Toxicology, 60 (1990) 161--172 Elsevier Scientific Publishers Ireland Ltd.
Effect of cadmium on membrane potential in isolated rat hepatocytes Jos6e Martel, Michel Marion and Francine Denizeau Department of Chemistry, Universit~ du Quebec ?t Montrbal, Montrdal, Quebec (Canada) (Received June 1st, 1989; accepted September 1st, 1989)
Summary The effect of cadmium (Cd) on rat hepatocytes upon short term exposure was studied by focusing on the integrity of mitochondria and on the possible consequences of its disturbance, such as alterations in plasma membrane potential and loss of cell viability. Changes in the potential of mitochondrion and plasma membranes were monitored using [3H]triphenylmethylphosphonium (TPMP ÷) and [~4C]SCN- probes, respectively. Isolated rat hepatocytes were exposed to increasing CdCI 2 concentrations for short time periods (30--120 min). Cd measurement by atomic absorption showed that the cells efficiently accumulated Cd, as did mitochondria in situ. In CdCl2-treated cultures, it was observed that the release of TPMP ÷, which revealed a drop in the mitochondrial membrane potential, was time- and concentration-dependent, and that the first significant efflux was caused by a 30-min exposure to 89 gM CdCI 2. No significant change in plasma membrane potential, as judged from the increase in the uptake of SCN-, was detected after 30 min, suggesting the greater precocity of the mitochondrial attack. Finally, the release of lactate dehydrogenase (LDH) occurred only after 2 h of exposure, reflecting ultimate stages of cell injury induced by Cd. These results suggest that Cd induces an alteration in mitochondrial function in hepatocytes which may lead to the loss of plasma membrane potential and cell viability. The study therefore adds further evidence of the role of mitochondria as primary targets in Cd-induced cytotoxicity.
Key words: Cadmium; Cytotoxicity; Membrane potential; Mitochondria; Hepatocytes
Introduction I t h a s b e e n k n o w n f o r m a n y y e a r s t h a t c a d m i u m ( C d ) is a n e x t r e m e l y t o x i c e n v i r o n m e n t a l c o n t a m i n a n t h a v i n g a l o n g h a l f - l i f e i n m a n . T h e g r e a t e s t a c c u mulation occurs in the liver and kidney, which represent critical target organs [2,3]. A l t h o u g h t h e t o x i c i t y o f t h i s m e t a l h a s b e e n well e s t a b l i s h e d in v i v o  a n d i n v i t r o , t h e m e c h a n i s m s i n v o l v e d still r e m a i n u n c l e a r . S o m e h y p o t h e s e s i m p l y m i t o c h o n d r i a as t h e m o s t s e n s i t i v e t a r g e t s : t h e i n j u r i o u s e f f e c t s o f C d o n m i t o chondria-linked functions have been demonstrated with regard to mitochondrial
Address correspondence to: Dr. Francine Denizeau, D6partement de Chimie, Universit6 du Quebec Montr6al, Case postale 8888, succursale A, Montr6al, Qu6bec H3C 3P8, Canada. 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
enzymes [5--10], cell respiration [11--13], the lactate/pyruvate ratio [14--16] and energy-rich compound levels [17,18]. The integrity of mitochondria can be followed by mitochondrial membrane potential probes, such as TPMP*, in various cell types [19--21]. Hoek et al.  have initiated the use of T P M P ÷ in isolated hepatocytes for this particular purpose. This lipophilic cation accumulates in the mitochondrial matrix due to its high negative potential ; the loss of this potential will cause the release of T P M P ÷ from the organelle, and subsequently from the cells. Using a similar approach, the plasma membrane potential can be monitored by measuring the distribution of a permeant anion, SCN-, which is excluded from cells maintaining an intact potential [19,23]. A fall in the plasma membrane potential can therefore be followed by an increase in the uptake of SCN- by the cells. The aim of the present study was to extend the work on the involvement of mitochondria in Cd toxicity. For this purpose, freshly isolated hepatocytes were exposed to increasing CdC12 concentrations for short time periods (30--120 min). First, the accumulation of Cd by hepatocytes and mitochondria was monitored by atomic absorption spectrophotometry. Changes in mitochondrial membrane potential were followed with T P M P ÷. In parallel, perturbations in the plasma membrane potential were monitored using the SCN- probe. Cell viability was also assessed by the leakage of LDH into the extracellular medium . Thus, the present study examines the effect of Cd on hepatocytes upon short-term exposure by focusing on the integrity of mitochondria and on the possible consequences of its disturbance, such as alterations in the plasma membrane potential and the loss of cell viability. Materials and methods
