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Effect of 2-acetylaminofluorene on intracellular free Ca2+ in isolated rat hepatocytes
Toxicology, 71 (1992) 21-33 Elsevier Scientific Publishers Ireland Ltd.
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Effect of 2-acetylaminofluorene on intracellular free C a 2+ in isolated rat hepatocytes S. Lefebvre, M. M a r i o n a n d F. D e n i z e a u * D~partement de Chimie, Universitk du Qubbec a Montrkal. QuObec (Canada) (Received May 22nd, 1991; accepted August 13th, 1991)
Summary The effect of 2-acetylaminofluorene (2-AAF) on the intracellular free Ca 2+ ([Ca2÷]i} and viability of isolated rat hepatocytes has been investigated using the fluorescent probes quin 2 and propidium iodide respectively. At the highest concentration tested (224 ~tM), 2-AAF produces an elevation of [Ca2+]i which shows a biphasic profile. A small initial increase is observed during the first 5 min; this is followed by a considerable rise which reaches up to 2.5 times the control value at 15 min. These changes in intracellular calcium are not accompanied by detectable alterations in cell viability. In order to determine the mechanisms by which this effect of 2-AAF takes place, three calcium antagonists, namely verapamil, TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) and ruthenium red (RuR), have been used. The results suggest that the first phase is dependent upon internal Ca 2+ store mobilization, while the second phase seems to be related to Ca 2+ entry from the extracellular space. The data obtained with RuR further indicate that mitochondria may be involved in the perturbation of calcium homeostasis caused by 2-AAF. In addition, in the experiments involving antagonists, no consistent pattern emerges that suggests a close relationship between intracellular Ca 2+ levels and cell viability. The present study provides further information on the mechanisms by which the well-known hepatotoxin 2-AAF may interact with liver cells. It also shows that when these cells are exposed to a toxin, short-term changes in [Ca2+]i may not be accompanied by loss of cell viability, and conversely, that changes in cell viability may occur without alterations in [Ca2+] i.
Key words." 2-Acetylaminofluorene; Calcium; Hepatocytes; Quin-2; Cell viability
Introduction The importance of Ca 2+ in the cell is well established. Intracellular free Ca 2+ is implicated in a wide variety of cellular processes such as hormonal response, enzymatic activation and cell division [1-3]. The intracellular free Ca 2÷ concentration is regulated within narrow limits exhibiting very low levels in the cytosol (10 - 7 tO 10 -8 M) as compared to a relatively high extracellular concentration (10 - 3 M ) . Correspondence to." Dr. Francine Denizeau, D6partement de Chimie, Universit6 du Qu4bec h Montr6al, Case Postale 8888, Succursale A, Montr6al, Qu6bec, Canada H3C 3P8. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
22 The regulation of the intracellular free Ca 2+ concentration in most cells is known to take place at three major sites: the plasma membrane, the endoplasmic reticulum (ER) and the mitochondria. Thus, a dysfunction in any of the systems involved may result in a non-physiological increase in cytosolic free Ca 2+ which can lead to cell injury. It has been shown that Ca 2+ may play a critical role in the mechanisms of chemically induced cytotoxicity. This has been extensively investigated especially in hepatocytes [4-8] although it still remains a subject of controversy. Many studies performed with different toxins have provided evidence of an alteration of function at the level of either of the Ca 2+ regulating systems. For example, Orrenius et al. have demonstrated that the metabolism of menadione impairs the ability of rat liver mitochondria to take up and retain Ca 2+ [9]. Moore and coworkers have shown that different toxic agents like carbon disulfide, carbon tetrachloride and other chlorinated halothanes can inhibit the endoplasmic reticulum calcium pump [10,11]. They proposed that this inhibition may represent the primary event in the cytotoxicity induced by these xenobiotics. The disturbance of Ca 2+ fluxes at the level of the plasma membrane in the presence of toxic agents has been suggested in many studies. Tsokos-Kuhn et al. have demonstrated the inhibition of the liver plasma membrane Ca 2+ ATPase by acetaminophen and bromobenzene in vivo [12], while Mold4us et al. have shown the same phenomenon in vitro [13]. Similar observations have been reported by Orrenius et al. with menadione [14]. Previous data obtained with the well-known hepatotoxin acetaminophen have led to the proposal that the alkylation of membrane-bound proteins is likely to be an important molecular event in the process that renders the cell unable to control its cytosolic Ca 2+ concentration. Indeed, acetaminophen, after undergoing oxidation by cytochrome P-450, produces alkylating species that interact with macromolecules including membrane proteins. The plasma membrane Ca 2+, Mg 2+ ATPase has been proposed as being one of the target proteins [12], although other plasma membrane Ca 2+ translocases may be affected. Such interactions are believed to lead to an elevation of intracellular free Ca 2÷ in the liver in vivo [15]. Therefore, it appears that alkylating agents may represent a threat to proper maintenance of cell Ca 2÷ homeostasis. In the light of the previous findings, it becomes relevant to further investigate the effect of xenobiotics, in particular of alkylating agents such as acetaminophen on Ca 2+ metabolism in liver cells. Among these agents, 2-acetylaminofluorene (2-AAF) is a well known hepatocarcinogen that shares many similarities with acetaminophen both in its chemical structure and its bioactivation by cytochrome P-450 which represents an important step in the formation of alkylating species [16]. The aim of this study was therefore to investigate the effect of 2-AAF on the intracellular free calcium and viability of isolated rat hepatocytes. By using the pharmacological agents verapamil, TMB-8, (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate), and ruthenium red (RuR), an attempt was made to determine at what level perturbation takes place. The results obtained provide further information on the mechanisms by which 2-AAF may exert its deleterious effects on liver cells.
24 magnetic stirring and the cuvette chamber was maintained at 37°C. The fluorescence signal was calibrated by addition of 50 ~M digitonin; after 20 min, maximum fluorescence (Fmax) was obtained. This was followed by the addition of 6 mM E G T A (ethyleneglycol-bis(13-aminoethylether)-N,N'tetraacetic acid) to obtain the minimum fluorescence (Fmi,). The intracellular free calcium concentration ([Ca2+]i) was evaluated using the following equation [Ca2+]i = g d ( F - Fmin)/(Fma x - F). assuming a dissociation constant (Kd) value of 115 nM as determined by Tsien et al. [20]. The possibility of having interference in the fluorescence measurement of quin 2 was tested by establishing calibration curves with Ca2+-EGTA according to Tsien et al. [26]. Tests carried out in our laboratory have shown that none of the compounds used (DMSO, 2-AAF, verapamil, TMB-8 and RuR) has an effect on the dissociation constant (Kd) of the quin 2-Ca 2+ complex.
Viability measurement using propidium iodide
Viability of the hepatocytes was monitored in a continuous fashion by a fluorometric assay using propidium iodide as described by Gores et al. [21]. Briefly, the cells (105/ml) were incubated at 37°C in the modified Hanks' medium described above. An aliquot of this suspension was transferred to a quartz cuvette. Fluorescence intensity was taken at an excitation wavelength of 515 nm and an emission wavelength of 620 nm. Immediately after addition of 1 #M propidium iodide, an initial fluorescence measurement was made (A). At the end of experiment, 400 #M digitonin was added to permeabilize the cells and a final maximum fluorescence (B) was obtained after 20 min. Hepatocyte viability was evaluated using the following relationship V = 100 x ( B - X ) / ( B - A ) where V is the viability in percent and X the fluorescence measured at any moment during the experiment. In one series of experiments, hepatocyte viability was monitored by measuring lactate dehydrogenase (LDH) activity in the extracellular medium according to Mold6us et al. [22].
