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Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, Rosario, Argentina
(Received January 6, 2006; accepted March 17, 2006)
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Abstract |
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; Bouchard et al., 1993; Jacquemin et al., 1993; Crocenzi et al., 2001). The cholestatic properties of estrogens are suggested to be mediated by their D-ring glucuronide conjugates, such as estradiol 17ß-glucuronide. (Meyers et al., 1980; Vore, 1987). In contrast, A-ring (3-glucuronide) metabolites are choleretic (Meyers et al., 1980). Therefore, patients with obstetric cholestasis excrete more D- versus A-ring glucuronides in urine (Adlercreutz et al., 1974), suggesting that D-ring glucuronides may be involved in its pathogenesis. Moreover, in a recent work, our group demonstrated that ursodeoxycholate, a bile salt that prevents estrogen cholestasis, decreases hepatic formation of EE 17ß-glucuronide in the rat (Sanchez Pozzi et al., 2003).
Galactosamine (GAL) is a hepatotoxic drug that markedly depletes hepatic UDP-glucuronic acid (UDP-GA) levels, whereas extrahepatic UDP-GA is minimally affected (Gregus et al., 1988). At a dose of 350 mg/kg b.wt. or higher, GAL induces liver injury, as indicated by a pronounced increase in the serum levels of enzyme markers of hepatic damage, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) (Decker and Keppler, 1972; Markov et al., 1991; Gujral et al., 2003). We postulate that if EE 17ß-glucuronide formation is a relevant step in the pathogenesis of EE-induced cholestasis, a low dose of GAL, enough to decrease UDP-GA synthesis and yet nontoxic, would prevent the alteration in bile flow induced by the estrogen.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-hydroxysteroid dehydrogenase, sulfatase type H-1 from Helix pomatia, ß-glucuronidase type B-1 from bovine liver, NADPH, and dimethyl sulfoxide were purchased from Sigma Chemical Co. (St. Louis, MO). Tauroursodeoxycholate (TUDC) was a generous gift from Prodotti Chimici e Alimentari S.p.A. (Genoa, Italy). This bile salt was 99% pure when examined by HPLC. All other reagents were of the highest analytical grade and used as supplied. Animals. Adult male Wistar rats weighing 300 to 350 g were used throughout. Animals were maintained on a standard diet and water ad libitum and housed in a temperature- (21-23°C) and humidity- (45-50%) controlled room under a constant 12-h light/dark cycle. All animals received humane care, according to the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health (publication 25-28, revised 1996).
Effect of GAL on Hepatic UDP-GA Levels. Animals were divided into three groups: 1) GAL, injected with GAL (a single 200 mg/kg b.wt. dose, i.p.), 2) GAL + U, injected with GAL (200 mg/kg b.wt.) and uridine (a single 0.9 g/kg b.wt. dose, i.p.), and 3) C, control group injected with saline, vehicle of GAL and U. Rats were sacrificed by exsanguination under sodium pentobarbital anesthesia 1, 2, or 18 h after treatment. Livers were quickly excised and placed in liquid nitrogen. A piece of frozen liver was finely ground in a mortar containing liquid nitrogen. One gram of liver powder was homogenized in 20 ml of precooled 150 mM KCl-10 mM KH2PO4 buffer (pH 7.0). The homogenate was heated in a boiling water bath for 5 min and centrifuged (5000g, 10 min). The supernatant was filtered (0.2 µm; Nalge Nunc International, Rochester, NY) to remove glycogen. A 20-µl aliquot of the filtrate was immediately chromatographed by HPLC using a 5-µm C18 column [Luna C18(2); Phenomenex Inc., Torrance, CA]. The eluent consisted of methanol/water [50:50 (v/v)] containing 10 mM KH2PO4 and 5 mM tetrabutylammonium hydrogen-sulfate (pH 7.0). The flow rate was 1 ml/min. UDP-GA was monitored by UV absorption at 260 nm (Alary et al., 1992).
