated with anti Ya, Ybl, Ybr, Yc or Yp antisera for 2 h. Subsequently, the nitrocellulose membranes were probed with secondary antibody conjugated to alkaline phosphatase during 1 h. The different subunits of GST were then detected by the alkaline phosphatase color reaction using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Immunoreactive protein bands were quantified by densitometry (Shimadzu, CS-9000). Statistical Analysis Results are presented as means f SD. Statistical analysis was performed using the Newman-Keuls multiple-range test (28), which included ANOVA. Values of p < 0.05 were considered to be statistically significant.
Results Liver-body weight ratio The effect of SL administration on the liver-body weight ratio are presented in Table 1. SL produced a significant increase in the ratio at all doses tested. Recovery of cytosolic protein was similar in all groups. Table I Effect of SL treatment on Ihfer-body wt ratio. CONTROL Liver WV body wt x 100
3.97 f 0.16
Cytoaolic protein recovery (mgla liver)
4a.q * 4.3
SLW
SL,
Sk0
4.79 f 0.19 -
4.82 f 0.47
49.6 f 5.2
47.7 f 4.2
l
4.79 f 0.21 ** 49.4 f 3.9
SL was administered at daily doses of 50, 100 and 200 @Iovkg body wt (Sk, SLlm and SL, respectiveiy)for 3 consacutivedays. Data are expressed as mean f SD of 4 to 5 rats par group. * Siinificantly dtfferentfrom controlgroup (pO.05). *t Significantlydiierant from controlgroup (p4.01).
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GST activity in liver, jejunum and colon Fig. 1 illustrates the effect of SL pretreatment on GST activity towards CDNB in the different tissues. As can be observed there is a dose-dependent increase of GST activity in liver, but not in jejunum, where the three doses of SL exhibited similar inducing effect. No significant differences were observed in colonic GST activity between SL-treated and control rats.
C
SL50
%oo
SL200
Fig. 1 GST activities in liver, jejunum and colon from control and SL-pretreated rata. SL was administered at daily doses of 50, 100 and 200 pmol/kg body wt (Sk,, SLloo and SL, respectively) for 3 consecutive days. Data are expressed as mean i SD of 4 to 5 rats per group. * Significantly different from control (~~0.05). * Significantly different from control, SLWand SLIW (peO.01).
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Estimation of relative content of GST subunits To evaluate the effect of SL treatment on enzyme subunit composition, pooled cytosols of liver, jejunum and colon from control and SLzm rats were subjected to western blotting. As shown in Fig. 2, immunoblot analysis revealed more Ya immunoreactive protein (140 %) in liver and jejunum (45 %) from SL-treated rats when compared with controls. Western blot analysis also showed that Ybr, ybz and Yc were not significantly affected by SL in liver, while jejunum exhibits weak immunoreactive bands in control animals that were not affected by the steroid. Contents of Ya, Ybr, yb2 and Yc subunits were hardly detected in colon (even loading a sixteen-fold higher quantity of cytosolic protein than for liver) and were not modified by SL (image not shown).
Ya
Ybl
Yb2 Yc
1
2
3
4
Fig. 2 Effect of SL at the dose of 200 pmoUkg body wt on the relative content of GST subunits in pooled cytosols of liver and jejunum detected by Western blot. Lanes 1 and 2 represent GST subunits of liver from control and SL-treated rats respectively and lanes 3 and 4 represent GST subunits of jejunum from control and SLtreated rats respectively. Each lane was loaded with 1.5 pg of total cytosolic protein for liver samples or 12 Fg for jejunum samples. lmmunoreactive protein bands were quantified by densitometry. Values of the corresponding areas (arbitrary units) are: Liver: Ya: control, 1855; SL, 4455; Yb,: control, 4471; SL, 5465; Y&: control, 3428; SL, 3435; Yc: control, 837; SL. 849; and Jejunum: Ya: control, 2798; SL, 4060. The areas of jejunal Yb,, Y& and Yc from control and SL-treated rats were not determined. In contrast to the pattern of distribution of the above mentioned subunits, constitutive was better detected in colon and jejunum than in liver (Fig. 3). As can be observed,
Yp SL
Induction of GST by Spironolactone
22!xl
administration colon.
