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Wy-14,643 but not 2-ethylhexanol increases intracellular free calcium in cultured Kupffer cells
Toxicology Letters, 59 (1991) 239-244 Q 1991 Elsevier Science Publishers B.V. 0378-4274/91/$ 3.50
239
TOXLET 02670
Wy- 14,643 but not 2-et~yl~exanol increases intracellular free calcium in cultured Kupffer cells
Taizo Hijioka, Barbara J. Keller and Ronald G. Thurman Laboratory
of Hepatobiology
and Toxicology,
Department
of Pharmacology,
University of North Carolina at
Chapel Hill, Chapel Hill, NC (U.S. A.)
(Received 25 June 1991) (Accepted 13 September 1991) Key words: Wy-14,643; 2-Ethylhexanol; Calcium; Kupffer cells SUMMARY Hypolip~d~ic drugs and phthahc ester plasticizers induce peroxisomes and cause hepatocelluiar carcinoma in rodents by mechanisms which remain unknown. Recent evidence from this laboratory suggests that many agents in this class of chemicals are uncouplers of mitochondrial oxidative phosphorylation both in vitro and in vivo. Uncoupling of oxidative phosphorylation decreases ATP required for ion pumps and could thereby indirectly increase intracellular free calcium. The goal of these experiments, therefore, was to compare the effect of the potent uncoupler and non-genotoxic carcinogen Wy-14,643 with the weaker agent 2-ethylbexanol on intracellular free calcium in cultured Kupffer cells. Kupffer cells, the resident hepatic macrophages, are activated by calcium and release a variety of mitogenic growth factors that may modulate cell proliferation. In this study, the cytosolic free calcium concentration in Fura-Z-loaded cultured Kupffer cells was increased significantly from 78 + 11 to 838 + 112 nM following incubation with Wy-14,643 (1.25 mM). The increase in intracellular calcium due to Wy-14,643 was both time- and dose-dependent, At equimolar concentrations, ethylhexanol had no effect on intracellular calcium (65 + 20 nM). However, at higher concentrations (3 mM), ethylhexanol also increased intracellular calcium. These data suggest that elevation of intracellular calcium in Kupffer cells may be involved in the mechanism of action of this interesting class of non-genotoxic carcinogens.
INTRODUCTION
Peroxisome proliferators are a class of compounds composed largely of phthalic ester plasticizers and lipid-lowering drugs which are non-genotoxic carcinogens in
Address for correspondence: Dr. Ronald G. Thurman, Laboratory of Hepatobiology and Toxicology, Department of Ph~a~logy, CB# 7365, Faculty Laboratory Office Building, University of North Carohna at Chapel Hilt, Chapel Hilt, NC 27599-7365, U.S.A.
240
rats and mice [l]. Key findings from our laboratory showed that 2-ethylhexanol, a major metabolite of the plasticizer di(ethylhexy1) phthalate, was an uncoupler of oxidative phosphorylation in isolated mitochondria and in the perfused rat liver [2]. Moreover, when the effects of several structurally dissimilar peroxisome proliferators on isolated mitochondria were compared, all compounds studied uncoupled oxidative phosphorylation to varying degrees [3]. Interestingly, weak tumor-causing agents like valproate were weak uncouplers [3], whereas the more potent carcinogen Wy14,643 exhibited more pronounced effects [4]. Further, chronic treatment with Wy14,643 caused uncoupling in vivo assessed by an increase in oxygen uptake and inhibition of urea synthesis, a process highly dependent on ATP, by the perfused liver [4]. Based on these findings, we propose that highly lipophilic plasticizers and lipidlowering drugs accumulate in mitochondrial membranes, uncouple oxidative phosphorylation and alter cellular energetics, leading to increases in intracellular calcium. Kupffer cells, the resident hepatic macrophages, are activated by calcium and release a variety of mitogenic cytokines [5] and eicosanoids [6] which may play a role in cell proliferation and tumor formation. Therefore, the purpose of this study was to compare the effects of a potent (WY-14,643) and a weak (ethylhexanol) non-genotoxic carcinogen on intracellular calcium levels in cultured Kupffer cells. METHODS