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). Effect of GAL on Biliary Secretory Function. Animals were divided into six experimental groups: 1) control, receiving the vehicle of EE, propylene glycol (0.5 ml/kg b.wt. s.c.), and the vehicle of both GAL and uridine, saline (0.5 ml/kg b.wt. i.p.), for 5 consecutive days. 2) EE, administered daily with EE (5 mg/kg b.wt. s.c.) and saline for 5 days. 3) EE + GAL, coadministered daily with EE, GAL (200 mg/kg b.wt. i.p.), and saline for 5 days. 4) EE + GAL + U, coadministered daily with EE, GAL, and uridine (0.9 g/kg b.wt. i.p.) for 5 days. 5) GAL, coadministered daily with GAL and propylene glycol for 5 days. 6) U, coadministered daily with uridine and propylene glycol for 5 days.
Surgical procedures were made on the 6th day, i.e., 1 day after administration of the last EE dose. Bile collection started between 9:00 AM and 11:00 AM to minimize the influence of circadian variations on bile flow. Animals were anesthetized and maintained under this condition throughout the experiment. A middle abdominal incision was made, and the common bile duct was cannulated using a PE-10 polyethylene tubing. Tracheal cannulation was systematically performed to remove bronchial secretion induced by the anesthetic. Body temperature was maintained at 37.0-38.5°C with a warming lamp.
Basal Bile Secretion Studies. Thirty minutes after bile duct cannulation, bile was collected for two 15-min periods, one of which was collected in tubes containing 1 ml of 6% 5-sulfosalicylic acid for total glutathione (reduced + oxidized) determination. To evaluate biliary 
secretion, basal bile was collected under liquid Vaseline Petroleum Jelly to avoid equilibration with atmospheric CO2. At the end of bile collection, the animals were sacrificed by exsanguination, and the livers were removed and weighed. Bile flow was determined by gravimetry, assuming a bile density of 1.0 g/ml. Basal bile was assayed for total and individual bile salt content. Total bile salt concentration was assessed using the 3-hydroxysteroid dehydrogenase procedure (Talalay, 1960). Individual bile salts were determined by HPLC (Waters, Milford, MA) as reported (Tietz et al., 1984) using authentic standards. Total glutathione was measured by the recycling method of Tietze (1969). concentration in bile was measured immediately after bile collection in an automated blood-gas analyzer (Compact 1; AVL Medical Instruments AG, Schaffhausen, Switzerland). Biliary excretion rates were calculated as the product between bile flow and biliary concentration. Activities of the enzyme markers of hepatocellular injury, AST (EC 2.6.1.1
[EC]
), ALT (EC 2.6.1.2
[EC]
), and ALP (EC 3.1.3.1
[EC]
), were determined in plasma using commercial kits (Wiener Lab, Rosario, Argentina).
TUDC Infusion Studies. Rats from the same six experimental groups were infused with TUDC to determine the secretory rate maximum (SRM) of this bile salt into bile. TUDC was chosen because of its extremely low toxicity, in contrast to other naturally occurring bile salts, whose apparent maximum transport is largely dependent on their cytotoxicity rather than by saturation of their canalicular transport system (Hardison et al., 1981). The SRM of TUDC was assessed by infusing the bile salt intravenously, dissolved in 2% bovine serum albumin in saline, at stepwise-increasing rates (2.0, 2.5, 5.0, 6.5, 12.0, and 16.0 µmol/min/100 g b.wt.). Each infusion rate was maintained for 20 min, and bile samples were collected every 10 min for 120 min. SRM was calculated as the mean of the three highest consecutive secretory rates recorded over the whole infusion period (Hardison et al., 1981). The bile-salt-independent fraction of bile flow (BSIF) was estimated in these animals by the conventional extrapolation to zero bile salt output of the regression line between bile flow and bile salt output, as stimulated by TUDC infusion (Erlinger, 1994).
Statistical Analysis. Results were expressed as mean ± S.E. One-way analyses of variance, followed by Newman-Keuls test, were performed for multiple comparison among groups. The regression line analysis was done by the least-square method. The significance of the differences between slopes and between y-intercepts was assayed by covariance analysis. Values of p < 0.05 were considered to be statistically significant.
