Toxicological Sciences 69, 460-469 (2002)
Copyright © 2002 by the Society of Toxicology
SYSTEMS TOXICOLOGY |
Effect of Cadmium on Bromosulfophthalein Kinetics in the Isolated Perfused Rat Liver System
,1
* 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 |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bromosulfophthalein (BSP) is a relatively nontoxic organic anion used as an in vivo indicator of liver performance. Elimination of BSP via the biliary system following iv injection requires dissociation from albumin in plasma, translocation across the sinusoidal membrane, conjugation with glutathione within the hepatocyte, translocation across the bile canalicular membrane, and excretion in bile. The effects of cadmium (Cd), anin vivo hepatotoxicant in rats, on BSP kinetics in the isolated perfused rat liver (IPRL) were studied to investigate the interaction between liver toxicity and BSP kinetics. Livers were isolated from male Fisher 344 rats. After a 30-min period for acclimation to the IPRL system, livers were dosed with Cd (as cadmium acetate), in the presence of 0.25% bovine serum albumin, to give initial concentrations of 10 and 100 µM. Sixty min after Cd dosing, the IPRL system was dosed with BSP to give an initial concentration of 150 µM and the elimination kinetics of BSP from the perfusion medium were monitored. Cadmium concentrations in livers at the end of the experiments were 60 ± 4 and 680 ± 210 µmol/kg for the 10 and 100 µM doses, respectively. Exposure to 10 µM Cd for 60 min resulted in a reduction in bile flow, no significant effect on lactate dehydrogenase (LDH) leakage, and slight effects on BSP clearance. Similar studies following exposure to 100 µM Cd showed a dramatic decrease in bile flow with complete cholestasis 60 min after Cd addition. LDH leakage into perfusion medium at the end of the experiment was less than 10%, indicating that Cd affected bile production well before the liver showed significant signs of necrosis. Clearance of BSP from the perfusion medium was dramatically reduced. Taken together, the data indicate that Cd has a significant effect on the kinetics of BSP in the IPRL and the dominant effects were mediated through the cholestatic effect of Cd.
Key Words: cadmium; bromosulfophthalein; isolated perfused rat liver; biokinetics; cholestasis.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The liver plays an important role in the metabolism and excretion of xenobiotics. Bromosulfophthalein (BSP) is a cholephilic nontoxic dye that is commonly used to evaluate hepatic function. Elimination of BSP from liver sinusoids into bile involves dissociation from plasma binding proteins in the sinusoids, translocation across the basolateral membrane of hepatocytes, intracellular conjugation with glutathione to form the BSP-glutathione conjugate (BSP-SG), and/or binding to cytosolic protein, and ultimately translocation of BSP and/or BSP-SG across the bile canalicular membrane (Chutlani et al., 1965; Colon et al., 1974
). The alteration of any of these processes as a result of exposure to a xenobiotic compound will be reflected in the kinetics of BSP clearance. Thus, monitoring BSP kinetics in the extracellular, intracellular, and biliary spaces following treatment with a xenobiotic compound will indicate which, if any, of these processes are affected, thereby providing insight into the mechanism of action of the chemical under investigation. Used in this way, BSP kinetics serves as a probe of toxic mechanisms, much as lactate dehydrogenase (LDH) release serves as a probe of membrane integrity.
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 |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Chemicals.
Acetonitrile, cadmium acetate, taurocholic acid, BSP, and low endotoxin bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). Heparin was purchased from Elkins-Sinn, Inc. (Cherry Hill, NJ). All reagents used for liver perfusion and analytical methods were of high analytical grade.
Animals.
Male Fisher F-344 rats (Charles River Laboratories), 220–340 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.
|
Liver surgery and perfusion.
Surgery was performed as previously described (Toxopeus and Frazier, 1999
). Briefly, animals were anesthetized by im injection of 90 mg/kg ketamine HCl and 10 mg/kg xylazine. The abdomen was opened through a midline and one transversal incision and the bile duct was cannulated. Heparin sodium solution (0.5 ml; 500 U/ml in 0.9% NaCl) was injected via the abdominal vena cava to prevent blood clotting. The portal vein was cannulated and the liver was perfused with Krebs-Ringer solution (pH 7.4; saturated with 95% O2 and 5% CO2; 37°C) at a flow rate of 25 ml/min. After cannulating the superior vena cava through the right atrium of the heart, the inferior vena cava was ligated and the liver was carefully removed.
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 (7.37–7.42). 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).
|
Samples of perfusion medium were collected from the reservoir from 5–150 min after Cd dosing to evaluate Cd uptake by the liver. In addition, samples of perfusion medium were collected from the reservoir from 2–90 min post-BSP dosing to determine BSP clearance from perfusion medium. Bile samples were collected over 15-min intervals throughout the studies to be analyzed for BSP/BSP-SG concentrations. Perfusion medium samples for LDH activity were collected every 30 min.
