* Operational Toxicology Branch, Human Effectiveness Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio 454337400;
Department of Physics, Wright State University, Dayton, Ohio 45435; and
ManTech Environmental Technology, Inc., Dayton, Ohio 45433
Received September 3, 2002; accepted December 6, 2002
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ABSTRACT |
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Key Words: cadmium; hepatocytes; in vitro; albumin; kinetics.
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INTRODUCTION |
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The liver is the primary target organ following acute systemic cadmium (Cd2+) exposure (Dudley et al., 1982). Uptake of Cd2+ into the liver is critical to the development of the overall toxicity induced by this heavy metal. Approximately half of the Cd2+ absorbed systemically is rapidly accumulated in the liver of rats (Perry et al., 1970
; Sasser and Jarboe, 1977
). This rapid accumulation by the liver results in reduced availability of Cd2+ to other organs such as kidney and testes, which are more sensitive to its toxic actions.
Direct metabolic conversion of Cd2+, such as oxidation, reduction, or alkylation, is not known to exist. After entering the blood, Cd2+ can bind to plasma proteins (albumin, globulins, etc.), metallothionein (Mt), or directly to erythrocytes. The divalent Cd2+ ions bind to anionic groups on proteins and other molecules through charge interactions and with sulfhydryl groups through coordinate-covalent binding. Cadmium has a particularly high affinity for the sulfhydryl groups of albumin and Mt (Nordberg et al., 1985). Therefore, Cd2+ circulates in the blood primarily bound to albumin and traces of Mt (Foulkes and Blanck, 1990
; Roberts and Clark, 1988
). Presumably, this affinity for protein sulfhydryl groups results in Cd2+ binding to a multitude of cellular proteins, both within cells and on cell surfaces.
Frazier and Kingsley (1976, 1977
) have shown using kinetic studies in the isolated perfused rat liver that the uptake of Cd2+ by the liver involves a combination of simple diffusion and carrier mediated processes. Isolated hepatocytes have been used as a model for examining Cd2+ kinetics and toxicity (Din and Frazier, 1985
; Stacey et al., 1980
; Stacey and Klaassen, 1980
). Cadmium uptake in isolated hepatocyte suspension and plated cultures has been shown to be rapid, biphasic, and energy-independent (Blazka and Shaikh, 1992
; Stacey and Klaassen, 1980
). However, these studies conducted Cd2+ exposures under nonphysiological conditions with respect to plasma albumin concentrations. Furthermore, Cd2+ uptake kinetics in freshly isolated suspension cultures have been shown to differ from those observed in primary cultures of rat hepatocytes (Kukongviriyapan and Stacey, 1989
). These differences are probably due to the compromised integrity of the plasma membrane during hepatocyte isolation (Gautum et al., 1987
; Wanson et al., 1977
).
The objective of the present study was to investigate the effects of physiological concentrations of serum albumin (600 µM) on the kinetics of Cd2+ uptake in primary cultures of rat hepatocytes. Previous studies have shown that Cd2+ can bind to plasma albumin from a variety of species (Suzuki et al., 1986; Nordberg and Nordberg, 1987
) and to approximately the same extent (Goumakos et al., 1991
). The use of rat serum albumin (RSA) was considered, but limited availability and excessive cost made it not feasible to conduct a study of the magnitude planned. Based on these observations, and the fact that most previous work investigating Cd2+ uptake kinetics in vitro were conducted utilizing bovine serum albumin (BSA) at nonphysiological concentrations, BSA was chosen as the source of albumin to conduct in vitro Cd2+ uptake studies in primary hepatocytes exposed and cultured under more physiological conditions with respect to albumin concentration. The influence of albumin binding on the concentration of free Cd2+ in tissue culture dosing buffer was investigated in independent binding studies. The binding data were used to interpret the uptake kinetics of Cd2+. These kinetic data are critical for the development of a BBK model for Cd2+ to support accurate predictions of hepatic dosimetry in rats and to extrapolate this dosimetry estimate to humans.