Isolation of Kupffer cells
Livers from fed, female Sprague-Dawley rats (240-280 g) were isolated under pentobarbital anesthesia (60 mg/kg, i.p.) and perfused via the portal vein for 10 min with Krebs-Ringer-HEPES buffer containing 115 mM NaCl, 5 mM KCl, 1 mM KH,PO,, 25 mM HEPES, 1 mM CaCl, and 0.016% collagenase (pH 7.4) followed by 10 min of perfusion with calcium-free buffer containing 0.5 mM EGTA. Liver cells were dispersed by gentle shaking in phosphate-buffered saline (pH 7.4, 4°C) and the nonparenchymal cell fraction was separated by centrifugation through Percoll gradients based on the method of Pertoft and Smedsrod [7]. Non-parenchymal cells were resuspended in RPM1 1640 culture medium containing 10% heat-inactivated fetal calf serum, 10 mM HEPES, 100 U/ml penicillin G, 100 ,@ml streptomycin sulfate and 0.25 ,@ml amphotericin B (2.0 x lo6 cells/ml), then seeded onto glass coverslips and cultured at 37°C in a 5% CO* atmosphere. After 1 h, non-adherent cells were removed by replacement with fresh culture medium. Cells were subsequently incubated for 1 h with 1 pm latex beads which were phagocytized by every flat cell on the coverslips, verifying that they were Kupffer cells [8]. Measurement of intracellular calcium
Kupffer cells that had been cultured for 3 days were incubated with Krebs-RingerHEPES buffer containing 5 yM Fura-2/AM, 0.06% Pluronic F127, 1 mM MgSO,, 5 mM glucose and 2% bovine serum albumin at 37°C for 15 min and allowed to equilibrate to 25°C for 30 min. Coverslips plated with Kupffer cells were rinsed and placed
241
in chambers with Krebs-Ringer-HEPES containing 1 mM MgSO, and 5 mM glucose at 25°C. Changes in fluorescence intensity of Fura- at excitation wavelengths of 340 and 380 nm were monitored in individual Kupffer cells using the Spex Analytical System interfaced with a Nikon diaphot inverted microscope [9]. Each value was corrected by subtracting the system dark noise and autofluorescence assessed by quenching Fura- fluorescence with Mn2’. Intracellular free calcium concentration was determined by measuring the ratio of Fura- fluorescence when excited at either 340 or 380 nm [lo]. Ratio values were converted to calcium concentrations by comparison with a standard curve determined for Fura- pentapotassium salt in buffered salt solutions containing calcium and EGTA in ratios calculated to give free calcium concentrations ranging from 0 to 1000 nM [ 10,111. Materials Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid) was obtained from Chemsyn Science Laboratories (Lenexa, KA), 2-ethylhexanol was purchased from Aldrich (Milwaukee, WI) and Fura- acetoxymethyl ester and Pluronic F127 were acquired from Molecular Probes Inc. (Eugene, OR). All other chemicals and reagents were of the highest available purity from standard commercial sources. RESULTS
Cultured Kupffer cells were incubated with various concentrations of Wy-14,643 for up to 50 min (Fig. 1). Culture medium containing 0.4 mM Wy-14,643 did not affect intracellular free calcium levels; however, higher concentrations of Wy-14,643
I
c
600
-
400
-
,;+ 0m
Minutes
Fig. 1.Effect of Wy-14,643 on intracellular free calcium levels in cultured Kupffer cells. Kupffer cells were isolated and cultured for 2-3 days as described in Methods. Wy-14,643 was dissolved in DMSO and added to the culture medium at the concentrations indicated. Cytosolic free calcium concentration in Kupffer cells ([Ca*‘],) was assessed fluorometrically using Fura- as described in Methods. Each trace represents the calcium concentration in one representative Kupffer cell.
