Biliary Secretory Function in Rats Chronically Intoxicated with Aluminum

Marcela A. Gonzalez*, Marcelo G. Roma{dagger}, Claudio A. Bernal{ddagger}, Maria de Lujan Alvarez{dagger} and Maráia C. Carrillo{dagger},1

* Human Physiology Area, Litoral National University, {dagger} Institute of Experimental Physiology (IFISE)-CONICET, National University of Rosario, and {ddagger} Bromatology and Nutrition Area, Litoral National University, Argentine

Received November 28, 2003; accepted January 13, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of a chronic aluminum (Al) exposure on biliary secretory function, with special emphasis on hepatic handling of non–bile salt organic anions, was investigated. Male Wistar rats received, intraperitoneally, either 27 mg/kg body weight of Al, as Al hydroxide [Al (+) rats], or the vehicle saline [Al (–) rats] three times a week for 3 months. Serum and hepatic Al levels were increased by the treatment (~9- and 4-fold, respectively). This was associated with enhanced malondialdehyde formation (+110%) and a reduction in GSH content (–17%) and in the activity of the antioxidant enzymes catalase (–84%) and GSH peroxidase (–46%). Bile flow (–23%) and the biliary output of bile salts (–39%), cholesterol (–43%), and proteins (–38%) also decreased. Compartmental analysis of the plasma decay of the model organic anion bromosulphophthalein revealed that sinusoidal uptake and canalicular excretion of the dye were significantly decreased in Al (+) rats (–53 and –43%, respectively). Expression of multidrug resistance–associated protein 2 (Mrp2), the main, multispecific transporter involved in the canalicular excretion of organic anions, was also decreased (–40%), which was associated with a significant decrease in the cumulative biliary excretion of the Mrp2 substrate, dinitrophenyl-S-glutathione (–50%). These results show that chronic Al exposure leads to oxidative stress, cholestasis, and impairment of the hepatic handling of organic anions by decreasing both sinusoidal uptake and canalicular excretion. The alteration of the latter process seems to be causally related to impairment of Mrp2 expression. We have addressed some possible mechanisms involved in these deleterious effects.

Key Words: aluminum; liver; biliary function; multidrug resistance–associated protein 2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aluminum (Al) is one of the most abundant metals in the earth's crust. Human exposure to Al has been increasing over the last decades. This element appears mainly in food products and in drinking water derived from both natural sources and treatment methods (Yokel and McNamara, 2001Go). The major contributors to the current human exposure are food products such as grain products, processed cheese, and salt (Nieboer et al., 1995Go). Also, pharmaceutical products and medical treatments can be an important way to incorporate high levels of Al, either as an active compound or as a contaminant. For example, Al hydroxide is used as an antacid and as a phosphate binder in vaccines, and antiperspirant use has been shown to contribute significantly to Al bioavailability (Yokel and McNamara, 2001Go). Finally, bioavailability by inhalation of airborne soluble Al was shown to be about 1.5% in the industrial environment (Yokel and McNamara, 2001Go).

Al has the potential to be toxic for humans. Patients on dialysis (Alfrey et al., 1976Go) or on long-term treatment with total parenteral nutrition (Klein, 1993Go) have been shown to accumulate this metal in different organs. The human toxicological effects include encephalopathy (Alfrey et al., 1976Go), bone disease (Ward et al., 1978Go), and anemia (Short et al., 1980Go). Finally, Al is a possible contributing factor in Alzheimer's disease (Campbell, 2002Go).