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MATERIALS AND METHODS |
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Animals.
Male Fischer 344 rats (225300 g) were obtained from Charles River Laboratories. Animals were fed rat chow (Purina) ad libitum and had unrestricted access to purified water. The animals used in this study were handled 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.
Rat liver perfusion.
Male Fischer 344 rats were anesthetized ip with 1 ml/kg of a mixture of ketamine (70 mg/ml; Parke-Davis, Moris Plains, NJ) and xylazine (6 mg/ml; Mobay Corp., Shawnee, KS) prior to undergoing the in situ liver perfusion. Livers were perfused at a flow rate of 20 ml/min at 37°C using an antigrade two-step perfusion method described by Seglen (1976) as previously modified (DelRaso and Frazier, 1999
). The first perfusion step resulted in blood removal from the liver using Hanks balanced salt solution (HBSS; pH 7.2), lacking calcium and magnesium, supplemented with 15 mM 4-(2-hydroxyethl)-1-piperazineethanesulfonic acid (HEPES), heparin (2.0 U/ml) and ethylenebis(oxyethylenenitrilo)-tetraacetic acid (EGTA; 0.5 mM). The second perfusion step resulted in liver digestion, using complete HBSS buffer (pH 7.2) supplemented with 15 mM HEPES and collagenase (0.26 U/ml; based on Wunsche U/mg).
Hepatocyte enrichment and culture.
Viable primary rat hepatocytes were enriched from the crude hepatocyte suspension by low speed centrifugation (50 g) for 3 min, repeated three times. Typical viabilities of isolated hepatocytes ranged from 80 to 90% with yields of 200 to 300 million cells, as determined by trypan blue dye exclusion (Story et al., 1983). For cell culture studies, primary hepatocytes (2 to 3 x 108 cells) were adjusted to a cell density of 1.2 x 106 cell/ml in Chee modified MEM culture medium (pH 7.2; Gibco, Grand Island, NY) supplemented with HEPES (10 mM), insulin/transferrin/sodium selenite solution (final concentration 5 µg/ml, 5 µg/ml, and 5 ng/ml, respectively) and dexamethasone (0.4 mg/ml). For all Cd2+ uptake studies, 1.0 ml of cells was seeded in 6-well (1.2 x 106 cells/well) culture plates previously coated with rat tail collagen (Upstate Biotechnology, Lake Placid, NY) at 2.6 mg/cm2. After 3 h of incubation in a 95% air/5% CO2 atmosphere at 37°C, to allow for attachment, rat hepatocytes were re-fed with 2.0 ml of Chee culture medium lacking dexamethasone. Hepatocytes were cultured and treated according to the experimental schedule outlined in Figure 1
.
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LDH assay.
Lactate dehydrogenase (LDH) enzyme activity was assayed in hepatocyte homogenates and culture medium to determine plasma membrane integrity. LDH enzyme activity in primary hepatocytes was determined using a DuPont ACA V discrete clinical analyzer (DuPont, Huffman Estates, IL). Measurements of LDH enzymatic activity were monitored at 37°C by reflectance spectrophotometry at 340 nm. The change in absorbance at 340 nm due to the formation of reduced nicotinamide adenine dinucleotide (NADH) is proportional to the rate of product formed, and, hence, enzymatic activity. The enzyme activities determined in medium were expressed as International Units (U) of Biochemistry (conversion of 1 mmol of substrate to product at 37°C per minute) per liter and reported as a percentage of total LDH (intracellular plus medium) content.
Protein assay.
Cell protein content in plated cultures was determined by the Lowry method (Lowry et al., 1951), using the BCA Protein Assay Kit (PIERCE, Rockford, IL). Cell sonicates (25 ml) were diluted 1:4 with DPBS. Aliquots (10 ml) of diluted cell sonicates and BCA assay kit reagent (200 ml) were placed in a 96-well plate and read spectrophotometrically at a wavelength of 562 nm in a THERMOmax Microplate Reader. Concentrations were expressed as mg protein/ml of original lysate.
