Involvement of Mrp2 in Hepatic and Intestinal Disposition of Dinitrophenyl-S-glutathione in Partially Hepatectomized Rats
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《毒物学科学杂志》
Institute of Experimental Physiology, National University of Rosario, S2002LRL-Rosario, Argentina
ABSTRACT
The ability of the liver and small intestine for secretion of dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), into bile and lumen, respectively, as well as expression of Mrp2 in both tissues, were assessed in 70–75% hepatectomized rats. An in vivo perfused intestinal model was used. A single iv dose of 30 μmol/kg b.w. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered and its glutathione conjugate, DNP-SG, was determined by HPLC in bile and intestinal perfusate. One and seven days after hepatectomy, biliary excretion of DNP-SG was decreased by 90 and 50% with respect to shams, respectively, when expressed per mass unit. In contrast, intestinal excretion was increased by 63% or unchanged one and seven days post-hepatectomy, respectively. Tissue content of DNP-SG 5 min after CDNB administration was substantially decreased in liver and significantly increased in intestine, one day post-hepatectomy. Western and immunofluorescence studies revealed preserved levels and localization of Mrp2 in both tissues from hepatectomized animals, irrespective of the time analyzed. In spite of preserved expression of Mrp2, the higher availability of DNP-SG in intestinal cells, likely as a consequence of increased glutathione-S-transferase-mediated conjugation of CDNB, may explain the in vivo findings. Further experiments in isolated hepatocytes suggested that decreased synthesis of DNP-SG rather than altered canalicular transport is responsible for the substantial impairment in excretion of this compound into bile. Taken together, these results indicate that the intestine may partially compensate for liver DNP-SG disposition, particularly shortly after surgery, while liver capability is recovering.
Key Words: Mrp2; 1-chloro-2,4-dinitrobenzene; dinitrophenyl-S-glutathione; liver regeneration; glutathione; glutathione-S-transferase.
INTRODUCTION
In various forms of liver injury such as viral hepatitis, toxic or drug-induced liver injury, and surgical resection, the functional liver mass is restored by tissue regeneration. Partial hepatectomy in rats represents a useful model to study regulation of liver function during the regeneration process (Rahman and Hodgson, 2000). Irrespective of whether liver cell function is preserved under this experimental model, the substantial loss of liver mass suggests a temporary impairment in the overall metabolic and secretory function. Extrahepatic tissues may play a role as an alternative site for biotransformation and apical transport of xenobiotics. On this regard, it was reported that glutathione S-transferase (GST) activity, expressed per mass unit, was increased in small intestine and decreased in liver two days after partial hepatectomy (Carnovale et al., 1995). Decreased enzyme activity in liver was associated with downregulation of GST expression at transcriptional level (Lee and Boyer, 1993). We previously observed that another phase II enzyme, UDP-glucuronosyltransferase (UGT), followed a similar behavior. Though UGT specific activity was preserved in the remaining liver, the substantial decrease in liver mass led to a significant decrease in overall glucuronidation capability (Catania et al., 1998). As described for GST, we found that UGT specific activity, as well as expression detected by Western blotting, was increased in small intestine in these same rats, suggesting a partial compensatory role while liver mass is recovering.
Alteration in the activity of drug transporters after partial hepatectomy may also lead to changes in toxicity of their substrates. The basolateral organic anion transporter 1 (Oatp1) was found to be unchanged and Oatp2 only slightly decreased 24 h after partial hepatectomy in crude liver plasma membrane (Vos et al., 1999). These same authors reported that the expression of canalicular multidrug resistance-associated protein 2 (Mrp2) was preserved in hepatectomized animals, whereas P-glycoprotein was substantially upregulated, likely as a consequence of increased expression of the Mdr1b component. Gerloff et al. (1999) reported data on expression of rat liver transporters, detected in microsomal membranes, at different periods post-hepatectomy. Overall, the authors found differential regulation of basolateral and canalicular organic anion transporters in the regenerating liver. Whereas microsomal content of Mrp2 was preserved, expression of Oatp1 and Oatp2 significantly decreased. This latter finding would suggest decreased ability for the uptake of organic anions at the basolateral level. Recently, Chang et al. (2004) reported that Mrp2 expression in liver was decreased in 90% but not in 70% hepatectomized rats, clearly indicating a dependence with liver mass removal. The evidence on downregulation of basolateral transporters together with the loss of liver mass would indicate an impairment in the overall capability for transport of organic anions from blood to bile.
Mrp2 plays an important role in elimination of potentially toxic endo- and xenobiotics, including bilirubin, hormones, drugs, and carcinogens, primarily as their glucuronide, glutathione or sulfate conjugates (Buchler et al., 1996; Paulusma et al., 1996). Mrp2 also mediates the active transport of oxidized (GSSG) and reduced (GSH) glutathione into bile (Knig et al., 1999; Rebbeor et al., 2002). It was demonstrated that Mrp2 is also present on the apical surface of the rat enterocyte (Mottino et al., 2000). The data indicated that Mrp2 protein is preferentially localized in the proximal intestine and gradually decreases from the jejunum to the distal ileum and that its expression is highest at the tip region of the villus. Mrp2 thus follows a similar pattern of distribution along the intestine and the villus axis as the conjugating enzymes in the rat. Jejunum is also the main site for transport of glutathione conjugates from the serosal to the mucosal side of the intestinal epithelium (Gotoh et al., 2000). Clearly, conjugating enzymes and Mrp2 may act coordinately to metabolize and secrete xenobiotics into the intestinal lumen (Catania et al., 2004). Whether this coordinated action may lead to a compensatory increase in intestinal conjugation of xenobiotics and subsequent Mrp2-mediated secretion of conjugated derivatives while liver capability is restoring is not known. Because phase II enzyme activities are increased in small intestine from two-third partially hepatectomized rats (Carnovale et al., 1995; Catania et al., 1998) it was of interest to explore if Mrp2 expression and activity are also increased. We thus evaluated the hepatic and intestinal Mrp2 levels and their respective secretory activity for dinitrophenyl-S-glutathione (DNP-SG), an Mrp2 substrate generated endogenously after systemic administration of 1-chloro-2,4-dinitrobenzene (CDNB), in hepatectomized animals. The analysis of disposition of DNP-SG in this experimental model allowed us to simultaneously evaluate conjugation and apical excretion capabilities for both tissues.
MATERIALS AND METHODS
Chemicals. Leupeptin, phenylmethylsulfonyl fluoride, pepstatin A, 1-chloro-2,4-dinitrobenzene (CDNB), NADPH, collagenase, glutathione, and glutathione reductase were obtained from Sigma Chemical Co (St. Louis, MO). 2-Methylbutane was obtained from Acros Organics (Pittsburgh, PA) and 2-vinylpyridine was obtained from Fluka Chemical Corp (Milwaukee, WI). All other chemicals and reagents were commercial products of analytical grade purity and used as supplied.
Animals and surgical procedures. Adult Male Wistar rats (320–370 g) were used throughout. Animals had free access to food and water and
Sham (n = 4), PH1 (n = 4), and PH7 (n = 4) were used in in vivo experiments for assessment of blood levels (5 min after CDNB administration), and biliary and intestinal excretion of DNP-SG (every 10 and 15 min respectively, for 60 min), and for determination of liver and jejunum mass.