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 1–14 days, iv) increased serum bile salts, which was associated with a reduction in bile flow (Klein et al., 1988Go) and an alteration in the glycine-to-taurine bile salt ratio (Klein et al., 1989aGo). A reduction in cytochrome P450 levels in microsomes has also been described (Jeffery et al., 1987Go). 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, 2000Go). The multidrug resistance–associated 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., 1999Go; Paulusma et al., 1999Go). Excretion of the last two compounds has been shown to be a driving force in the so-called "bile salt–independent fraction of the bile flow" (Ballatori and Truong, 1992Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatments.
Male Wistar rats (300–360 g) were used throughout. All the experimental protocols were performed according to the Guide for Care and Use of Laboratory Animals (National Institutes of Health publication 25-28, revised 1996). Rats were housed with a 12 h light/dark cycle and controlled temperature (21–25°C), and they were fed ad libitum with a standard rodent diet and water. After 7–10 days of acclimatization, rats were assigned randomly to two different groups of similar mean body weight. One group received Al (as Al hydroxide) at the dose of 27 mg/kg body weight, ip, in 0.5 ml of saline, three times a week for 90 consecutive days [Al (+) rats]. A second group received the vehicle alone with an identical administration schedule [Al (–) rats]. This protocol was designed according to the experimental model of Al intoxication developed in the Department of Pathological Anatomy, School of Odontology, University of Buenos Aires (Degiorgis et al., 1987Go). Al hydroxide was used for Al supply because it is one of the main Al-containing components of drinking water (Fulton et al., 1989Go) and is a major ingredient in antacid and vaccine formulations (Lione, 1985Go).

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 acid–reactive substances (TBARS) colorimetric method of Ohkawa et al. (1979)Go. 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, 1959Go). 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, 1966Go) and expressed as nmol/min/mg protein. CAT and GSH-Px activities were assessed by the methods of Beers and Sizer (1952)Go and Paglia and Valentine (1967)Go, respectively. Protein levels in each fraction were determined as previously described (Lowry et al., 1951Go).

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 non–bile 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:

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:

where D is the dose of BSP administered.

Plasma clearance (Clp) was calculated using the following equation:

where AUC is the area under the curve and can be calculated using the following equation:

Half-times for the rapid (t1/2 {alpha}) and the slow (t1/2 ß) phases of BSP plasma decay were calculated as follows:

The fractional transfer rates were calculated as previously described (Richards et al., 1959Go) using the following equations:

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)Go. 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., 1984Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After 90 days of Al treatment, no animal deaths occurred and no evident toxic symptoms were observed in either Al (+) or Al (–) rats. At the end of the treatment, the mean body weight of Al (+) rats was not statistically different from that of Al (–) rats [337 ± 4 g vs. 335 ± 10 g, respectively; p > 0.05]. Increases in the Al levels of plasma and liver were observed after the 90 days of Al administration. Plasma Al concentrations increased from 9 ± 4 µg/l (control rats) to 750 ± 50 µg/l, and the hepatic Al content increased from 26 ± 9 µg/g liver weight (control rats) to 122 ± 12 µg/g liver weight.

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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TABLE 1 Effect of A1 Treatment on Hepatic TBARS Content and the Status of Hepatic Antioxidant Defenses

 
Table 2 gives the basal bile flow and the basal excretion rate of different bile constituents after 90 days of Al treatment. Bile flow was decreased by 23% in Al (+) rats. Similarly, the biliary outputs of proteins, cholesterol, and bile salts were significantly lower in Al (+) rats compared with Al (–) rats.


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TABLE 2 Effect of A1 Treatment on Bile Flow and Biliary Output of Several Biliary Solutes

 
The efficiency of the different steps involved in the hepatic handling of the non–bile acid organic anion model, BSP, was assessed by pharmacokinetic analysis. Plasma decay of BSP was slower in Al (+) rats, as shown by the increase in the half-times for the rapid and slow phases of plasma decay (Table 3). Al treatment reduced significantly the plasma clearance of BSP (–59%), which was accompanied by a significant reduction in the fractional rate of hepatic BSP uptake (r12, –53%) and in the fractional rate of the biliary excretion of the dye (r3, –43%). A clear tendency existed for the fractional rate of hepatic BSP reflux to be lower, but, due to high interindividual variability, the difference did not achieve statistical significance (Table 3).


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TABLE 3 Effect of A1 Treatment on Pharmacokinetic Parameters Derived from the Bicompartmental Analysis of Plasma Decay of BSP

 
The model organic anion BSP was selectively cleared by the liver, conjugated with GSH within the hepatocytes in a process catalyzed by a GST isoenzyme, and subsequently excreted into bile. Upon analyzing the biliary excretion of total, free, and conjugated BSP, a decrease in the total excretion of the dye, without any change in the conjugated-to-total BSP ratio, was observed in Al (+) rats (Table 4).