Treatment with Ca 2+ antagonists
For the investigation of the effect of verapamil, hepatocytes or quin 2-loaded hepatocytes were preincubated in the presence of 200 #M verapamil (dissolved in DMSO) for 10 min prior to analysis. In the study using TMB-8, hepatocytes or quin 2-loaded hepatocytes were preincubated in the presence of 0.6 mM TMB-8 (dissolved in Tris buffer pH 7.4) for 10 min before analysis. Finally, in the experiments that tested the effect of RuR, hepatocytes or quin 2-loaded hepatocytes were preincubated with 25 t~M RuR (dissolved in modified Hanks' medium) for 10 min prior to fluorescence measurements.
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Results
Effect of 2 - A A F concentration on hepatocyte intracellular free Ca 2+ The effect of 2-AAF on the intracellular free Ca 2+ in hepatocytes was first determined. For this study, cell suspensions were exposed to three different 2-AAF concentrations (56, 112 and 224/zM). These concentrations were chosen due to the fact that they did not induce cytotoxicity over the incubation period as determined by L D H leakage measurement (results not shown). Intracellular free Ca 2÷ was monitored using quin 2 (see Materials and Methods) during the first 20 min of exposure. As shown in Fig. 1, 56 #M 2-AAF did not cause any significant increase in intracellular free Ca 2+ over the 20-min period. However, at a concentration of 112 /~M, 2-AAF produces a slight initial increase of intracellular free Ca 2+ that is maintained for the rest of the incubation time. The most striking change in cytosolic free Ca 2÷ is obtained with 224 #M 2-AAF. In this case, the small elevation observed during the first 5 min is similar to that obtained with 112/~M 2-AAF; however, this small elevation is followed by a considerable rise which reaches up to 2.5-times the control value at 15 rain. This biphasic response suggests that there may be more than one mechanism implicated in the perturbation of cytosolic free Ca 2+ by 2-AAF. In the light of these observations, the latter concentration (224/zM 2-AAF) was selected for the subsequent studies with verapamil, TMB-8 and RuR.
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Time (rain) Fig. 1. Effect of different concentrations of 2-AAF on intracellular free Ca -'+ of hepatocytes. At time = 0.224 #M (O), 112 #M ( I ) or 56 #M 2-AAF (/~) was added to quin 2-loaded hepatocytes. Determinations were carried out as described in the Materials and Methods section. Data represent means ± S.E. for at least three experiments with different cell preparations. Initial cytosolic free Ca 2÷ was 141.7 n M ± 7.1 (S.E.) for experiments in which extracellular free Ca 2+ was measured (including those of the other Figures).
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Fig. 2. Effect of verapamil pretreatment on intracellular free Ca e+ (A), and cell viability (B) of hepatocytes exposed to 2-AAF. In (A), quin 2-loaded hepatocytes were exposed to 224 #M 2-AAF (O). For (11), 2-AAF was added to quin 2-loaded hepatocytes pretreated with verapamil as described in Materials and Methods. Intracellular free Ca 2÷ was determined as described in Fig. 1. In (B), viability of hepatocytes exposed to 224 #M 2-AAF was assessed using propidium iodide (see Materials and Methods). Control hepatocytes (O). For (11), 2-AAF was added to verapamil pretreated hepatocytes. Data represent means ± S.E. for at least three experiments with different cell preparations.
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Fig. 3. Effect of TMB-8 pretreatment on intracellular free Ca if (A) . and on cell viability (B) of hepatocytes exposed to 2-AAF. In (A), quin 2-loaded hepatocytes were exposed to 224 PM 2-AAF (0). For (W). 2-AAF was added to quin 2-loaded hepatocytes pretreated with TMB-8 (see Materials and Methods section). Intracellular free Ca2+ was determined as described for Fig. I. In (B), viability of hepatocytes exposed to 224 1M 2-AAF was assessed using propidium iodide as described for Fig. 2. Control hepatocytes (0). For (r). 2-AAF was added to hepatocytes pretreated with TMB-8. Data represent means f SE. for at least three experiments with different cell preparations.
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Modulation of the effect of 2-AAF by verapamil Verapamil is a calcium-entry blocker whose site of action is known to be the plasma membrane. Thus, an elevation of cytosolic free Ca 2+ caused by Ca 2+ entry from the extracellular medium should be antagonized in the presence of verapamii. Therefore, the effect of verapamil on the 2-AAF-induced cytosolic free Ca 2+ increase was tested. The results are shown in Fig. 2A. As can be seen, a rise in intracellular free Ca 2+ takes place during the first 5 min of exposure; this is followed by a diminution to approximately the resting level. In these experiments, verapamil alone had no detectable effect on Ca 2+ levels nor on cell viability, as determined with propidium iodide (data not shown). These data suggest that the elevation in intracellular free Ca 2+ produced by 2-AAF is likely to result from a biphasic process involving first a mobilization from intracellular stores and a subsequent entry of Ca 2+ from the extracellular medium. However, it must be noted that cell viability is preserved up to 6 min of exposure to both 2-AAF and verapamil (Fig. 2B). Beyond this point, cell viability is rapidly lost (91.8-36.1%) while Ca 2+ levels remain constant (Fig. 2A).
Modulation of the effect of 2-AAF by TMB-8 TMB-8 is known to inhibit Ca 2+ mobilization from the endoplasmic reticulumassociated Ca 2+ pools; thus, an elevation of intracellular free Ca 2+ attributable to internal Ca 2+ store mobilization should be prevented by this agent. Figure 3A presents the [Ca2+]i levels in the presence of 2-AAF and TMB-8. As can be seen, TMB-8 completely inhibits the rise in cytosolic free Ca 2+ produced by 2-AAF. Moreover, it has little effect on cell viability (88.6-73.3'V,,, Fig. 3B). Again in these experiments, the drug alone had no significant effect either on [Ca2+]i or cell viability. The previous results indicate that internal Ca 2+ stores would be implicated in the response of hepatocytes to 2-AAF.
Modulation of the effect of 2-AAF by RuR R u R is a hexavalent cation which blocks Ca 2+ uptake through the mitochondrial uniport. In our assays, when used as a single agent, it does not produce any change in [Ca2÷]i levels nor in cell viability (data not shown). However, it affects the initial augmentation of intracellular free Ca 2+ produced by 2-AAF, as depicted in Fig. 4A. It can be observed that the intracellular free Ca 2÷ rise is enhanced within the first 5 min. Furthermore, the second phase of Ca 2+ elevation detected in the 2-AAF-treated cells is attenuated to a great extent when hepatocytes are preincubated with RuR. There is no concomitant loss of cell viability for RuR-treated hepatocytes exposed to 2-AAF during the exposure period (Fig. 4B). These results suggest that mitochondria may also be implicated in the calcium-mediated response of hepatocytes to 2-AAF. Discussion The perturbation of cell calcium homeostasis by 2-AAF has been investigated in this study. The results shown in Fig. 1 indicate that the lowest concentration of 2-AAF used (56 #M), does not alter cytosolic free Ca 2+, at least over a short-term
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Fig. 4. Effect of ruthenium red (RuR) pretreatment on intracellular free Ca’+ (A). and on cell viability (6) of hepatocytes exposed to Z-AAF. In (A), 2-loaded hepatocytes were exposed to 224 PM 2-AAF (0). For (W). 2-AAF was added to quin 2-loaded hepatocytes pretreated with RuR (see Materials Methods). Intracellular free CaZC was determined as described for Fig. 1. For (B). viability of hepdtocytes exposed to 224 pM 2-AAF was assessed using pidium iodide as described for Fig. 2. Control hepatocytes (0). For (m). 2-AAF was added to hepatocytes pretreated with RuR. Data represent means l for at least three experiments with different cell preparations.