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 |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Concentration of Cadmium in Perfusion Medium and Liver Tissue
Samples of perfusion medium were taken at different times following Cd treatment and the Cd concentration determined by atomic absorption spectrophotometry. The nominal initial Cd concentration in perfusion medium was either 10 µM or 100 µM Cd. The final concentration was 7.6 ± 0.7 µM or 50 ± 3 µM Cd, respectively (Fig. 2
). Cadmium lost from the perfusion medium was accumulated in the liver over the course of the experiment giving final tissue concentrations of 60 ± 4 and 680 ± 210 µmol/kg or 7.9 and 13.6 times greater than the concentration present in the perfusion medium at the end of the experiment, respectively.
|
Liver Viability
LDH leakage.
To assess liver toxicity, LDH leakage into perfusion medium was monitored throughout the experimental studies. LDH activity in samples taken from control and Cd treated experiments were not significantly different. Figure 3
shows a slight (nonsignificant) increase in LDH leakage for the 10 µM and 100 µM Cd treatments compared to control. However, in all cases, LDH leakage remained low at the end of the 3 h perfusions.
|
Bile flow.
Another viability parameter used to evaluate liver integrity is bile flow (Fig. 4
). Control livers produced an initial bile flow of 1.1 ± 0.1 µl/min/g liver, with no statistically significant change up to t = 60 min. Following BSP dosing at t = 60 min, bile flow increased slightly, peaking at t = 90 min, due to the choleretic effects of BSP, and finally a gradual decrease to 1.0 ± 0.2 µl/min/g liver at the end of the 180 min perfusion. Note, these trends were observed in all individual perfusion experiments; however, the combined data do not indicate a statistically significant change at any time relative to the control value observed during the equilibration period due to variability between individual experiments.
|
For 10 µM Cd experiments, bile flow showed the same pattern as the control up to t = 90 min. However a significant decrease in bile flow (p < 0.05) was observed after 105 min of Cd treatment and continued to decrease to 0.3 ± 0.2 µl/min/g liver at the end of the experiment.
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-SG Concentration in Perfusion Medium
Figure 5B
illustrate BSP-SG conjugate concentrations in the perfusion medium for control, 10 µM, and 100 µM Cd treatments. BSP-SG conjugate appeared in the perfusion medium reservoir 10 min after dosing with BSP in all studies. In control experiments, BSP-SG concentrations increase up to a maximum of 12 ± 6 µM at t = 90 min (30 min after dosing) followed by a slight but not statistically significant decrease to 8 ± 6 µM at the end of the experiment. For 10 µM and 100 µM Cd treatment experiments, BSP-SG concentration in perfusion medium was significantly increased above control values at t = 105 min and continued to increase to 36 ± 15 µM and 40 ± 18 µM, respectively, by the end of the experiment. BSP-SG concentrations in perfusion medium at the end of the experiment were not statistically different between the two Cd treatments.
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.
|
BSP-SG Conjugate Concentration in Bile
Results for BSP-SG (Fig. 6B
) also indicate that the concentration peaks between t = 75 and 90 min (17.6 ± 2.1 x 103 and 13.2 ± 2.9 x 103 µM for controls and 10 µM Cd, respectively) with no significant differences in the concentrations of the conjugate between control and 10 µM Cd treatments at any time. During the same interval of time (t = 75 and 90 min), the average concentrations of BSP-SG in perfusion medium for control and 10 µM Cd treatment were 9.5 ± 4.5 and 13 ± 4 µM respectively. The concentration ratio of BSP-SG between bile and perfusion medium for control and treatment in this interval was much greater than that for BSP, approximately 1789 and 1015 respectively.
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.
|
Cumulative Excretion of BSP-SG Conjugate in Bile
Figure 7B
illustrates the cumulative concentration of BSP-SG conjugate in bile. A significant difference in the total BSP-SG excreted between control (19.01 ± 1.9 µmol) and 10 µM Cd treated livers (9.3 ± 3.5 µmol) is observed. In control experiments 63% of the 30 µmol of BSP added to the perfusion medium was converted into BSP-SG and excreted into the bile by the end of the experiment. In contrast in 10 µM Cd treated experiments only 30% of the BSP was metabolized and excreted via bile.
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.
|
Net Disposition of BSP
The overall disposition of BSP in these studies is summarized in Table 3
. In general, the total recovery of BSP was good, varying from 81.0 to 95.3%. The most obvious effects of Cd treatment are the reduced elimination of BSP from the perfusion medium, the decrease in BSP and BSP-SG elimination in bile, and the increase in the BSP-SG conjugate in the perfusion medium. An additional observation is the effect of Cd treatment on total metabolism of BSP to BSP-SG conjugate. Summing up the total BSP-SG in the system at the end of the experiment (BSP-SG in the perfusion medium plus the bile plus the liver) and expressing the sum as a percentage of the total BSP recovered, 81.3% of the BSP was converted to BSP-SG in control experiments, but only 72.0 and 30.7% was converted in the 10 and 100 µM Cd treatments, respectively. Thus, the overall metabolic activity was significantly reduced in the Cd treated livers.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Viability and Bile Flow
LDH is a useful indicator of liver toxicity resulting from plasma membrane damage and increased cellular permeability. An important observation is that LDH release into the perfusion medium was not significantly different among control and Cd treated IPRL experiments, indicating good liver viability and membrane integrity in all experiments. However, bile flow decreased significantly in Cd treated livers, suggesting that Cd affects hepatic transport and biliary secretory processes well before causing significant plasma membrane damage to hepatocytes.