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TABLE 4 Effect of A1 Treatment on BSP Biliary Excretion

 
Since the canalicular transfer of BSP, the rate-limiting step in its overall transfer from plasma to bile, was significantly impaired by Al treatment, we assessed both expression and function of Mrp2, the main canalicular carrier involved in this process. Al induced a decrease in the Mrp2 levels (–40%), as quantified by densitometric analysis of the Western blotting bands (Fig. 1). Transport activity of this protein was further evaluated in vivo by analyzing the time course of the biliary excretion of the Mrp2 substrate DNP-SG. DNP-SG is produced intracellularly by GST-mediated conjugation of CDNB, which freely permeates the sinusoidal membrane, with GSH; this avoids the influence of changes in basolateral transporter activity on its secretory process, so that DNP-SG's biliary excretion reflects either or both GST-conjugating activity or Mrp2 transport efficiency. Figure 2 shows that Al treatment both reduced and delayed the biliary secretion of DNP-SG. As a consequence, both the peak of DNP-SG biliary excretion and cumulative DNP-SG biliary secretion (throughout the experimental period) were decreased by 50% compared with Al (–) groups. Since GST-conjugating activity towards CDNB was only decreased by 35% (Table 1), changes in CDNB conjugation cannot fully account for the decrease in DNP-SG output, with the remaining drop due to impaired transport activity.



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FIG. 1. Upper panel: Western blotting of Mrp2 protein content in total plasma membrane of Al (–) and Al (+) rats (bands of two representative animals from each group). Lower panel: Densitometric analysis of Mrp2 immunoreactive bands in Al (–) rats (open bar) and in Al (+) rats (solid bar) is also shown. *p < 0.05.

 


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FIG. 2. Effects of Al on the time course of biliary excretion rate of DNP-SG. Inset shows cumulative DNP-SG biliary excretion throughout the experimental period in Al (–) rats (open bar) and in Al (+) rats (solid bar).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was conducted to elucidate the mechanisms involved in the impairment of bile secretory function induced by chronic administration of Al to rats.

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., 1989Go; El-Maraghy et al., 2001Go). This finding was attributed to the presence of Al in hepatic giant cells (Bertholf et al., 1989Go). 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 salt–dependent fraction. A similar result was observed when Al was administered subchronically (i.e., over 14 days; Klein et al., 1988Go), 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., 1989bGo). 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., 1988Go), no reported data are available on the changes induced by Al on the hepatic handling of non–bile 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, 2002Go; Takikawa, 2002Go).

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, 2002Go). 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, 1976Go), 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., 1980Go), 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., 2003Go) and GSH-Px (El-Maraghy et al., 2001Go). 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., 2003Go). The effect is associated with decreased levels of hepatic antioxidant defenses including GSH (Abubakar et al., 2003Go; El-Maraghy et al., 2001Go ), CAT (Abubakar et al., 2003Go.), and GSH-Px (El-Maraghy et al., 2001Go). 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, 2001Go).

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., 2000Go). 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., 1999Go; Trauner et al., 1997Go). 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, 1984Go; Marzolo et al., 1990Go), 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)Go proposed that Al exerts its hepatotoxic effect by affecting protein synthesis, an assertion confirmed by others showing that the hepatic levels of numerous phase I–detoxifying enzyme proteins decreased after Al exposure (Fulton and Jeffery, 1994Go; Jeffery et al., 1987Go). The reduction in the hepatic levels of several enzymes reported in this study (Table 1) confirms and extends this concept to phase II–detoxifying 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., 2000Go). 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., 1999Go), a key determinant of the so-called bile salt–independent fraction of the bile flow (Ballatori and Truong, 1992Go).

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.


    ACKNOWLEDGMENTS
 
This work was supported by a research grant from the Universidad Nacional del Litoral, Cursos de Acción para la Investigación y Desarrollo (CAI+D 2002, Código Proy, 17–116), Secretaría de Ciencia y Técnica, UNL. We thank Stella Mahieu, María del Cármen Contini, and researchers from IFISE (Instituto de Fisiología Experimental) for their valuable collaboration in the use of technical methodologies.


    NOTES
 

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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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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