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30 exposure. At concentrations of 112 and 224 #M, an elevation in intracellular free Ca 2+ is observed: while a slight initial and sustained elevation of cell Ca 2+ is obtained with 112 #M 2-AAF, a biphasic response occurs with 224 /~M 2-AAF. This biphasic response is characterized by a marked increase of intracellular free Ca 2+ after 10 min of exposure. The profiles observed suggest that there may be more than one mechanism implicated in the perturbation of Ca 2+ homeostasis by 2-AAF. Moreover, in contrast to what has been suggested for a number of other toxins, the rise in Ca 2+ is not associated with loss of cell viability. In order to determine the site(s) of Ca 2+ deregulation induced by 2-AAF, three Ca 2+ antagonists have been used. Verapamil was the first Ca 2+ antagonist tested. This compound is known to block Ca 2+ entry across the plasma membrane catalyzed by potential-operated Ca -,+ channels in excitable cells [23]. Despite the fact that hepatocytes are a cell type which does not present many of these Ca 2+ channels, it has been previously reported that verapamil inhibits the transport of Ca 2+ across the hepatocyte plasma membrane [24,25]. Many studies have demonstrated a cytoprotective effect of this compound in the case of exposure to different toxic agents known to elevate cytosolic free Ca 2+ [25,26]. The cytoprotective effect of verapamil is related to its ability to prevent Ca 2+ accumulation by the cell. The results obtained in this study show that verapamil provokes a re-establishment of the cytosolic free Ca 2+ to approximately the resting level at t = 5 min, after the initial increase induced by 2-AAF. But unexpectedly, this re-establishment to normal level is rapidly accompanied by a progressive loss of cell viability (Figs. 2A and B). Because verapamil has no effect on the initial rise in cytosolic free Ca -'+, it can be hypothesized that, during this phase, the change is related to an internal Ca 2+ store mobilization. The further elevation may be due to an entry from the extracellular medium which is prevented by the presence of verapamil. The blockade of the second phase of 2-AAF-induced Ca -~+ elevation surprisingly parallels an increase in cell sensitivity. These results are somewhat contradictory to the previous investigations in which verapamil protected against cytotoxicity. This unpredictable phenomenon could be explained by the fact that the multidrug resistance glycoprotein (Gp 170) is normally expressed in liver cells [27,29], and can act as an efflux pump for structurally unrelated cytotoxic agents and drugs [28,30]. It has been proposed that Gp 170 can be a 'natural' mechanism of defense in a variety of cells, including hepatocytes [28,30]. On the other hand, it has been shown that Gp 170 is inhibited by verapamil and other Ca 2+ antagonists [31,33]. Thus, there is a possibility that Gp 170 participates in the extrusion of 2-AAF that has been taken up by the hepatocytes. In the presence of verapamil, this extrusion process may be impaired, thereby causing a potentiation of the cytotoxic effect of 2-AAF and loss of viability. The second Ca 2+ antagonist used was TMB-8. This compound can interfere with Ca 2+ mobilization from intracellular stores like the endoplasmic reticulum. It appears to function by stabilizing membrane-bound Ca 2+ and by inhibiting the binding of calmodulin to membrane-bound Ca 2+ ATPase in a variety of tissues [34,35]. As seen in Fig. 3A, TMB-8 completely inhibits the elevation of intracellular free Ca 2+. Assuming that the action of TMB-8 in hepatocytes is similar to that observed in other cell types, the contribution of internal Ca -,+ stores would be important in
31
the response of hepatocytes to 2-AAF. Moreover, these results coupled with those obtained in the experiments using verapamil, lead to the hypothesis that the first phase of 2-AAF-induced cytosolic Ca 2÷ elevation may trigger the second phase of elevation attributed to the entry of Ca 2÷ from the extracellular medium. In the last series of experiments, R u R was used to determine the possible implication of mitochondria in the 2-AAF-induced elevation in cytosolic free Ca 2÷. RuR is a hexavalent cationic dye that has been reported to inhibit, in a non-competitive manner, the inflow of Ca 2÷ into mitochondria via their Ca 2÷ uniport [36,37]. The results obtained with this Ca 2÷ antagonist (Figs. 4A and B) show that the intracellular free Ca 2÷ rise is enhanced during the first phase (first 5 min) and that the second phase is almost totally eliminated. This indicates that mitochondria, in the initial moments of exposure to 2-AAF, could act by 'pumping in' Ca 2÷ to antagonize the rise caused by 2-AAF. Another possible effect of R u R is the inhibition of Ca 2÷ release from internal stores (sarcoplasmic reticulum), as demonstrated in muscle cells [38]. It has not been established to what extent such a mechanism may be pertinent in the case of liver cells. Nonetheless, in our experiments, when the hepatocytes are preincubated with RuR, the 2-AAF-induced rise in [Ca2+]i is enhanced rather than being reduced, indicating that if there is any inhibition by R u R on the release of Ca 2÷ from internal stores, it does not appear to play a major role. R u R has also been shown to inhibit plasma membrane Ca 2+ ATPase in red blood cell and smooth muscle plasma membrane [39,40]. However, in intact cells, it has only a partial effect: in red blood cells, R u R inhibits Ca 2÷ efflux to 60°/,, at 1 m M concentration [40]. If a similar inhibition takes place in hepatocytes, the enhanced rise in Ca 2÷ seen with R u R could be the consequence of reduced Ca 2÷ ATPase activity. However, as judged from the previously published data, at the concentration of R u R used in our experiments (25/zM), the inhibition of the enzyme is not likely to occur to a significant degree. It is also to be noted that R u R alone did not affect basal Ca 2÷ levels. Therefore, the proposal that mitochondria may play a role in 2-AAF-induced Ca 2÷ pertubation still remains valid. The results of earlier experiments carried out by Olafsdottir et al. [41], and in which RuR was used in the assessment of the role of mitochondria in the injury caused to hepatocytes by the Ca 2÷ ionophore A23187, also support the present interpretation. In conclusion, the data show that 2-AAF can perturb Ca 2÷ homeostasis in isolated hepatocytes. This perturbation cannot be associated with the cytotoxic effect of this compound under our exposure conditions, since it is not accompanied by a detectable loss of viability. Furthermore, this perturbation seems to involve more than one site, including the plasma membrane and internal Ca 2÷ stores. However, further work is required in order to describe more precisely how 2-AAF exerts its deleterious effects on hepatocytes and to explore the possible link between 2-AAF bioactivation and the perturbation of Ca 2+ homeostasis. References A. Binet, B. Berthon and M. Claret, Hormone-induced increase in free cytosolic calcium and glycogen phosphorylase activation in rat hepatocytes incubated in normal and low-calcium media. Biochem. J., 228 (1985) 565.