increased Yp level in liver (118 %) and jejunum
Vol. 63, No. 26, 1998
(145 %), exhibiting
no effect in
123456 Fig. 3 Effect of SL at the dose of 200 pmollkg body wt on the relative content of Yp subunit in the three tissues detected by Western blot. Equal amounts of total cytosolicprotein were loaded in all lanes (12 pg). Lanes 1 and 2 represent hepatic cytosol from controland SL-treated rats respectively;lanes 3 and 4 represent jejunal cytosolfrom control and SL-treated rats respectivelyand lanes 5 and 6 represent colonic cytosol from controland SL-treated rats respectively.lmmunoreactiveprotein bands were quantified by densitometry.Values of the correspondingareas (arbitrary units) are: control liver, 147; SL liver, 320; controljejunum, 676; SL jejunum, 1657; controlcolon, 1815; SL colon, 1990.
Discussion GST system can be induced by a variety of endogenous compounds such as cholesterol (29) or hormones (30) and by xenobiotics (9). The induction is also tissue-specific, since the same xenobiotic treatment evokes a different response from one tissue to another (3 1, 32). Additionally, even in response to a single xenobiotic, different subunits show different degrees of inducibility in a given tissue (1,33,34). The inducibility of GST isoenqmes has been exhaustively studied for the classical inducing agents, phenobarbital and 3-methylcholanthrene, in hepatic (33-35) and extrahepatic tissues (32, 36, 37). SL presents a rather different chemical structure when compared with them but shares similarities in the inducing effect on several Phase I and some Phase II biotransformation systems (13). In fact, the steroid has been effective in inducing UDPglucuronosyltransferase activity toward endogenous and exogenous substrates and the effect was observed not only in liver but also in jejunum (17, 38). We here observed that total activity of GST, another important Phase II detoxifying system, is also increased in response to SL. The effect was observed in liver and jejunum but not in colon. While liver exhibited a dose-dependent effect, jejunum seemed to be maximally induced at the dose of 50 pmol/kg body wt but the magnitude of increase was lower than in liver. Because not all GST subunits are induced by a given agent (9), only weak increases in specific activity are often observed when enzyme activity is measured toward the prototypic substrate CDNB (25). Differential induction of specific enzyme subunits is more readily detected by measuring changes in their levels by Western blot. In the present work, Ya protein was more induced in liver (140 %) than in jejunum (45 %), while Yp was similarly induced in both tissues (118 and 145 % respectively). However, the contribution of the increase in Yp to the total hepatic
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GST activity would be negligible since it is known that this subunit is not present in adult rat hepatocytes (39). Normally only traces are detected in liver cytosolic preparations due to contamination with biliary epithelial cells (39). Consequently, the increase in Ya rather than in Yp content, conjointly with the enhancement in the liver to body w-t ratio seem to provide the liver of SL-treated animals with a higher potentiality for GST-mediated reactions. Intestine is a rich source of Yp since this subunit provides about 40 % of total cytosolic enzyme activity in normal conditions (40). The present results show a significant induction of Yp in response to SL administration, even greater than that of Ya. Thus, contrarily to what was observed in liver, the Yp induction in jejunum suggests a higher contribution to the total GST activity. It is well known that environmental factors affect the development of cancers. Even the diet contains a large number of (pre)carcinogens (41). The detoxication systems, such as GSTs, can minimize carcinogenecity by lowering the biological activity and increasing its excretability (1,2,42), thus preventing the damage of DNA in the epithelial cells of the intestinal tract (43). In consequence, the high Yp level in &treated rats may contribute significantly to the detoxication of carcinogenic compounds that enter the body via gastrointestinal tract. In colon, Yp subunit represents the only one clearly detected among the different subunits tested in the current study. Absence of induction of GST activity in this tissue was paralleled with the absence of increase in the subunit level. Similarly, colonic GST was hardly induced by a variety of agents (43). The reason by which SL increases Yp content in jejunum and liver but not in colon is not known, however it is possible that the time and doses of SL treatment used in this study were inadequate to shown some effects in the colonic mucosa. Alternatively, it could be a consequence of differences in the number of receptors and/or in the subsequent mechanisms involved in gene activation. In fact, it was postulated that the differential induction of GST in aorta vs. liver could be due to tissue-specific differences in transcription factors (44). In conclusion, SL was able to increase GST enzyme activity, mainly by induction of Ya subunit in liver andYp subunit in jejunal mucosa, without affecting colonic GST. These findings contribute not only to the knowledge of the regulatory features of GST in the different rat tissues, but also to the side effects of SL treatment when used as a diuretic agent as was demonstrated for Phase I reactions ( 13).
