* Human Physiology Area, Litoral National University, Institute of Experimental Physiology (IFISE)-CONICET, National University of Rosario, and
Bromatology and Nutrition Area, Litoral National University, Argentine
Received November 28, 2003; accepted January 13, 2004
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ABSTRACT |
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Key Words: aluminum; liver; biliary function; multidrug resistanceassociated protein 2.
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INTRODUCTION |
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Al has the potential to be toxic for humans. Patients on dialysis (Alfrey et al., 1976) or on long-term treatment with total parenteral nutrition (Klein, 1993
) have been shown to accumulate this metal in different organs. The human toxicological effects include encephalopathy (Alfrey et al., 1976
), bone disease (Ward et al., 1978
), and anemia (Short et al., 1980
). Finally, Al is a possible contributing factor in Alzheimer's disease (Campbell, 2002
).
It has been demonstrated in experimental animals that Al exposure has an important impact on liver function. Rats subchronically intoxicated with Al (5 mg/kg body weight/day for 114 days, iv) increased serum bile salts, which was associated with a reduction in bile flow (Klein et al., 1988) and an alteration in the glycine-to-taurine bile salt ratio (Klein et al., 1989a
). A reduction in cytochrome P450 levels in microsomes has also been described (Jeffery et al., 1987
). Alterations of bile secretory function due to chronic Al exposure have not been studied yet. They could be considerably different from those occurring under short-term exposure, since exacerbation of the deleterious effect, due to hepatic accumulation of Al or other potentially toxic biliary constituents, or, conversely, compensatory mechanisms may occur. Therefore, using a rat model, we have studied the interaction between chronic exposure to Al and bile secretory function, with special emphasis on organic anion biliary transport.
Bile formation and biliary excretion of cholephilic xenobiotics are determined by the ability of the hepatocyte to transport solutes. Several transport systems have been identified both in the sinusoidal and in the canalicular membranes, and most of them have been functionally characterized by molecular cloning (Jansen, 2000). The multidrug resistanceassociated protein 2 (Mrp2), located in the canalicular membrane, mediates the rate-limiting step in the biliary secretion of different organic anions, including glutathione (GSH)-S-conjugates of leukotriene-C4 or bromosulphophthalein (BSP), glucuronide conjugates of bilirubin, estrogens, and bile salts, and both reduced and oxidized GSH (Konig et al., 1999
; Paulusma et al., 1999
). Excretion of the last two compounds has been shown to be a driving force in the so-called "bile saltindependent fraction of the bile flow" (Ballatori and Truong, 1992
). Therefore, our second aim was to investigate the functional status of Mrp2, due to its dual, key role both in the generation of bile flow and in the biliary transport of organic anions.
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MATERIALS AND METHODS |
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After treatment, rats from control and treated groups were sacrificed by exsanguination, and samples of liver were obtained to determine lipid peroxidation; hepatic GSH content; glutathione-S-transferase (GST), catalase (CAT), and glutathione peroxidase (GSH-Px) activities; and Mrp2 protein levels. Another set of animals from both experimental groups was used to perform pharmacokinetic studies. Finally, a different group of rats was used to study the transport activity of Mrp2.
Al content in plasma and liver was determined by electrothermical atomic absorption spectroscopy (graphite oven and Perkin-Elmer Spectrometer 5000, Boston, MA, USA).
Oxidative stress measurement.
The amount of aldehydic products generated by lipid peroxidation in liver tissue was quantified by the thiobarbituric acidreactive substances (TBARS) colorimetric method of Ohkawa et al. (1979). The colored products resulting from the reaction between TBA and the hydrolyzed, peroxidized lipids were extracted with n-butanol, and the color was measured at 532 nm. TBARS were expressed in terms of malondialdehyde (MDA) levels (nmoles/g liver weight) by using a standard curve of 1,1,3,3-tetramethoxypropane, which is converted mole for mole into MDA.
Liver homogenates in 5% TCA were used to measure total GSH levels as the main nonprotein sulphidryl compounds, as described previously (Ellman, 1959). GST, CAT, and GSH-Px activities were assessed in liver cytosolic fractions. The cytosol fraction was obtained by ultracentrifugation (100,000 x g) of liver homogenates (20%, w/v) prepared in 1 mM EDTA, 30 mM Na2HPO4, and 250 mM sucrose buffer, pH 7.4. GST activity was assayed using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate (Goldstein and Combes, 1966
) and expressed as nmol/min/mg protein. CAT and GSH-Px activities were assessed by the methods of Beers and Sizer (1952)
and Paglia and Valentine (1967)
, respectively. Protein levels in each fraction were determined as previously described (Lowry et al., 1951
).
