* ManTech Environmental Technology, Dayton, Ohio 45433-7400;
Department of Physics, Wright State University, Dayton, Ohio 45324; and
Operational Toxicology Branch, Air Force Research Laboratory, Bldg. 79, 2856 G St., Wright Patterson Air Force Base, Ohio 45433-7400
Received January 9, 2002; accepted July 2, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: cadmium; bromosulfophthalein; isolated perfused rat liver; biokinetics; cholestasis.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The uptake of BSP at the basolateral membrane by hepatocytes seems to be mediated by a carrier transport process since saturation, competitive inhibition, and counter transport have been observed (Winkler, 1965). It has been shown that BSP undergoes conjugation with glutathione in the liver and a considerable fraction of BSP excreted into the bile is in the conjugated form (Whelan et al., 1970
). Conjugation of BSP is an important determinant of the rate of BSP excretion by facilitating the transport of BSP from hepatocytes into bile. However, conjugation is not obligatory for biliary excretion (Whelan et al., 1970
). Glutathione (GSH) is a cofactor in the conjugation reaction and is the most abundant low molecular weight peptide in hepatocytes. Concentrations in mammalian liver are 4 to 8 mM; with nearly all glutathione present as reduced GSH; less than 5% is present in the oxidized form (GSSG; Will, 1999
). Intracellular GSH levels are regulated by a complex mechanism involving control of synthesis, transport, and utilization. GSH is an essential cellular component and prolonged failure to maintain adequate intracellular levels is detrimental to the cell (Will, 1999
). Conjugation of BSP with glutathione (to form BSP-SG) is catalyzed by hepatic glutathione S-transferases (Whelan et al., 1970
). Glutathione S-transferases (GSTs) are a family of isoenzymes that conjugate GSH with electrophilic compounds and serve in detoxification of xenobiotics, e.g., drugs, environmental pollutants, pesticides, herbicides, and carcinogens.
Cadmium was selected as the test chemical for this study based on known effects of Cd on relevant hepatic functions. Cadmium (Cd) is not an essential element but accumulates in the environment as a result of industrial practices, and it has a long biological half-life in humans (Goyer and Cherian, 1995). In the body, it accumulates primarily in the liver and kidney and results in both hepatic and renal tubular damage (Goyer and Cherian, 1995
). The production of reactive oxygen species and oxidative tissue damage has been associated with Cd hepatotoxicity. It has been demonstrated that Cd produces dose- and time-dependent increases in intracellular glutathione concentrations during chronic environmental or occupational exposures at low doses. However, at high-level acute Cd exposures, significant glutathione depletion occurs (Chin and Templenton, 1993
). Furthermore, Cd is known to cause a reduction in glutathione content in isolated hepatocytes (Stacey et al., 1980
). Cd is transported into bile through the canalicular membranes via the carrier mediator canalicular isoform of multidrug resistance protein (cMrp; Sugawara et al., 1997
). Cytosolic metallothionein (MT) strongly binds Cd and once MT synthesis is induced in the liver, the transport of Cd via bile is suppressed. Therefore, hepatobiliary excretion of Cd is not a primary route for removal of Cd from the liver following exposure (Sugawara et al., 1997
).
Glutathione plays a role in protection against cadmium induced hepatocellular damage; however, its role in cadmium induced cholestasis remains unclear (Muller and Ohnesorge, 1982). Cholestatic injury or intrahepatic cholestasis is characterized by arrested bile flow and is an important form of chemical induced hepatic injury. Intrahepatic cholestasis may result from impaired formation of canalicular bile or impaired flow of bile. Flow may be impaired by altered fluidity of the bile, impaired canalicular contractility, or by lesions of the ductal system that affect normal flow (Hyman, 1999
).