Acknowledgements This work was supported by a research grant from Consejo National Cientificas y TCcnicas (CONICET), Argentina.
de Investigaciones
References 1. T.D. BOYBR, Hepatology 9 486-496 (1989). 2. B. KBTTIXEX, D.J. MEYER and A.G. CLARK, Glutathione Conjugation: Mechanisms and Biological Signzjicance, H. Sies and B. Ketterer @Ids.), 73-135, Academic Press, London (1988). 3. I. LISTOWSKY, Hepatic Transport and Bile Secretion: Physiology and Pathophysiologv, N. Tavoloni and P.D. Berk @is.), 397-405, Raven Press Ltd, New York (1993). 4. B. MANNBRVIK and U.H. DANIELSON, CRC Crit. Rev. Biochem. 23 283-337 (1988). 5. B. MANNBRVIK, P. ALIN, C. GUTHBNBERG, H. JENSSON, M.K. TAHIR, M. WORHOLM and H. JGRNVALL, Proc. Natl. Acad. Sci. U.S.A. 82 7202-7206 (1985).
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6. D.J. MEYER, K.S. GILMORE, B. COLES, K. DALTON, P.B. I-IULPERT and B. KETTERER, Biochem. J. 274 409-414 (1991). 7. B. MANNERVIK and H. JENSSON, J. Biol. Chem. 257 9909-9912 (1982). 8. T.D. BOYER, W.C. KENNEY and D. ZAKIM, B&hem. Pharmacol. 32 1843-1850 (1983). 9. C.B. PICKETT and A.Y.H. LU, Amm Rev. Biochem. 58 743-764 (1989). 10. A.H. CONNEY, Pharmacol. Rev. 19 317-366 (1967). 11. P. MAZEL and D. PESSAYRE, Advances in Modern Toxicology, vol. I, part I: New Concepts in Safty Evaluation, M.A. Mehlman, R.E. Shapiro and H. Blumenthal (Eds.), 307-343, Hemisphere Publishing Company, New York (1976). 12. J.R. MITCHELL and D.J. JOLLOWS, Gastroenterology 68 392-410 (1975). 13. H.R. OCHS, D.J. GREENBLATT, G. BODEM and T.W. SMITH, Am. Heart J. 96 389-400 (1978). 14. D.R. FELLER and M.C. GERALD, Biochem. Pharmacol. 20 1991-2000 (1971). 15. B. STRIPP, M.E. HAMRICK, N.G. ZAMPAGLIONE and J.R. GILLETE, J. Pharmacol. Exp. Ther. 176 766-771 (1971). 16. A.D. MOTTINO, E.E. GUIBERT and E.A. RODRIGUEZ GARAY, Biochem. Pharmacol. 38 851-853 (1989). 17. V.A. CATANIA, M.G. LUQUITA, M.C. CARRILLO and A.D. MOTTINO, Can. J. Physiol. Pharmacol. 68 1385-1387 (1990). 18. B. SOLYMOSS and G. ZIGMOND, Can. J. Physiol. Pharmacol. 51319-323 (1973). 19. J.P. MIGUET, D. VUITTON, A. THEBAULT-LUCAS, C. JOANNE and D. DHUMEAUX, Gastroenterology 78 996- 1000 ( 1980). 20. G. ZSIGMOND and B. SOLYMOSS, J. Pharmacol. Exp. Ther. 183 499-507 (1972). 