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19 20
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H. Sugita, S. lshiura, K. Kamakura, H. Nakase, H. Hagiwara, I. Nonaka and K. Tomomatsu, Caactivated neutral protease in physiological and pathological conditions, in S. Ebashi et al. (Eds.), Calcium Regulation in Biological Systems, Academic Press, Tokyo, 1984, p. 243. M. Poenie, J. Alderton, R. Steinhardt and R. Tsien, Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science, 233 (1986) 886. P. Nicotera, D. Mc Conkey, S.A. Svensson, G. Bellomo and S. Orrenius, Correlation between cytosolic Ca 2÷ concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicology, 52 (1988) 55. F.A.X. Schanne, A.B. Kane, E.E. Young and J.L. Farber, Calcium dependence of toxic cell death: A final common pathway. Science, 206 (1979) 700. M.T. Smith, H. Thor and S. Orrenius, Toxic injury to isolated hepatocytes is not dependent on extracellular calcium. Science, 213 (1981) 1257. M.W. Fariss and D.J. Reed, Mechanism of chemical-induced toxicity. 11. Role ofextracellular calcium. Toxicol. Appl. Pharmacol., 79 (1985) 296. J.F. Nagelkerke, P. Dogterom, H.J.G.M. De Bont and G.J. Mulder, Prolonged high intracellular free calcium concentrations induced by ATP are not immediatly cytotoxic in isolated rat hepatocytes. Biochem. J., 263 (1989) 347. G. Bellomo, S.A. Jewell and S. Orrenius, The metabolism of menadione impairs the ability of rat liver mitochondria to take up and retain calcium. J. Biol. Chem., 257 (1982) 11558. L. Moore, Inhibition of liver endoplasmic reticulum calcium pump by CCI 4 and release of a sequestered calcium pool. Biochem. Pharmacol., 35 (1986) 4131. L. Moore, 1,I-Dichloroethylene inhibition of liver endoplasmic reticulum calcium pump function. Biochem. Pharmacol., 31 (1982) 1463. J.O. Tsokos-Kuhn, H. Hughes, C.V. Smith and J.R. Mitchell, Alkylation of the liver plasma membrane and inhibition of the Ca 2+ ATPase by acetaminophen. Biochem. Pharmacol., 37 (1988) 2125. D.J. Porubek, M. Rundgren, P.J. Harvison, S.D. Nelson and P. Mold6us, Investigation of mechanisms of acetaminophen toxicity in isolated rat hepatocytes with the acetaminophen analogues 3,5-dimethylacetaminophen and 2,6-dimethylacetaminophen. Mol. Pharmacol., 31 (1987) 647. P. Nicotera, M. Moore, F. Mirabelli, G. Bellomo and S. Orrenius, Inhibition of hepatocytes plasma membrane Ca 2÷ ATPase activity by menadione metabolism and its restoration by thiols. FEBS Lett., 181 (1985) 149. J.O. Tsokos-Kuhn, Evidence in vivo for elevation of intracellular free Ca ~÷ in the liver after diquat, acetaminophen and CCI 4. Biochem. Pharmacol., 38 (1989) 3061. J.G. Sipes and A.J. Gandolfi, Biotransformation of toxicant, in CD. Klaassen, M.O. Amdur and J. Doull (Eds.), Toxicology: The Basic Science of Poisons, Mac Millan Publishing Company, New York, 1986, p. 65. P.O. Seglen, Preparation of isolated rat liver cells, Methods Cell. Biol., 13 (1976) 29. F. Denizeau, M. Marion, G. Chevalier and M.G. C6t6, Inability ofchrysotile fibers to modulate the 2-acetylaminofluorene-induced UDS in primary cultures of rat hepatocytes. Mutat. Res., 155 (1985) 83. M.K. Patterson, Measurement of growth and viability of cells in culture, in W.B. Jakoby and 1.H. Pastan (Eds.), Methods in Enzymology, Vol. LVIII, Academic Press, New York, 1979, p. 141. R.Y. Tsien, T. Pozzan and T.J. Rink, Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell. Biol., 94 (1982) 325. G.J. Gores , A.L. Nieminen , K.E. Fleishman, T.L. Dawson, B. Herman and J.J. Lemasters, Extracellular acidosis delays onset of cell death in ATP-depleted hepatocytes. Am. J. Physiol. (Cell Physiol.), 24 (1988) C315. P. Mold6us, J. H6gberg and S. Orrenius, Isolation and use of liver cell, in S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. LII, part C, Academic Press, New York, 1979, p. 60. R.A. Janis and A. Scriabine, Sites of action of Ca 2÷ channel inhibitor. Biochem. Pharmacol., 32 (1983) 3499. B.P. Hughes, S.E. Milton, G.J. Barrit and A.M. Auld, Studies with verapamil and nifepidine provide evidence for the presence in the liver cell plasma membrane of two types of Ca 2+ inflow transporter which are dissimilar to potential-operated Ca 2÷ channels. Biochem. Pharmacol., 35 (1986) 3045.
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E.J. Landon, R.K. Jaiswal, R.J. Naukam and B.V. Rama Sastry, Effects of calcium channel blocking agents on membrane microviscosity and calcium in the liver of the carbon tetrachloride treated rat. Biochem. Pharmacol., 33 (1984) 3353. R.D. Smith, P.S. Wolf, R.J. Regan and S.R. Jolly, Cytoprotective effects of CEB's in hepatic injury, in L. Will. Shahab (Ed.), The Emergence of Drugs which Block Calcium Entry, Springer-Verlag, New York , 1988, p. 61. F. Thiebaut, T. Tsuruo, H. Hamada, M.M. Gottesman, 1. Pastan and M.C. Willingham, Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc. Natl. Acad. Sci., 84 (1987) 7735. Y. Kamimoto, Z. Gatmaitan, J. Hsu and I.M. Arias, The function of Gp 170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles. J. Biol. Chem., 264 (19891 11693. S.S. Thorgeirsson, B.E. Huber, S. Sorrell, A. Fojo, 1. Pastan and M.M. Gottesman, Expression of the multidrug-resistant gene in hepatocarcinogenesis and regenerating rat liver. Science, 236 (1987~ 1120. N. Kartner and V. Ling, Multidrug resistance in cancer. Sci. Am., 260(3) (19891 44. M. Naito and T. Tsuruo, Competitive inhibition by verapamil of ATP-dependant high affinity vincristine binding to the plasma membrane of multidrug-resistance K562 cells without calcium ion involvement. Cancer Res., 49 (1989) 1452. H.M. Coley, P.R. Twentyman and P. Workman, Improved cellular accumulation is characteristic of anthracyclines which retain high activity in multidrug resistant cell lines. Alone or in combination with verapamil or cyclosporin A. Biochem. Pharmacol., 38 (1989) 4467. J.A. Plumb , R. Milroy and S.B. Kaye, The activity of verapamil as a resistance modifier in vitro in drug resistant h u m a n tumour cell lines is not stereospecific. Biochem. Pharmacol., 39 (1990) 787. R.J. Smith and S.S. Iden, Phorbol myristate acetate-induced release of granule enzymes from human neutrophils: inhibition by the calcium antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride. Biochem. Biophys. Res. Commun., 91 (1979) 263. N.E. Owen and M . L Villereal, Effects of the intracellular Ca 2+ antagonist TMB-8 on serum stimulated Na ÷ influx in h u m a n fibroblasts. Biochem. Biophys. Res. Commun., 109 (1982) 762. K.C. Reed and F.L. Bygrave, The inhibition ofmitochondrial calcium transport by lanthanides and ruthenium red. Biochem. J., 140 (1974) 143. C.E. Thomas and D.J. Reed, Effect ofextracellular Ca 2+ omission on isolated hepatocytes. II. Loss of mitochondrial membrane potential and protection by inhibitors of uniport Ca 2+ transduction. J. Pharmacol. Exp. Ther., 245 (1988) 501. Y. Kanamura, L. Raeymaekers and R. Casteels, Effects of doxorubicin and ruthenium red on intracellular Ca 2+ stores in skinned rabbit mesenteric smooth-muscle fibers. Cell. Calcium, 10 (1989) 433. L. Missiaen, H. De Smedt, G. Droogmans, F. Wuytack, Raeymaekers and R. Casteels, Ruthenium red and compound 48/80 inhibit the smooth-muscle plasma-membrane Ca 2+ pump via interaction with associated polyphosphoinositides. Biochim. Biophys. Acta., 1023 (1990) 449. S. Sarkadi, i. Szsz, A. Gerloczy and G. Gardor, Transport parameters and stoichiometry of active calcium ion extrusion in intact h u m a n red cells. Biochim. Biophys. Acta., 464 (1977) 93. K. Olafsdottir, G.A. Pascoe and D.J. Reed, Mitochondrial glutathione status during Ca 2+ ionophore-induced injury to isolated hepatocytes. Arch. Biochem. Biophys., 263 (1988) 226.