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Results |
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Biliary Excretion of EE Metabolites. Two hours after injection of [3H]EE, cumulative biliary excretion of total [3H] decreased approximately by 10% in GAL-treated rats (C, 74 ± 2%; GAL, 66 ± 2% of the injected dose; p < 0.05) and was restored to C values by U (GAL + U, 75 ± 3% of the injected dose; p < 0.05 versus GAL). A comparative HPLC analysis of untreated bile samples versus samples preincubated with ß-glucuronidase or sulfatase + ß-glucuronidase led to the identification of one peak as a sulfate derivative and the other two as glucuronides. One of the glucuronide peaks was identified as EE 17ß-glucuronide. GAL treatment produced a decrease of approximately 40% in the excretion of this compound (C, 5.9 ± 0.6%; GAL, 3.4 ± 0.4% of the injected dose; p < 0.05), which was restored by U coadministration (GAL + U, 5.8 ± 0.5%; p < 0.05 versus GAL). The excretion of the other glucuronide was also decreased by GAL (C, 13.8 ± 1.3%; GAL, 8.2 ± 0.9%, of the injected dose; p < 0.05) and restored to C values by U administration (GAL + U, 14.0 ± 1.4%; p < 0.05 versus GAL). The excretion of the sulfate derivative was not modified by the treatments (C, 18 ± 2%; GAL, 20 ± 2%; GAL+U, 21 ± 2%).
Serum Markers of Hepatocellular Integrity. As shown in Table 1, neither ALT nor AST was affected by GAL administration, indicating that the protocol used to study GAL action on biliary secretory function was not toxic. Neither of these serum markers was modified by EE, in agreement with previous results (Bouchard et al., 1993; Crocenzi et al., 2001; Sanchez Pozzi et al., 2003). However, EE treatment led to a 40% increase in ALP serum activity, a biochemical marker of cholestasis (Table 1), which was not restored by GAL coadministration.
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Effect of GAL on Basal Bile Flow and Bile Salt Output. Table 1 shows that GAL partially prevented EE-induced reduction in bile flow, whereas uridine coadministration blocked this preventive effect. Table 1 also shows that conjoint EE and GAL treatment partially restored the excretion of the individual bile salts decreased by EE, with excretion rates of cholate and chenodeoxycholate reaching statistical significance. Uridine coadministration blocked the protective effect of GAL only for cholate excretion. The counteracting effect of uridine was not evident for total bile salt secretion, most probably due to a nonsignificant increase in the biliary excretion of muricholate, the main biliary bile salt in the rat.
Effect of GAL on the Impairment of the Hepatic Handling of TUDC Induced by EE. Table 2 shows the effect of the different treatments on SRM of the nontoxic bile salt TUDC. This parameter was diminished by EE treatment (-88%) and partially restored by GAL coadministration (+125% compared with EE group). Uridine coadministration abolished GAL effects.
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Effect of GAL on the Decrease of the BSIF Induced by EE. As can be seen in Fig. 2, a significant decrease in BSIF, as estimated by the y-intercept of the regression line between bile flow and bile salt output under TUDC infusion, was found in EE-treated rats (0.56 µl/min/g liver wt. versus 1.93 µl/min/g liver wt. in control; p < 0.05). GAL partially prevented this impairment (1.11 µl/min/g liver wt., p < 0.05 versus EE), and U coadministration blocked GAL effect (0.69 µl/min/g liver wt., p < 0.05 versus GAL). None of the treatments modified the slope of the regression lines, which represents the choleretic efficiency of the secreted bile salts.
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, compounds thought to be the major driving forces for BSIF (Hardison and Wood, 1978; Ballatori and Truong, 1992), were assessed. As shown in Table 1, basal biliary excretions of both glutathione and were significantly diminished by EE pretreatment. Impairment in the biliary excretion of was significantly but only partially prevented by GAL. Although there was a trend toward increased glutathione output in the EE-treated group receiving also GAL, the differences were not statistically significant; nevertheless, the eventual recovery of this parameter was insignificant compared with control values.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Some evidence points toward EE 17ß-glucuronide as the putative mediator. Indeed, this and other D-ring glucuronide conjugates such as estradiol 17ß-glucuronide induce acute cholestasis (Meyers et al., 1980; Vore, 1987). Furthermore, in a previous work, we demonstrated that ursodeoxycholate, a bile salt that prevents EE cholestasis, decreases the formation of the 17ß-glucuronide derivative of EE (Sanchez Pozzi et al., 2003).