242
Control
Ethylhexanol
Wy- 14,643
Fig. 2. Effect of equimolar ~n~ntrations of Wy-14,643 and ethylhexanol on intracellular free calcium levels in cultured Kupffer cells. Kupffer cells were isolated and cultured for 2-3 days as described in Methods. Cells were incubated with Wy-14,643 or ethylhexanol (1.25 mM) and cytosolic free calcium concentrations were assessed as described in Methods. *P < 0.05. Mean + SEM, n = 4 cells/group.
elevated intracellular calcium in a time- and dose-dependent manner {Fig. 1). Intracellular free calcium con~ntrations increased rapidly to values over 900 nM at 20,40
Concentration,
mM
Fig. 3. Effect of Wy-14,643 and ethylhexanol concentrations on intracellular free calcium in Kupffer cells. Conditions as in Fig. 2. Each point represents average data from 211 individual Kupffer cells.
243
and 45 min following addition of 3.0, 2.0 and 1.25 mM Wy-14,643, respectively. In most cells, calcium levels remained elevated for about 10 min and then declined (Fig. 1). This decrease in intracellular fluorescence was most likely due to leakage of Furaacross the cellular membrane (Fig. 1). Incubations with concentrations of Wy-14,643 greater than 3 mM increased intracellular calcium rapidly to values over 3000 nM indicative of toxicity (data not shown). At equimolar concentrations (1.25 mM), Wy-14,643 but not ethylhexanol elevated intracellular free calcium significantly in isolated Kupffer cells (P < 0.05; Fig. 2). A dose-response with ethylhexanol and Wy-14,643 is shown in Figure 3. Wy-14,643 had a more pronounced effect on intracellular calcium than did ethylhexanol; however, 3 mM ethylhexanol did significantly increase intracellular calcium levels (Fig. 3). DISCUSSION
Recently, Keller and colleagues showed that many non-genotoxic carcinogens (i.e. peroxisome-proliferating agents) uncoupled oxidative phosphorylation in isolated rat liver mitochondria to varying degrees [3]. Interestingly, weak tumor-causing agents like ethylhexanol were weak uncouplers whereas the more potent compound, Wy14,643, exhibited more pronounced effects [2,4]. Therefore, we hypothesize that peroxisome proliferators, which are highly lipophilic, accumulate in mitochondrial membranes where they uncouple oxidative phosphorylation and interfere with cellular energetics and ion gradients. As a consequence of mitochondrial uncoupling, the ATP supply for ion pumps would be decreased, which could increase levels of intracellular calcium indirectly. Therefore, in this study a comparison was made between the effects of Wy-14,643 and 2-ethylhexanol on intracellular free calcium concentrations in cultured Kupffer cells. Indeed, incubation of Kupffer cells with Wy-14,643 increased intracellular calcium (Figs. 1 and 2). The observation that higher concentrations of Wy-14,643 led to elevation of intracellular calcium at earlier time points can most likely be explained by a time-dependent accumulation of Wy-14,643 in the mitochondrial membrane. When a critical concentration of Wy-14,643 was reached, uncoupling occurred followed by dissipation of the ion gradient across the mitochondrial membrane leading to release of calcium into the cytosol. Interestingly, at equimolar concentrations up to 2 mM, Wy-14,643 but not ethylhexanol elevated intracellular free calcium in Kupffer cells (Fig. 3). This observation is consistent with the finding that Wy-14,643 is a better uncoupler than ethylhexanol [2,4]. Therefore, the extent to which these chemicals dissipate the mitochondrial proton gradient in vitro may correlate with their potencies in vivo. More work is needed to determine if Kupffer cells are involved in the mechanism of this interesting class of hepatotoxic chemicals and if simple predictive tests can be developed based on uncoupling or changes in intracellular calcium levels in Kupffer cells.