Sham (n = 3) and PH1 (n = 4) were used for assessment of liver and intestinal content of DNP-SG, 5 min after CDNB administration.
Sham (n = 3) and PH1 (n = 6) were used for assessment of basal bile flow, basal GSH and GSSG biliary excretion and tissue content, and immunofluorescence and GST activity in liver and intestine. These animals were sacrificed after basal bile collection.
Sham (n = 4), PH1 (n = 4), PH2 (n = 4), and PH7 (n = 4) were used in Western blotting analysis of Mrp2.
Sham (n = 3) and PH1 (n = 3) were used for assessment of Mrp2 transport activity in isolated hepatocytes.
Biliary and intestinal excretion and tissue content of DNP-SG. The rats were anesthetized with urethane (1000 mg/kg b.w. ip) and thus maintained throughout. Body temperature was measured with a rectal probe, and maintained at 37°C with a heating lamp. The femoral vein and the common bile duct were cannulated with polyethylene tubing (PE50 and PE10, respectively). Intestinal excretion studies were performed using the in situ single-pass perfusion technique (Gotoh et al., 2000). Briefly, the intestine was perfused with isotonic phosphate buffered saline, pH = 7.35, from the upper jejunum to the end of distal jejunum (about 50 cm in length) with a peristaltic pump at a rate of 0.4 ml/min. After a 30-min stabilization period, a single bolus of CDNB (30 μmol/kg b.w. in 1:19 dimethylsulfoxide:saline, iv) was administered. Bile and intestinal perfusate were collected at 10- and 15-min intervals, respectively, for 60 min. A blood sample was collected 5 min after CDNB injection from the tail vein and immediately centrifuged to separate serum. Saline was administered intravenously throughout the experiment to replenish body fluids. Bile, intestinal perfusate, and serum sample were treated with 70% (v/v) HClO4 (50 μl per ml of sample) and centrifuged at 3500 x g for 5 min. DNP-SG content was determined in the supernatants by HPLC as described (Mottino et al., 2001).
Hepatic and intestinal content of DNP-SG was evaluated in a different set of animals 5 min after iv administration of a single bolus of CDNB (30 μmol/kg in 1:19 dimethylsulfoxide:saline). The animals were sacrificed by cardiac puncture and the liver and proximal jejunum were removed, rinsed with ice-cold saline, and weighed. One gram of each organ was homogenized in two volumes of phosphate buffer saline, pH = 7.35. The homogenates thus obtained were treated with HClO4 as described above, centrifuge, and the supernatant analyzed by HPLC.
Basal GSH and GSSG biliary excretion and content in liver and intestine. The common bile duct was cannulated with polyethylene tubing (PE10) under urethane anesthesia. After a 30-min stabilization period, bile was collected for 10 min in pre-weighed tubes containing 0.1 ml of 10% sulfosalicylic acid for determination of total and oxidized glutathione. Bile flow was determined gravimetrically, assuming a density of 1 g/ml. At the end of the bile collection period, the animals were sacrificed by cardiac puncture and the liver and proximal jejunum were removed, rinsed with cold saline, and homogenized (20% w/v in saline). Two volumes of the homogenate were mixed with 1 volume of 10% sulfosalicylic acid, centrifuged at 5000 x g for 5 min, and the supernatant immediately used in glutathione species assay. Total glutathione (GSH + GSSG) and GSSG in bile and in liver and intestinal homogenates were determined spectrophotometrically by using the recycling method of Tietze (1969), as modified by Griffith (1980).
Western blot studies of Mrp2. The liver was perfused in situ with ice-cold saline through the portal vein and crude plasma membranes were prepared by differential centrifugation as described (Meier et al., 1984). The whole small intestine was divided into four equal segments (about 25 cm each) and carefully rinsed with ice-cold saline. The most proximal segment, starting from the pylorus, was named A, while the most distal segment close to the ileo-cecal valve was named D. Brush border membranes were prepared from each segment as described (Mottino et al., 2000). Protein concentration in membrane preparations was measured using bovine serum albumin as a standard (Lowry et al., 1951). Western blot detection of Mrp2 content was performed as previously described by using a monoclonal antibody to human Mrp2 (1:2500, M2 III-6, Alexis Biochemicals, Carlsbad, CA) (Mottino et al., 2000).
Immunofluorescence studies. For in situ immunodetection of Mrp2, slices (5 μm) from liver and proximal jejunum were prepared with a Zeiss Microm HM500 microtome cryostat, air dried for 2 h, and fixed for 10 min with cold acetone (–20°C). Double labeling of Mrp2 and ZO-1 in liver was performed by using monoclonal anti-human Mrp2 (1:100) and rabbit anti-human ZO-1 (1:50, Zymed Laboratories Inc., San Francisco, CA) antibodies as described (Mottino et al., 2002). The images were captured on a Zeiss Pascal LSM confocal system attached to a Zeiss Axioplan 2 imaging microscope. Densitometric analysis of confocal images was performed as described (Crocenzi et al., 2003a), using the software Scion Image beta 4.02 for Windows (Scion). The variances of the Mrp2 fluorescence curves were then calculated, and compared statistically by using the Mann-Whitney test; any difference between groups thus reflects changes in localization. For Mrp2 detection in small intestine, tissue sections were incubated overnight with the monoclonal Mrp2 (1:100) antibody followed by incubation with Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratory, Inc., West Grove, PA) (1:200) for 2 h. The images were captured on a Zeiss Axiovert 25 CFL inverted microscope. To ensure comparable staining and image capture performance for PH and sham groups, intestinal and liver slices were prepared the same day, mounted on the same glass slide, and subjected to the staining procedure and microscopy analysis simultaneously.
GST activity. Cytosolic fractions from liver and proximal jejunum were obtained by ultracentrifugation as previously described (Siekevitz, 1962). Protein concentration in cytosols was measured using bovine serum albumin as a standard (Lowry et al., 1951). Glutathione conjugating activity towards CDNB was assayed by a reported procedure (Habig et al., 1974) except that GSH concentration was raised to 250 mM and CDNB was added to the incubation mixture as a 300 mM solution in dimethylsulfoxide. 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 a linear function of time and protein concentration.
Mrp2 transport activity in isolated hepatocytes. Hepatocytes were isolated by collagenase perfusion and mechanical dissociation (Seglen, 1973). The cells, suspended in Krebs-Henseleit Ringer-Hepes buffer, pH 7.40, were used for determination of DNP-SG content and excretion rate. Protein concentration in the suspensions was determined using bovine serum albumin as standard (Lowry et al., 1951). Cell viability was determined by trypan blue exclusion and was always greater than 87%. Pre-loading of the hepatocytes with DNP-SG was performed by incubating the cells with CDNB (100 μM in Krebs-Henseleit Ringer-Hepes buffer, pH 7.40) as described (Oude Elferink et al., 1989). Aliquots of cell suspensions (7 x 104 cells) were taken, loaded in test tubes (Beckman-type 0.4 ml polyethylene tubes) containing a lysis solution (ClNa 3 M, triton X-100 0.1%) and a silicone layer (Wacker-Chemie GmbH, Munich, Germany), and incubated at 37°C for 0, 30, 60, and 90 s in CDNB free buffer. At the end of the incubation period, the suspensions were centrifuged at 9000 x g for 20 s. DNP-SG was determined in supernatants and cells by HPLC. Initial excretion rate was estimated as the slope of the regression curve of the amount of DNP-SG present in the supernatants per mg of hepatocyte protein vs. time.