Basal bile flow and pharmacokinetic studies.
At 90 days of treatment, rats were anesthetized with acepromazine (1 mg/kg body weight, ip) and ketamine (100 mg/kg body weight, ip). The bile duct and the right femoral vein were cannulated with polyethylene tubing PE 10 and PE 50, respectively. Body temperature was maintained throughout at 37 ± 0.5°C with a heating lamp to avoid hypothermic variations of bile secretory function. After 20 min of stabilization, spontaneously secreted bile was collected for 15 min in preweighed vials and on ice. Bile flow was determined gravimetrically, by assuming a bile density of 1 g/ml, and expressed as µl/min/100 g body weight. Biliary excretion outputs were calculated as the product of bile flow and solute concentration values.
After these basal studies, pharmacokinetic studies were carried out to characterize the functional status of the nonbile salt organic anion transport systems by using the Mrp2 substrate BSP; for this purpose, rats of both groups received a BSP injection (6 mg/100 g body weight, iv). Blood samples were collected every 2 min over a 30-min time period, and bile samples were collected every 10 min over a 60-min time period. Plasma and bile BSP concentrations were determined spectrophotometrically at 580 nm, after the addition of 0.5 M NaOH. The percentage of biliary BSP recovery was estimated by the percentage ratio between the dye excreted in bile samples throughout the experimental period and the amount of BSP injected.
In vivo pharmacokinetic studies of BSP plasma decay were carried out to assess the fractional transfer rates for the transport of the dye from plasma to liver (hepatic uptake, r12), liver to plasma (sinusoidal efflux, r21), and liver to bile (canalicular excretion, r3), and other pharmacokinetic parameters. For this purpose, the plasma dye concentration data (Cp), plotted against time (t), were fitted by a least-squares curve-fitting technique to the following biexponential equation:
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Preliminary triexponential fits failed to show any improvement in the goodness of fit, as judged by the Akaike's criterion. Therefore, a two-compartmental model with an open-ended biliary outflow was considered physiologically realistic for compartmental analysis.
Volume of distribution of the dye is given by the following equation:
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Plasma clearance (Clp) was calculated using the following equation:
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Half-times for the rapid (t1/2 ) and the slow (t1/2 ß) phases of BSP plasma decay were calculated as follows:
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The fractional transfer rates were calculated as previously described (Richards et al., 1959) using the following equations:
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Free and GSH-conjugated BSP (GSH-BSP) were separated from each other by reverse-phase high-performance thin-layer chromatography (RP-HPTLC), by adapting the method for HPLC of Snel et al. (1993). Briefly, samples and standards (BSP and GSH-BSP) (< 1 µl) were spotted on RP-HPTLC plates (Sigma-Aldrich Corp., St. Louis, MO). The mobile phase consisted of 10 mM sodium phosphate buffer, pH 6: acetonitrile (80:30, v:v). Chromatography was performed with an Eastman Chromagram chamber plate set (Distillation Products Industries, Rochester, NY). After 1 h, bands were visualized under NH3 atmosphere and immediately digitized. Densitometry of resulting bands was performed by image analysis using GelPro Analyzer 3.0 software (Cybernetics, Silver Spring, MA, USA).
Mrp2 protein levels.
Expression of Mrp2 was assessed by Western blotting. Samples of liver tissue were taken from multiple liver lobes immediately after perfusion with ice-cold saline, snap-frozen in liquid nitrogen, and stored at 70°C until assay. Immunoblotting of Mrp2 was carried out on mixed membrane fractions obtained as described previously (Meier et al., 1984). Preparations were loaded onto 10% SDS-polyacrylamide gels and subjected to electrophoresis. After electrotransfer, nitrocellulose membranes were probed with a monoclonal, mouse anti-Mrp2 (MC-206; Kamiya Biomedical Co., Seattle, WA) at 1:2000 dilution for 1 h. The immune complex was detected by incubation with HRP-linked secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) at a 1:2000 dilution for 1 h. Immunoreactive bands were detected using a chemiluminescence kit (ECL+Plus, Amersham Pharmacia Biotech) exposed to Bio-Max MR-2 films (Sigma-Aldrich Corp.) for 5 min and quantified by densitometry (Shimadzu CS-9000, Shimadzu Corp., Kyoto, Japan).
Transport activity of Mrp2.