The isolated perfused rat liver (IPRL) is a useful experimental system for evaluating hepatic function without the influence of other organ systems, undefined plasma constituents, and neural-hormonal effects. Hepatic architecture, cell polarity, and bile flow are preserved in the IPRL. Furthermore, the IPRL allows repeated sampling of the perfusate and permits easy exposure of the liver to different concentrations of test chemical (Gores et al., 1986). In this study, we used the IPRL system to investigate the effect of a hepatotoxicant, cadmium, on the kinetics and metabolism of BSP in the liver.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals.
Male Fisher F-344 rats (Charles River Laboratories), 220340 g, were used as liver donors (Table 1). All animals had free access to food and water. All animal studies described in this report were conducted in accordance with the principles stated in the "Guide for the Care and Use of Laboratory Animals," National Research Council, 1996, and the Animal Welfare Act of 1966, as amended.
|
The excised liver was connected to the recirculating perfusion system (for details of the perfusion system see Toxopeus and Frazier, 1999). In most experiments, the liver was perfused with 200 ml Krebs-Ringer buffer supplemented with 0.25% BSA (w/v) at a flow rate of 40 ml/min at 37°C in a recirculating system. The perfusion medium was equilibrated with 95% O2/5% CO2 to maintain the pH in the physiological range (7.377.42). Sodium taurocholate (18 mg/ml) was continuously infused into the perfusion medium at a rate of 1 ml/h to maintain bile secretion.
Experimental design.
In all experiments, the liver was allowed to equilibrate in the perfusion system for 30 min before treatments were initiated. All times are reported relative to the end of the recovery period, i.e., t = 0 at the end of the recovery period. In control perfusions, BSP was added to the perfusion reservoir at t = 60 min to attain a concentration of 150 µM BSP in the perfusion medium. In treatment studies, cadmium acetate was added to the perfusion reservoir at t = 0 min to attain a concentration of 10 or 100 µM Cd and BSP was added at t = 60 min to attain a concentration of 150 µM BSP in the perfusion medium (Fig. 1).
|
Analytical Methods
LDH analysis.
LDH is an indicator of membrane integrity in general and leakage of LDH into perfusion medium indicate that the hepatocellular plasma membrane has been severely damaged. LDH activity in perfusion medium was determined as previously described by Korzeniewski and Callewaert (1983). Analyses were performed using a microplate reader (Molecular Devices Sunnyvale, CA) set at 492 nm wavelength. The percentage LDH leakage was calculated as the percentage of activity in perfusion medium at time t (APM(t)) relative to the total LDH activity. The total LDH activity is the activity in the perfusion medium at the end of the experiment (APM(t = 150)) plus the activity in the liver calculated from the liver homogenate (AL(t = 150)). Therefore,
![]() | (1) |
Bile flow measurements.
Bile was collected in preweight microcentrifuge tubes over intervals of 15 min. The bile flow (QB) was calculated as:
![]() | (2) |
HPLC analysis.
BSP and BSP-SG conjugate were analyzed by reverse phase HPLC using a Luna 5µ C18 (2) (250 x 4.60 mm, 5µ micron) analytical column (Phenomenex, Torrance, CA), and a photodiode array detector (Model 996; Waters, Milford, MA) set at a wavelength of 580 nm. The mobile phase consisted of 0.2 % triethylamine, 0.01 % NH4OH, 24 % acetonitrile, and 76 % deionized water adjusted to pH 10.2 with phosphoric acid. The pump (Model 510; Waters, Milford, MA) was set at a rate of 0.5 ml/min. Perfusion medium samples were diluted 1:10 and bile samples were diluted 1:100 with water. All samples were filtered using a 1 ml syringe fitted with a 0.45 µm pore/13 mm filter unit (Millex-HV13; Millipore Corporation, Bedford, MA).
To determine the concentration of BSP and BSP-SG conjugate in liver tissue, 1 g of wet liver was homogenized in 3 ml of ddH2O with a mechanically driven Teflon glass homogenizer (TRI-R Stir R-K43, TRI-R Instruments, Rockville Center, NY). An aliquot (500 µl) of liver homogenate was mixed with acetone (500 µl) and centrifuged for 20 min at 11000 rpm using an Eppendrorf centrifuge (Model 5415 C; Brinkmann Instruments Inc.). The extract was mixed with 500 µl of acetonitrile, centrifuged again for 20 min at 11000 rpm and the supernatant was evaporated to complete dryness under nitrogen. After drying, the sample was dissolved in 500 µl of ddH2O and injected in the HPLC system for analysis.