Statistical analysis. Data are presented as means ± SD. Statistical analysis was performed using one-way ANOVA, followed by the Bonferroni test, unless otherwise stated. Values of p < 0.05 were considered to be statistically significant.
RESULTS
Liver and Small Intestine Mass
Table 1 shows that liver mass was significantly decreased by liver resection one day after surgery and returned to the normal value after seven days. Because 70 to 75% of the liver was removed during surgery, the data on liver mass for the PH1 group, which represented about 50% of the normal value, confirmed the efficient regeneration of the liver tissue by 24 h after surgery. The mass of the small intestine corresponding to the 50 cm segment perfused in vivo for transport studies was not affected by liver resection either at one or seven days post-surgery, as also indicated in Table 1. This portion of the small intestine mainly corresponds to proximal and distal jejunum, where the highest expression and activity of Mrp2 were reported (Gotoh et al., 2000; Mottino et al., 2000).
Biliary and Intestinal Excretion of DNP-SG
Panel A in Figure 1 shows that biliary excretion of DNP-SG was substantially impaired in PH1 with respect to sham rats, particularly during the first periods of bile collection, and that this measure was partially recovered in the PH7 group. Cumulative biliary excretion of DNP-SG was decreased by 90 and 50% in PH1 and PH7, respectively, in response to surgery (see inset in Fig. 1A). In contrast, intestinal excretion of DNP-SG was significantly increased in PH1 (+63%, see inset in Fig. 1B) whereas PH7 group did not show any difference with respect to shams.
Expression and Localization of Mrp2
Figure 2 shows Western blot study of Mrp2 in plasma membranes from liver and proximal jejunum (equivalent to segment B) at one, two, and seven days post-hepatectomy. Neither liver nor jejunum exhibited any change in Mrp2 levels detected by densitometry in response to liver resection when referred to sham animals (see right panels). We also evaluated Mrp2 expression in segments A, C, and D in response to hepatectomy. Neither of these segments exhibited any change in Mrp2 levels as revealed by Western blotting (data not shown). Because liver cell polarity and expression of tight junction proteins, necessary for proper apical localization of canalicular transporters, are temporarily altered during regeneration (Takaki et al., 2001), we also evaluated localization of Mrp2 in liver by confocal microscopy. Red immunofluorescence detection of Mrp2, shown in gray in upper panels in Figure 3, indicated a similar pattern of distribution between PH1 and sham groups. Preserved localization of Mrp2 at the canalicular membrane level was further confirmed by performing double immunofluorescence labeling with antibodies against Mrp2 and ZO-1, the tight junction protein ZO-1 being used as a marker of the limits of canaliculi. Middle panels in Figure 3 show that in PH1, as well as in sham liver, the Mrp2 red staining (shown in gray) is completely delimited by the green lines corresponding to ZO-1 (shown in white). Densitometric analysis of distribution of canalicular Mrp2 (see Figure 4), followed by statistical comparison of the variance obtained from both curves, indicated a similar profile for PH1 and sham groups. Immunoflourescence detection of Mrp2 in jejunum indicated localization at the periphery of the intestinal villus (see arrows in bottom panels in Figure 3) in both sham and PH1 groups, as previously reported for normal rats (Mottino et al., 2000).
Serum, Liver, and Intestinal Content of DNP-SG and GST Activity
Since secretion of DNP-SG was changed in liver and intestine from PH1 group in spite of preserved expression and localization of Mrp2, we further explored whether alterations in transport of the Mrp2 substrate resulted from changes in DNP-SG cellular availability. Table 2 shows that tissue content of DNP-SG detected 5 min after CDNB administration was significantly decreased in liver (–70%) and increased (+113%) in jejunum from PH1 group, whereas serum levels did not differ between groups. Table 2 also shows that cytosolic GST activity was slightly but significantly decreased (–36%) in liver and increased (+25%) in jejunum from these same animals.
Biliary Excretion and Hepatic and Intestinal Content of Glutathione Species
It is well accepted that biliary secretion of GSSG and GSH is mainly mediated by Mrp2 at the canalicular level (Buchler et al., 1996; Knig et al., 1999; Paulusma et al., 1996, 1999). We therefore examined GSSG and GSH biliary excretion rate, as well as their intracellular levels, in animals from PH1 and sham groups. Data from Table 3 indicate that biliary excretion and intrahepatic level of GSSG were normal in hepatectomized animals when expressed per g of liver. In contrast, excretion rate of GSH was significantly decreased (–69%), and GSH intrahepatic level increased (+203%), one day post-hepatectomy. Table 3 also shows that levels of GSSG and GSH in proximal jejunum were not affected by hepatectomy.
Excretion of DNP-SG by Isolated Hepatocytes
To further explore the origin of deficient biliary excretion of DNP-SG by the regenerating liver, we performed experiments in isolated hepatocytes. It was demonstrated that CDNB is efficiently taken up by hepatocytes and subsequently converted in its glutathione conjugated derivative in this model (Oude Elferink et al., 1989). Intracellular synthesis of DNP-SG could result, at least in part, from a non-enzymatic process since it was found to be completed in less than 30 s. Table 4 shows that cells from hepatectomized animals excreted DNP-SG at a similar rate as sham hepatocytes. Intracellular level of the metabolite was also similar between groups. Thus, in contrast to what was observed in in vivo experiments, availability of DNP-SG for subsequent excretion was preserved together with transport activity itself. This set of experiments strongly suggests that Mrp2 intrinsic activity is preserved shortly after hepatectomy.
DISCUSSION
The small intestine represents the principal site of absorption for ingested compounds, whether dietary, therapeutic, or toxic. Many of these compounds, once internalized into the enterocyte, become substrates for apical efflux transporters, which extrude them back into the lumen. The major efflux transporters are members of the ABC superfamily proteins such as P-glycoprotein and Mrp2 (Chan et al., 2004; Dietrich et al., 2003). Together with intracellular metabolizing enzymes, they constitute a defense against toxic injury. For example, lipophilic compounds, such as CDNB, undergo conjugation with glutathione before being recognized as a Mrp2 substrate. Though contribution of the small intestine to overall DNP-SG disposition was minimal in normal conditions, intestinal participation could be more relevant under conditions of downregulation of hepatic Mrp2 or alternatively, under conditions of decreased liver mass with preserved expression of Mrp2. In the current study we found that biliary excretion of DNP-SG, expressed per mass unit, was substantially decreased in hepatectomized rats one day after surgery and that excretion of this compound by the small intestine duplicated its normal value. As a result, contribution of the intestine to xenobiotic disposition became as important as the liver one and may play a compensatory role early after hepatectomy.