Effective Concentration 50% (EC50) Determinations
In order to evaluate the effect of BSA on Cd2+ toxicity, the Cd2+ exposure-response relationship was evaluated in the presence or absence of BSA. Primary rat hepatocytes were seeded into rat tail collagen-coated 96-well culture plates and allowed to attach for 3 h. Following the 21-h recovery period, primary hepatocyte cultures were exposed to Cd2+ in TGS buffer lacking BSA (1.8 to 18 µM Cd2+) or containing 600 µM BSA (18 to 320 µM Cd2+) for 1 h at 37°C. Cell viability was evaluated by the MTT assay at two time points: (1) immediately after the Cd2+ treatment, and (2) 23 h after the 1-h exposure. Exposure-response curves, expressed as percent of control optical density, were generated using SigmaPlotTM graphics package (Jandel Scientific, San Rafael, CA). Cadmium EC50 values were determined by nonlinear regression analysis of exposure-response curves, using SigmaStatTM statistical package (Jandel Scientific, San Rafael, CA).
Binding Studies
Cadmium-BSA binding studies were conducted in TGS buffer containing 600 µM BSA spiked with stable Cd2+ (32 to 8000 µM) and 109Cd2+ (0.4 µCi/ml). Samples were incubated at 37°C for 1 h, and aliquots of each Cd2+/BSA solution (450 µl) were centrifuged in Centrifree microseparation devices (Amicon, Inc., Beverly, MA), using a 33° fixed-angle rotor at 37°C for 15 min at 2000 g in an RC-5B centrifuge (Sorvall, Newtown, CT). Aliquots of the Cd2+/BSA solutions and their respective ultrafiltrates were counted for 5 min, using a Cobra Quantum gamma counter (Packard, Meriden, CT). The binding of Cd2+ to the plastic microseparation devices was minimized by treating the sample reservoirs and filtrate cups with a 5% (wt/vol) solution of cetyltrimethylammonium bromide (CTAB; Aldrich, Milwaukee, WI) overnight at room temperature.
Binding data were analyzed by Scatchard analysis (Scatchard, 1949), where the ratio of bound to free Cd2+ is plotted versus the bound Cd2+ concentration. Empirical fitting of the data provide estimates of the binding affinity (expressed as the dissociation constant [Kd µM] and the capacity [n: number of binding sites per molecule of BSA]).
Cadmium Uptake Studies
Following the 21-h recovery period, hepatocytes cultured in 6-well plates were exposed to Cd2+ in TGS buffer lacking BSA (0.42 to 180 µM Cd2+) or containing 600 µM BSA (32 to 1000 µM Cd2+) at 37°C. Concentration ranges for Cd2+ uptake studies were based on acute cytotoxicity. Concentrations of Cd2+ resulting in greater than 10% leakage of the total intracellular LDH immediately following a 1-h exposure were not used. To determine Cd2+ uptake rates, 1.0 ml of Cd2+-containing exposure buffer radiolabeled with 109Cd2+ (30.4 µCi/ml TGS buffer) was placed in each well and the cells incubated at 37°C. At specific time points, from 15 s to 60 min, radiolabeled TGS buffer was removed from each well and the wells were washed rapidly two times with ice cold phosphate-buffered saline (PBS). Following PBS washes, cells were solubilized by adding 1 ml of 0.2 N NaOH/0.2% SDS solution to each well. After removing a 25 µl aliquot for protein determination, the amount of incorporated radiolabeled metal was determined by gamma spectrometry. Data were converted to nmol Cd2+/ml of lysate, using the specific activity of the exposure buffer as determined for each dosing solution, and normalized by dividing by the protein concentration (mg protein/ml cell lysate). Data are expressed as nmol Cd2+/mg cellular protein.
