Regulation of hepatic P-gp expression and activity by genistein in rats.

P-glycoprotein (P-gp) is an ABC transporter exhibiting high pharmacotoxicological relevance by extruding a wide range of cytotoxic compounds out of the cells. Previously, we demonstrated that the phytoestrogen genistein (GNT) modulates P-gp expression in hepatocellular carcinoma in vitro. Although several beneficial effects (e.g., antioxidant, antimutagenic, anticancer) have been attributed to GNT, the molecular mechanisms have not been totally elucidated. In the present work, we evaluated the effect of GNT on P-gp expression in rat liver, kidney and ileum. We found that GNT (5 mg/kg daily s.c. 3 days) increased hepatic P-gp expression and also Mdr1a (one of the genes encoding P-gp) mRNA levels. Renal and intestinal P-gp remained unchanged after GNT treatment. Hepatic P-gp activity measured with rhodamine-123 and digoxin, both well-known P-gp substrates, was also increased. In vitro experiments using hepatocyte primary cell culture demonstrated that inhibition of ER-α with ICI182/780 did not prevent Mdr1a mRNA up-regulation by GNT (10 µM). In contrast, Mdr1a induction was suppressed after pregnane X receptor (PXR) inhibition by sulforaphane and knockdown of this nuclear receptor. These findings were confirmed in vivo by using the PXR antagonist ketoconazole. In conclusion, we demonstrated the induction of hepatic P-gp expression and activity by GNT in vivo, with PXR being a likely mediator. This suggests that GNT, at concentrations observed in plasma of individuals consuming the phytoestrogen in the diet or through supplements, could affect the clearance of relevant P-gp substrates of therapeutic use as well as toxicity of environmental and food toxicants.

Molecular basis of perinatal changes in UDP-glucuronosyltransferase activity in maternal rat liver.

The molecular basis of perinatal changes occurring in major UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform belonging to family 2, was analyzed in rat liver. Nonpregnant, pregnant (19-20 days of pregnancy), and two groups of postpartum animals corresponding to early and middle stages of lactation (2-4 and 10-12 days after delivery, respectively) were studied. UGT activity determined in UDP-N-acetylglucosamine-activated microsomes revealed that bilirubin, p-nitrophenol, and ethynylestradiol (17beta-OH and 3-OH) but not androsterone and estrone glucuronidation rates, were decreased in pregnant rats. Decreased enzyme activities returned to control values after delivery. p-Nitrophenol, androsterone, and estrone conjugation rate increased in postpartum rats. Western blot analysis performed with anti-peptide-specific (anti-1A1, 1A5, 1A6, and 2B1) antibodies revealed decreased levels of all family 1 isoforms and UGT2B1 during pregnancy. In postpartum animals, protein level recovered (1A5 and 2B1) or even increased (1A1 and 1A6) with respect to control rats. Northern blot analysis suggested that expression of UGT proteins is down-regulated at a post-translational level during pregnancy and that increased levels of 1A1 and 1A6 observed in postpartum rats were associated to increased mRNA. To establish whether prolactin is involved in up-regulation of UGT1A1 and 1A6 postpartum, ovariectomized rats were treated with 300 microg of ovine prolactin per day for 7 days. The data indicated that prolactin was able to increase expression of UGT1A6 (protein and mRNA) but not 1A1. Thus, prolactin is the likely mediator of the increased expression of UGT1A6 observed in maternal liver postpartum.

Quantitative and qualitative gender-related differences in jejunal glutathione S-transferase in the rat effect of testosterone administration.

Gender-related differences and the regulation by testosterone of glutathione S-transferase were studied in rat jejunum. We analyzed enzyme activity and the relative content of GST subunits. Four experimental groups of adult rats were studied: normal males, castrated males, castrated males injected with testosterone and normal females. Glutathione S-transferase activity was assayed using 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Differences in subunit composition among groups were evaluated by western blot analysis. The results demonstrated that 1-chloro-2,4-dinitrobenzene conjugation rate is higher in normal males than in normal females and castrated males. Testosterone administration to castrated males raised the activity up to the level observed in normal males. No significant difference in glutathione S-transferase activity towards 1,2-dichloro-4-nitrobenzene was observed among groups. Western blot analysis revealed that males and females differ in all subunits tested that is, rGSTA2, rGSTM1, rGSTM2 and rGSTP1, and that testosterone regulates the content of rGSTM1, rGSTM2 and rGSTP1. In conclusion, jejunal GST shows a gender-dependent regulation affecting both enzyme activity and subunit composition, and testosterone appears to be one of the factors involved.