Determination of Cd concentration in buffer and liver tissue.
Flame atomic absorption spectrophotometry (GBC Scientific Equipment Inc., Arlington Heights, IL) was used for quantitative analysis of Cd concentrations in perfusion medium and liver tissue. Samples from 10 µM Cd treatment were filtered before analysis. For experiments with 100 µM Cd treatments, samples were filtered then diluted 1:10 in 0.5% nitric acid. For liver tissue preparation, 1 g of wet liver was homogenized in 3 ml of 70% nitric acid and digested to complete dryness in a vacuum oven at 110°C. The digestate was dissolved in 3 ml of 0.5% nitric acid and filtered (Millex-HV13; Millipore Corporation, Bedford, MA). For livers treated with 100 µM Cd, digested samples were diluted 1:10 with ddH2O.
Statistics.
Comparison of data was performed by one-way ANOVA followed by the Tukey test with p 0.05 as the level of significance.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
A dramatic effect on bile flow was observed when perfused livers were treated with 100 µM Cd. In these studies, the initial bile flow at t = 0 was 1.0 ± 0.2 µl/min/g liver and rapidly decreased to 0.1 µl/min/g liver 45 min after Cd treatment. Bile flow stopped completely 60 min after Cd dosing indicating significant functional liver toxicity.
BSP Clearance from Perfusion Medium
HPLC analysis of perfusion medium allows for the identification and quantification of BSP and BSP-SG separately. In control experiments, BSP concentration in perfusion medium decreased from 134 ± 16 µM 2 min after BSP dosing to 6 ± 5 µM 90 min after dosing (Fig. 5A). This corresponds to 95% clearance of BSP from perfusion medium. Treatment with 10 µM Cd resulted in a slight decrease in clearance from 137 ± 2 µM at 2 min after dosing to 23 ± 11 µM BSP at the end of the experiment (85% clearance). Livers treated with 100 µM Cd exhibited a significant decrease in BSP clearance compared to the controls, with the concentration decreasing from 141 ± 8 µM to 96 ± 17 µM (only 30% clearance). Following the 100 µM Cd dose, BSP clearance from the perfusion medium totally ceased 30 min after BSP dosing (t = 90 min).
|
BSP Concentration in Bile
Figure 6A shows the concentration of BSP in bile as determined by HPLC. The BSP concentration in bile peaks between 15 and 30 min after BSP dosing (t = 75 and 90 min) in both control and 10 µM Cd treated IPRL preparations (4.1 ± 0.9 x 103 µM and 3.8 ± 0.75 x 103 µM respectively). No statistically significant differences (p
0.05) between data for control and 10 µM Cd exposed livers were found. In the interval from t = 75 to t = 90 min, the average concentration of BSP in the perfusion medium in control and 10 µM treatment experiments was 56.5 ± 7 and 68.5 ± 17 µM, respectively. Thus, the concentration ratios of bile to perfusion medium are approximately 72.6 and 55.5 for the control and 10 µM treatment group, respectively. No data were obtained for the 100 µM Cd treatment group due to complete cholestasis at the time of BSP dosing.
|
Cumulative Excretion of BSP in Bile
The cumulative excretion of BSP was determined by multiplying the BSP concentration in bile by the bile volume in each sampling interval and summing. The experimental data are illustrated in Fig. 7A. A total of 3.6 ± 0.7 µmol of BSP are excreted in bile in control experiments versus 2.2 ± 0.4 µmol in the 10 µM Cd treated livers, a statistically significant decrease.