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 8–14 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.
|
The strategy of using BSP kinetics in the IPRL system as an experimental probe for exploring mechanisms of hepatotoxicity should be useful for investigating other xenobiotic chemicals that impact BSP kinetic processes. This study also suggests an approach for investigating mechanistic interactions between chemicals in complex mixtures. However, for this strategy to be fully implemented, it is essential to develop appropriate biologically based kinetic models for BSP to quantitatively evaluate hepatotoxic effects of chemical at the level of specific kinetic parameters that characterize the fundamental kinetic processes. Such models are currently under development.
|
ACKNOWLEDGMENTS |
|---|
The authors would like to acknowledge TSgt Gerri Miller for her valuable assistance during the perfusions, and Lt Eric Styron and Mr. Dan Pollard for their technical assistance in the analysis of Cd, BSP, and BSP-glutathione conjugate. This work was financially supported by the Air Force Office of Scientific Research (2312A202). Technical support was provided by ManTech Geo-Centers Joint Venture F41624-96-C-9010.
|
NOTES |
|---|
1 To whom correspondence should be addressed. Fax: (937) 255-1474. E-mail: john.frazier{at}wpafb.af.mil.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bagchi, D., Bagchi, M., Hassoun, E. A., and Stohs, S. J. (1996). Cadmium-induced excretion of urinary lipid metabolites, DNA damage, glutathione depletion, and hepatic lipid peroxidation in Sprague-Dawley rats. Biol. Trace Elem. Res. 52, 143–154.[Web of Science][Medline]
Ballatori, N., and Truong, A. T. (1989). Relation between biliary glutathione excretion and bile acid-independent bile flow. Am. J. Physiol. 256, G22–G30.[Medline]
Chin, T. A., and Templenton, D. M. (1993). Protective elevations of glutathione and metallothionein in cadmium-exposed mesangial cells. Toxicology 77, 145–156.[Web of Science][Medline]
Chuttani, H. K., Gupta, P. S., Gulati, S., and Gupta, D. H. (1965). Acute copper sulfate poisoning. Am. J. Med. 39, 849–854.[Web of Science][Medline]
Colon, A. R., Pardo, V., and Sandberg, D. H. (1974). Experimental Reye’s Syndrome induced by viral potentiation of chemical toxin. In Reye’s Syndrome (J. D. Pollack, Ed.), pp. 194–214. 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, 511–517.[Web of Science][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. 389–412. Academic Press, San Diego, CA.
Hyman, J. (1999). Hepatotoxicity: The Adverse Effects of Drugs and Others Chemicals on the Liver, 2nd ed., pp. 295–323. Lippincott Williams & Wilkins, Philadelphia, PA.
Kang, Y. J. (1992). Exogenous glutathione decreases cellular cadmium uptake and toxicity. Drug Metab. Dispos. 20, 714–718.[Abstract]
Korzeniewski, C., and Callewaert, D. M. (1983). An enzyme-release assay for natural cytotoxicity. J. Immunol. Methods 64, 313–320.[Web of Science][Medline]
Kullak-Ublick, G. A. (1999). Regulation of organic anion and drug transporters of the sinusoidal membrane. J. Hepatol. 31, 563–573.[Web of Science][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, 65–79.[Web of Science][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, 734–739.[Web of Science][Medline]
Muller, L., and Ohnesorge, F. K. (1982). Different response of liver parenchymal cells from starved and fed rats to cadmium. Toxicology 25, 141–150.[Web of Science][Medline]
Perrin, D. D., and Watt, A. E. (1971). Complex formation of zinc and cadmium with glutathione. Biochim. Biophys. Acta 230, 96–104.[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, 470–480.[Web of Science][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, 267–283.[Web of Science][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, 87–94.[Web of Science][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, 336–339.[Web of Science][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, 161–178.[Web of Science][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, 1833–1839.[Web of Science][Medline]
Webb, M., and Cain, K. (1982). Functions of metallothionein. Biochem. Pharmacol. 31, 137–142.[Web of Science][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, 542–557.[Web of Science][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. 6.1.1–6.1.18. 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. 551–566. FA Davis, Philadelphia.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. W. Laschke, M. D. Menger, Y. Wang, G. Lindell, B. Jeppsson, and H. Thorlacius Sepsis-associated cholestasis is critically dependent on P-selectin-dependent leukocyte recruitment in mice Am J Physiol Gastrointest Liver Physiol, May 1, 2007; 292(5): G1396 - G1402. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||