2 1. J.M. PELLEGRINO, A.D. MO‘ITINO, J.V. RODRIGUEZ and E.A. RODRIGUEZ GARAY, Experientia 38 112-l 13 (1982). 22. L.M. PINKUS, J.N. KETLEY and B. JAKOBY, Biochem. Pharmacol. 26 2359-2363 (1977). 23. P. SIEKEVITZ, Methods Enzymol. 5 6 l-68 (1962). 24. A.G. GORNALL, C.J. BARDAWILL and M.M. DAVID, J. Biol. Chem. 177 75 l-766 (1949). 25. W.H. HABIG, M.J. PABST and W.B. JAKOBY, J. Biol. Chem. 249-7 130-q 139 (1974). 26. U.K. LAEMMLI, Nature 227 680-685 (1970). 27. H. TOWBIN, T. STAEHELIN and J. GORDON, Proc. Natl. Acad. Sci. USA 76 4350-4354 (1979). 28. R.J. TALLARIDA and R.B. MURRAY, Manual ofpharmacologic calculations with computer programs. Springer-Verlag, New York (1987). 29. E. HIETANEN, M. AHOTUPA, A. HEIKELA and M. LAITINEN, Drug-Nun. Interact .1313327 (1982). 30. L. STAFFAS, L. MANKOWITZ, M. SGDESTRGM, A. BLANCK, I. PORSCHHALLSTROM, c. SUNDBERG, B. MANNERvIK, B. om, J. RYDSTROM and J.w. DePIERRE, Biochem .J. 286 65-72 (1992). 3 1. D.J. MEYER, J.M. HARRIS, K.D. GILMORE, B. COLES, T.W. KENSLER and B. KETTERER, Carcinogenesis 14 567-572 (1993). 32. M. DEBERL, T. IGARASHI and T. SATOH, Biochem. Biophys. Acta 1158 175-180 (1993). 33. T. IGARASIE, N. IROKAWA, S. ONO, S. OHMORI, K. UENO, H. KIT’AGAWA and T. SATOH, Xenobiotica 17 127- 137 ( 1987). 34. R.M.E. VOS, M.C. SNOEK, W.J.H. Van BERKEL, F. MULLER and P.J. Van BLADEREN, Biochem. Pharmacol. 37 1077-1082 (1988). 35. V.D.H. DING and C.B. PICKETT, Arch. Biochem. Biophys. 240 553-559 (1985). 36. G. CLIFTON and N. KAPLOWITZ, Biochem. Pharmacol. 27 687-688 (1978). 37. D. SHEEHAN, C.M. RYLE and T.J. MANTLE, Biochem. J. 219 687-688 (1984). 38. A.D. MOTTINO, E.E. GUIBERT and E.A. RODRIGUEZ GARAY, B&hem. Pharmacol. 41 1075-1077 (1991).
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39. M. PAROLA, M.E. BIOCCA, G. LEONARDUZZI, E. ALBANO, M.U. DIANZANI, K.S. GILMORE, D.J. MEYER, B. KETTERER, T.F. SLATER and K.H. CHEESEMAN, Biochem. J. 291641-647 (1993). 40. K. TAHIR, N. t)ZER and B. MANNERVIK, Biochem. J. 253 759-764 (1988). 41. B.N. AMES, Science 221 1256-1264 (1983). 42. V.L. SPARNINS, P.L. VENEGAS and L.W. WAITENBERG, J. Natl. Cancer Inst 68 493-496 (1982). 43. W.A.NIJI-IOFF,G.M. GROENand W.H.M. PETERS, Intemat. J. Oncol. 3 1131-1139 (1993). B.B. WEKSLER and A.J. DANNENBERG, Biochem. 44. K. KASHFI, J.A. RIMARACHIN, Pharmacol. 47 1903- 1907 ( 1994).