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Effect of 2-acetylaminofluorene on intracellular free C a 2+ in isolated rat hepatocytes S. Lefebvre, M. M a r i o n a n d F. D e n i z e a u * D~partement de Chimie, Universitk du Qubbec a Montrkal. QuObec (Canada) (Received May 22nd, 1991; accepted August 13th, 1991)
Summary The effect of 2-acetylaminofluorene (2-AAF) on the intracellular free Ca 2+ ([Ca2÷]i} and viability of isolated rat hepatocytes has been investigated using the fluorescent probes quin 2 and propidium iodide respectively. At the highest concentration tested (224 ~tM), 2-AAF produces an elevation of [Ca2+]i which shows a biphasic profile. A small initial increase is observed during the first 5 min; this is followed by a considerable rise which reaches up to 2.5 times the control value at 15 min. These changes in intracellular calcium are not accompanied by detectable alterations in cell viability. In order to determine the mechanisms by which this effect of 2-AAF takes place, three calcium antagonists, namely verapamil, TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) and ruthenium red (RuR), have been used. The results suggest that the first phase is dependent upon internal Ca 2+ store mobilization, while the second phase seems to be related to Ca 2+ entry from the extracellular space. The data obtained with RuR further indicate that mitochondria may be involved in the perturbation of calcium homeostasis caused by 2-AAF. In addition, in the experiments involving antagonists, no consistent pattern emerges that suggests a close relationship between intracellular Ca 2+ levels and cell viability. The present study provides further information on the mechanisms by which the well-known hepatotoxin 2-AAF may interact with liver cells. It also shows that when these cells are exposed to a toxin, short-term changes in [Ca2+]i may not be accompanied by loss of cell viability, and conversely, that changes in cell viability may occur without alterations in [Ca2+] i.
Key words." 2-Acetylaminofluorene; Calcium; Hepatocytes; Quin-2; Cell viability
Introduction The importance of Ca 2+ in the cell is well established. Intracellular free Ca 2+ is implicated in a wide variety of cellular processes such as hormonal response, enzymatic activation and cell division [1-3]. The intracellular free Ca 2÷ concentration is regulated within narrow limits exhibiting very low levels in the cytosol (10 - 7 tO 10 -8 M) as compared to a relatively high extracellular concentration (10 - 3 M ) . Correspondence to." Dr. Francine Denizeau, D6partement de Chimie, Universit6 du Qu4bec h Montr6al, Case Postale 8888, Succursale A, Montr6al, Qu6bec, Canada H3C 3P8. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
22 The regulation of the intracellular free Ca 2+ concentration in most cells is known to take place at three major sites: the plasma membrane, the endoplasmic reticulum (ER) and the mitochondria. Thus, a dysfunction in any of the systems involved may result in a non-physiological increase in cytosolic free Ca 2+ which can lead to cell injury. It has been shown that Ca 2+ may play a critical role in the mechanisms of chemically induced cytotoxicity. This has been extensively investigated especially in hepatocytes [4-8] although it still remains a subject of controversy. Many studies performed with different toxins have provided evidence of an alteration of function at the level of either of the Ca 2+ regulating systems. For example, Orrenius et al. have demonstrated that the metabolism of menadione impairs the ability of rat liver mitochondria to take up and retain Ca 2+ [9]. Moore and coworkers have shown that different toxic agents like carbon disulfide, carbon tetrachloride and other chlorinated halothanes can inhibit the endoplasmic reticulum calcium pump [10,11]. They proposed that this inhibition may represent the primary event in the cytotoxicity induced by these xenobiotics. The disturbance of Ca 2+ fluxes at the level of the plasma membrane in the presence of toxic agents has been suggested in many studies. Tsokos-Kuhn et al. have demonstrated the inhibition of the liver plasma membrane Ca 2+ ATPase by acetaminophen and bromobenzene in vivo [12], while Mold4us et al. have shown the same phenomenon in vitro [13]. Similar observations have been reported by Orrenius et al. with menadione [14]. Previous data obtained with the well-known hepatotoxin acetaminophen have led to the proposal that the alkylation of membrane-bound proteins is likely to be an important molecular event in the process that renders the cell unable to control its cytosolic Ca 2+ concentration. Indeed, acetaminophen, after undergoing oxidation by cytochrome P-450, produces alkylating species that interact with macromolecules including membrane proteins. The plasma membrane Ca 2+, Mg 2+ ATPase has been proposed as being one of the target proteins [12], although other plasma membrane Ca 2+ translocases may be affected. Such interactions are believed to lead to an elevation of intracellular free Ca 2÷ in the liver in vivo [15]. Therefore, it appears that alkylating agents may represent a threat to proper maintenance of cell Ca 2÷ homeostasis. In the light of the previous findings, it becomes relevant to further investigate the effect of xenobiotics, in particular of alkylating agents such as acetaminophen on Ca 2+ metabolism in liver cells. Among these agents, 2-acetylaminofluorene (2-AAF) is a well known hepatocarcinogen that shares many similarities with acetaminophen both in its chemical structure and its bioactivation by cytochrome P-450 which represents an important step in the formation of alkylating species [16]. The aim of this study was therefore to investigate the effect of 2-AAF on the intracellular free calcium and viability of isolated rat hepatocytes. By using the pharmacological agents verapamil, TMB-8, (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate), and ruthenium red (RuR), an attempt was made to determine at what level perturbation takes place. The results obtained provide further information on the mechanisms by which 2-AAF may exert its deleterious effects on liver cells.
24 magnetic stirring and the cuvette chamber was maintained at 37°C. The fluorescence signal was calibrated by addition of 50 ~M digitonin; after 20 min, maximum fluorescence (Fmax) was obtained. This was followed by the addition of 6 mM E G T A (ethyleneglycol-bis(13-aminoethylether)-N,N'tetraacetic acid) to obtain the minimum fluorescence (Fmi,). The intracellular free calcium concentration ([Ca2+]i) was evaluated using the following equation [Ca2+]i = g d ( F - Fmin)/(Fma x - F). assuming a dissociation constant (Kd) value of 115 nM as determined by Tsien et al. [20]. The possibility of having interference in the fluorescence measurement of quin 2 was tested by establishing calibration curves with Ca2+-EGTA according to Tsien et al. [26]. Tests carried out in our laboratory have shown that none of the compounds used (DMSO, 2-AAF, verapamil, TMB-8 and RuR) has an effect on the dissociation constant (Kd) of the quin 2-Ca 2+ complex.
Viability measurement using propidium iodide
Viability of the hepatocytes was monitored in a continuous fashion by a fluorometric assay using propidium iodide as described by Gores et al. [21]. Briefly, the cells (105/ml) were incubated at 37°C in the modified Hanks' medium described above. An aliquot of this suspension was transferred to a quartz cuvette. Fluorescence intensity was taken at an excitation wavelength of 515 nm and an emission wavelength of 620 nm. Immediately after addition of 1 #M propidium iodide, an initial fluorescence measurement was made (A). At the end of experiment, 400 #M digitonin was added to permeabilize the cells and a final maximum fluorescence (B) was obtained after 20 min. Hepatocyte viability was evaluated using the following relationship V = 100 x ( B - X ) / ( B - A ) where V is the viability in percent and X the fluorescence measured at any moment during the experiment. In one series of experiments, hepatocyte viability was monitored by measuring lactate dehydrogenase (LDH) activity in the extracellular medium according to Mold6us et al. [22].