Induction of phase II biotransformation reactions in rat jejunum during lactation. Possible involvement of prolactin.

The effect of lactation on UDP-glucuronosyltransferase (UGT) and Glutathione S-transferase (GST) activities was studied in jejunum from mother rats, 14 (LM14) and 21 (LM21) days after delivery. p-Nitrophenol glucuronidation rate was increased in LM14 and LM21 rats while conjugation of bilirubin and estrone was not affected and androsterone glucuronidation was decreased. Additional studies, including Western blotting and microsomal lipid analysis, revealed that the enhancement in p-nitrophenol UGT activity is most likely associated with an inductive process rather than with a modification in enzyme constraint. GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was also increased in LM14 and LM21 while activity towards 1,2-dichloro-4-nitrobenzene (DCNB) was not affected. Western blotting revealed a significant increase in the cytosolic content of mu (rGSTM2) and pi (rGSTP1) class subunits in LM14 and LM21 groups, while the alpha class subunit rGSTA2 remained unchanged. To evaluate the potential modulatory role of prolactin on the same enzyme systems, ovariectomized rats were treated with ovine prolactin (oPRL) at doses of 100, 200 and 300 microg/100 g body wt. per day for 4 days. Hormone administration affected UGT activities towards p-nitrophenol and androsterone and GST activity towards CDNB in a way and magnitude consistent with those produced in lactating rats, while conjugation of estrone, bilirubin and DCNB were unchanged. Western blotting data were also consistent with those of lactating rats. These results indicate that UGT and GST activities are increased in rat jejunum during lactation, due to induction of some specific isoforms, and that prolactin is the likely mediator of these effects.

Prolactin increases the hepatic content of mu-class subunits of glutathione S-transferase in the rat.

Hepatic glutathione S-transferase (GST) activity is increased in postpartum female rats, a phenomenon that depends on the lactation stimulus. Here we evaluated the effect of prolactin (PRL) administration on hepatic enzyme activity and on the expression of the major subunits of the alpha- (rGSTA1, rGSTA2, rGSTA3) and mu-classes (rGSTM1, rGSTM2). A similar study was conducted in lactating (LM) and in nonlactating (NLM) mother rats 14 days after delivery and in virgin female rats (V). Ovine PRL (oPRL) was administered to ovariectomized rats at daily doses of 75, 150, 200, and 300 microg/100 g b.wt. (PRL1, PRL2, PRL3, and PRL4, respectively) for 4 consecutive days. GST activity was measured using 1-chloro-2,4-dinitrobenzene as substrate. The relative content of the different subunits was determined by Western blot. oPRL produced a dose-dependent increase in GST activity (60% at the highest dose). Subunit analysis performed in PRL2 and PRL4 revealed a substantial enhancement in rGSTM2 and to a lesser extent in rGSTM1, in response to oPRL. The effect was also dose-dependent. alpha-Class subunits were increased only slightly after hormone treatment. A 60% increase in GST activity was observed for LM relative to NLM and V. As was observed for PRL treatment, the increase was associated with changes in the expression of mu-class subunits whereas alpha-class subunits were not affected by lactation. Taken together these data would indicate a role of PRL in regulating GST activity postpartum via an increase in the content of mu-class subunits, particularly rGSTM2.

Enhancement of intestinal UDP-glucuronosyltranferase activity in partially hepatectomized rats.