ELSEVIER
DIFFERENTLdL INDUCTION OF GLUTATHIONE S-TRANSFERASE SUBUNITS BY SPIRONOLACTONE IN BAT LIVER, JEJUNUM AND COLON Viviana A. Catania ‘, Marcel0 G. Luquita, Emique J. Sanchez Pozzi and Aldo D. Mottino Jnstituto de Fisiologia Experimental, CONJCET. Facultad de Ciencias Bioqtimicas y Farmaceuticas, U.N.R. Suipacha 570. (2000) Rosario. ARGENTINA (Received
in tinal form October
8, 1998)
Summary The effect of spironolactone pretreatment on ghrtathione S-transferase activity and on the relative content of the principal subunits (Ya, Yc, Ybr, m and Yp or 1, 2, 3, 4 and 7 respectively) was studied in rat liver, jejunum and colon. Male Wistar rats were injected with spironolactone i.p. at daily doses of 50, 100 and 200 p.mol/kg body wt for 3 consecutive days. Glutathione S-transferase activities were assayed using 1-chloro-2,4-dinitrobenzene as substrate. Changes in subunit composition were evaluated by Western blot analysis in rats treated with the highest dose of spironolactone. The results demonstrated a dose-dependent increase in enzyme activity in liver, while in jejunum the three tested doses exhibited the same magnitude of induction. No significant difference in ghrtathione S-transferase activity was observed between control and treated rats for the colon. Immunoblot analysis revealed more Ya and Yp protein in liver (140 and 118 % increase respectively) and jejunum (45 and 145 % increase respectively) from treated rata. While Ya and Yp relative contents were similar in jejumun, the latter subunit slightly contributed to total GST in liver, even in SL-treated animals. The inducer produced no change in subunit composition in colon. Jn conclusion, spironolactone was able to increase glutathione S-transferase activity mainly by induction of Ya subunit in liver and Yp subunit in jejunal mucosa, without affecting colonic enzyme. Key Words:
glutathione
S-transferase,
liver, jejunum,
colon, spironolactone
The ghrtathione S-transferases (GSTs) are a group of multifunctional enzymes that catalyze the nucleophilic attack of glutathione (GSH) on electrophilic centers in a wide variety of endogenous or exogenous organic molecules (1). In addition to their detoxifying properties, GSTs play an important role in the binding and intracellular traffic of several endogenous compounds. GSTs are primarily cytosolic and exhibit broad and overlapping substrate specificities (2-4). The isoenzymes, constituted by homo- or heterodhners, have been grouped into four speciesindependent classes: Q, u, rc and 8, based on their substrate and inhibitor binding properties, their immunological cross-reactivities and the similarity of their primary structures, as determined by ’ Correspondence
2286
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recombinant DNA analysis (5, 6). An invariable feature of GSTs is that no heterodimers exist between subunits of the different classes. The o-class GSTs formed by the Ya and Yc subunits (or subunits 1 and 2, respectively) have a similar substrate specificities and are able to form heterodimers with each other (7, 8). The u-class formed by Ybr and Ybr subunits (or subunits 3 and 4, respectively) differs from the YaiYc isozymes in their substrate specificities and form heterodimers with each other but not the Ya/yc subunits (8). Yp subunit (or subunit 7) belongs to n-class GSTs. GST isoenzymes show unique patterns of tissue-specific distribution (2, 3) and are differentially induced by xenobiotics, carcinogens and other drugs (9). On the other hand, it is known that the administration of agents which increase the activity of the hepatic drugmetabolizing enzymes may modify pharmacological effects (10) or metabolic-mediated hepatotoxicity of drugs given therapeutically (11, 12). Thus, it appears important to establish the existence of inducing properties of commonly used therapeutic agents. Spironolactone (SL), a widely used diuretic agent (13), increases the activity of several Phase I and Phase II metabolizing systems (13- 17), and accelerates biotransformation or elimination of numerous compounds (13, 18) in experimental animals. The inducer effect was also evidenced in humans when the steroid was administered as a diuretic in patients with alcoholic cirrhosis (19). Regarding the inducer properties of SL on GST system, the data available up to now are indirect evidences about increases in the uptake and metabolism of bromosulfophthalein in rats treated with the steroid, phenomena that depend in part on the intrahepatic content of GSTs (20, 21). Based on these previous findings, we focused our attention on the evaluation of the effect of SL pretreatment on total GST activity and on the relative level of the principal subunits in rat liver. Due to the significant role of the intestine as the first place for metabolism of xenobiotics that enter the body via the gastrointestinal tract, the studies were also performed in jejunum and colon.