|
BSP and BSP-SG Conjugate in Liver
The concentration of BSP and BSP-SG conjugate were determined in the liver at the end of each experiment. The hepatic concentrations of both parent and conjugate (Table 2) were very low in all cases as compared to the concentration of BSP and BSP-SG in bile.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The most significant effect of Cd on BSP kinetics (see below) was mediated through the reduction of bile flow. Livers treated with 100 µM Cd showed a severe cholestatic effect 45 min after Cd treatment, and complete stoppage of bile flow at 60 min. Several mechanisms have been proposed to account for the induction of cholestasis. They include alteration in the cytoskeleton of the hepatocyte, changes in the tight junction structure, disorders of the membranes carriers and ion pumps, alterations in membrane permeability and fluidity, and precipitation in bile canaliculi (Miura et al., 1997). It is possible that Cd may injure interhepatic tight junctions, increasing leakage from biliary canaliculi, leading to loss of hepatocyte polarity and dissipation of osmotic gradients. This effect can influence secretion of bile via both bile acid-dependent flow and bile acid-independent flow (Tuchweber et al., 1986
). Injury to tight junctions contributes to cholestasis by permitting regurgitation of bile into the sinusoids (Tuchweber et al., 1986
). Damage to tight junctions and leakage of canalicular bile back into the sinusoids could be possible in these experiments and could explain the higher concentrations of BSP and BSP-SG found in perfusion medium in experiments where the liver was treated with Cd. However, the possibility also exists that Cd may directly affect the bile duct by causing inflammation or damage to biliary epithelial cells, thus obstructing bile flow and consequently causing liver cholestasis.
For normal production of bile, the organic anion transporter multidrug resistance protein (Mrp2) and the bile salt export pump (Bsep) are essential (Sugawara et al., 1996). It may be possible that Cd affects these two transport proteins resulting in the cholestatic effect. Endotoxin-induced cholestasis is correlated with a strong down regulation of canalicular Mrp2 and decreased mRNA levels and protein levels of Bsep in rat liver. In the case of endotoxin treatment, Mrp1 and Mrp3 appear to compensate for the decreased transport activity of Mrp2 enhancing the efflux of organic anions into sinusoidal space (Kullak-Ublick, 1999
). In the situation of Cd induced cholestasis, a decrease in Mrp2 transport function would decrease transport of BSP and BSP-SG into bile, increase intrahepatic concentration of the two chemical species and subsequently increase efflux of BSP and BSP-SG back into the perfusion medium. This cascade of events could also account for the higher concentrations of BSP and BSP-SG conjugate in the perfusion medium from perfused livers treated with Cd.
Another mechanism for the Cd effect on bile formation may involve GSH and GSH conjugates. Glutathione in bile serves as one of the osmotic driving forces in bile acid-independent bile formation (Ballatori and Troung, 1989). It is entirely possible that the choleretic effect of BSP at 15 and 30 min after BSP addition is due to the high level of BSP-SG output into the bile. On the other hand, studies using rat kidney fibroblasts indicates that GSH forms a complex with Cd (Kang, 1992). Therefore, the cholestatic effect of Cd observed in livers treated with 10 and 100 µM Cd could be due in part to low intracellular concentrations of free GSH resulting in reduced biliary formation and low BSP clearance from perfusion medium.
Cd Concentration in Liver
It is well known that Cd accumulates in the liver after in vivo treatment with ionic Cd. In our experiments Cd concentration in livers treated with 10 and 100 µM Cd were 814 times higher than the concentration in perfusion medium, confirming that Cd accumulates in liver in the IPRL system.
Biliary excretion of Cd is a possible route for Cd clearance from the liver. However, Sugawara et al. (1997) suggested that even though hepatobiliary excretion of Cd is mediated via carrier transport systems, including the canalicular isoform of the multidrug resistance protein (cMrp) and the GSH carrier, disruption of these transport systems does not enhance the accumulation of Cd in the liver. Therefore, they proposed that hepatobiliary excretion of Cd is not a significant route for Cd removal from the liver following exposure to high Cd doses. In the IPRL studies reported here, very little Cd is excreted in the bile (data not shown).