To evaluate whether a temporary hepatic insufficiency may affect intestinal glucuronidation, we determined UDP-glucuronosyltransferase activity towards bilirubin and p-nitrophenol in rat jejunum and liver after partial hepatectomy. Enzyme assays were performed in native, and in UDP-N-acetylglucosamine- or palmitoyl lysophosphatidylcholine-activated microsomes at different times post-hepatectomy. Content of enzyme was analyzed by Western blot. Microsomal cholesterol/phospholipid ratio, phospholipid and total fatty acid classes were also determined to evaluate the possible influence on enzyme activity. The results show that while hepatic microsomes exhibited no change in UDP-glucuronosyltransferase activity (for both substrates) with respect to shams at any time of study, intestinal activities increased significantly 48 h after surgery, returning to sham values 96-h post-hepatectomy. Western blotting confirmed the increase (about 50% for both substrates 48-h post-hepatectomy) in intestinal UDP-glucuronosyltransferase activity. No variations were observed in hepatic and intestinal microsomal lipid composition in agreement with the absence of modification in the percent of activation by palmitoyl lysophosphatidylcholine. In conclusion, jejunum but not liver, was able to produce a compensatory increase in conjugation capacity during a transitory loss of hepatic mass. The phenomenon is associated to a modification in the amount of UDP-glucuronosyltransferase, rather than to changes in the characteristics of the enzyme environment.

Differential induction of glutathione S-transferase subunits by spironolactone in rat liver, jejunum and colon.

The effect of spironolactone pretreatment on glutathione S-transferase activity and on the relative content of the principal subunits (Ya, Yc, Yb1, Yb2 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 micromol/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 glutathione 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 rats. While Ya and Yp relative contents were similar in jejunum, 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. In 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.

Ovine prolactin increases hepatic UDP-glucuronosyltransferase activity in ovariectomized rats.

The potential role of prolactin in modulating hepatic UDP- glucuronosyltransferase (UGT) activity was studied. Ovariectomized adult female rats were treated with ovine prolactin (oPRL) at doses of 150, 200, 260, and 310 micrograms/100 g b.wt. per day, for 4 days. Enzyme assays were performed in native and activated microsomes with p-nitrophenol as substrate. Activation was achieved either by including UDP-N-acetylglucosamine in the incubation mixtures or by preincubating native microsomes with palmitoyl-lysophosphatidylcholine. Data obtained with UDP-N-acetylglucosamine as activator showed that increasing doses of oPRL produced a progressive increase in enzyme activity up to a maximum of about 35% over basal values. Immunoblotting of microsomal protein with anti-UGT antiserum revealed also a dose-dependent increase in the immunoreactive protein. A kinetic method for measuring glucuronidating enzyme content confirmed the result of the immunoblot. oPRL induced minor changes in the physicochemical properties of the microsomal membrane. Consistent with this observation, studies performed with palmitoyl-lysophosphatidylcholine as activator showed no change in UGT latency, suggesting that the functional characteristics of the enzyme were not substantially affected by oPRL. The current data support the conclusion that prolactin may act as a modulator of UGT activity by increasing the amount of enzyme.

Enhancement of intestinal bilirubin UDP-glucuronosyltransferase activity by modification of microsomal lipid composition.

Microsomal membranes from rat small intestine exhibit a higher cholesterol/phospholipid ratio and a lower phosphatidyl-choline/sphingomyelin ratio than those of the liver, which could negatively influence membrane-bound enzymes like bilirubin UDP-glucuronosyltransferase. To study the effect of in vitro modifications in the lipid composition of intestinal microsomes on bilirubin glucuronidating activity, two strategies were employed. On one hand, microsomal lipids were modified in order to mimic those of the liver tissue; on the other hand, cholesterol content of microsomal membranes was increased or decreased with respect to the normal value. Lipid changes were carried out by both an enzyme-mediated and a detergent-mediated procedure. Irrespective of the methodology employed, when a depletion in the cholesterol content was produced, enzyme activity increased about 40%, and when lipid composition approached that of the liver tissue, which not only decreased cholesterol but also modified phospholipid classes, enzyme activity increased about 80%. Both lipid modifications produced a 'fluidification' of microsomal membranes measured by fluorescence anisotropy of 1,6-diphenylhexatriene, being the effect of the approach to the liver higher than that of the decrease of cholesterol. In turn, the enrichment in cholesterol of microsomal membranes led to a decrease of enzyme activity of about 20% and to a 'rigidization' of the membranes. The present findings suggest that in rat intestine, bilirubin glucuronidation is strongly influenced by microsomal lipids. In particular, there seems to be an inverse association between enzyme activity and the cholesterol content of membrane.

Gender-related differences in the amount and functional state of rat liver UDP-glucuronosyltransferase.