Uptake Rate Analysis
At specific time points up to 60 min after the addition of Cd2+, the amount of radiolabeled Cd2+ associated with hepatocytes was determined. Initially, the total amount of Cd2+ associated with cells (nmol/mg cell protein) was plotted versus time. The data suggested that two uptake components are active; one resulted in a rapid uptake and equilibration within the first minute (Component I) and the second in a slower linear process that occurred throughout the 1-h study (Component II). For each separate experiment, cadmium uptake rates associated with Component II (RII: nmol/min/mg cell protein) were determined from the slopes obtained from linear regression analysis of Cd2+ uptake (nmol/mg cell protein) from 15 to 60 min using SigmaPlotTM (Fig. 2A). The amount of Cd2+ accumulated via Component I (CdI, rapidly internalized and cell membrane associated) at a particular time point (T) was calculated by subtracting Component II (CdII) from the total Cd2+ (CdTot) accumulation curve (Fig. 2B
) according to the following equation,
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Statistics.
Differences of means were compared by a one-way analysis of variance (ANOVA). Means found to be significant by ANOVA were compared using a Student-Newman-Keuls pairwise multiple comparison procedure with Type I error level held at p < 0.05. All statistical analyses were conducted with SigmaStatTM (Jandel Scientific, San Rafael, CA).
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RESULTS |
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DISCUSSION |
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Albumin, at a physiological concentration of approximately 600 µM, is a critical macromolecule in plasma and plays an important role in binding and transporting Zn2+ and other metals. In fact, Cd2+ has a high affinity for anionic and sulfhydryl groups on albumin and Mt (Nordberg et al., 1985) and circulates in the blood primarily bound to these macromolecules (Foulkes and Blanck, 1990
; Roberts and Clark, 1988
). In the case of Zn, 98% of the exchangeable fraction of serum Zn2+ is bound to albumin (Giroux and Henkin, 1972
). Although there are numerous potential Zn2+ binding sites on albumin, the Zn2+ concentration in blood does not appear to exceed the albumin concentration under physiological conditions. This suggests that only one high-affinity Zn2+ binding site on albumin is occupied under physiological conditions. Previous studies have shown that Cd2+ can bind to rat and human plasma albumin (Goumakos et al., 1991
; Nordberg and Nordberg, 1987
; Suzuki et al., 1986
). The Scatchard analysis of equilibrium dialysis data in a previous study has indicated two high-affinity Cd2+ binding sites and one high-affinity Zn2+ binding site on human and canine albumin (Goumakos et al., 1991
). In the present study, Scatchard analysis of Cd2+-BSA binding data agreed with the previous binding studies and indicated two high affinity Cd2+ binding sites on BSA. Scatchard analysis also indicated a dissociation constant of 15.2 µM for the BSA-Cd2+ complex. This is lower than a previously reported dissociation constant of 38 µM for a human albumin-Cd2+ complex (Aoki and Suzuki, 1987
). These data suggest that although different species of albumin appear to have similar numbers of binding sites, they do not appear to have the same binding affinity for Cd2+. Thus, when using in vitro binding in in vivo BBK models it may be necessary to utilize binding parameters specifically evaluated for the albumin species of interest.
Data presented in the present study indicated that transport of Cd2+ into rat hepatocytes occurred as a two-component process that is hypothesized to involve binding to the plasma membrane and internalization. The proposed first component (Component I) was observed to be rapid reaching a steady state in less than one minute. We hypothesized that failure to derive a rate for this rapid component of Cd2+ uptake was due to rapid equilibrium binding of Cd2+ to the outer surface of the plasma membrane. This component, unlike Component II, was found to be sensitive to washes containing the metal chelator EGTA. This additional finding supports the notion that Component I Cd2+ uptake was associated with surface binding.