Material and Methods Chemicals SL and GSH were purchased from Sigma Chemical dinitrobenzene (CDNB) was from Tokyo Kasei Kogyo Co. against rat Ya, Ybr, m, Yc and Yp subunits of GST were (Dublin, Ireland). All other reagents were of the highest grade
Co (St. Louis, MO). l-chloro-2,4Ltd. (Japan). Polyclonal antibodies obtained from Biotrin International commercially available.
Animals and treatment Adult male Wistar rats (340-400 g) were used. They were maintained ad libitum on a standard laboratory pellet diet until the moment of sacrifice and were allowed free access to water and saline solution during treatment. Groups of rats were injected with SL i.p. at daily doses of 50 (SLsO), 100 (SLrss) and 200 (SL& pmovkg body wt, dissolved in propylene glycol, for 3 consecutive days prior to the experiments as was previously described (16, 17) with the last dose administered 18 h before sacrifice. Control rats (C) were injected with propylene glycol (vehicle of SL) according to the same schedule described above. All animals received humane care as outlined in the “Guide for the Care and Use of Laboratory Animals” (NH publication no. 86-23, revised 1985).
Preparation of cytosolic fractions All animals were (50 mgikg body weight) The livers were perfused were promptly removed.
killed by bleeding after cardiac puncture under per&barbital anesthesia between 10 and 11 a.m., to avoid possible effects of diurnal variations. in situ with iced 0.9 % NaCl solution through the portal vein, and then The ratio liver to body wt was calculated. Mucosa samples were obtained
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from the proximal jejunum (40 cm) and the whole colon. The segments were isolated, washed with ice-cold saline solution, opened leugthwise and then everted. The mucus was carefully removed by blotting with moist tissue paper. Mucosa was fIeed from the underlying muscular layer by scrapping with a glass slide on an ice-cold surface (22). Homogenates from the three tissues (25 % w/v) were prepared in 0.15 M Tris-HCI buffer, pH 7.40. Cytosolic fractions were obtained by ultracentrifugation at 105000 g for 1 h at 4 “C as previously described (23). Protein content was determined by the biuret reaction (24), with bovine serum albumin as standard. Enzyme assay GST activities toward CDNB were assayed by a reported procedure (25) except that GSH concentration was raised to 5 mM and CDNl3 concentration was 1.25 mM added in dimethyl sulfoxide. Assays were routinely performed at 37 ‘C, and in 0.13 M sodium phosphate buffer pH 6.50 to decrease the background due to non-enzymatic conjugation. Under these experimental conditions, enzyme activities were linear function of time and protein concentration. Western blot analysis SDS-PAGE (10 %) was performed under reducing conditions according to the method of Laemmli (26). Proteins were transferred onto nitrocellulose sheets by the method of Towbin et al. (27). The nitrocellulose sheets were then incubated with anti Ya, Ybl, Ybr, Yc or Yp antisera for 2 h. Subsequently, the nitrocellulose membranes were probed with secondary antibody conjugated to alkaline phosphatase during 1 h. The different subunits of GST were then detected by the alkaline phosphatase color reaction using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Immunoreactive protein bands were quantified by densitometry (Shimadzu, CS-9000). Statistical Analysis Results are presented as means f SD. Statistical analysis was performed using the Newman-Keuls multiple-range test (28), which included ANOVA. Values of p < 0.05 were considered to be statistically significant.