In the present work, we provide further, more compelling evidence to support the role of glucuronidation in the development of EE cholestasis. We administered GAL, a compound that selectively decreases hepatocyte UDP-GA levels, and found a protective effect in cholestasis. Due to its toxic nature, it was important to rule out any harmful effect of GAL at the dose administered here. Indeed, at doses of 350 mg/kg b.wt. or higher, this compound produces hepatotoxicity, as a consequence of the decrease in mRNA synthesis as a result of shortage of uridine nucleotides. This decrease is fully reverted by the administration of uridine (Keppler et al., 1974). In our experiments, we used a daily dose of 200 mg/kg b.wt. in a compromise between UDP-GA depletion and toxicity. Serum levels of the enzyme markers of hepatotoxicity (ALT and AST) were not affected by this dose of GAL, excluding overt toxicity in our protocol. In addition, none of the functional parameters evaluated such as bile flow, biliary excretions of bile salts and glutathione, or SRM of TUDC was modified by GAL administration. However, this dose was able to produce a partial decrease (-85%) in hepatic UDP-GA content and a concomitant decrease in biliary excretion of EE glucuronides by 40%. Because production of estrogen glucuronide was not totally abolished, a remaining cholestatic effect of EE in GAL-cotreated rats was expected. Uridine coadministration, which restored hepatic UDP-GA levels, reverted totally or partially the effects of GAL on bile flow, SRM of TUDC, and BSIF, confirming that shortage of UDP-GA was responsible for GAL effects.
Impairment of bile salt secretion by EE can be attributed to both diminution of the bile salt pool size (Crocenzi et al., 2001) and diminished expression of the canalicular bile salt export pump (Bsep) (Lee et al., 2000). The quantitative and qualitative alterations induced by EE in the bile salt pool are complex and probably result from simultaneous inhibition of the activity of microsomal cholesterol 7--hydroxylase (Cyp7a1) (Davis et al., 1986; Koopen et al., 1999) and preservation of the mitochondrial sterol 27-hydroxylase (Cyp27a1) (Kern et al., 1977). Cyp7a1 is the enzyme that catalyzes the key, rate-limiting step of the classic major pathway of the overall bile salt synthesis, whereas Cyp27a1 catalyzes the first step in the alternative pathway of bile salt synthesis, which produces selectively chenodeoxycholate and the bile salts derived from its further hepatic and/or intestinal conversion, i.e., muricholate and hiodeoxycholate (Koopen et al., 1999). These metabolic effects led to a preservation of the biliary excretion of both muricholate and hiodeoxycholate and a decrease in the biliary excretion of the other bile salts by the estrogen. Our finding that GAL reverted this effect of EE, showing a control-like pattern of biliary bile salt output, suggests that EE-glucuronide can be involved in these metabolic effects of EE.
Experiments performed with infusion of TUDC revealed that GAL also prevented, although to a lower extent, the alteration in Bsep transport activity induced by EE, thus implicating, at least partially, EE-17ß-glucuronide as a mediator. It is well accepted that SRM estimates the total number of active transporters at the canalicular membrane. Diminution of Bsep activity by EE may result from an inhibitory action of cholestatic derivatives of EE, as was observed with estradiol 17ß-glucuronide (Stieger et al., 2000) or, alternatively, from a reduction in Bsep protein expression (Lee et al., 2000). Either or both of these factors may indeed explain the decreased SRM of TUDC in EE group as previously reported (Crocenzi et al., 2001). The mechanisms involved in Bsep expression decrease are poorly understood; acute administration of the endogenous estrogen derivative estradiol-17ß-glucuronide was reported to produce a rapid and reversible endocytic internalization of Bsep with concomitant reduction in activity (Crocenzi et al., 2003). Persistent exposure to estrogen cholestatic metabolites like EE 17ß-glucuronide, as can be expected to occur after subacute administration of EE, may lead to delivery of Bsep-containing vesicles to the lysosomal compartment, followed by lysosomal degradation; this phenomenon was hypothesized to be responsible for Mrp2 down-regulation several hours after endotoxin administration (Kubitz et al., 1999) and after bile duct ligation (Paulusma et al., 2000).