Intracellular protein binding most likely plays the major role in Cd accumulation in the liver. MT is an intracellular sulfhydryl-rich metal binding protein with a high affinity for Cd (Webb and Cain, 1982). In addition, other sulfhydryl-rich proteins will bind Cd. Thus, a combination of Cd binding to metallothionein and other sulfhydryl containing intracellular proteins and limited biliary excretion of Cd are sufficient to account for the high accumulation of Cd in the liver.
BSP Excretion
This study investigated the effect of Cd treatment on the kinetics of BSP in the IPRL system. Experimental data show a significant dose-dependent difference in the clearance of total BSP from the perfusion medium between control and Cd treated livers. Furthermore, HPLC analysis of perfusion medium demonstrates higher concentrations of both the parent BSP and the GSH conjugated, BSP-SG, in the perfusion medium following Cd treatment when compared to control studies. These data suggest that Cd toxicity may inhibit either the uptake of BSP at the sinusoidal membrane and/or the excretion of both BSP and BSP-GS at the biliary canalicular membrane. In addition, the complete cessation of BSP clearance from the perfusion medium at the 100 µM concentration provides circumstantial evidence that Cd may block the metabolism of BSP to the BSP-SG conjugate.
Although the concentration of BSP and BSP-SG in bile following the 10 µM Cd treatment was not significantly different when compared to control experiments, the cumulative excretion of BSP in bile decreased in treated livers. Of the 30 µmol of BSP added to the perfusion medium, only a total of 11.5 µmol of BSP (BSP + BSP-SG) was recovered in the bile from livers treated with 10 µM Cd versus 22.6 µmol recovered in control experiments. Furthermore, the cumulative excretion of BSP and BSP-SG conjugate in bile was 40 and 51% lower, respectively, in livers treated with 10 µM Cd when compared to controls. These differences in the cumulative excretion of BSP and BSP-SG are most likely due to the reduced bile formation caused by the cholestatic effects of Cd. However we cannot discard the possibility that Cd may inhibit BSP uptake at the sinusoidal membrane and/or inhibit the rate of conjugation of BSP to BSP-SG.
Impaired BSP Metabolism
The data clearly indicate that impaired bile formation has a major effect on BSP clearance. However, cholestasis alone cannot account for the cessation of BSP clearance from the perfusion medium in the 100 µM Cd treatment studies. Even under the conditions of complete cholestasis, where no BSP is eliminated in the bile, BSP should continue to be taken up by the liver and be metabolized to the BSP-SG conjugate. The data imply that metabolic conversion of BSP to the BSP-SG conjugate also ceases. Previous studies have shown that a single high dose of Cd can reduce the GSH concentration in rat livers by 65 % in vivo (Bagchi et al., 1996). Other studies suggest that this GSH depletion may be a consequence of the production of reactive oxygen species at a rate exceeding the ability to regenerate reduced glutathione (Li et al., 1993
). Loss of intracellular GSH also could be due to Cd binding to GSH (Perrin and Watt, 1971
) with subsequent efflux into the bile or perfusate, conversion of the reduced GSH to the oxidized form GSSG or membrane damage resulting in GSH leakage (Strubelt et al., 1996
). Whatever the specific mechanism for the reduction in intracellular GSH, this effect offers another possible explanation for impaired clearance of BSP, i.e., that Cd may deplete GSH, the required cosubstrate for glutathione transferase, thus inhibiting the BSP conjugation reaction and subsequently reducing or completely blocking the clearance of BSP, depending on the extent of GSH depletion.