Results Liver-body weight ratio The effect of SL administration on the liver-body weight ratio are presented in Table 1. SL produced a significant increase in the ratio at all doses tested. Recovery of cytosolic protein was similar in all groups. Table I Effect of SL treatment on Ihfer-body wt ratio. CONTROL Liver WV body wt x 100
3.97 f 0.16
Cytoaolic protein recovery (mgla liver)
4a.q * 4.3
SLW
SL,
Sk0
4.79 f 0.19 -
4.82 f 0.47
49.6 f 5.2
47.7 f 4.2
l
4.79 f 0.21 ** 49.4 f 3.9
SL was administered at daily doses of 50, 100 and 200 @Iovkg body wt (Sk, SLlm and SL, respectiveiy)for 3 consacutivedays. Data are expressed as mean f SD of 4 to 5 rats par group. * Siinificantly dtfferentfrom controlgroup (pO.05). *t Significantlydiierant from controlgroup (p4.01).
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GST activity in liver, jejunum and colon Fig. 1 illustrates the effect of SL pretreatment on GST activity towards CDNB in the different tissues. As can be observed there is a dose-dependent increase of GST activity in liver, but not in jejunum, where the three doses of SL exhibited similar inducing effect. No significant differences were observed in colonic GST activity between SL-treated and control rats.
C
SL50
%oo
SL200
Fig. 1 GST activities in liver, jejunum and colon from control and SL-pretreated rata. SL was administered at daily doses of 50, 100 and 200 pmol/kg body wt (Sk,, SLloo and SL, respectively) for 3 consecutive days. Data are expressed as mean i SD of 4 to 5 rats per group. * Significantly different from control (~~0.05). * Significantly different from control, SLWand SLIW (peO.01).
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Estimation of relative content of GST subunits To evaluate the effect of SL treatment on enzyme subunit composition, pooled cytosols of liver, jejunum and colon from control and SLzm rats were subjected to western blotting. As shown in Fig. 2, immunoblot analysis revealed more Ya immunoreactive protein (140 %) in liver and jejunum (45 %) from SL-treated rats when compared with controls. Western blot analysis also showed that Ybr, ybz and Yc were not significantly affected by SL in liver, while jejunum exhibits weak immunoreactive bands in control animals that were not affected by the steroid. Contents of Ya, Ybr, yb2 and Yc subunits were hardly detected in colon (even loading a sixteen-fold higher quantity of cytosolic protein than for liver) and were not modified by SL (image not shown).
Ya
Ybl
Yb2 Yc
1
2
3
4
Fig. 2 Effect of SL at the dose of 200 pmoUkg body wt on the relative content of GST subunits in pooled cytosols of liver and jejunum detected by Western blot. Lanes 1 and 2 represent GST subunits of liver from control and SL-treated rats respectively and lanes 3 and 4 represent GST subunits of jejunum from control and SLtreated rats respectively. Each lane was loaded with 1.5 pg of total cytosolic protein for liver samples or 12 Fg for jejunum samples. lmmunoreactive protein bands were quantified by densitometry. Values of the corresponding areas (arbitrary units) are: Liver: Ya: control, 1855; SL, 4455; Yb,: control, 4471; SL, 5465; Y&: control, 3428; SL, 3435; Yc: control, 837; SL. 849; and Jejunum: Ya: control, 2798; SL, 4060. The areas of jejunal Yb,, Y& and Yc from control and SL-treated rats were not determined. In contrast to the pattern of distribution of the above mentioned subunits, constitutive was better detected in colon and jejunum than in liver (Fig. 3). As can be observed,
Yp SL
Induction of GST by Spironolactone
22!xl
administration colon.
increased Yp level in liver (118 %) and jejunum
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(145 %), exhibiting
no effect in
123456 Fig. 3 Effect of SL at the dose of 200 pmollkg body wt on the relative content of Yp subunit in the three tissues detected by Western blot. Equal amounts of total cytosolicprotein were loaded in all lanes (12 pg). Lanes 1 and 2 represent hepatic cytosol from controland SL-treated rats respectively;lanes 3 and 4 represent jejunal cytosolfrom control and SL-treated rats respectivelyand lanes 5 and 6 represent colonic cytosol from controland SL-treated rats respectively.lmmunoreactiveprotein bands were quantified by densitometry.Values of the correspondingareas (arbitrary units) are: control liver, 147; SL liver, 320; controljejunum, 676; SL jejunum, 1657; controlcolon, 1815; SL colon, 1990.