It was previously reported that hepatic Mrp2 expression was essentially unchanged shortly after two-third hepatectomy (Gerloff et al., 1999; Vos et al., 1999) suggesting preserved capability for canalicular transport of conjugated compounds by the remaining liver. Our Western blot study confirmed preserved expression of liver Mrp2 up to seven days after liver resection. Adult hepatocytes are normally quiscent, and within one day after partial hepatectomy, the remaining cells enter into proliferative phase to regenerate this organ (LaBrecque, 1994), with concomitant alteration in cell polarity and tight junction integrity (Takaki et al., 2001). This could in turn affect normal localization and function of Mrp2. Altered localization of Mrp2 at the canalicular level may coexist with preserved expression detected in canalicular or mixed plasma membranes by Western blotting, as was demonstrated to occur in different experimental conditions of acute cholestasis (Crocenzi et al., 2003a; Mottino et al., 2002; Rost et al., 1999). Our current data on immunofluorescence detection of Mrp2 indicate preserved localization at the canalicular level in PH1 group. Because endocytic internalization of canalicular transporters associated with acute models of cholestasis may occur in a very short time (Crocenzi et al., 2003a,b; Haussinger et al., 2000; Mottino et al., 2002), we also evaluated Mrp2 localization in liver from hepatectomized rats 1 and 2 h after surgery. We found no changes with respect to sham animals (images not shown). The data thus indicate normal localization of Mrp2 in spite of alterations in polarity of tight junction structures described for the regenerating liver.
In contrast to Western and immunofluorescence studies, transport studies showed a substantial decrease in biliary excretion of DNP-SG by the regenerating liver. Whereas decreased conjugation was previously demonstrated by in vitro assessment of cytosolic GST activity (Carnovale et al., 1995) as a consequence of decreased levels of GST isoforms involved in CDNB conjugation (Lee and Boyer, 1993), no studies explored the in vivo formation of DNP-SG in hepatectomized animals. Because the impairment in DNP-SG formation in vivo was of higher magnitude than the decrease registered for GST activity in vitro, it is possible that restrictions in availability of GST substrates or alternatively, the presence of GST inhibitors account for this discrepancy. Because CDNB is assumed to freely enter the cells due to its lipophilic nature, and GSH liver content is increased, rather than decreased, in PH1 animals (Table 3), it is unlikely that availability of GST substrates represented a limiting factor. The current data indicate that DNP-SG was formed and secreted at comparable rates between sham and PH1 groups in isolated hepatocytes, a model in which cells are rinsed with buffer likely leading to washout of accumulated inhibitors. Though it is not possible to exclude that in vivo Mrp2-mediated transport of DNP-SG is inhibited by alternative Mrp2 substrates such as bilirubin glucuronides in PH1 group, the substantial decrease in liver DNP-SG content detected 5 min after CDNB administration indicated impairment in CDNB conjugation as a main cause of altered biliary secretion. This in turn, likely resulted from decreased GST levels together with a potential inhibitory action on its activity. Bile salts or bilirubin, which were demonstrated to transiently increase in serum post-hepatectomy (Vos et al., 1999) and to inhibit GST activity in vitro (Fukai et al., 1989; Singh et al., 1988), are good candidates to explain in vivo inhibition of DNP-SG formation.
Previous in vivo studies clearly showed impaired transport of GSH into bile shortly after hepatectomy (Huang et al., 1998; Vos et al., 1999; Yang and Hill, 2001). This finding was tentatively explained by the occurrence of competitive inhibition by other Mrp2 substrates (Vos et al., 1999), or alternatively by the fact that a more specific GSH transporter, different from Mrp2, is affected in regenerating liver (Yang and Hill, 2001). Huang et al. (1998) also reported that GSH levels increased in liver from hepatectomized animals, though the authors proposed an increased synthesis as a more plausible explanation. Our current study further demonstrated that GSSG liver content and biliary excretion were not affected one day after hepatectomy. GSSG as well as glutathione conjugates are well recognized Mrp2 substrates exhibiting higher affinity toward Mrp2 than GSH (Knig et al., 1999). Preserved biliary excretion of GSSG in PH1 group would thus agree with preserved expression and localization of Mrp2 at the canalicular level.
Western and immunofluorescence studies indicate normal expression and localization of Mrp2 in small intestine from hepatectomized rats. In spite of this, intestinal excretion of DNP-SG was significantly increased in hepatectomized rats one day after surgery. It is possible that higher proportion of CDNB reached extrahepatic tissues as a consequence of the significant decrease in liver mass. This, together with increased levels of cytosolic GST, as detected in vitro, could explain the increased intestinal disposition of DNP-SG in the PH1 group. From the current data it could be speculated that iv administration of 30 μmol/kg b.w. of CDNB and subsequent formation of DNP-SG did not saturate intestinal Mrp2 capability, as increased production of the conjugate in PH1 animals led to higher intestinal secretion. In support of this possibility we previously observed that saturation of DNP-SG transport in the isolated intestinal sac model occurred at a excretion rate of about 20 nmol/min/g of tissue (Mottino et al., 2001) and, according to the current data, the maximal excretion rate registered in PH1 group was 1 nmol/min/g of tissue.
Data from the current experiments show that intestinal excretion of DNP-SG represents less than 10% of biliary excretion in sham rats. However, because of the impairment in intrahepatic formation of the conjugated derivative of CDNB in PH1 animals, cumulative intestinal excretion of DNP-SG by 60 min was about the same as biliary excretion, which in turn was about one-tenth that of sham rats (see insets in Figs. 1A and 1B). Based on the data in Table 1, the whole organ contribution to DNP-SG disposition could be estimated as about the same for jejunum and liver in PH1 rats. In contrast, seven days after surgery, liver mass was totally restored in hepatectomized animals and cumulative biliary excretion of DNP-SG was about half the sham value when expressed per g of tissue. In consequence, the estimated overall capacity for DNP-SG excretion in PH7 rats was substantially recovered when compared with PH1 animals. We postulate that the intestine may represent an organ of relevance for metabolism of CDNB and posterior secretion of DNP-SG shortly after hepatectomy. This tissue may thus contribute to attenuate toxicity of compounds incorporated systemically, until liver mass and intrinsic metabolic capability are restored. Our data contrast with previous findings by Dietrich et al. indicating an impairment in intestinal expression of Mrp2 in bile duct-ligated rats as well as in patients with biliary obstruction (Dietrich et al., 2004). These authors demonstrated an inhibitory action of interleukin 1 (IL-1), released in response to the liver inflammatory process, on intestinal Mrp2 expression. It is known that downregulation of hepatic transporters involves cytokine cascades as initiating mediators in different animal models of cholestasis (Trauner et al., 1999). It is possible that differences in Mrp2 expression in intestine in response to bile duct ligation vs. 70–75% liver resection depend in part on the magnitude in cytokines production and systemic release between the two experimental models. In support of this possibility, it was recently reported a significantly increased IL-6 plasma levels in 90% hepatectomized rats but not in 70% hepatectomized rats, in association with a decrease in expression of hepatic Mrp2 (Chang et al., 2004).
In conclusion, though intestinal Mrp2 expression was preserved in hepatectomized rats, the increased conjugation of CDNB, leading to higher availability of the Mrp2 substrate for its subsequent excretion, may partially compensate for liver dysfunction, particularly shortly after surgery, while liver capability is recovering.
ACKNOWLEDGMENTS
This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas and Fundación Antorchas, Argentina. We express our gratitude to Drs. J. Elena Ochoa and José M. Pellegrino for their invaluable technical assistance.