Reduced ATP
Studies in Eisai hyperbilirubinuric (EHB) rats showed that biliary excretion of Cd from the liver is highly regulated by ATP-driven anion transport in rats (Vos et al., 1999). Secretion of cholephilic compounds from hepatocytes is largely dependent on two ATP-binding cassette protein superfamilies, the P-glycoprotein subfamily and the multidrug resistance protein (MRP) subfamily (Vos et al., 1999
). Mrp1 and Mrp2 are able to transport multivalent anionic conjugates such as bilirubin diglucuronide and GSH conjugates (Vos et al., 1999
). It is possible that Cd reduces ATP levels in the hepatocyte affecting the function of ATP-dependent hepatic transport systems thus reducing the ability of the liver to excrete BSP and BSP-SG conjugated into bile. Previous studies conducted by Strubelt et al.(1996) using the isolated perfused rat liver showed that Cd treatments at 10, 30, or 100 µM Cd reduce hepatic ATP concentration to less than 20% of controls.
Conclusions
These studies demonstrate that monitoring of BSP kinetics in the extracellular, intracellular, and biliary spaces in the IPRL system is a useful tool for elucidating mechanisms of hepatotoxicity. The overall kinetic pathways for BSP in the IPRL are illustrated in Figure 8 and the potential sites of Cd toxicity are indicated. This study shows that Cd elicits multidimensional dose-dependent effects on the kinetics of BSP and the BSP-SG conjugate in the IPRL system. Evaluation of Cd dosimetry in the livers indicated that the concentration of cadmium in the liver increased proportionately with dose and was significantly higher than the concentration in the perfusion medium, probably due to the binding of cadmium to intracellular proteins. A significant cholestatic effect on bile flow was observed when livers were treated with Cd (Lesion 1 in Fig. 8
) and this effect is probably the major contributor to the reduction in BSP clearance. No significant changes in the percentage of LDH leakage into the perfusion medium were observed throughout all experiments; thus, the effects of Cd are not due to overt necrosis of hepatocytes. Some of the effects on BSP kinetics observed could be due to effects of Cd either directly on transport systems and/or indirectly through the depletion of ATP (Lesion 2). The high dose Cd study also implies that the conjugation reaction may be inhibited by Cd (Lesion 3) possibly through depletion of the cosubstrate GSH. An increase in BSP-SG in perfusion medium was observed in livers treated with Cd suggesting that Cd may damage hepatic tight junctions (Lesion 4) and/or biliary transport systems inhibiting biliary excretion of BSP-SG (Lesion 2) resulting in BSP-SG efflux back into the perfusion medium.
|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ballatori, N., and Truong, A. T. (1989). Relation between biliary glutathione excretion and bile acid-independent bile flow. Am. J. Physiol. 256, G22G30.
Chin, T. A., and Templenton, D. M. (1993). Protective elevations of glutathione and metallothionein in cadmium-exposed mesangial cells. Toxicology 77, 145156.[ISI][Medline]
Chuttani, H. K., Gupta, P. S., Gulati, S., and Gupta, D. H. (1965). Acute copper sulfate poisoning. Am. J. Med. 39, 849854.[ISI][Medline]
Colon, A. R., Pardo, V., and Sandberg, D. H. (1974). Experimental Reyes Syndrome induced by viral potentiation of chemical toxin. In Reyes Syndrome (J. D. Pollack, Ed.), pp. 194214. Grune and Stratton, New York.
Gores, G. J., Kost, L. J., and LaRusso, N. F. (1986). The isolated perfused rat liver: Conceptual and practical considerations. Hepatology 6, 511517.[ISI][Medline]
Goyer, R. A., and Cherian, M. G. (1995). Renal effects of metals. In Metal Toxicology (R. A. Goyer, C. D. Klaassen, and M. P. Waalkes, Eds.), pp. 389412. Academic Press, San Diego, CA.
Hyman, J. (1999). Hepatotoxicity: The Adverse Effects of Drugs and Others Chemicals on the Liver, 2nd ed., pp. 295323. Lippincott Williams & Wilkins, Philadelphia, PA.