Mrp2 transport activity was evaluated by administering CDNB (10 µmol/kg body weight, iv, in saline). CDNB permeates the sinusoidal membrane freely and is further conjugated with GSH by the GST system, so that its GSH derivate, dinitrophenyl-S-GSH (DNP-SG), is selectively excreted into bile by Mrp2. After CDNB administration, bile samples were collected at 10-min intervals for 60 min, and the biliary excretion rate of DNP-SG was calculated as the product of bile flow and the DNP-SG biliary concentration, as measured spectrophotometrically at 335 nm.
Statistical analysis.
All the values were expressed as mean ± SE. Variables were statistically compared by the Student's t-test. A value of p < 0.05 was considered significantly different.
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RESULTS |
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The levels of lipid peroxidation in liver tissue and the status of hepatic antioxidant defenses following Al treatment are given in Table 1. Lipid peroxidation, as measured in terms of TBARS, was significantly increased in Al (+) rats. Conversely, the total GSH content and the activities of the antioxidant enzymes GST, GSH-Px, and CAT were significantly reduced in Al (+) rats.
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DISCUSSION |
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Exposure to Al for 3 months induced a significant (4-fold) increase in hepatic Al content, similar to previous studies where Al was administered parenterally (Bertholf et al., 1989
; El-Maraghy et al., 2001
). This finding was attributed to the presence of Al in hepatic giant cells (Bertholf et al., 1989
). The hepatic Al accumulation was accompanied by a decrease in bile flow and in bile salt secretion, a primary driving force for bile formation, suggesting that, at least in part, bile flow decrease was due to a reduction in its bile saltdependent fraction. A similar result was observed when Al was administered subchronically (i.e., over 14 days; Klein et al., 1988
), but not when Al was given enterally, a fact attributed to the lack of Al accumulation in liver when using this way of administration (Klein et al., 1989b
). Our finding that bile secretory function was similarly affected 90 days after Al administration as it was with a shorter administration period (14 days) indicates that no further compensatory mechanism takes place to counteract the alterations in the bile secretory mechanisms occurring early after Al exposure.
Whereas the alterations in the bile salt secretory function induced by Al intoxication had been evaluated previously (Klein et al., 1988), no reported data are available on the changes induced by Al on the hepatic handling of nonbile salt organic anions. This information is highly relevant, though, since these transport systems are involved in the biliary elimination of potentially toxic endo- and xenobiotics, including bilirubin, glucuronidated and sulfated bile salts, leukotriene C4, medicaments, and numerous dietary constituents (Klaassen, 2002
; Takikawa, 2002
).
To determine possible alterations in these transport systems induced by Al administration, we evaluated the hepatic handling of the model organic anion BSP, a relatively nontoxic organic anion that is widely used as an indicator of liver function. For this purpose, kinetics analysis of the plasma disappearance of dye, which provides a quantitative estimation of the efficiency by which transport events are functioning in the hepatocyte in vivo, was carried out. BSP is selectively taken up by the liver through an Na+-independent transport mechanism (mediated by the organic anion transporting polypeptide [OATP] families), conjugated with GSH within the hepatocytes by GST, and subsequently excreted into bile by the multispecific, ATP-dependent canalicular transporter Mrp2 (Takikawa, 2002). The pharmacokinetic analysis indicated that both sinusoidal uptake and canalicular excretion of the dye were impaired by Al administration, as assessed by the decrease in the fractional transfer rates for the transport of the dye from plasma to liver (r12) and from liver to bile (r3), respectively (Table 3). The alteration in these transport steps, particularly the impairment of the canalicular transfer of the dye (which is the rate-limiting step in the overall transfer of BSP from blood to bile), explains the decrease in the total excretion of the dye (Table 4). Changes in BSP intrahepatic conjugation, a metabolic step that facilitates its canalicular transfer (Barnhart and Combes, 1976
), seem not to account for the alteration of the transport step, as no change in the conjugated-to-total BSP ratio was observed in bile (Table 4).
Although conjugating activity of GST, the enzyme involved in GSH-S conjugation of the dye, was impaired by Al (Table 1), increased transit time throughout the hepatocyte due to defective excretion, which increase chances for dye conjugation, may have compensated for the decrease in the conjugating activity. However, a differential effect of Al on transport efficiency of free and conjugated BSP cannot be ruled out. For example, an impairment of bile salt output, like that occurring in Al-treated rats, may selectively decrease free BSP biliary output (Gregus et al., 1980), thus explaining the lack of change in BSP output under conditions of impaired BSP conjugation. Therefore, the alteration in the canalicular transfer of the dye is more likely accounted for by an alteration in the activity/content of its canalicular transporter Mrp2. Western blot analysis and the further assessment of functional activity of Mrp2 confirmed this assertion. Indeed, the hepatic level of Mrp2 was decreased in Al (+) rats, which was accompanied by a decreased excretion of the specific Mrp2 substrate DNP-SG.