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ABSTRACT
The ability of the liver and small intestine for secretion of dinitrophenyl-S-glutathione (DNP-SG), a substrate for multidrug resistance-associated protein 2 (Mrp2), into bile and lumen, respectively, as well as expression of Mrp2 in both tissues, were assessed in 70–75% hepatectomized rats. An in vivo perfused intestinal model was used. A single iv dose of 30 μmol/kg b.w. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered and its glutathione conjugate, DNP-SG, was determined by HPLC in bile and intestinal perfusate. One and seven days after hepatectomy, biliary excretion of DNP-SG was decreased by 90 and 50% with respect to shams, respectively, when expressed per mass unit. In contrast, intestinal excretion was increased by 63% or unchanged one and seven days post-hepatectomy, respectively. Tissue content of DNP-SG 5 min after CDNB administration was substantially decreased in liver and significantly increased in intestine, one day post-hepatectomy. Western and immunofluorescence studies revealed preserved levels and localization of Mrp2 in both tissues from hepatectomized animals, irrespective of the time analyzed. In spite of preserved expression of Mrp2, the higher availability of DNP-SG in intestinal cells, likely as a consequence of increased glutathione-S-transferase-mediated conjugation of CDNB, may explain the in vivo findings. Further experiments in isolated hepatocytes suggested that decreased synthesis of DNP-SG rather than altered canalicular transport is responsible for the substantial impairment in excretion of this compound into bile. Taken together, these results indicate that the intestine may partially compensate for liver DNP-SG disposition, particularly shortly after surgery, while liver capability is recovering.
Key Words: Mrp2; 1-chloro-2,4-dinitrobenzene; dinitrophenyl-S-glutathione; liver regeneration; glutathione; glutathione-S-transferase.
INTRODUCTION
In various forms of liver injury such as viral hepatitis, toxic or drug-induced liver injury, and surgical resection, the functional liver mass is restored by tissue regeneration. Partial hepatectomy in rats represents a useful model to study regulation of liver function during the regeneration process (Rahman and Hodgson, 2000). Irrespective of whether liver cell function is preserved under this experimental model, the substantial loss of liver mass suggests a temporary impairment in the overall metabolic and secretory function. Extrahepatic tissues may play a role as an alternative site for biotransformation and apical transport of xenobiotics. On this regard, it was reported that glutathione S-transferase (GST) activity, expressed per mass unit, was increased in small intestine and decreased in liver two days after partial hepatectomy (Carnovale et al., 1995). Decreased enzyme activity in liver was associated with downregulation of GST expression at transcriptional level (Lee and Boyer, 1993). We previously observed that another phase II enzyme, UDP-glucuronosyltransferase (UGT), followed a similar behavior. Though UGT specific activity was preserved in the remaining liver, the substantial decrease in liver mass led to a significant decrease in overall glucuronidation capability (Catania et al., 1998). As described for GST, we found that UGT specific activity, as well as expression detected by Western blotting, was increased in small intestine in these same rats, suggesting a partial compensatory role while liver mass is recovering.
Alteration in the activity of drug transporters after partial hepatectomy may also lead to changes in toxicity of their substrates. The basolateral organic anion transporter 1 (Oatp1) was found to be unchanged and Oatp2 only slightly decreased 24 h after partial hepatectomy in crude liver plasma membrane (Vos et al., 1999). These same authors reported that the expression of canalicular multidrug resistance-associated protein 2 (Mrp2) was preserved in hepatectomized animals, whereas P-glycoprotein was substantially upregulated, likely as a consequence of increased expression of the Mdr1b component. Gerloff et al. (1999) reported data on expression of rat liver transporters, detected in microsomal membranes, at different periods post-hepatectomy. Overall, the authors found differential regulation of basolateral and canalicular organic anion transporters in the regenerating liver. Whereas microsomal content of Mrp2 was preserved, expression of Oatp1 and Oatp2 significantly decreased. This latter finding would suggest decreased ability for the uptake of organic anions at the basolateral level. Recently, Chang et al. (2004) reported that Mrp2 expression in liver was decreased in 90% but not in 70% hepatectomized rats, clearly indicating a dependence with liver mass removal. The evidence on downregulation of basolateral transporters together with the loss of liver mass would indicate an impairment in the overall capability for transport of organic anions from blood to bile.
Mrp2 plays an important role in elimination of potentially toxic endo- and xenobiotics, including bilirubin, hormones, drugs, and carcinogens, primarily as their glucuronide, glutathione or sulfate conjugates (Buchler et al., 1996; Paulusma et al., 1996). Mrp2 also mediates the active transport of oxidized (GSSG) and reduced (GSH) glutathione into bile (Knig et al., 1999; Rebbeor et al., 2002). It was demonstrated that Mrp2 is also present on the apical surface of the rat enterocyte (Mottino et al., 2000). The data indicated that Mrp2 protein is preferentially localized in the proximal intestine and gradually decreases from the jejunum to the distal ileum and that its expression is highest at the tip region of the villus. Mrp2 thus follows a similar pattern of distribution along the intestine and the villus axis as the conjugating enzymes in the rat. Jejunum is also the main site for transport of glutathione conjugates from the serosal to the mucosal side of the intestinal epithelium (Gotoh et al., 2000). Clearly, conjugating enzymes and Mrp2 may act coordinately to metabolize and secrete xenobiotics into the intestinal lumen (Catania et al., 2004). Whether this coordinated action may lead to a compensatory increase in intestinal conjugation of xenobiotics and subsequent Mrp2-mediated secretion of conjugated derivatives while liver capability is restoring is not known. Because phase II enzyme activities are increased in small intestine from two-third partially hepatectomized rats (Carnovale et al., 1995; Catania et al., 1998) it was of interest to explore if Mrp2 expression and activity are also increased. We thus evaluated the hepatic and intestinal Mrp2 levels and their respective secretory activity for dinitrophenyl-S-glutathione (DNP-SG), an Mrp2 substrate generated endogenously after systemic administration of 1-chloro-2,4-dinitrobenzene (CDNB), in hepatectomized animals. The analysis of disposition of DNP-SG in this experimental model allowed us to simultaneously evaluate conjugation and apical excretion capabilities for both tissues.
MATERIALS AND METHODS
Chemicals. Leupeptin, phenylmethylsulfonyl fluoride, pepstatin A, 1-chloro-2,4-dinitrobenzene (CDNB), NADPH, collagenase, glutathione, and glutathione reductase were obtained from Sigma Chemical Co (St. Louis, MO). 2-Methylbutane was obtained from Acros Organics (Pittsburgh, PA) and 2-vinylpyridine was obtained from Fluka Chemical Corp (Milwaukee, WI). All other chemicals and reagents were commercial products of analytical grade purity and used as supplied.
Animals and surgical procedures. Adult Male Wistar rats (320–370 g) were used throughout. Animals had free access to food and water and
Sham (n = 4), PH1 (n = 4), and PH7 (n = 4) were used in in vivo experiments for assessment of blood levels (5 min after CDNB administration), and biliary and intestinal excretion of DNP-SG (every 10 and 15 min respectively, for 60 min), and for determination of liver and jejunum mass.