Materials The sources of materials and chemicals were as follows: collagenase, Williams' medium E (WME), fetal bovine serum (FBS) and gentamicin (10 mg/ml), Gibco Canada; carbonyl cyanide m-chlorophenylhydrazone (CCCP), digitonin and valinomycin, Sigma Chemical Co., St. Louis, MO, USA. The following radioisotopes were obtained from New England Nuclear Canada Ltd: [methyl-3H] methyltriphenylphosphonium iodide (35.4 Ci/mmol) and 3H20 (5 mCi/ml). Potassium [14C] thiocyanate (58 mCi/mmol) was purchased from Amersham Canada Ltd and [carboxyl-X4C]inulin (3.32 /aCi/mg) from ICN Radiochemicals, Irvine, CA. All other reagents were of analytical grade. Preparation and incubation o f hepatocytes The hepatocytes were prepared by collagenase perfusion according to Seglen  with slight modifications . Male Sprague--Dawley rats (150--300 g) were anaesthetized with chloral hydrate (600 m g / k g , i.p.) prior to surgery. Briefly, the procedure involves an in situ two-step perfusion of the liver through the portal vein. Preperfusion with Ca2÷-free Hank's balanced salt solution is followed by perfusion with collagenase (60 U/ml) in a Ca2÷-containing Hank's medium. Liver cells are released in Williams' Medium E supplemented with 10% fetal bovine
serum and 0.005% gentamicin (WME-10% FBS). The cell suspension is filtered succesively through 250/~m and 50 gm nylon mesh and then centrifuged (50 g for 2 min). The pellet is resuspended in WME-10% FBS medium and centrifuged through Hank's medium containing 2% of bovine serum albumin (Hank's-BSA). After resuspension of the pellet in Hank's-BSA, a differential count was performed. Cell viability was routinely in the range of at least 85% as determined by exclusion of trypan blue . Hepatocytes were centrifuged and resuspended in WME-10% FBS medium. The cells (2.5 ml) were seeded in 35-mm Petri dishes (Corning) (1.3 x 106 cells) or in 60-mm Petri dishes (4.4 × 106 cells) and incubated in an atmosphere of water-saturated 95% 0 2 - - 5 % CO 2. Prior to any treatment, a time period of 2 h was allowed to permit attachment of viable hepatocytes to culture dishes. After this period, unattached cells were removed by washing the samples three times with Hank's medium and all further incubations were performed at 37°C in WME supplemented with 0.1% FBS. Unless specified, 3 culture dishes were used for each treatment and all experiments were carried out with 3 different cell preparations. Cd accumulation by cells and mitochondria Four 60-mm Petri dishes were used for each condition. They were incubated with concentrations of CdC12, ranging from 18 to 225 /~M. Aliquots of a stock solution of CdC12 (in 5% H N O 3) was added to the culture medium to obtain the desired final concentrations. After Cd treatment, the cells were washed as described above, and pooled in 4 ml of fractionation medium (125 mM sucrose, 60 mM KC1 and 3 mM HEPES, pH 7.1 at 22°C). Aliquots (250 /~l) were taken for protein determination and measurement of total cellular Cd after acid digestion (500 /~l of 8 N HNO3, final concentration, were diluted to 2 N HNO 3 just before taking measurements). Cd was measured by atomic absorption spectroscopy on a spectrophotometer (Varian AA-1475) coupled to a graphite furnace. The amount of cell protein was determined by the micro Bradford procedure using bovine serum albumin as the standard . The rest of the cell suspension was treated with digitonin to permeabilize the plasma membrane . After 2 min of agitation with 0.0015% digitonin, the suspension was diluted with 10 ml of fractionation medium and centrifuged (500 g for 5 min). The digitonin treatment did not affect mitochondria, as judged from the absence of significant activity of the mitochondrial markers glutamic dehydrogenase or adenylate kinase (intra-membrane space) in the supernatant . The pellet was washed (500 g for 5 min) with an homogenizing buffer (250 mM sucrose and 50 mM HEPES, pH 7.4 at 4°C). The pellet was resuspended in 0.4 ml of the same buffer and the cells were disrupted with a motor-driven Potter homogenizer (15 up-down strokes). Mitochondria were isolated by conventional differential centrifugation . The pellet obtained after centrifugation at 8000 g represents a mitochondriarich fraction, as evaluated by the markers glutamic dehydrogenase and adenylate kinase . This pellet was resuspended in 1 ml of homogenizing buffer and samples (250/~l) were taken for determination of mitochondrial Cd and protein, as previously described. 163
Measurement of changes in mitochondrial membrane potential Changes in the mitochondrial membrane potential were assessed by determining the loss of [3H] T P M P ÷ from the cells into the culture medium . This was performed with Cd as well as with agents having a known action on membrane potential to test the reliability of the system, namely, the mitochondrial uncoupler C C C P , the K ÷ ionophore valinomycin and high K ÷ medium. Thirty-five millimeter Petri dishes were used. The cultures were exposed to 0.5 /aCi/ml [3HITPMP ÷ for 30 min, and washed 3 times with H a n k ' s medium. Fresh medium containing either CdCI z (18--225 /aM), 40/aM CCCP, or 10/ag/ml valinomycin was added and the cultures were incubated for various time periods. High K ÷ medium contained 120 mM KC1 and 24 mM KHCO3, instead of N a H C O 3, to give a final K* concentration of 150 mM. At the end of the incubation, the medium was discarded and the cells were washed as previously and lysed by treatment with 2 ml of 0.3 N N a O H at 37°C for 1 h. The intracellular [3H]TPMP ÷ was measured in 100-/al aliquots by liquid scintillation counting (LSC). The quantity of cell protein was determined as before.
