Introduction

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During lactation, the xenobiotics taken by the mother may represent a risk to the infant, either by direct incorporation into the breast milk (reviewed in Welch and Findlay, 1981) or by affecting the various processes by which milk's natural components are synthesized and secreted (Walsh and Neville, 1994). Decreasing systemic concentration of active metabolites in the mother through an enhanced hepatic biotransformation and subsequent excretion into bile may attenuate that risk. In the rat, bile flow and biliary excretion of some xenobiotics are increased during lactation (Klaassen and Strom, 1978; Kilpatrick et al., 1980; Bolt et al., 1984; Muraca et al., 1984). In addition, it was demonstrated that the increase in bile secretory function during the postpartum period is modulated by prolactin (PRL)¹ (Liu et al., 1992; Ganguly et al., 1993).

The availability of xenobiotics to be secreted in bile is dependent on the activity of biotransformation systems. UDP-glucuronosyltransferase and glutathione S-transferase (GST), the most important phase II detoxifying systems, increase significantly their activities in female rats postpartum (Borlakoglu et al., 1993; Luquita et al., 1994; Luquita et al., 1995). UDP-glucuronosyltransferase-mediated conjugation of p-nitrophenol was stimulated by PRL, suggesting a role of the hormone in regulating enzyme activity after delivery (Luquita et al., 1996). However, the regulation of GST system under the same circumstances is not known.

GST isoenzymes are primarily cytosolic, exhibit broad, overlapping substrate specificities [reviewed in Listowsky (1993) and Hayes and Pulford (1995)], and are organized as both homo- and heterodimers constituted by at least 12 different subunits. The major subunits are classified in -, µ-, -, and -classes based on their gene family. In rat liver, the most important isoforms belong to - and µ-classes. -Class includes principally rGSTA1, rGSTA2, and rGSTA3 subunits, and µ-class includes rGSTM1 and rGSTM2 subunits (reviewed in Hayes and Pulford, 1995). Regulation of GSTs are associated strongly with modulation of the expression of the different subunits.

The present study was undertaken to determine whether PRL may modulate hepatic content of GST in the rat. For this purpose, we analyzed enzyme activity and the expression of the major subunits in response to hormone administration. We also analyzed the effect of lactation on the same parameters to establish a potential association with the effect of PRL administration.

	Materials and Methods

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Chemicals. Ovine PRL (oPRL) and reduced glutathione (GSH) were purchased from Sigma Chemical Co. (St. Louis, MO). 1-Chloro-2,4-dinitrobenzene (CDNB) was from Tokyo Kasei Kogyo Co. Ltd. (Japan). Polyclonal antibodies against rGSTA1, rGSTA2, rGSTA3, rGSTM1, and rGSTM2 subunits of GST were obtained from Biotrin International (Dublin, Ireland). The rGSTA1 and rGSTA2 subunits are conjointly detected by this commercial antiserum (rGSTA1+rGSTA2). All other reagents were of the highest grade commercially available.

Postpartum Animals. Adult female Wistar rats weighing 230 to 330 g were used. They were fed ad libitum on a standard laboratory pellet diet and were allowed free access to water. Three experimental groups were studied: virgin rats (V), and two groups of mother rats that were sacrificed 14 days after delivery because at this time the maximal increase in GST activity was observed (Borlakoglu et al., 1993; Luquita et al., 1995). In one group (to evaluate the possible effect of the pregnancy-delivery event), the animals were separated from their litters immediately after delivery (nonlactating mothers, NLM), while in the other group they were allowed to lactate (lactating mothers, LM). Litters were standardized to 10 pups. The animals were about 18 to 19 weeks old at the time of sacrifice.