Treatment with Ca 2+ antagonists
For the investigation of the effect of verapamil, hepatocytes or quin 2-loaded hepatocytes were preincubated in the presence of 200 #M verapamil (dissolved in DMSO) for 10 min prior to analysis. In the study using TMB-8, hepatocytes or quin 2-loaded hepatocytes were preincubated in the presence of 0.6 mM TMB-8 (dissolved in Tris buffer pH 7.4) for 10 min before analysis. Finally, in the experiments that tested the effect of RuR, hepatocytes or quin 2-loaded hepatocytes were preincubated with 25 t~M RuR (dissolved in modified Hanks' medium) for 10 min prior to fluorescence measurements.
25
Results
Effect of 2 - A A F concentration on hepatocyte intracellular free Ca 2+ The effect of 2-AAF on the intracellular free Ca 2+ in hepatocytes was first determined. For this study, cell suspensions were exposed to three different 2-AAF concentrations (56, 112 and 224/zM). These concentrations were chosen due to the fact that they did not induce cytotoxicity over the incubation period as determined by L D H leakage measurement (results not shown). Intracellular free Ca 2÷ was monitored using quin 2 (see Materials and Methods) during the first 20 min of exposure. As shown in Fig. 1, 56 #M 2-AAF did not cause any significant increase in intracellular free Ca 2+ over the 20-min period. However, at a concentration of 112 /~M, 2-AAF produces a slight initial increase of intracellular free Ca 2+ that is maintained for the rest of the incubation time. The most striking change in cytosolic free Ca 2÷ is obtained with 224 #M 2-AAF. In this case, the small elevation observed during the first 5 min is similar to that obtained with 112/~M 2-AAF; however, this small elevation is followed by a considerable rise which reaches up to 2.5-times the control value at 15 rain. This biphasic response suggests that there may be more than one mechanism implicated in the perturbation of cytosolic free Ca 2+ by 2-AAF. In the light of these observations, the latter concentration (224/zM 2-AAF) was selected for the subsequent studies with verapamil, TMB-8 and RuR.
300 0
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=a
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Time (rain) Fig. 1. Effect of different concentrations of 2-AAF on intracellular free Ca -'+ of hepatocytes. At time = 0.224 #M (O), 112 #M ( I ) or 56 #M 2-AAF (/~) was added to quin 2-loaded hepatocytes. Determinations were carried out as described in the Materials and Methods section. Data represent means ± S.E. for at least three experiments with different cell preparations. Initial cytosolic free Ca 2÷ was 141.7 n M ± 7.1 (S.E.) for experiments in which extracellular free Ca 2+ was measured (including those of the other Figures).
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.-
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Fig. 2. Effect of verapamil pretreatment on intracellular free Ca e+ (A), and cell viability (B) of hepatocytes exposed to 2-AAF. In (A), quin 2-loaded hepatocytes were exposed to 224 #M 2-AAF (O). For (11), 2-AAF was added to quin 2-loaded hepatocytes pretreated with verapamil as described in Materials and Methods. Intracellular free Ca 2÷ was determined as described in Fig. 1. In (B), viability of hepatocytes exposed to 224 #M 2-AAF was assessed using propidium iodide (see Materials and Methods). Control hepatocytes (O). For (11), 2-AAF was added to verapamil pretreated hepatocytes. Data represent means ± S.E. for at least three experiments with different cell preparations.
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15 20
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Fig. 3. Effect of TMB-8 pretreatment on intracellular free Ca if (A) . and on cell viability (B) of hepatocytes exposed to 2-AAF. In (A), quin 2-loaded hepatocytes were exposed to 224 PM 2-AAF (0). For (W). 2-AAF was added to quin 2-loaded hepatocytes pretreated with TMB-8 (see Materials and Methods section). Intracellular free Ca2+ was determined as described for Fig. I. In (B), viability of hepatocytes exposed to 224 1M 2-AAF was assessed using propidium iodide as described for Fig. 2. Control hepatocytes (0). For (r). 2-AAF was added to hepatocytes pretreated with TMB-8. Data represent means f SE. for at least three experiments with different cell preparations.
01
28
Modulation of the effect of 2-AAF by verapamil Verapamil is a calcium-entry blocker whose site of action is known to be the plasma membrane. Thus, an elevation of cytosolic free Ca 2+ caused by Ca 2+ entry from the extracellular medium should be antagonized in the presence of verapamii. Therefore, the effect of verapamil on the 2-AAF-induced cytosolic free Ca 2+ increase was tested. The results are shown in Fig. 2A. As can be seen, a rise in intracellular free Ca 2+ takes place during the first 5 min of exposure; this is followed by a diminution to approximately the resting level. In these experiments, verapamil alone had no detectable effect on Ca 2+ levels nor on cell viability, as determined with propidium iodide (data not shown). These data suggest that the elevation in intracellular free Ca 2+ produced by 2-AAF is likely to result from a biphasic process involving first a mobilization from intracellular stores and a subsequent entry of Ca 2+ from the extracellular medium. However, it must be noted that cell viability is preserved up to 6 min of exposure to both 2-AAF and verapamil (Fig. 2B). Beyond this point, cell viability is rapidly lost (91.8-36.1%) while Ca 2+ levels remain constant (Fig. 2A).
Modulation of the effect of 2-AAF by TMB-8 TMB-8 is known to inhibit Ca 2+ mobilization from the endoplasmic reticulumassociated Ca 2+ pools; thus, an elevation of intracellular free Ca 2+ attributable to internal Ca 2+ store mobilization should be prevented by this agent. Figure 3A presents the [Ca2+]i levels in the presence of 2-AAF and TMB-8. As can be seen, TMB-8 completely inhibits the rise in cytosolic free Ca 2+ produced by 2-AAF. Moreover, it has little effect on cell viability (88.6-73.3'V,,, Fig. 3B). Again in these experiments, the drug alone had no significant effect either on [Ca2+]i or cell viability. The previous results indicate that internal Ca 2+ stores would be implicated in the response of hepatocytes to 2-AAF.
Modulation of the effect of 2-AAF by RuR R u R is a hexavalent cation which blocks Ca 2+ uptake through the mitochondrial uniport. In our assays, when used as a single agent, it does not produce any change in [Ca2÷]i levels nor in cell viability (data not shown). However, it affects the initial augmentation of intracellular free Ca 2+ produced by 2-AAF, as depicted in Fig. 4A. It can be observed that the intracellular free Ca 2÷ rise is enhanced within the first 5 min. Furthermore, the second phase of Ca 2+ elevation detected in the 2-AAF-treated cells is attenuated to a great extent when hepatocytes are preincubated with RuR. There is no concomitant loss of cell viability for RuR-treated hepatocytes exposed to 2-AAF during the exposure period (Fig. 4B). These results suggest that mitochondria may also be implicated in the calcium-mediated response of hepatocytes to 2-AAF. Discussion The perturbation of cell calcium homeostasis by 2-AAF has been investigated in this study. The results shown in Fig. 1 indicate that the lowest concentration of 2-AAF used (56 #M), does not alter cytosolic free Ca 2+, at least over a short-term
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Fig. 4. Effect of ruthenium red (RuR) pretreatment on intracellular free Ca’+ (A). and on cell viability (6) of hepatocytes exposed to Z-AAF. In (A), 2-loaded hepatocytes were exposed to 224 PM 2-AAF (0). For (W). 2-AAF was added to quin 2-loaded hepatocytes pretreated with RuR (see Materials Methods). Intracellular free CaZC was determined as described for Fig. 1. For (B). viability of hepdtocytes exposed to 224 pM 2-AAF was assessed using pidium iodide as described for Fig. 2. Control hepatocytes (0). For (m). 2-AAF was added to hepatocytes pretreated with RuR. Data represent means l for at least three experiments with different cell preparations.