Sham (n = 3) and PH1 (n = 4) were used for assessment of liver and intestinal content of DNP-SG, 5 min after CDNB administration.
Sham (n = 3) and PH1 (n = 6) were used for assessment of basal bile flow, basal GSH and GSSG biliary excretion and tissue content, and immunofluorescence and GST activity in liver and intestine. These animals were sacrificed after basal bile collection.
Sham (n = 4), PH1 (n = 4), PH2 (n = 4), and PH7 (n = 4) were used in Western blotting analysis of Mrp2.
Sham (n = 3) and PH1 (n = 3) were used for assessment of Mrp2 transport activity in isolated hepatocytes.
Biliary and intestinal excretion and tissue content of DNP-SG. The rats were anesthetized with urethane (1000 mg/kg b.w. ip) and thus maintained throughout. Body temperature was measured with a rectal probe, and maintained at 37°C with a heating lamp. The femoral vein and the common bile duct were cannulated with polyethylene tubing (PE50 and PE10, respectively). Intestinal excretion studies were performed using the in situ single-pass perfusion technique (Gotoh et al., 2000). Briefly, the intestine was perfused with isotonic phosphate buffered saline, pH = 7.35, from the upper jejunum to the end of distal jejunum (about 50 cm in length) with a peristaltic pump at a rate of 0.4 ml/min. After a 30-min stabilization period, a single bolus of CDNB (30 μmol/kg b.w. in 1:19 dimethylsulfoxide:saline, iv) was administered. Bile and intestinal perfusate were collected at 10- and 15-min intervals, respectively, for 60 min. A blood sample was collected 5 min after CDNB injection from the tail vein and immediately centrifuged to separate serum. Saline was administered intravenously throughout the experiment to replenish body fluids. Bile, intestinal perfusate, and serum sample were treated with 70% (v/v) HClO4 (50 μl per ml of sample) and centrifuged at 3500 x g for 5 min. DNP-SG content was determined in the supernatants by HPLC as described (Mottino et al., 2001).
Hepatic and intestinal content of DNP-SG was evaluated in a different set of animals 5 min after iv administration of a single bolus of CDNB (30 μmol/kg in 1:19 dimethylsulfoxide:saline). The animals were sacrificed by cardiac puncture and the liver and proximal jejunum were removed, rinsed with ice-cold saline, and weighed. One gram of each organ was homogenized in two volumes of phosphate buffer saline, pH = 7.35. The homogenates thus obtained were treated with HClO4 as described above, centrifuge, and the supernatant analyzed by HPLC.
Basal GSH and GSSG biliary excretion and content in liver and intestine. The common bile duct was cannulated with polyethylene tubing (PE10) under urethane anesthesia. After a 30-min stabilization period, bile was collected for 10 min in pre-weighed tubes containing 0.1 ml of 10% sulfosalicylic acid for determination of total and oxidized glutathione. Bile flow was determined gravimetrically, assuming a density of 1 g/ml. At the end of the bile collection period, the animals were sacrificed by cardiac puncture and the liver and proximal jejunum were removed, rinsed with cold saline, and homogenized (20% w/v in saline). Two volumes of the homogenate were mixed with 1 volume of 10% sulfosalicylic acid, centrifuged at 5000 x g for 5 min, and the supernatant immediately used in glutathione species assay. Total glutathione (GSH + GSSG) and GSSG in bile and in liver and intestinal homogenates were determined spectrophotometrically by using the recycling method of Tietze (1969), as modified by Griffith (1980).
Western blot studies of Mrp2. The liver was perfused in situ with ice-cold saline through the portal vein and crude plasma membranes were prepared by differential centrifugation as described (Meier et al., 1984). The whole small intestine was divided into four equal segments (about 25 cm each) and carefully rinsed with ice-cold saline. The most proximal segment, starting from the pylorus, was named A, while the most distal segment close to the ileo-cecal valve was named D. Brush border membranes were prepared from each segment as described (Mottino et al., 2000). Protein concentration in membrane preparations was measured using bovine serum albumin as a standard (Lowry et al., 1951). Western blot detection of Mrp2 content was performed as previously described by using a monoclonal antibody to human Mrp2 (1:2500, M2 III-6, Alexis Biochemicals, Carlsbad, CA) (Mottino et al., 2000).
Immunofluorescence studies. For in situ immunodetection of Mrp2, slices (5 μm) from liver and proximal jejunum were prepared with a Zeiss Microm HM500 microtome cryostat, air dried for 2 h, and fixed for 10 min with cold acetone (–20°C). Double labeling of Mrp2 and ZO-1 in liver was performed by using monoclonal anti-human Mrp2 (1:100) and rabbit anti-human ZO-1 (1:50, Zymed Laboratories Inc., San Francisco, CA) antibodies as described (Mottino et al., 2002). The images were captured on a Zeiss Pascal LSM confocal system attached to a Zeiss Axioplan 2 imaging microscope. Densitometric analysis of confocal images was performed as described (Crocenzi et al., 2003a), using the software Scion Image beta 4.02 for Windows (Scion). The variances of the Mrp2 fluorescence curves were then calculated, and compared statistically by using the Mann-Whitney test; any difference between groups thus reflects changes in localization. For Mrp2 detection in small intestine, tissue sections were incubated overnight with the monoclonal Mrp2 (1:100) antibody followed by incubation with Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratory, Inc., West Grove, PA) (1:200) for 2 h. The images were captured on a Zeiss Axiovert 25 CFL inverted microscope. To ensure comparable staining and image capture performance for PH and sham groups, intestinal and liver slices were prepared the same day, mounted on the same glass slide, and subjected to the staining procedure and microscopy analysis simultaneously.
GST activity. Cytosolic fractions from liver and proximal jejunum were obtained by ultracentrifugation as previously described (Siekevitz, 1962). Protein concentration in cytosols was measured using bovine serum albumin as a standard (Lowry et al., 1951). Glutathione conjugating activity towards CDNB was assayed by a reported procedure (Habig et al., 1974) except that GSH concentration was raised to 250 mM and CDNB was added to the incubation mixture as a 300 mM solution in dimethylsulfoxide. 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 a linear function of time and protein concentration.
Mrp2 transport activity in isolated hepatocytes. Hepatocytes were isolated by collagenase perfusion and mechanical dissociation (Seglen, 1973). The cells, suspended in Krebs-Henseleit Ringer-Hepes buffer, pH 7.40, were used for determination of DNP-SG content and excretion rate. Protein concentration in the suspensions was determined using bovine serum albumin as standard (Lowry et al., 1951). Cell viability was determined by trypan blue exclusion and was always greater than 87%. Pre-loading of the hepatocytes with DNP-SG was performed by incubating the cells with CDNB (100 μM in Krebs-Henseleit Ringer-Hepes buffer, pH 7.40) as described (Oude Elferink et al., 1989). Aliquots of cell suspensions (7 x 104 cells) were taken, loaded in test tubes (Beckman-type 0.4 ml polyethylene tubes) containing a lysis solution (ClNa 3 M, triton X-100 0.1%) and a silicone layer (Wacker-Chemie GmbH, Munich, Germany), and incubated at 37°C for 0, 30, 60, and 90 s in CDNB free buffer. At the end of the incubation period, the suspensions were centrifuged at 9000 x g for 20 s. DNP-SG was determined in supernatants and cells by HPLC. Initial excretion rate was estimated as the slope of the regression curve of the amount of DNP-SG present in the supernatants per mg of hepatocyte protein vs. time.
