Institute of Experimental Physiology, National University of Rosario, S2002LRL-Rosario, Argentina
1 To whom correspondence should be addressed at Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531, S2002LRL-Rosario, Santa Fe, Argentina. Fax: 54-341-4399473. E-mail: amottino{at}unr.edu.ar
Received September 20, 2004; accepted November 29, 2004
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ABSTRACT |
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Key Words: Mrp2; 1-chloro-2,4-dinitrobenzene; dinitrophenyl-S-glutathione; liver regeneration; glutathione; glutathione-S-transferase.
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INTRODUCTION |
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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 (König 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.
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MATERIALS AND METHODS |
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Animals and surgical procedures. Adult Male Wistar rats (320370 g) were used throughout. Animals had free access to food and water and received humane care as outlined in the NIH guidelines for the Care and Use of Laboratory Animals. Partial hepatectomy (PH) was performed according to the procedure of Higgins and Anderson (1931) under ether anesthesia. A midline incision was made in the subxyphoid area and the median, right, and left lateral lobes were removed (7075% of the liver). Control rats were subjected to a sham operation with gentle manipulation of the liver lobes. Experiments were performed 1, 2, or 7 days after surgery (PH1, PH2, and PH7 groups, respectively) according to:
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.
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RESULTS |
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DISCUSSION |
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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 (König 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 1ß (IL-1ß), 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. 7075% 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.
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ACKNOWLEDGMENTS |
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