In the present study, the slower uptake phase (Component II) of Cd2+ correlated well with previously reported studies of Cd2+ uptake in rat hepatocytes. In fact, the Component II uptake rates obtained in the present study in primary hepatocytes exposed to Cd2+ in TGS buffer lacking albumin were similar to that obtained by Stacey and Klaassen (1980) in primary hepatocytes exposed to Cd2+ in saline. Furthermore, Failla et al. (1979)
have shown that the quantity of Cd2+ accumulated by hepatocytes is directly proportional to both the level of metal and the duration of the incubation. These authors showed that Cd2+ accumulated linearly by this slower phase of uptake for 7 to 8 h in monolayers of primary hepatocytes.
In vitro studies have indicated that cellular Cd2+and other chemical uptake and toxicity are reduced in the presence of albumin (Frazier, 1995; Gulden et al. 2002
; Klug et al., 1988
; Seibert et al., 2002
). Frazier (1995)
has shown that as the albumin concentration increases the Cd2+ exposure-response curve shifts to the right, indicating an apparent increased resistance to Cd2+ toxicity. In the present study, the exposure of rat hepatocytes to Cd2+ in solutions containing physiological albumin (600 µM) also resulted in a shift in the exposure-response curve to the right when compared to similar Cd2+ exposures in the absence of albumin. Therefore, the observed Cd2+ EC50, determined under more physiological conditions, was found to be 65.5 ± 2.4 µM. This value was 4-fold greater than that of the EC50 observed under BSA-free conditions (14.3 ± 3.9 µM). However, when this EC50 was recalculated, using the amount of free Cd2+ in the BSA-containing exposure buffer as the dose metric, the EC50 was reduced by a factor of 62 to 1.05 ± 0.36 µM, an approximate 14-fold reduction compared to the EC50 observed under BSA-free conditions. Interestingly, when these exposure-response curves were plotted using intracellular Cd2+ (nmol/mg) as the dose metric, instead of the Cd2+ concentration in the exposure buffer, no significant difference in the calculated EC50 was observed. In addition, when the slopes of the Cd2+ exposure-response curve derived from Cd2+-exposed rat hepatocytes in the presence and absence of BSA were observed they were similar. This suggests that the mechanism of toxicity was not affected by the presence of BSA. These findings indicate that the protective effect of albumin resulted from a reduction of the Cd2+ uptake and was not due to some direct beneficial effect of albumin on hepatocytes. This is supported by the recent work of Gulden et al. (2002)
, where it was demonstrated that albumin had no other effect than to bind organochlorine pesticides and chlorophenols in vitro, and that cytotoxicity was based solely on the free concentration of these chemicals. The data further suggest that significant Cd2+-induced hepatotoxicity, regardless of exposure conditions, occurs when an apparent intracellular threshold of approximately 0.7 nmol/mg is exceeded.
Previous ex vivo studies investigating hepatic Cd2+ transport using isolated perfused rat liver are consistent with the hypothesis that Cd2+ uptake into the liver occurs via a combination of simple diffusion and a carrier-mediated process (Frazier and Kingsley, 1976, 1977
; Kingsley and Frazier, 1979
). A temperature-sensitive, facilitated-carrier transport mechanism operating in conjunction with passive diffusion in the uptake of Cd2+ and Zn2+ has also been reported in other systems (Bobilya et al., 1992
; Failla and Cousins, 1978
; Failla et al., 1979
; Frazier, 1981
; Stacey and Klaassen, 1980
, 1981
). In the present study, the rate of Cd2+ transport via Component II was non linearly related to the concentration of free Cd2+ in the exposure medium (see Fig. 10B
). This result suggests that more than one transport process was involved in Cd2+ uptake and is consistent with the previous studies on Cd2+ transport mentioned above.