PRL Treatment. Adult female Wistar rats weighing 230 to 280 g were ovariectomized at 14 to 15 weeks of age and randomly divided into five experimental groups: control rats (OVX, which were sacrificed 25 days after ovariectomy) and rats receiving total daily s.c. doses of 75, 150, 200, and 300 µg of oPRL/100 g b.wt. (PRL1, PRL2, PRL3, and PRL4 groups, respectively) injected every 8 h for 4 consecutive days, starting 21 days after ovariectomy. The last administration was made 4 to 5 h before sacrifice. Animals from the OVX group were injected with the vehicle of oPRL (0.9% NaCl/25 mM Tris-HCl, buffered to pH 7.60). Treatment of ovariectomized rats with oPRL demonstrated to be an appropriate postpartum model to study the regulation of bile secretory function (Liu et al., 1992; Liu et al., 1995) and hepatic UDP-glucuronosyltransferase activity (Luquita et al., 1996). In fact, although oPRL is less potent than rat PRL in the rat, its greater stability has resulted in its wide use experimentally. Infusion of 300 µg of oPRL/100 g b.wt./day i.v. for 7 days resulted in plasma levels of 750 ng/ml oPRL, whereas plasma PRL readily achieves levels of 200 to 700 ng/ml in maternal rats during suckling (Ganguly et al., 1993, 1997). Therefore, the present s.c. administration of 75 to 300 µg of oPRL/100 g b.wt. for 4 days is well within the physiologic range of PRL.

Preparation of Cytosolic Fractions and Enzyme Assay. Cytosolic fractions were obtained as described (Luquita et al., 1995). Protein content was determined by the method of biuret (Gornall et al., 1949), with bovine serum albumin as standard. GST activities toward CDNB were assayed as described (Habig et al., 1974) with minor modifications (Luquita et al., 1995).

Western Blot Analysis. Immunoblot studies and densitometry were performed as described (Catania et al., 1995). In preliminary experiments, liver cytosol from virgin female rats demonstrated a linear relationship between antibody response and protein amount in the range of 1 to 5 µg.

Statistical Analysis. Results are presented as means ± S.D. Statistical analysis was performed using the Newman-Keuls multiple-range test (Tallarida and Murray, 1986), which includes analysis of variance. Values of P < .05 were considered to be statistically significant.

	Results

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GST Activity. Figure 1 shows the GST activity of rat liver cytosols using CDNB as a substrate for each of the different treatment groups. Hepatic cytosolic fractions from LM exhibited higher activities (approximately a 60% increase) than V or NLM rats. Treatment of ovariectomized animals with increasing doses of oPRL produced a progressive increase in GST activity up to the dose of 200 µg/100 g b.wt. The subsequent dose (300 µg/100 b.wt.) did not further enhance enzyme activity. Thus, the maximal increase in GST activity obtained was about 60%.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   CDNB conjugation by liver cytosols from the different experimental groups. Data are expressed as the mean value ± S.D. of six to seven rats per group. One-way analysis of variance followed by a Newman-Keuls test were applied separately to both experimental sets (both graphs). PRL1, PRL2, PRL3, and PRL4 correspond to ovariectomized rats injected with oPRL at daily doses of 75, 150, 200, and 300 µg/100 g b.wt. for 4 consecutive days, respectively. a, significantly different from V and NLM (P < .01). b, significantly different from OVX and PRL1 (P < .05). c, significantly different from OVX and PRL1 (P < .01). d, significantly different from PRL2 (P < .05).

Relative Content of GST Subunits. The cytosols from V, LM, and NLM rat liver were analyzed by Western blot as shown in Fig. 2. The relative composition of the GST subunits was estimated by densitometry. Bar graph of the same figure shows a higher content for rGSTM1 (about a 40% increase) and rGSTM2 (about a 100% increase) proteins in cytosol from LM relative to NLM and V rats. The last two groups did not show significant differences in µ-class subunits. The Western blot image in Fig. 2 also shows that the relative contents of -class subunits (rGSTA1+rGSTA2 and rGSTA3) were similar in all groups (densitometry not shown). Because of the inability of the antiserum to discriminate between rGSTA1 and rGSTA2, it was not possible to rule out an inverse regulation of both subunits, thus leading to an invariable intensity of the immunoprecipitation band. However, it seems likely that the global content of -class subunits is not affected in response to the suckling stimulus.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Western blot analysis of GST subunits in the different experimental groups. The upper image is a typical one and shows the immunoreactive bands of major subunits from a pooled cytosol prepared with two to three animals per group. Densitometry revealed substantial differences in the content of rGSTM1 and rGSTM2, but not in -class subunits (data not shown), in response to lactation or after administration of oPRL to ovariectomized rats. The bar graph shows mean values ± S.D. of the relative areas (arbitrary units) of µ-class subunits of three different pools of cytosols, each prepared with two to three animals per group. PRL2 and PRL4 correspond to ovariectomized rats injected with oPRL at daily doses of 150 and 300 µg/100 g b.wt. for 4 consecutive days, respectively. a, significantly different from V and NLM (P < .05). b, Significantly different from OVX (P < .05). c, significantly different from PRL2 (P < .05).