Kang, Y. J. (1992). Exogenous glutathione decreases cellular cadmium uptake and toxicity. Drug Metab. Dispos. 20, 714718.[Abstract]
Korzeniewski, C., and Callewaert, D. M. (1983). An enzyme-release assay for natural cytotoxicity. J. Immunol. Methods 64, 313320.[ISI][Medline]
Kullak-Ublick, G. A. (1999). Regulation of organic anion and drug transporters of the sinusoidal membrane. J. Hepatol. 31, 563573.[ISI][Medline]
Li, W., Zhao, Y., and Chou, I. N. (1993). Alterations in cytoskeletal protein sulfhydryls and cellular glutathione in cultured cells exposed to cadmium and nickel ions. Toxicology 77, 6579.[ISI][Medline]
Miura, H., Tazuma, S., Yamashita, G., Hatsushika, S., and Kajiyama, G. (1997). Effect of cholestasis induced by organic anion on the lipid composition of hepatic membrane subfractions and bile in rats. J. Gastroenterol. Hepatol. 12, 734739.[ISI][Medline]
Muller, L., and Ohnesorge, F. K. (1982). Different response of liver parenchymal cells from starved and fed rats to cadmium. Toxicology 25, 141150.[ISI][Medline]
Perrin, D. D., and Watt, A. E. (1971). Complex formation of zinc and cadmium with glutathione. Biochim. Biophys. Acta 230, 96104.[ISI][Medline]
Stacey, N. H., Cantilena, L. R., Jr., and Klaassen, C. D. (1980). Cadmium toxicity and lipid peroxidation in isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 53, 470480.[ISI][Medline]
Strubelt, O., Kremer, J., Tilse, A., Keogh, R., Pentz, R., and Younes, M. (1996). Comparative studies of the toxicity of mercury, cadmium and copper toward the isolated perfused rat liver. J. Toxicol. Environ. Health 47, 267283.[ISI][Medline]
Sugawara, N., Lai, Y.-R., Arizono, K., and Ariyoshi, T. (1996). Biliary excretion of exogenous cadmium and endogenous copper and zinc in the Eisai hyperbilirubinuric (EHB) rat with a near absence of biliary glutathione. Toxicology 112, 8794.[ISI][Medline]
Sugawara, N., Lai, Y.-R., Arizono, K., Kitajima, T., and Inoue, H. (1997). Lack of biliary excretion of Cd linked to an inherent defect of the canalicular isoform of multidrug resistance protein (cMrp) does not abnormally stimulate accumulation of Cd in the Eisai hyperbilirubinemic (EHB) rat liver. Arch. Toxicol. 71, 336339.[ISI][Medline]
Toxopeus, C., and Frazier, J. M. (1999). The isolated perfused rat liver and its use in the study of chemical kinetics: Quality and performance parameters. Air Force Technical Report No. AFRL-HE-WP-TR-1998-0134.
Tuchweber, B., Weber, A., Roy, C. C., and Yousef, I. M. (1986). Mechanisms of experimentally induced intrahepatic cholestasis. Prog. Liver Dis. 8, 161178.[ISI][Medline]
Vos, T. A., Ros, J. E., Havinga, R., Moshage, H., Kuipers, F., Jansen, P. L., and Muller, M. (1999). Regulation of hepatic transport systems involved in bile secretion during liver regeneration in rats. Hepatology 29, 18331839.[ISI][Medline]
Webb, M., and Cain, K. (1982). Functions of metallothionein. Biochem. Pharmacol. 31, 137142.[ISI][Medline]
Whelan, G., Hoch, J., and Combes, B. (1970). A direct assessment of the importance of conjugation for biliary transport of sulfobromophthalein sodium. J. Lab. Clin. Med. 75, 542557.[ISI][Medline]
Will, Y. (1999) The glutathione pathway. In Current Protocols in Toxicology (M. D. Maines, L. G. Costa, D. J. Reed, S. Sassa, and I. G. Sipes, Eds.), pp. 6.1.16.1.18. John Wiley and Sons, Corvallis, OR.
Winkler, K. (1965). The kinetics of bromosulfophthalein elimination during continuous infusion in man. In The Biliary System (W. Taylor, Ed.), pp. 551566. FA Davis, Philadelphia.