As noted above, when the exposure concentration of Cd2+ is expressed as the calculated free Cd2+ based on the binding studies, the Component II Cd2+ uptake rate in the presence of BSA was increased when compared to the uptake rate at similar free Cd+2 exposure concentrations in the absence of BSA. We have hypothesized that this behavior may have resulted from diffusion-limited, nonequilibrium conditions occurring near the cell surface that in effect results in higher free Cd2+ concentrations at the cell surface in the presence of BSA (Fig. 11). Support for this hypothesis is found in the work of Weisiger et al. (1991)
. They expanded a sinusoidal transport model to investigate hepatic uptake of protein-bound ligands. These authors found that an earlier standard sinusoidal transport model (Baker and Bradley, 1966
; Goresky et al., 1973
) failed to fit their biological data, which showed increased uptake rates in the presence of increasing albumin concentrations for equivalent free concentrations of an anionic ligand (oleate). They determined that the standard model was deficient because it did not consider the presence of diffusion through an unstirred water layer. This layer is always found adjacent to membranes and often can be a major factor in determining the uptake rate in experimental systems (Barry and Diamond, 1984
; Weisiger et al., 1989
). In an extension of the standard sinusoidal transport model, Bass and Pond (1988)
incorporated an unstirred fluid layer near the cell surface across which ligand must diffuse to reach the cell. In their model, when the free concentration of the ligand in the bulk medium is equal in the presence and absence of albumin, the free concentration of ligand near the transporting membrane is higher in the presence of albumin. This occurs because free ligand near the membrane can arise both from diffusion of free ligand directly through the unstirred layer, and also from release of bound ligand that has diffused to the membrane surface. Thus, in this situation, the binding between ligand and albumin is not in equilibrium near the membrane surface. The extra free ligand near the membrane in the presence of albumin essentially occurs because there is enhanced transport of ligand through the unstirred layer, namely, the normal diffusion of free ligand plus the additional diffusion of bound ligand. Although the diffusion of bound ligand is fairly slow, due to the lower diffusion constant of albumin relative to free ligand, the potentially high concentration of bound ligand in the bulk medium can provide a significant increase to net delivery of free ligand to the membrane surface. It is the concentration of free ligand near the membrane surface that determines the rate of transport through the membrane. This type of model was found to explain the albumin dependence of the kinetic data of Weisiger et al. (1991)
and is consistent with our Cd2+ uptake data in the presence and absence of albumin.
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Furthermore, the results of the analysis of the concentration dependence of Component II Cd2+ uptake is also consistent with the unstirred layer hypothesis. Given the existence of the unstirred layer, in the presence of BSA, the linear transport rate constant (P) should increase and the concentration at half maximum saturable transport (KT) should decrease. The data indicate that P did increase significantly. On the other hand, while there was a trend for KT to decrease, the difference is not statistically significant. Unfortunately, the experimental design did not allow for a good statistical comparison of KT between treatments. Additional uptake experiments at free Cd2+ concentrations near the KT would be required to resolve this issue.
In summary, our data indicated that the exposure of primary rat hepatocytes to Cd2+ in the presence of physiological concentrations of BSA (600 µM) altered the cadmium dose-response relationship, indicating less toxicity when compared to Cd2+ exposures in the absence of BSA. However, the Cd2+ dose-response relationships were similar when viability was plotted against the amount of Cd2+ internalized by rat hepatocytes, independent of the presence of BSA. These data suggest that the apparent reduced hepatotoxicity is due to the binding of Cd2+ to BSA in the exposure buffer with a consequent reduction in the concentration of free Cd2+ available for transport into the hepatocytes. The present study also indicates that the internalization rate of Cd2+ via Component II in rat hepatocytes exposed in the presence of BSA was enhanced compared to the internalization rate in the absence of BSA at corresponding free Cd2+ concentrations in the bulk medium. Taken together, our data suggest that the presence of BSA alters the rate of Cd2+ uptake, but not the dose-response relationships when the internal dose is used as the dose metric. We hypothesize that the effects of BSA on kinetics result from diffusion-limited, nonequilibrium conditions occurring near the cell surface, and resulting in higher free Cd2+ concentrations at the cell surface. This hypothesis is consistent with the hepatic-uptake model proposed by Bass and Pond (1988) that incorporated this feature into their model.
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ACKNOWLEDGMENTS |
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NOTES |
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