	Discussion

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Increased GST activity has been demonstrated previously in liver (Borlakoglu et al., 1993; Luquita et al., 1995) and jejunum (Luquita et al., 1995) of postpartum rats. Hepatic CDNB conjugating activity was maximal 14 days after delivery, and the suckling stimulus was necessary to evidence the effect (Luquita et al., 1995). Thus, GST activities at 4 or 7 days postpartum did not differ from those of control rats. We report here that administration of oPRL to ovariectomized rats produced a dose-dependent induction of GST activity after 4 days of treatment. The maximal percent increase was similar to that described in postpartum rats 2 weeks after delivery (Fig. 1). The current findings thus reinforce the previous hypothesis regarding a positive modulatory effect of PRL on the metabolism of xenobiotics postpartum (Luquita et al., 1996), particularly those related to conjugation reactions. Western blot analysis of cytosols revealed that for both lactating and PRL-treated rats, increased GST activities were attributable mainly to an increase in the content of subunits belonging to µ-class, particularly rGSTM2.

In most studies characterizing the inducibility of GSTs, attention has focused on the increased catalytic activity. However, the binding properties of GSTs also may provide cytoprotection against active xenobiotics by buffering their intracellular concentrations, particularly under conditions in which the concentrations are very high (reviewed in Boyer, 1989 and Listowsky, 1993). Similarly, the intracellular fate of endogenous compounds such as bilirubin, steroids, thyroid hormones, and bile acids seems to be influenced by the GST content. For instance, bile acid binding by cytosolic proteins has been proposed to have an important role in both intracellular transport and protection from potentially toxic effects of bile acids (Takikawa et al., 1986). It has been demonstrated that increased bile secretory function in the rat during lactation is associated with an increase in hepatic bile acid transport, with PRL being responsible for the effect (Liu et al., 1992). Particularly, it was found that the hormone stimulates the Na⁺-dependent uptake of taurocholate (TC) in hepatocytes (Ganguly et al., 1993). It also has been demonstrated that cholic acid, the unconjugated precursor of TC, binds preferentially to - and µ-classes of GST with similar affinity (Takikawa et al., 1986). In consequence, it is possible that the higher content of subunits of µ-class present in cytosol from lactating and PRL-treated rats is important for modulating the intracellular trafficking of bile acids postpartum.

The mechanism by which oPRL increases GST activity and rGSTM1 and rGSTM2 content is not known. It has been shown that PRL increases the mRNA encoding the Na⁺-TC cotransport polypeptide (ntcp) (Liu et al., 1995) and that this occurs via transcriptional regulation of the ntcp promoter by PRL (Ganguly et al., 1997). Further studies are needed to determine whether a similar mechanism is involved in increasing expression of the µ-class subunits by PRL.

In summary, total GST activity is increased in lactating female rats postpartum, primarily reflecting an increased expression of the µ-class subunits. The phenomenon was not observed when the suckling stimulus was not present. Administration of exogenous PRL exhibited a positive modulatory effect on these same subunits and on total GST activity in such a way that the maximal increase was similar to that reported for lactating rats. Taken together, these data would indicate a role for PRL in regulating GSTs postpartum.