244
ACKNOWLEDGEMENT
This work was supported in part by a grant from NIEHS (ES-04325). REFERENCES 1 Reddy, J.K. and Lalwai, N.D. (1989) C’arcinogenesis by hepatic peroxisome prohferators: evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Biochem. 7, 305-310. 2 Keller, B.J., Liang, D. and Thurman, R.G. (1991) 2-Ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria. Toxicol. Lett. 57, 113-120. 3 Keller, B.J. and Thurman, R.G. (1990) Non-genotoxic carcinogens are uncouplers of mitochondriai oxidative phospho~lation. Pha~acologist 32, 333. 4 Keller, B.J., Marsman, D.S., Popp, J.A. and Thurman, R.G. (1991) The nongenotoxic carcinogen WY-14,643 is an uncouplter of oxidative phosphorylation in isolated rat liver mitochondria and in vivo. Toxicologist 11, 187. 5 Decker, T., Lohmann-Matthes, M.L., Karck, U., Peters, T. and Decker, K. (1989) Comparative study of cytotoxicity, tumor necrosis factor, and prostaglandin release after stimulation of rat Kupffer cells, murine Kupffer cells, and murine inflammatory liver macrophages. J. Leukocyte Biol. 45, 139146. 6 Birmelin, M. and Decker, K. (1984) Synthesis of prostanoids and cyclic nucleotides by phagocytosing rat Kupffer cells. Eur. J. Biochem. 142,219~225. 7 Pertoft, H. and Smedsrod, B. (1987) Separation and characterization of liver cells. In: T.G. Pretlow II and T.P. Pretlow (Eds.), Cell Separation: Methods and Selected Applications, Vol. 4, pp. l-24, Academic Press, New York. 8 Doolittle, M., Bohman, R., Durstenfeld, A. and Cascarano, J. (1987) Identification and characterization of liver nonparenchymal cells by flow cytometry, Hepatology 7,696703. 9 Salm, A.K. and McCarthy, K.D. (1990) Norepinephrine-evoked calcium transients in cultured cerebral type I astroglia. Clia 3, 529538. 10 Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) A new generation of Ca*’ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440-3450. 11 Hirning, L.D., Fox, A.P., McCleskey, W., Olivera, B.M., Thayer, S.A., Miller, R.J. and Tsien, R.W. (1988) Dominant role of N-type Ca” channels in evoked release of norepinephrine from sympathetic neurons. Science 239,57-61.
239
TOXLET 02670
Wy- 14,643 but not 2-et~yl~exanol increases intracellular free calcium in cultured Kupffer cells
Taizo Hijioka, Barbara J. Keller and Ronald G. Thurman Laboratory
of Hepatobiology
and Toxicology,
Department
of Pharmacology,
University of North Carolina at
Chapel Hill, Chapel Hill, NC (U.S. A.)
(Received 25 June 1991) (Accepted 13 September 1991) Key words: Wy-14,643; 2-Ethylhexanol; Calcium; Kupffer cells SUMMARY Hypolip~d~ic drugs and phthahc ester plasticizers induce peroxisomes and cause hepatocelluiar carcinoma in rodents by mechanisms which remain unknown. Recent evidence from this laboratory suggests that many agents in this class of chemicals are uncouplers of mitochondrial oxidative phosphorylation both in vitro and in vivo. Uncoupling of oxidative phosphorylation decreases ATP required for ion pumps and could thereby indirectly increase intracellular free calcium. The goal of these experiments, therefore, was to compare the effect of the potent uncoupler and non-genotoxic carcinogen Wy-14,643 with the weaker agent 2-ethylbexanol on intracellular free calcium in cultured Kupffer cells. Kupffer cells, the resident hepatic macrophages, are activated by calcium and release a variety of mitogenic growth factors that may modulate cell proliferation. In this study, the cytosolic free calcium concentration in Fura-Z-loaded cultured Kupffer cells was increased significantly from 78 + 11 to 838 + 112 nM following incubation with Wy-14,643 (1.25 mM). The increase in intracellular calcium due to Wy-14,643 was both time- and dose-dependent, At equimolar concentrations, ethylhexanol had no effect on intracellular calcium (65 + 20 nM). However, at higher concentrations (3 mM), ethylhexanol also increased intracellular calcium. These data suggest that elevation of intracellular calcium in Kupffer cells may be involved in the mechanism of action of this interesting class of non-genotoxic carcinogens.