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30 exposure. At concentrations of 112 and 224 #M, an elevation in intracellular free Ca 2+ is observed: while a slight initial and sustained elevation of cell Ca 2+ is obtained with 112 #M 2-AAF, a biphasic response occurs with 224 /~M 2-AAF. This biphasic response is characterized by a marked increase of intracellular free Ca 2+ after 10 min of exposure. The profiles observed suggest that there may be more than one mechanism implicated in the perturbation of Ca 2+ homeostasis by 2-AAF. Moreover, in contrast to what has been suggested for a number of other toxins, the rise in Ca 2+ is not associated with loss of cell viability. In order to determine the site(s) of Ca 2+ deregulation induced by 2-AAF, three Ca 2+ antagonists have been used. Verapamil was the first Ca 2+ antagonist tested. This compound is known to block Ca 2+ entry across the plasma membrane catalyzed by potential-operated Ca -,+ channels in excitable cells [23]. Despite the fact that hepatocytes are a cell type which does not present many of these Ca 2+ channels, it has been previously reported that verapamil inhibits the transport of Ca 2+ across the hepatocyte plasma membrane [24,25]. Many studies have demonstrated a cytoprotective effect of this compound in the case of exposure to different toxic agents known to elevate cytosolic free Ca 2+ [25,26]. The cytoprotective effect of verapamil is related to its ability to prevent Ca 2+ accumulation by the cell. The results obtained in this study show that verapamil provokes a re-establishment of the cytosolic free Ca 2+ to approximately the resting level at t = 5 min, after the initial increase induced by 2-AAF. But unexpectedly, this re-establishment to normal level is rapidly accompanied by a progressive loss of cell viability (Figs. 2A and B). Because verapamil has no effect on the initial rise in cytosolic free Ca -'+, it can be hypothesized that, during this phase, the change is related to an internal Ca 2+ store mobilization. The further elevation may be due to an entry from the extracellular medium which is prevented by the presence of verapamil. The blockade of the second phase of 2-AAF-induced Ca -~+ elevation surprisingly parallels an increase in cell sensitivity. These results are somewhat contradictory to the previous investigations in which verapamil protected against cytotoxicity. This unpredictable phenomenon could be explained by the fact that the multidrug resistance glycoprotein (Gp 170) is normally expressed in liver cells [27,29], and can act as an efflux pump for structurally unrelated cytotoxic agents and drugs [28,30]. It has been proposed that Gp 170 can be a 'natural' mechanism of defense in a variety of cells, including hepatocytes [28,30]. On the other hand, it has been shown that Gp 170 is inhibited by verapamil and other Ca 2+ antagonists [31,33]. Thus, there is a possibility that Gp 170 participates in the extrusion of 2-AAF that has been taken up by the hepatocytes. In the presence of verapamil, this extrusion process may be impaired, thereby causing a potentiation of the cytotoxic effect of 2-AAF and loss of viability. The second Ca 2+ antagonist used was TMB-8. This compound can interfere with Ca 2+ mobilization from intracellular stores like the endoplasmic reticulum. It appears to function by stabilizing membrane-bound Ca 2+ and by inhibiting the binding of calmodulin to membrane-bound Ca 2+ ATPase in a variety of tissues [34,35]. As seen in Fig. 3A, TMB-8 completely inhibits the elevation of intracellular free Ca 2+. Assuming that the action of TMB-8 in hepatocytes is similar to that observed in other cell types, the contribution of internal Ca -,+ stores would be important in
31
the response of hepatocytes to 2-AAF. Moreover, these results coupled with those obtained in the experiments using verapamil, lead to the hypothesis that the first phase of 2-AAF-induced cytosolic Ca 2÷ elevation may trigger the second phase of elevation attributed to the entry of Ca 2÷ from the extracellular medium. In the last series of experiments, R u R was used to determine the possible implication of mitochondria in the 2-AAF-induced elevation in cytosolic free Ca 2÷. RuR is a hexavalent cationic dye that has been reported to inhibit, in a non-competitive manner, the inflow of Ca 2÷ into mitochondria via their Ca 2÷ uniport [36,37]. The results obtained with this Ca 2÷ antagonist (Figs. 4A and B) show that the intracellular free Ca 2÷ rise is enhanced during the first phase (first 5 min) and that the second phase is almost totally eliminated. This indicates that mitochondria, in the initial moments of exposure to 2-AAF, could act by 'pumping in' Ca 2÷ to antagonize the rise caused by 2-AAF. Another possible effect of R u R is the inhibition of Ca 2÷ release from internal stores (sarcoplasmic reticulum), as demonstrated in muscle cells [38]. It has not been established to what extent such a mechanism may be pertinent in the case of liver cells. Nonetheless, in our experiments, when the hepatocytes are preincubated with RuR, the 2-AAF-induced rise in [Ca2+]i is enhanced rather than being reduced, indicating that if there is any inhibition by R u R on the release of Ca 2÷ from internal stores, it does not appear to play a major role. R u R has also been shown to inhibit plasma membrane Ca 2+ ATPase in red blood cell and smooth muscle plasma membrane [39,40]. However, in intact cells, it has only a partial effect: in red blood cells, R u R inhibits Ca 2÷ efflux to 60°/,, at 1 m M concentration [40]. If a similar inhibition takes place in hepatocytes, the enhanced rise in Ca 2÷ seen with R u R could be the consequence of reduced Ca 2÷ ATPase activity. However, as judged from the previously published data, at the concentration of R u R used in our experiments (25/zM), the inhibition of the enzyme is not likely to occur to a significant degree. It is also to be noted that R u R alone did not affect basal Ca 2÷ levels. Therefore, the proposal that mitochondria may play a role in 2-AAF-induced Ca 2÷ pertubation still remains valid. The results of earlier experiments carried out by Olafsdottir et al. [41], and in which RuR was used in the assessment of the role of mitochondria in the injury caused to hepatocytes by the Ca 2÷ ionophore A23187, also support the present interpretation. In conclusion, the data show that 2-AAF can perturb Ca 2÷ homeostasis in isolated hepatocytes. This perturbation cannot be associated with the cytotoxic effect of this compound under our exposure conditions, since it is not accompanied by a detectable loss of viability. Furthermore, this perturbation seems to involve more than one site, including the plasma membrane and internal Ca 2÷ stores. However, further work is required in order to describe more precisely how 2-AAF exerts its deleterious effects on hepatocytes and to explore the possible link between 2-AAF bioactivation and the perturbation of Ca 2+ homeostasis. References A. Binet, B. Berthon and M. Claret, Hormone-induced increase in free cytosolic calcium and glycogen phosphorylase activation in rat hepatocytes incubated in normal and low-calcium media. Biochem. J., 228 (1985) 565.