Statistical analysis. Data are presented as means ± SD. Statistical analysis was performed using one-way ANOVA, followed by the Bonferroni test, unless otherwise stated. Values of p < 0.05 were considered to be statistically significant.
RESULTS
Liver and Small Intestine Mass
Table 1 shows that liver mass was significantly decreased by liver resection one day after surgery and returned to the normal value after seven days. Because 70 to 75% of the liver was removed during surgery, the data on liver mass for the PH1 group, which represented about 50% of the normal value, confirmed the efficient regeneration of the liver tissue by 24 h after surgery. The mass of the small intestine corresponding to the 50 cm segment perfused in vivo for transport studies was not affected by liver resection either at one or seven days post-surgery, as also indicated in Table 1. This portion of the small intestine mainly corresponds to proximal and distal jejunum, where the highest expression and activity of Mrp2 were reported (Gotoh et al., 2000; Mottino et al., 2000).
Biliary and Intestinal Excretion of DNP-SG
Panel A in Figure 1 shows that biliary excretion of DNP-SG was substantially impaired in PH1 with respect to sham rats, particularly during the first periods of bile collection, and that this measure was partially recovered in the PH7 group. Cumulative biliary excretion of DNP-SG was decreased by 90 and 50% in PH1 and PH7, respectively, in response to surgery (see inset in Fig. 1A). In contrast, intestinal excretion of DNP-SG was significantly increased in PH1 (+63%, see inset in Fig. 1B) whereas PH7 group did not show any difference with respect to shams.
Expression and Localization of Mrp2
Figure 2 shows Western blot study of Mrp2 in plasma membranes from liver and proximal jejunum (equivalent to segment B) at one, two, and seven days post-hepatectomy. Neither liver nor jejunum exhibited any change in Mrp2 levels detected by densitometry in response to liver resection when referred to sham animals (see right panels). We also evaluated Mrp2 expression in segments A, C, and D in response to hepatectomy. Neither of these segments exhibited any change in Mrp2 levels as revealed by Western blotting (data not shown). Because liver cell polarity and expression of tight junction proteins, necessary for proper apical localization of canalicular transporters, are temporarily altered during regeneration (Takaki et al., 2001), we also evaluated localization of Mrp2 in liver by confocal microscopy. Red immunofluorescence detection of Mrp2, shown in gray in upper panels in Figure 3, indicated a similar pattern of distribution between PH1 and sham groups. Preserved localization of Mrp2 at the canalicular membrane level was further confirmed by performing double immunofluorescence labeling with antibodies against Mrp2 and ZO-1, the tight junction protein ZO-1 being used as a marker of the limits of canaliculi. Middle panels in Figure 3 show that in PH1, as well as in sham liver, the Mrp2 red staining (shown in gray) is completely delimited by the green lines corresponding to ZO-1 (shown in white). Densitometric analysis of distribution of canalicular Mrp2 (see Figure 4), followed by statistical comparison of the variance obtained from both curves, indicated a similar profile for PH1 and sham groups. Immunoflourescence detection of Mrp2 in jejunum indicated localization at the periphery of the intestinal villus (see arrows in bottom panels in Figure 3) in both sham and PH1 groups, as previously reported for normal rats (Mottino et al., 2000).
Serum, Liver, and Intestinal Content of DNP-SG and GST Activity
Since secretion of DNP-SG was changed in liver and intestine from PH1 group in spite of preserved expression and localization of Mrp2, we further explored whether alterations in transport of the Mrp2 substrate resulted from changes in DNP-SG cellular availability. Table 2 shows that tissue content of DNP-SG detected 5 min after CDNB administration was significantly decreased in liver (–70%) and increased (+113%) in jejunum from PH1 group, whereas serum levels did not differ between groups. Table 2 also shows that cytosolic GST activity was slightly but significantly decreased (–36%) in liver and increased (+25%) in jejunum from these same animals.
Biliary Excretion and Hepatic and Intestinal Content of Glutathione Species
It is well accepted that biliary secretion of GSSG and GSH is mainly mediated by Mrp2 at the canalicular level (Buchler et al., 1996; Knig et al., 1999; Paulusma et al., 1996, 1999). We therefore examined GSSG and GSH biliary excretion rate, as well as their intracellular levels, in animals from PH1 and sham groups. Data from Table 3 indicate that biliary excretion and intrahepatic level of GSSG were normal in hepatectomized animals when expressed per g of liver. In contrast, excretion rate of GSH was significantly decreased (–69%), and GSH intrahepatic level increased (+203%), one day post-hepatectomy. Table 3 also shows that levels of GSSG and GSH in proximal jejunum were not affected by hepatectomy.
Excretion of DNP-SG by Isolated Hepatocytes
To further explore the origin of deficient biliary excretion of DNP-SG by the regenerating liver, we performed experiments in isolated hepatocytes. It was demonstrated that CDNB is efficiently taken up by hepatocytes and subsequently converted in its glutathione conjugated derivative in this model (Oude Elferink et al., 1989). Intracellular synthesis of DNP-SG could result, at least in part, from a non-enzymatic process since it was found to be completed in less than 30 s. Table 4 shows that cells from hepatectomized animals excreted DNP-SG at a similar rate as sham hepatocytes. Intracellular level of the metabolite was also similar between groups. Thus, in contrast to what was observed in in vivo experiments, availability of DNP-SG for subsequent excretion was preserved together with transport activity itself. This set of experiments strongly suggests that Mrp2 intrinsic activity is preserved shortly after hepatectomy.
DISCUSSION
The small intestine represents the principal site of absorption for ingested compounds, whether dietary, therapeutic, or toxic. Many of these compounds, once internalized into the enterocyte, become substrates for apical efflux transporters, which extrude them back into the lumen. The major efflux transporters are members of the ABC superfamily proteins such as P-glycoprotein and Mrp2 (Chan et al., 2004; Dietrich et al., 2003). Together with intracellular metabolizing enzymes, they constitute a defense against toxic injury. For example, lipophilic compounds, such as CDNB, undergo conjugation with glutathione before being recognized as a Mrp2 substrate. Though contribution of the small intestine to overall DNP-SG disposition was minimal in normal conditions, intestinal participation could be more relevant under conditions of downregulation of hepatic Mrp2 or alternatively, under conditions of decreased liver mass with preserved expression of Mrp2. In the current study we found that biliary excretion of DNP-SG, expressed per mass unit, was substantially decreased in hepatectomized rats one day after surgery and that excretion of this compound by the small intestine duplicated its normal value. As a result, contribution of the intestine to xenobiotic disposition became as important as the liver one and may play a compensatory role early after hepatectomy.