INTRODUCTION
Peroxisome proliferators are a class of compounds composed largely of phthalic ester plasticizers and lipid-lowering drugs which are non-genotoxic carcinogens in
Address for correspondence: Dr. Ronald G. Thurman, Laboratory of Hepatobiology and Toxicology, Department of Ph~a~logy, CB# 7365, Faculty Laboratory Office Building, University of North Carohna at Chapel Hilt, Chapel Hilt, NC 27599-7365, U.S.A.
240
rats and mice [l]. Key findings from our laboratory showed that 2-ethylhexanol, a major metabolite of the plasticizer di(ethylhexy1) phthalate, was an uncoupler of oxidative phosphorylation in isolated mitochondria and in the perfused rat liver [2]. Moreover, when the effects of several structurally dissimilar peroxisome proliferators on isolated mitochondria were compared, all compounds studied uncoupled oxidative phosphorylation to varying degrees [3]. Interestingly, weak tumor-causing agents like valproate were weak uncouplers [3], whereas the more potent carcinogen Wy14,643 exhibited more pronounced effects [4]. Further, chronic treatment with Wy14,643 caused uncoupling in vivo assessed by an increase in oxygen uptake and inhibition of urea synthesis, a process highly dependent on ATP, by the perfused liver [4]. Based on these findings, we propose that highly lipophilic plasticizers and lipidlowering drugs accumulate in mitochondrial membranes, uncouple oxidative phosphorylation and alter cellular energetics, leading to increases in intracellular calcium. Kupffer cells, the resident hepatic macrophages, are activated by calcium and release a variety of mitogenic cytokines [5] and eicosanoids [6] which may play a role in cell proliferation and tumor formation. Therefore, the purpose of this study was to compare the effects of a potent (WY-14,643) and a weak (ethylhexanol) non-genotoxic carcinogen on intracellular calcium levels in cultured Kupffer cells. METHODS
Isolation of Kupffer cells
Livers from fed, female Sprague-Dawley rats (240-280 g) were isolated under pentobarbital anesthesia (60 mg/kg, i.p.) and perfused via the portal vein for 10 min with Krebs-Ringer-HEPES buffer containing 115 mM NaCl, 5 mM KCl, 1 mM KH,PO,, 25 mM HEPES, 1 mM CaCl, and 0.016% collagenase (pH 7.4) followed by 10 min of perfusion with calcium-free buffer containing 0.5 mM EGTA. Liver cells were dispersed by gentle shaking in phosphate-buffered saline (pH 7.4, 4°C) and the nonparenchymal cell fraction was separated by centrifugation through Percoll gradients based on the method of Pertoft and Smedsrod [7]. Non-parenchymal cells were resuspended in RPM1 1640 culture medium containing 10% heat-inactivated fetal calf serum, 10 mM HEPES, 100 U/ml penicillin G, 100 ,@ml streptomycin sulfate and 0.25 ,@ml amphotericin B (2.0 x lo6 cells/ml), then seeded onto glass coverslips and cultured at 37°C in a 5% CO* atmosphere. After 1 h, non-adherent cells were removed by replacement with fresh culture medium. Cells were subsequently incubated for 1 h with 1 pm latex beads which were phagocytized by every flat cell on the coverslips, verifying that they were Kupffer cells [8]. Measurement of intracellular calcium
Kupffer cells that had been cultured for 3 days were incubated with Krebs-RingerHEPES buffer containing 5 yM Fura-2/AM, 0.06% Pluronic F127, 1 mM MgSO,, 5 mM glucose and 2% bovine serum albumin at 37°C for 15 min and allowed to equilibrate to 25°C for 30 min. Coverslips plated with Kupffer cells were rinsed and placed
241