As previously reported, EE impaired BSIF by diminishing the biliary excretion of glutathione (Bouchard et al., 1994) and (Alvaro et al., 1997), two compounds thought to be mainly responsible for this fraction of bile flow. Impairment of BSIF by EE was also partially protected by GAL, which reverted the effects of the estrogen on excretion but not on glutathione output. Because Western blot analysis demonstrated that anion exchanger 2, the canalicular transporter of , is preserved in EE cholestasis (Alvaro et al., 1997), a functional alteration of the transporter could be responsible for excretion impairment. GAL prevention of estrogen-induced alteration in excretion suggests that EE-glucuronide could participate in the alleged functional alteration of the transporter, although the mechanism remains unknown. Our finding that GAL was not effective in preventing glutathione secretory failure indicates that alteration of glutathione excretion induced by EE could be independent of EE-glucuronide formation.
In conclusion, GAL administration partially prevented EE cholestasis via a mechanism that involves a decrease in UDP-GA availability. The consequent decrease in EE 17ß-glucuronide formation most likely accounted for this prevention, supporting the hypothesis that EE 17ß-glucuronide plays a key role in the cholestasis induced by its unconjugated parent compound.
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Footnotes |
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Parts of this study were presented in the Biannual Meeting of the International Association for the Study of the Liver (IASL), March 16-20, 2004; Salvador da Bahia, Brazil.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: EE, ethinylestradiol; GAL, galactosamine; UDP-GA, UDP-glucuronic acid; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; TUDC, tauroursodeoxycholate; HPLC, high-performance liquid chromatography; U, uridine; SRM, secretory rate maximum; BSIF, bile-salt-independent fraction of bile flow; C, control; Bsep, bile salt export pump.
Address correspondence to: Dr. Enrique J. Sánchez Pozzi, Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET—Universidad Nacional de Rosario. Suipacha 570 (2000) Rosario, Argentina. E-mail: esanchez{at}unr.edu.ar
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-ethinylestradiol on the neutral and acidic pathways of bile salt synthesis in the rat. J Lipid Res 40: 100-108.This article has been cited by other articles:
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  A. D. Mottino, T. Hoffman, F. A. Crocenzi, E. J. Sanchez Pozzi, M. G. Roma, and M. Vore Disruption of function and localization of tight junctional structures and Mrp2 in sustained estradiol-17beta-D-glucuronide-induced cholestasis Am J Physiol Gastrointest Liver Physiol, July 1, 2007; 293(1): G391 - G402. [Abstract] [Full Text] [PDF]
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) or in combination with galactosamine (200 mg/kg b.wt. per day for 5 days, i.p.; EE + GAL, 
) or galactosamine + uridine (0.9 g/kg b.wt. per day for 5 days, i.p.; EE + GAL + U, 
). Animals receiving only galactosamine (GAL, 
) or uridine (U, 
) or controls receiving only the vehicle (propylene glycol; Control, 
) are also shown. Regression analysis yielded the following equations: Control: y = 0.0046 ± 0.0002 x + 1.94 ± 0.06; GAL: y = 0.0046 ± 0.0001 x + 1.80 ± 0.04; U: y = 0.0049 ± 0.0002 x + 1.76 ± 0.10; EE: y = 0.0043 ± 0.0005 x + 0.56 ± 0.05; EE + GAL: y = 0.0041 ± 0.0003 x + 1.11 ± 0.03; EE + GAL +U: y = 0.0041 ± 0.0005 x + 0.69 ± 0.04. The correlation coefficient was >0.88 for all groups.