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H. Sugita, S. lshiura, K. Kamakura, H. Nakase, H. Hagiwara, I. Nonaka and K. Tomomatsu, Caactivated neutral protease in physiological and pathological conditions, in S. Ebashi et al. (Eds.), Calcium Regulation in Biological Systems, Academic Press, Tokyo, 1984, p. 243. M. Poenie, J. Alderton, R. Steinhardt and R. Tsien, Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science, 233 (1986) 886. P. Nicotera, D. Mc Conkey, S.A. Svensson, G. Bellomo and S. Orrenius, Correlation between cytosolic Ca 2÷ concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicology, 52 (1988) 55. F.A.X. Schanne, A.B. Kane, E.E. Young and J.L. Farber, Calcium dependence of toxic cell death: A final common pathway. Science, 206 (1979) 700. M.T. Smith, H. Thor and S. Orrenius, Toxic injury to isolated hepatocytes is not dependent on extracellular calcium. Science, 213 (1981) 1257. M.W. Fariss and D.J. Reed, Mechanism of chemical-induced toxicity. 11. Role ofextracellular calcium. Toxicol. Appl. Pharmacol., 79 (1985) 296. J.F. Nagelkerke, P. Dogterom, H.J.G.M. De Bont and G.J. Mulder, Prolonged high intracellular free calcium concentrations induced by ATP are not immediatly cytotoxic in isolated rat hepatocytes. Biochem. J., 263 (1989) 347. G. Bellomo, S.A. Jewell and S. Orrenius, The metabolism of menadione impairs the ability of rat liver mitochondria to take up and retain calcium. J. Biol. Chem., 257 (1982) 11558. L. Moore, Inhibition of liver endoplasmic reticulum calcium pump by CCI 4 and release of a sequestered calcium pool. Biochem. Pharmacol., 35 (1986) 4131. L. Moore, 1,I-Dichloroethylene inhibition of liver endoplasmic reticulum calcium pump function. Biochem. Pharmacol., 31 (1982) 1463. J.O. Tsokos-Kuhn, H. Hughes, C.V. Smith and J.R. Mitchell, Alkylation of the liver plasma membrane and inhibition of the Ca 2+ ATPase by acetaminophen. Biochem. Pharmacol., 37 (1988) 2125. D.J. Porubek, M. Rundgren, P.J. Harvison, S.D. Nelson and P. Mold6us, Investigation of mechanisms of acetaminophen toxicity in isolated rat hepatocytes with the acetaminophen analogues 3,5-dimethylacetaminophen and 2,6-dimethylacetaminophen. Mol. Pharmacol., 31 (1987) 647. P. Nicotera, M. Moore, F. Mirabelli, G. Bellomo and S. Orrenius, Inhibition of hepatocytes plasma membrane Ca 2÷ ATPase activity by menadione metabolism and its restoration by thiols. FEBS Lett., 181 (1985) 149. J.O. Tsokos-Kuhn, Evidence in vivo for elevation of intracellular free Ca ~÷ in the liver after diquat, acetaminophen and CCI 4. Biochem. Pharmacol., 38 (1989) 3061. J.G. Sipes and A.J. Gandolfi, Biotransformation of toxicant, in CD. Klaassen, M.O. Amdur and J. Doull (Eds.), Toxicology: The Basic Science of Poisons, Mac Millan Publishing Company, New York, 1986, p. 65. P.O. Seglen, Preparation of isolated rat liver cells, Methods Cell. Biol., 13 (1976) 29. F. Denizeau, M. Marion, G. Chevalier and M.G. C6t6, Inability ofchrysotile fibers to modulate the 2-acetylaminofluorene-induced UDS in primary cultures of rat hepatocytes. Mutat. Res., 155 (1985) 83. M.K. Patterson, Measurement of growth and viability of cells in culture, in W.B. Jakoby and 1.H. Pastan (Eds.), Methods in Enzymology, Vol. LVIII, Academic Press, New York, 1979, p. 141. R.Y. Tsien, T. Pozzan and T.J. Rink, Calcium homeostasis in intact lymphocytes: Cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell. Biol., 94 (1982) 325. G.J. Gores , A.L. Nieminen , K.E. Fleishman, T.L. Dawson, B. Herman and J.J. Lemasters, Extracellular acidosis delays onset of cell death in ATP-depleted hepatocytes. Am. J. Physiol. (Cell Physiol.), 24 (1988) C315. P. Mold6us, J. H6gberg and S. Orrenius, Isolation and use of liver cell, in S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. LII, part C, Academic Press, New York, 1979, p. 60. R.A. Janis and A. Scriabine, Sites of action of Ca 2÷ channel inhibitor. Biochem. Pharmacol., 32 (1983) 3499. B.P. Hughes, S.E. Milton, G.J. Barrit and A.M. Auld, Studies with verapamil and nifepidine provide evidence for the presence in the liver cell plasma membrane of two types of Ca 2+ inflow transporter which are dissimilar to potential-operated Ca 2÷ channels. Biochem. Pharmacol., 35 (1986) 3045.
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E.J. Landon, R.K. Jaiswal, R.J. Naukam and B.V. Rama Sastry, Effects of calcium channel blocking agents on membrane microviscosity and calcium in the liver of the carbon tetrachloride treated rat. Biochem. Pharmacol., 33 (1984) 3353. R.D. Smith, P.S. Wolf, R.J. Regan and S.R. Jolly, Cytoprotective effects of CEB's in hepatic injury, in L. Will. Shahab (Ed.), The Emergence of Drugs which Block Calcium Entry, Springer-Verlag, New York , 1988, p. 61. F. Thiebaut, T. Tsuruo, H. Hamada, M.M. Gottesman, 1. Pastan and M.C. Willingham, Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc. Natl. Acad. Sci., 84 (1987) 7735. Y. Kamimoto, Z. Gatmaitan, J. Hsu and I.M. Arias, The function of Gp 170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles. J. Biol. Chem., 264 (19891 11693. S.S. Thorgeirsson, B.E. Huber, S. Sorrell, A. Fojo, 1. Pastan and M.M. Gottesman, Expression of the multidrug-resistant gene in hepatocarcinogenesis and regenerating rat liver. Science, 236 (1987~ 1120. N. Kartner and V. Ling, Multidrug resistance in cancer. Sci. Am., 260(3) (19891 44. M. Naito and T. Tsuruo, Competitive inhibition by verapamil of ATP-dependant high affinity vincristine binding to the plasma membrane of multidrug-resistance K562 cells without calcium ion involvement. Cancer Res., 49 (1989) 1452. H.M. Coley, P.R. Twentyman and P. Workman, Improved cellular accumulation is characteristic of anthracyclines which retain high activity in multidrug resistant cell lines. Alone or in combination with verapamil or cyclosporin A. Biochem. Pharmacol., 38 (1989) 4467. J.A. Plumb , R. Milroy and S.B. Kaye, The activity of verapamil as a resistance modifier in vitro in drug resistant h u m a n tumour cell lines is not stereospecific. Biochem. Pharmacol., 39 (1990) 787. R.J. Smith and S.S. Iden, Phorbol myristate acetate-induced release of granule enzymes from human neutrophils: inhibition by the calcium antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride. Biochem. Biophys. Res. Commun., 91 (1979) 263. N.E. Owen and M . L Villereal, Effects of the intracellular Ca 2+ antagonist TMB-8 on serum stimulated Na ÷ influx in h u m a n fibroblasts. Biochem. Biophys. Res. Commun., 109 (1982) 762. K.C. Reed and F.L. Bygrave, The inhibition ofmitochondrial calcium transport by lanthanides and ruthenium red. Biochem. J., 140 (1974) 143. C.E. Thomas and D.J. Reed, Effect ofextracellular Ca 2+ omission on isolated hepatocytes. II. Loss of mitochondrial membrane potential and protection by inhibitors of uniport Ca 2+ transduction. J. Pharmacol. Exp. Ther., 245 (1988) 501. Y. Kanamura, L. Raeymaekers and R. Casteels, Effects of doxorubicin and ruthenium red on intracellular Ca 2+ stores in skinned rabbit mesenteric smooth-muscle fibers. Cell. Calcium, 10 (1989) 433. L. Missiaen, H. De Smedt, G. Droogmans, F. Wuytack, Raeymaekers and R. Casteels, Ruthenium red and compound 48/80 inhibit the smooth-muscle plasma-membrane Ca 2+ pump via interaction with associated polyphosphoinositides. Biochim. Biophys. Acta., 1023 (1990) 449. S. Sarkadi, i. Szsz, A. Gerloczy and G. Gardor, Transport parameters and stoichiometry of active calcium ion extrusion in intact h u m a n red cells. Biochim. Biophys. Acta., 464 (1977) 93. K. Olafsdottir, G.A. Pascoe and D.J. Reed, Mitochondrial glutathione status during Ca 2+ ionophore-induced injury to isolated hepatocytes. Arch. Biochem. Biophys., 263 (1988) 226.