in chambers with Krebs-Ringer-HEPES containing 1 mM MgSO, and 5 mM glucose at 25°C. Changes in fluorescence intensity of Fura- at excitation wavelengths of 340 and 380 nm were monitored in individual Kupffer cells using the Spex Analytical System interfaced with a Nikon diaphot inverted microscope [9]. Each value was corrected by subtracting the system dark noise and autofluorescence assessed by quenching Fura- fluorescence with Mn2’. Intracellular free calcium concentration was determined by measuring the ratio of Fura- fluorescence when excited at either 340 or 380 nm [lo]. Ratio values were converted to calcium concentrations by comparison with a standard curve determined for Fura- pentapotassium salt in buffered salt solutions containing calcium and EGTA in ratios calculated to give free calcium concentrations ranging from 0 to 1000 nM [ 10,111. Materials Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid) was obtained from Chemsyn Science Laboratories (Lenexa, KA), 2-ethylhexanol was purchased from Aldrich (Milwaukee, WI) and Fura- acetoxymethyl ester and Pluronic F127 were acquired from Molecular Probes Inc. (Eugene, OR). All other chemicals and reagents were of the highest available purity from standard commercial sources. RESULTS
Cultured Kupffer cells were incubated with various concentrations of Wy-14,643 for up to 50 min (Fig. 1). Culture medium containing 0.4 mM Wy-14,643 did not affect intracellular free calcium levels; however, higher concentrations of Wy-14,643
I
c
600
-
400
-
,;+ 0m
Minutes
Fig. 1.Effect of Wy-14,643 on intracellular free calcium levels in cultured Kupffer cells. Kupffer cells were isolated and cultured for 2-3 days as described in Methods. Wy-14,643 was dissolved in DMSO and added to the culture medium at the concentrations indicated. Cytosolic free calcium concentration in Kupffer cells ([Ca*‘],) was assessed fluorometrically using Fura- as described in Methods. Each trace represents the calcium concentration in one representative Kupffer cell.
242
Control
Ethylhexanol
Wy- 14,643
Fig. 2. Effect of equimolar ~n~ntrations of Wy-14,643 and ethylhexanol on intracellular free calcium levels in cultured Kupffer cells. Kupffer cells were isolated and cultured for 2-3 days as described in Methods. Cells were incubated with Wy-14,643 or ethylhexanol (1.25 mM) and cytosolic free calcium concentrations were assessed as described in Methods. *P < 0.05. Mean + SEM, n = 4 cells/group.
elevated intracellular calcium in a time- and dose-dependent manner {Fig. 1). Intracellular free calcium con~ntrations increased rapidly to values over 900 nM at 20,40
Concentration,
mM
Fig. 3. Effect of Wy-14,643 and ethylhexanol concentrations on intracellular free calcium in Kupffer cells. Conditions as in Fig. 2. Each point represents average data from 211 individual Kupffer cells.
243
and 45 min following addition of 3.0, 2.0 and 1.25 mM Wy-14,643, respectively. In most cells, calcium levels remained elevated for about 10 min and then declined (Fig. 1). This decrease in intracellular fluorescence was most likely due to leakage of Furaacross the cellular membrane (Fig. 1). Incubations with concentrations of Wy-14,643 greater than 3 mM increased intracellular calcium rapidly to values over 3000 nM indicative of toxicity (data not shown). At equimolar concentrations (1.25 mM), Wy-14,643 but not ethylhexanol elevated intracellular free calcium significantly in isolated Kupffer cells (P < 0.05; Fig. 2). A dose-response with ethylhexanol and Wy-14,643 is shown in Figure 3. Wy-14,643 had a more pronounced effect on intracellular calcium than did ethylhexanol; however, 3 mM ethylhexanol did significantly increase intracellular calcium levels (Fig. 3). DISCUSSION