Evaluation of alteration in plasma membrane potential Changes in the plasma membrane potential were monitored by the increase in the uptake of [14C]SCN-by the hepatocytes. The cultures were incubated with the positive control agents previously used with T P M P ÷, as well as with Cd. The cells (in 35-mm Petri dishes) were preincubated with 0.5/aCi/ml [~4C]SCN- for 30 min. Thereafter, high K ÷ medium, C C C P , valinomycin or CdC1 z were added and the incubation was continued for various time periods. At the end of the incubation, the medium was discarded and the cells were washed as previously and lysed with 600/al of 0.25o70 sodium dodecyl sulfate (SDS). Aliquots (550-/A) were taken for measurement of intracellular SCN- by LSC. The amount of cell protein was determined as described above.
Measurement of cellular volumes For each condition, intracellular waterspace was estimated in parallel incubations by a modification of the method of Baur et al. . Sixty millimeter Petri dishes were used. The cells were incubated with 2.0 /aCi/ml 3H20 and 0.3 /aCi/ml [carboxyl-~4C]inulin for 15 min. Incubations were terminated by discarding the medium and immediately adding 4.0 ml of 0.3 N N a O H . After cell lysis (at 37°C for 1 h), 200-/al aliquots were taken for determination of radioactivity by LSC. The amount of protein was determined as previously described.
Assessment of cell viability Viability of hepatocytes was monitored by measuring L D H activity in the extracellular medium, according to Mold6us et al. .
Statistical analysis Significance of differences between treated and control samples was assessed by A N O V A and the post-hoc Scheff6 F-test with a level of 95°7o.
Intracellular Cd accumulation T h e a c c u m u l a t i o n o f C d in h e p a t o c y t e s a n d in m i t o c h o n d r i a was first verified. F o r this p u r p o s e , cultures were e x p o s e d to increasing CdC12 c o n c e n t r a t i o n s ( 1 8 - 225/aM) for v a r i o u s t i m e p e r i o d s ( 3 0 - - 1 2 0 min). A s s h o w n in Fig. 1, the intracellular C d levels increase in a t i m e - a n d c o n c e n t r a t i o n - d e p e n d e n t m a n n e r , b o t h in h e p a t o c y t e s a n d in the m i t o c h o n d r i a l f r a c t i o n . T h e c o m p a r i s o n b e t w e e n the 2 c o m p a r t m e n t s can be m a d e with the d a t a expressed in 2 d i f f e r e n t ways. Statistical analysis o f the d a t a expressed in n m o l e s / mg o f p r o t e i n as in Fig. 1, shows t h a t there is no d i f f e r e n c e between the values o f the cellular a n d the m i t o c h o n d r i a l f r a c t i o n s . T h e a c c u m u l a t i o n o f m e t a l can also be expressed as a p e r c e n t a g e o f t o t a l cellular C d a c c u m u l a t i o n . T h e d a t a expressed this w a y (not shown) indicate t h a t a b o u t 10°70 o f the t o t a l cellular C d is l o c a t e d in m i t o c h o n d r i a after a 2-h exposure p e r i o d . C o n s i d e r i n g the fact t h a t the v o l u m e o f m i t o c h o n d r i a within the cell represents a b o u t 12070 o f the t o t a l v o l u m e , it can be c o n c l u d e d that, as a n average, the c o n c e n t r a t i o n o f C d in m i t o c h o n d r i a is c o m p a r a b l e to t h a t in the w h o l e cell.