It was previously reported that hepatic Mrp2 expression was essentially unchanged shortly after two-third hepatectomy (Gerloff et al., 1999; Vos et al., 1999) suggesting preserved capability for canalicular transport of conjugated compounds by the remaining liver. Our Western blot study confirmed preserved expression of liver Mrp2 up to seven days after liver resection. Adult hepatocytes are normally quiscent, and within one day after partial hepatectomy, the remaining cells enter into proliferative phase to regenerate this organ (LaBrecque, 1994), with concomitant alteration in cell polarity and tight junction integrity (Takaki et al., 2001). This could in turn affect normal localization and function of Mrp2. Altered localization of Mrp2 at the canalicular level may coexist with preserved expression detected in canalicular or mixed plasma membranes by Western blotting, as was demonstrated to occur in different experimental conditions of acute cholestasis (Crocenzi et al., 2003a; Mottino et al., 2002; Rost et al., 1999). Our current data on immunofluorescence detection of Mrp2 indicate preserved localization at the canalicular level in PH1 group. Because endocytic internalization of canalicular transporters associated with acute models of cholestasis may occur in a very short time (Crocenzi et al., 2003a,b; Haussinger et al., 2000; Mottino et al., 2002), we also evaluated Mrp2 localization in liver from hepatectomized rats 1 and 2 h after surgery. We found no changes with respect to sham animals (images not shown). The data thus indicate normal localization of Mrp2 in spite of alterations in polarity of tight junction structures described for the regenerating liver.
In contrast to Western and immunofluorescence studies, transport studies showed a substantial decrease in biliary excretion of DNP-SG by the regenerating liver. Whereas decreased conjugation was previously demonstrated by in vitro assessment of cytosolic GST activity (Carnovale et al., 1995) as a consequence of decreased levels of GST isoforms involved in CDNB conjugation (Lee and Boyer, 1993), no studies explored the in vivo formation of DNP-SG in hepatectomized animals. Because the impairment in DNP-SG formation in vivo was of higher magnitude than the decrease registered for GST activity in vitro, it is possible that restrictions in availability of GST substrates or alternatively, the presence of GST inhibitors account for this discrepancy. Because CDNB is assumed to freely enter the cells due to its lipophilic nature, and GSH liver content is increased, rather than decreased, in PH1 animals (Table 3), it is unlikely that availability of GST substrates represented a limiting factor. The current data indicate that DNP-SG was formed and secreted at comparable rates between sham and PH1 groups in isolated hepatocytes, a model in which cells are rinsed with buffer likely leading to washout of accumulated inhibitors. Though it is not possible to exclude that in vivo Mrp2-mediated transport of DNP-SG is inhibited by alternative Mrp2 substrates such as bilirubin glucuronides in PH1 group, the substantial decrease in liver DNP-SG content detected 5 min after CDNB administration indicated impairment in CDNB conjugation as a main cause of altered biliary secretion. This in turn, likely resulted from decreased GST levels together with a potential inhibitory action on its activity. Bile salts or bilirubin, which were demonstrated to transiently increase in serum post-hepatectomy (Vos et al., 1999) and to inhibit GST activity in vitro (Fukai et al., 1989; Singh et al., 1988), are good candidates to explain in vivo inhibition of DNP-SG formation.
Previous in vivo studies clearly showed impaired transport of GSH into bile shortly after hepatectomy (Huang et al., 1998; Vos et al., 1999; Yang and Hill, 2001). This finding was tentatively explained by the occurrence of competitive inhibition by other Mrp2 substrates (Vos et al., 1999), or alternatively by the fact that a more specific GSH transporter, different from Mrp2, is affected in regenerating liver (Yang and Hill, 2001). Huang et al. (1998) also reported that GSH levels increased in liver from hepatectomized animals, though the authors proposed an increased synthesis as a more plausible explanation. Our current study further demonstrated that GSSG liver content and biliary excretion were not affected one day after hepatectomy. GSSG as well as glutathione conjugates are well recognized Mrp2 substrates exhibiting higher affinity toward Mrp2 than GSH (Knig et al., 1999). Preserved biliary excretion of GSSG in PH1 group would thus agree with preserved expression and localization of Mrp2 at the canalicular level.
Western and immunofluorescence studies indicate normal expression and localization of Mrp2 in small intestine from hepatectomized rats. In spite of this, intestinal excretion of DNP-SG was significantly increased in hepatectomized rats one day after surgery. It is possible that higher proportion of CDNB reached extrahepatic tissues as a consequence of the significant decrease in liver mass. This, together with increased levels of cytosolic GST, as detected in vitro, could explain the increased intestinal disposition of DNP-SG in the PH1 group. From the current data it could be speculated that iv administration of 30 μmol/kg b.w. of CDNB and subsequent formation of DNP-SG did not saturate intestinal Mrp2 capability, as increased production of the conjugate in PH1 animals led to higher intestinal secretion. In support of this possibility we previously observed that saturation of DNP-SG transport in the isolated intestinal sac model occurred at a excretion rate of about 20 nmol/min/g of tissue (Mottino et al., 2001) and, according to the current data, the maximal excretion rate registered in PH1 group was 1 nmol/min/g of tissue.
Data from the current experiments show that intestinal excretion of DNP-SG represents less than 10% of biliary excretion in sham rats. However, because of the impairment in intrahepatic formation of the conjugated derivative of CDNB in PH1 animals, cumulative intestinal excretion of DNP-SG by 60 min was about the same as biliary excretion, which in turn was about one-tenth that of sham rats (see insets in Figs. 1A and 1B). Based on the data in Table 1, the whole organ contribution to DNP-SG disposition could be estimated as about the same for jejunum and liver in PH1 rats. In contrast, seven days after surgery, liver mass was totally restored in hepatectomized animals and cumulative biliary excretion of DNP-SG was about half the sham value when expressed per g of tissue. In consequence, the estimated overall capacity for DNP-SG excretion in PH7 rats was substantially recovered when compared with PH1 animals. We postulate that the intestine may represent an organ of relevance for metabolism of CDNB and posterior secretion of DNP-SG shortly after hepatectomy. This tissue may thus contribute to attenuate toxicity of compounds incorporated systemically, until liver mass and intrinsic metabolic capability are restored. Our data contrast with previous findings by Dietrich et al. indicating an impairment in intestinal expression of Mrp2 in bile duct-ligated rats as well as in patients with biliary obstruction (Dietrich et al., 2004). These authors demonstrated an inhibitory action of interleukin 1 (IL-1), released in response to the liver inflammatory process, on intestinal Mrp2 expression. It is known that downregulation of hepatic transporters involves cytokine cascades as initiating mediators in different animal models of cholestasis (Trauner et al., 1999). It is possible that differences in Mrp2 expression in intestine in response to bile duct ligation vs. 70–75% liver resection depend in part on the magnitude in cytokines production and systemic release between the two experimental models. In support of this possibility, it was recently reported a significantly increased IL-6 plasma levels in 90% hepatectomized rats but not in 70% hepatectomized rats, in association with a decrease in expression of hepatic Mrp2 (Chang et al., 2004).
In conclusion, though intestinal Mrp2 expression was preserved in hepatectomized rats, the increased conjugation of CDNB, leading to higher availability of the Mrp2 substrate for its subsequent excretion, may partially compensate for liver dysfunction, particularly shortly after surgery, while liver capability is recovering.
ACKNOWLEDGMENTS
This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas and Fundación Antorchas, Argentina. We express our gratitude to Drs. J. Elena Ochoa and José M. Pellegrino for their invaluable technical assistance.
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