Recently, Keller and colleagues showed that many non-genotoxic carcinogens (i.e. peroxisome-proliferating agents) uncoupled oxidative phosphorylation in isolated rat liver mitochondria to varying degrees [3]. Interestingly, weak tumor-causing agents like ethylhexanol were weak uncouplers whereas the more potent compound, Wy14,643, exhibited more pronounced effects [2,4]. Therefore, we hypothesize that peroxisome proliferators, which are highly lipophilic, accumulate in mitochondrial membranes where they uncouple oxidative phosphorylation and interfere with cellular energetics and ion gradients. As a consequence of mitochondrial uncoupling, the ATP supply for ion pumps would be decreased, which could increase levels of intracellular calcium indirectly. Therefore, in this study a comparison was made between the effects of Wy-14,643 and 2-ethylhexanol on intracellular free calcium concentrations in cultured Kupffer cells. Indeed, incubation of Kupffer cells with Wy-14,643 increased intracellular calcium (Figs. 1 and 2). The observation that higher concentrations of Wy-14,643 led to elevation of intracellular calcium at earlier time points can most likely be explained by a time-dependent accumulation of Wy-14,643 in the mitochondrial membrane. When a critical concentration of Wy-14,643 was reached, uncoupling occurred followed by dissipation of the ion gradient across the mitochondrial membrane leading to release of calcium into the cytosol. Interestingly, at equimolar concentrations up to 2 mM, Wy-14,643 but not ethylhexanol elevated intracellular free calcium in Kupffer cells (Fig. 3). This observation is consistent with the finding that Wy-14,643 is a better uncoupler than ethylhexanol [2,4]. Therefore, the extent to which these chemicals dissipate the mitochondrial proton gradient in vitro may correlate with their potencies in vivo. More work is needed to determine if Kupffer cells are involved in the mechanism of this interesting class of hepatotoxic chemicals and if simple predictive tests can be developed based on uncoupling or changes in intracellular calcium levels in Kupffer cells.
244
ACKNOWLEDGEMENT
This work was supported in part by a grant from NIEHS (ES-04325). REFERENCES 1 Reddy, J.K. and Lalwai, N.D. (1989) C’arcinogenesis by hepatic peroxisome prohferators: evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Biochem. 7, 305-310. 2 Keller, B.J., Liang, D. and Thurman, R.G. (1991) 2-Ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria. Toxicol. Lett. 57, 113-120. 3 Keller, B.J. and Thurman, R.G. (1990) Non-genotoxic carcinogens are uncouplers of mitochondriai oxidative phospho~lation. Pha~acologist 32, 333. 4 Keller, B.J., Marsman, D.S., Popp, J.A. and Thurman, R.G. (1991) The nongenotoxic carcinogen WY-14,643 is an uncouplter of oxidative phosphorylation in isolated rat liver mitochondria and in vivo. Toxicologist 11, 187. 5 Decker, T., Lohmann-Matthes, M.L., Karck, U., Peters, T. and Decker, K. (1989) Comparative study of cytotoxicity, tumor necrosis factor, and prostaglandin release after stimulation of rat Kupffer cells, murine Kupffer cells, and murine inflammatory liver macrophages. J. Leukocyte Biol. 45, 139146. 6 Birmelin, M. and Decker, K. (1984) Synthesis of prostanoids and cyclic nucleotides by phagocytosing rat Kupffer cells. Eur. J. Biochem. 142,219~225. 7 Pertoft, H. and Smedsrod, B. (1987) Separation and characterization of liver cells. In: T.G. Pretlow II and T.P. Pretlow (Eds.), Cell Separation: Methods and Selected Applications, Vol. 4, pp. l-24, Academic Press, New York. 8 Doolittle, M., Bohman, R., Durstenfeld, A. and Cascarano, J. (1987) Identification and characterization of liver nonparenchymal cells by flow cytometry, Hepatology 7,696703. 9 Salm, A.K. and McCarthy, K.D. (1990) Norepinephrine-evoked calcium transients in cultured cerebral type I astroglia. Clia 3, 529538. 10 Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) A new generation of Ca*’ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440-3450. 11 Hirning, L.D., Fox, A.P., McCleskey, W., Olivera, B.M., Thayer, S.A., Miller, R.J. and Tsien, R.W. (1988) Dominant role of N-type Ca” channels in evoked release of norepinephrine from sympathetic neurons. Science 239,57-61.