The basis for gender-dependent differences in rates of glucuronidation of xenobiotics is uncertain. To clarify this issue, the glucuronidation of p-nitrophenol was compared in liver microsomes from adult male and female rats. The activity of native UDP-glucuronosyltransferase was 47% higher in microsomes from male than from female rats. Immunoblotting of microsomal protein with anti-UDP-glucuronosyltransferase antiserum revealed 66% more immunoreactive protein in male microsomes. A kinetic method for measuring glucuronidating enzyme content confirmed the result of the immunoblot. Responses of UDP-glucuronosyltransferase to activation by palmitoyllysophosphatidylcholine or high pressure indicated that the activity of the enzyme was more latent in male than in female microsomes. Differences in enzyme latency could be due to differences in membrane structure. A comparison of microsomal fatty acid composition revealed significantly higher levels of oleic and linoleic acids and lower levels of stearic and docosahexaenoic acids in male than in female microsomes. The phospholipid composition, ratio of cholesterol:phospholipid, and membrane fluidity were similar in male and female microsomes. These results indicate that gender-dependent differences in UDP-glucuronosyltransferase activity are due to differences in both the amount and functional state of the enzyme.

Enhancement of cytosolic and microsomal glutathione S-transferase activities in liver and small intestine from female rats during lactation.

The influence of lactation on hepatic and intestinal glutathione S-transferase activities in mother rats was studied. Cytosolic and microsomal activities were assessed 7, 14 and 21 days after delivery, using 1-chloro-2,4-dinitrobenzene as substrate. Cytosolic and microsomal activities from liver and small intestine determined 7 days post partum did not differ from those of virgin female rats. The hepatic cytosolic activity was significantly increased with respect to that of virgin females 14 days after delivery and tended to revert to the control value on day 21 of lactation, whereas the intestinal activity was increased on day 14 and remained augmented even 21 days post partum. Although the respective microsomal activities showed percent increases higher than those of the cytosolic enzymes, they both exhibited a similar pattern of stimulation in response to lactation.

Effect of oral administration of ursodeoxycholic acid on rat hepatic and intestinal UDP-glucuronosyltransferase.

The effect of oral administration of the bile acid ursodeoxycholic acid on rat hepatic and intestinal microsomal UDP-glucuronosyltransferase was studied. The bile acid was administered during 8 days at a daily dose of 500 mg/kg body weight. Enzyme activity was assessed in native and activated microsomes, using bilirubin and p-nitrophenol as substrates. Activation was achieved either by including UDP-N-acetylglucosamine in the incubation mixture or by preincubating native microsomes with an optimal concentration of Lubrol Px. Irrespective of activation status of the microsomes, ursodeoxycholic acid treatment increased enzyme activities toward both substrates in intestine, but not in liver. The analysis of the degree of activation by Lubrol Px revealed that, at least for bilirubin, ursodeoxycholic acid decreased the latency of the intestinal enzyme. The analysis of the lipid composition of microsomes showed several changes in response to ursodeoxycholic acid in intestine but not in liver. Thus, a decrease in cholesterol/phospholipid ratio and an increase in the unsaturation index of total-lipid fatty acids, which correlated well with a membrane "fluidification," were observed. These modifications appear to be related to the lower latency of bilirubin UDP-glucuronosyltransferase in intestine from treated rats and could be responsible, at least in part, for the improvement of enzyme activity in this group. Whatever the mechanism involved, the increment of intestinal UDP-glucuronosyltransferase activities toward both substrates may be relevant as a complement to the hepatic enzymes in those liver diseases in which ursodeoxycholic acid is used as a therapeutic agent.

Analysis of p-nitrophenol glucuronidation in hepatic microsomes from lactating rats.