The underlying mechanisms by which Al affects bile production in general and organic anion transport status in particular are far from being completely understood and cannot be addressed using our results. However, our data point to the possible involvement of oxidative stress in Al-induced hepatotoxicity (Table 1), as indicated by an increase in TBARS, a finding causally linked to enhanced liver lipid peroxidation, and a decrease in the levels of antioxidant defenses, including GSH and the antioxidant enzymes CAT (Abubakar et al., 2003) and GSH-Px (El-Maraghy et al., 2001
). These results are in keeping with other studies showing that even lower (ip) Al doses and shorter administration periods induce a significant increase in hepatic reactive oxygen species (Abubakar et al., 2003
). The effect is associated with decreased levels of hepatic antioxidant defenses including GSH (Abubakar et al., 2003
; El-Maraghy et al., 2001
), CAT (Abubakar et al., 2003
.), and GSH-Px (El-Maraghy et al., 2001
). Furthermore, the decrease in GST activity we have reported could be a contributing factor since this enzyme has GSH-Px activity and is instrumental in detoxifying lipid hydroperoxides (Mari and Cederbaum, 2001
).
Oxidative stress alters the expression of canalicular transporters at a post-transcriptional level, serving as a potential mechanism of alteration of biliary secretory function. Indeed, incorporation of oxidizing agents into the perfusate of the isolated, perfused rat liver leads to cholestasis accompanied by a loss of immunoreactive Mrp2 from the canalicular membrane due, in turn, to internalization of the transporter in subapical vesicles (Schmitt et al., 2000). Sustained internalization as a consequence of long-lasting oxidative stress, likely to occur in our Al (+) rats, may lead to delivery of Mrp2 to the lysosomal compartment followed by degradation, as was shown to occur late in lipopolysaccharide-induced cholestasis (Kubitz et al., 1999
; Trauner et al., 1997
). This may explain our results showing a decreased expression of Mrp2 in Al (+) rats. A balance between exocytic insertion and endocytic internalization of canalicular transporters exists, and this balance may be disrupted either by inhibiting vesicle-mediated insertion of newly synthesized transporters or by stimulating their internalization, or both. Our results showing that Al (+) rats have an inhibited biliary excretion of protein and cholesterol, two compounds thought to reach bile mainly via a microtubule-dependent, vesicular mechanism (LaRusso, 1984
; Marzolo et al., 1990
), make the first possibility likely. However, a more direct action of Al in decreasing Mrp2 synthesis and/or in increasing Mrp2 degradation can not be excluded.
Berlyne et al. (1972) proposed that Al exerts its hepatotoxic effect by affecting protein synthesis, an assertion confirmed by others showing that the hepatic levels of numerous phase Idetoxifying enzyme proteins decreased after Al exposure (Fulton and Jeffery, 1994
; Jeffery et al., 1987
). The reduction in the hepatic levels of several enzymes reported in this study (Table 1) confirms and extends this concept to phase IIdetoxifying enzymes (GST) and other antioxidant enzymes (CAT and GSH-Px). The second possibility, the activation of lysosomal degradation of proteins by Al, seems more unlikely, since this metal was shown to inhibit rather than to activate the lysosomal proton pump (Zatta et al., 2000
). Regardless of the mechanism involved, the Al-induced impairment of the expression/function of Mrp2 and, presumably, other transport proteins involved in transhepatocellular transport of cholephilic compound may explain, at least in part, the cholestatic effect of this metal. Indeed, Mrp2 mediates canalicular secretion of GSH (Paulusma et al., 1999
), a key determinant of the so-called bile saltindependent fraction of the bile flow (Ballatori and Truong, 1992
).
In conclusion, our results show that chronic Al intoxication induces cholestasis and impairs hepatic transport of organic anions by decreasing both sinusoidal uptake and canalicular excretion. This latter event, which is the rate-limiting step in the overall transport from plasma to bile, is accounted for by a decreased expression of the main transporter involved in this process, Mrp2. Clearly, additional studies are needed to clarify the exact mechanism(s) through which Al exerts these deleterious effects.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Instituto de Fisiología Experimental (IFISE)-CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRL Rosario, Argentina. Fax: +54 341-4399473. E-mail: mcarill{at}fbioyf.unr.edu.ar
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