Discussion GST system can be induced by a variety of endogenous compounds such as cholesterol (29) or hormones (30) and by xenobiotics (9). The induction is also tissue-specific, since the same xenobiotic treatment evokes a different response from one tissue to another (3 1, 32). Additionally, even in response to a single xenobiotic, different subunits show different degrees of inducibility in a given tissue (1,33,34). The inducibility of GST isoenqmes has been exhaustively studied for the classical inducing agents, phenobarbital and 3-methylcholanthrene, in hepatic (33-35) and extrahepatic tissues (32, 36, 37). SL presents a rather different chemical structure when compared with them but shares similarities in the inducing effect on several Phase I and some Phase II biotransformation systems (13). In fact, the steroid has been effective in inducing UDPglucuronosyltransferase activity toward endogenous and exogenous substrates and the effect was observed not only in liver but also in jejunum (17, 38). We here observed that total activity of GST, another important Phase II detoxifying system, is also increased in response to SL. The effect was observed in liver and jejunum but not in colon. While liver exhibited a dose-dependent effect, jejunum seemed to be maximally induced at the dose of 50 pmol/kg body wt but the magnitude of increase was lower than in liver. Because not all GST subunits are induced by a given agent (9), only weak increases in specific activity are often observed when enzyme activity is measured toward the prototypic substrate CDNB (25). Differential induction of specific enzyme subunits is more readily detected by measuring changes in their levels by Western blot. In the present work, Ya protein was more induced in liver (140 %) than in jejunum (45 %), while Yp was similarly induced in both tissues (118 and 145 % respectively). However, the contribution of the increase in Yp to the total hepatic
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GST activity would be negligible since it is known that this subunit is not present in adult rat hepatocytes (39). Normally only traces are detected in liver cytosolic preparations due to contamination with biliary epithelial cells (39). Consequently, the increase in Ya rather than in Yp content, conjointly with the enhancement in the liver to body w-t ratio seem to provide the liver of SL-treated animals with a higher potentiality for GST-mediated reactions. Intestine is a rich source of Yp since this subunit provides about 40 % of total cytosolic enzyme activity in normal conditions (40). The present results show a significant induction of Yp in response to SL administration, even greater than that of Ya. Thus, contrarily to what was observed in liver, the Yp induction in jejunum suggests a higher contribution to the total GST activity. It is well known that environmental factors affect the development of cancers. Even the diet contains a large number of (pre)carcinogens (41). The detoxication systems, such as GSTs, can minimize carcinogenecity by lowering the biological activity and increasing its excretability (1,2,42), thus preventing the damage of DNA in the epithelial cells of the intestinal tract (43). In consequence, the high Yp level in &treated rats may contribute significantly to the detoxication of carcinogenic compounds that enter the body via gastrointestinal tract. In colon, Yp subunit represents the only one clearly detected among the different subunits tested in the current study. Absence of induction of GST activity in this tissue was paralleled with the absence of increase in the subunit level. Similarly, colonic GST was hardly induced by a variety of agents (43). The reason by which SL increases Yp content in jejunum and liver but not in colon is not known, however it is possible that the time and doses of SL treatment used in this study were inadequate to shown some effects in the colonic mucosa. Alternatively, it could be a consequence of differences in the number of receptors and/or in the subsequent mechanisms involved in gene activation. In fact, it was postulated that the differential induction of GST in aorta vs. liver could be due to tissue-specific differences in transcription factors (44). In conclusion, SL was able to increase GST enzyme activity, mainly by induction of Ya subunit in liver andYp subunit in jejunal mucosa, without affecting colonic GST. These findings contribute not only to the knowledge of the regulatory features of GST in the different rat tissues, but also to the side effects of SL treatment when used as a diuretic agent as was demonstrated for Phase I reactions ( 13).
Acknowledgements This work was supported by a research grant from Consejo National Cientificas y TCcnicas (CONICET), Argentina.
de Investigaciones
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