In the present study, hepatic p-nitrophenol glucuronidation was analyzed comparatively in virgin female, lactating mother and nonlactating mother rats (the last two groups 19-21 days post-partum). Enzyme assays were performed in native and activated microsomal suspensions. Activation was achieved either by including UDP-N-acetylglucosamine in the incubation mixtures or by preincubating native microsomes with optimal concentrations of Triton X-100 or palmitoyl-lysophosphatidylcholine. When UDP-N-acetylglucosamine was used as activator, enzyme activity increased in both lactating (about 80% increment) and nonlactating mothers (about 30% increment) as compared with virgin females. From an analysis of the degree of activation by Triton X-100 and palmitoyl-lysophosphatidylcholine, it can be inferred that the pregnancy-delivery event decreased the latency of UDP-glucuronosyltransferase activity that was detectable even 3 weeks post-partum, irrespective of whether suckling newborns were or were not kept with their mothers (lactating and nonlactating mothers, respectively). The estimation of apparent Vmax toward UDP-glucuronic acid in palmitoyl-lysophosphatidylcholine-activated microsomes, which allows an estimation of the amount of the enzyme, showed that lactation increased the number of catalytic units (about 40%). Hepatic UDP-glucuronic acid content was 70% higher in lactating rats than in other groups. The lipid composition and membrane fluidity (using 1,6-diphenyl-1,3,5-hexatriene as probe) were also analyzed in microsomes from all groups. A significant decrease in the unsaturation index that correlated with the rigidization of microsomal membranes was consistent with the changes in the degree of enzyme latency observed in lactating and nonlactating mothers. In conclusion, lactating rats exhibited enhanced p-nitrophenol UDP-glucuronosyltransferase activity as well as an increase in the hepatic content of UDP-glucuronic acid. These findings and the fact that lactation increased the liver to body weight ratio emphasize the role of the liver in the metabolism of planar phenolic derivatives in these circumstances.

Inhibition of rat liver microsomal bilirubin UDP-glucuronosyltransferase by ursodeoxycholic acid.

Ursodeoxycholic acid and its endogenous metabolite tauroursodeoxycholic acid inhibited in vitro the microsomal bilirubin UDP-glucuronosyltransferase from rat liver. The magnitude of the inhibition correlated well with the loss of integrity of microsomal vesicles, suggesting that bile salts needed to reach the lumen to exert their inhibitory effects. The endogenous bile acids cholic acid, chenodeoxycholic acid and deoxycholic acid also exhibited inhibitory effects on bilirubin glucuronidation in digitonin-disrupted microsomes. Ursodeoxycholic acid inhibitory capacity was similar to that of chenodeoxycholic acid and deoxycholic acid but greater than that of cholic acid, the major endogenous bile salt. Kinetic studies, performed in detergent-activated preparations, showed that the inhibitions produced by ursodeoxycholic and tauroursodeoxycholic acids were competitive toward both bilirubin and UDP-glucuronic acid. The estimated Ki(app) for both substrates did not differ statistically between ursodeoxycholic and tauroursodeoxycholic acids. Both bile salts were weak inhibitors toward bilirubin but rather strong inhibitors toward UDP-glucuronic acid.

Absence of hepatic p-nitrophenol UDP-glucuronosyltransferase induction by spironolactone in male rats: possible involvement of testosterone.

This study was performed to determine whether the lack of spironolactone induction of hepatic p-nitrophenol UDP-glucuronosyltransferase in male rats could be attributed to a presumed interaction between spironolactone and testosterone. The effect of spironolactone was evaluated in four experimental groups: normal females, normal males, castrated males, and castrated males that received testosterone. Enzyme activity was measured in native microsomes and in microsomes activated with UDP-N-acetylglucosamine or Triton X-100. When the nucleotide was included in the incubations, it was observed that enzyme activity in castrated male rats decreased to values approaching those obtained in normal females. Treatment of castrated animals with testosterone enhanced enzyme activity so that no significant difference existed between this group and normal males. This suggests that testosterone may act as an endogenous inducer of hepatic p-nitrophenol glucuronidation. It was also found that only females and castrated males showed an increase in enzyme activity in response to spironolactone treatment. Thus, the absence of an additive effect of endogenous or exogenous testosterone and spironolactone on UDP-glucuronosyltransferase activity suggests that these compounds could share a common induction mechanism, which appears to reach its maximal capacity in male rats. Possible explanations of this observation are discussed. From the analysis of enzyme activity in native and Triton X-100 activated microsomes, it can be postulated that spironolactone enzyme induction in female and castrated male rats could be attributed to an enhancement in the transferase synthesis rather than to an alteration of the membrane environment.

Differential effects of blood insulin levels on microsomal enzyme activities from hepatic and extrahepatic tissues of male rats.

Microsomal glutathione S-transferase, UDP-glucuronyl transferase, and aniline hydroxylase activities were determined in liver, renal cortex, and small intestine of control, streptozotocin-diabetic, alloxan-diabetic, and untreated insulin-injected male Wistar rats. Renal microsomal glutathione S-transferase activity showed a direct linear relationship with insulin blood levels, in agreement with our previous report on cytosolic glutathione S-transferase. This result suggests a possible regulatory mechanism of insulin that needs to be further examined. The hepatic microsomal UDP-glucuronyl transferase was only decreased in streptozotocin-diabetic rats and was not restored by insulin treatment. Intestinal UDP-glucuronyl transferase exhibited an opposite response in streptozotocin-treated animals that was not normalized by the administration of insulin. Hepatic aniline hydroxylase showed the same behaviour as intestinal UDP-glucuronyl transferase. These results suggest that streptozotocin and (or) its metabolites have a direct effect on hepatic and intestinal UDP-glucuronyl transferase activity and on hepatic aniline hydroxylase activity. On the other hand, insulin regulation of enzyme activity varies from one organ to another.

Intestinal phase II detoxification systems: effect of low-protein diet in weanling rats.

The effect of a low-protein diet (6% protein, LPD) in vivo on the in vitro activities of cytosolic and microsomal glutathione S-transferase (GST-c, GST-m) and microsomal UDP-glucuronyltransferase (UDP-GT) was studied in small intestine and liver of weanling male Wistar rats. LPD interrupted the normal curve of growth of the animals, which returned to control values after refeeding with a normal diet (27% protein, ND). Hepatic and intestinal GST-c increased in control rats in parallel with the growth curve. The microsomal enzymes did not show growth variations, except for intestinal UDP-GT activity which began to increase from the second day of ND. After 7 days of LPD there was an increase in GST-c (liver 35%, P less than 0.05; intestine 152%, P less than 0.01) and of UDP-GT (liver 58%, intestine 178%, P less than 0.05) which returned to control values after 2 days of refeeding with ND. GST-m did not show any variations in liver or intestine. The increase in GST-c, but not in GST-m, with nutritional stress suggests preferential induction of cytosolic enzymes in those enzymatic systems which are located in both positions. The increase in such enzymes after protein malnutrition could indicate an adaptive response by detoxification mechanisms, enhancing intestinal over hepatic capacity, perhaps because the intestine is the primary route of access for orally ingested xenobiotics.

Sex differences in spironolactone induction of rat intestinal and hepatic p-nitrophenol UDP-glucuronyltransferase.

In the present study we analyzed the effect of spironolactone administration on hepatic and intestinal p-nitrophenol-UDP-glucuronyltransferase activity. We used microsomal preparations from male and female Wistar rats to establish whether or not this effect was sex dependent. Enzyme activity was measured in the presence of UDP-N-acetylglucosamine, a presumed physiological activator of the enzyme. Female but not male microsomes showed an increase in enzyme activity of both hepatic and intestinal tissue preparations in response to the inducer pretreatment. In addition, the inducer effect observed in female rats showed a tissue-related difference, since percent increase in the intestinal enzyme activity was greater than that in the liver (127 and 52%, respectively). These results suggest that factors regulating enzyme activity or mechanisms involved in the inducer effect of spironolactone could be different in the intestinal mucosa in comparison to the liver. A possible explanation of sex-related response to spironolactone administration was discussed.

Possible role of blood insulin levels on glutathione S-transferase activities from different tissues of male rats.

The activity of in vitro glutathione S-transferase towards 1-chloro-2,4-dinitrobenzene was examined in liver, renal cortex, and small intestine (duodenum, jejunum, ileum) after the in vivo treatment of male Wistar rats with streptozotocin or alloxan. The studies were performed at 2, 10, 24, and 48 h and 7 and 15 days after streptozotocin treatment or 24 and 48 h after alloxan treatment. The results indicated that while the blood levels of insulin-glucose did not show variations, there were no alterations of the glutathione S-transferase activity in the tissues tested. On the other hand, when the treatments caused modifications on blood insulin-glucose levels, there were changes of glutathione S-transferase activity in all tissues (except in the ileum) in such a way that a direct relationship between plasma insulin levels and glutathione S-transferase activity could be demonstrated. These results were also confirmed through insulin administration to control and diabetic rats. The data demonstrate a possible regulation of glutathione S-transferase activity by blood insulin and (or) glucose levels in the tissues tested.