Toxicology Letters, 53 (1990) 115-120
115
Elsevier
TOXLET
02395
Perturbation of calcium homeostasis as a link between acute cell injury and carcinogenesis in the kidney
Spyridon Vamvakas and M.W. Anders Department of Pharmacology, University of Rochester, Rochester, NY (U.S.A.)
Key words: Calcium microscopy;
homeostasis;
ADP-ribosylation;
9( 1,2-Dichlorovinyl)-L-cysteine; Carcinogenesis;
Fluorescence
digital imaging
Cell death
Transport systems on the plasma membrane, the mitochondria, and the endoplasmic reticulum (ER) maintain cytosolic Ca*+ ([Ca*+]J at a very low concentration (x 100 nM) relative to the extracellular Ca*+ concentration ([Ca*+],), which is x 1 mM under physiological conditions. Uncontrolled and sustained increases in [Ca*+]i in chemical-induced toxicity can occur via an enhanced influx of [Ca*+], or by impairment of the intracellular buffering systems, or both. The role of Ca*+-mediated events in acute cell injury and cell death has been studied extensively. Although there are still questions, especially in distinguishing causal effects from epiphenomena, our understanding of this field has improved considerably. Activation of Ca*+ -dependent p hospholipases, proteases, and endonucleases as well as disruption of the cytoskeletal organization (Fig. 1) is involved in the acute injury and cell death associated with elevated [Ca*+]i [1,2]. Ca2+-activated phospholipases catalyze phospholipid breakdown to arachidonic acid and cytotoxic lysophosphohpids; moreover, deregulation of phospholipid turnover may disrupt the integrity of the cell membrane, further impairing the ability of the cell to maintain a normal ion homeostasis. Ca* + -dependent p roteases are involved in cytoskeletal organization, cleavage of receptors, regulation of mitosis, and proteolytic activation of protein kinase C. The use of intracellular Ca *+ chelators to buffer increases in [Ca*+]i delays bleb formation; similar effects can be achieved with selective inhibitors of
Address for correspondence: Elmwood
Avenue,
0378-4274/90/$3.50
Rochester,
S. Vamvakas,
Department
of Pharmacology,
University
NY 14642, U.S.A.
@ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
of Rochester,
601
116 CELL
DAMAGE I
YITOCHONDRIA
PLASMA MEUBRANE I
ENDOPLASYIC RETICULUM I
I
DIACYLGLYCEROL'j)
YEYBRANE
CYTO;KELETAL
DAYACE
ALTERATIONS
I
POLY
I-L
ADP-
RIBDSYLATION
CELL
DEATH
LONG-TERM IN CELLULAR
PERTURBATIONS HOMEOSTASIS
Fig. 1. Processes involved in Ca 2+-mediated acute and long-term cellular toxicity.
Ca2+-dependent proteases. Finally the Ca2+ ionophore A23 187 as well as glucocorticoids and phorbol esters, which facilitate the influx of [Ca2+], in immature thymocytes, cause chromatin condensation and DNA fragmentation. Induction of Ca2+dependent DNA strand-breaks has been demonstrated in mouse epidermal cells where inclusion of the Ca2+ chelator Quin-2 in the incubation system abolished the effect [3], and the toxicity of S-(pentachlorobutadienyl)glutathione and S-(dichlorovinyl)-L-cysteine (DCVC) in kidney cells is diminished by aurintricarboxylic acid, a selective inhibitor of Ca2+-dependent endonucleases [4; S. Vamvakas, unpublished data]. The acute effects of two well-established nephrotoxins on the Ca2+ homeostasis in kidney cells will be briefly described to illustrate two different pathways operating in the deregulation of this ion. Mercuric chloride (HgC12) produces acute necrosis in the proximal tubule in both rodents and humans_ [5]; 50 PM HgC12 induces an intial rise in [Ca2+]i to x 1000 nM, probably due to redistribution from the ER. After a rapid decrease, [Ca2+]i exhibits a second gradual increase due to an influx of [Ca2+], that is less marked but sustained. HgC12 does not interfere with the mitochondrial Ca2+ transport systems, as confirmed by the morphological picture of the injury. The necrotic phase is characterized by calcification in the proximal tubules consisting predominantly of calcium phosphate and hydroxylapatite deposits within the mitochondria, indicating that mitochondria are able to accumulate Ca2+ even at a very late stage in the course of HgClz toxicity.
117
In contrast, the key metabolite of the nephrocarcinogen trichloroethylene DCVC, which is nephrotoxic in vivo and cytotoxic to kidney cells in culture, appears to perturb primarily the mitochondrial Ca *+ homeostasis. The effects of DCVC on intracellular Ca*+ compartmentation have been studied with fluorescence digital imaging microscopy and the Ca*+ indicator fura- in single living kidney cells during the development of cell injury [6]. In the early stage of DCVC-induced toxicity in cultured kidney cells, the average [Ca*+]i increases 3-5-fold compared with control values. However the mitochondria, stained concomitantly with rhodamine 123, have significantly lower Ca*+ concentrations than the rest of the cell. This indicates impairment of the ability of the mitochondrial low-affinity, high-capacity Ca*+ buffering system to sequester Ca*+, although the mitochondrial membrane potential is still intact according to the staining with rhodamine 123. Plasma membrane blebs, collapse of the mitochondrial membrane potential, and loss of viability by propidium iodide staining are observed during later stages in the course of DCVC-induced toxicity. Although it has become increasingly evident in recent years that most renal carcinogens induce toxicity in proximal tubule epithelial cells and the toxic lesions precede and occur along with the development of neoplasia [7], it is still difficult to establish a causal link between chemically induced nephrotoxicity and the formation of kidney tumors and is even more difficult to characterize the involvement of Ca*+-dependent effects in these processes. A number of renal carcinogens, including nitrosamines, arylamines and amides, and polycyclic hydrocarbons, act via a direct genotoxic mechanism; either the parent compounds themselves or their metabolites interact with DNA, resulting in heritable alterations in target cells. On the other hand, non-mutagenic compounds, e.g. branched-chain hydrocarbons or the cationic chelator nitrilotriacetic acid (NTA), produce tumors primarily via extranuclear, epigenetic pathways. NTA is not biotransformed and does not react with DNA; it does, however, produce proximal tubule cell toxicity and may exert its nephrocarcinogenic action via alterations in the homeostasis of divalent cations such as Zn*+ and Ca*+ [7]. The role of genotoxicity versus epigenetic effects in the nephrocarcinogenicity of compounds that are intermediates between the two categories mentioned above, namely compounds that are weak genotoxins and potent nephrotoxins, is still unclear. Several halogenated alkenes such as hexachlorobutadiene, trichloroethylene and tetrachloroethylene are potent nephrotoxins [8]. The corresponding cysteine conjugates, which are the penultimate metabolites formed from these chloroalkenes in vivo, are cleaved by P-lyase to reactive intermediates in the kidney [9]. These cysteine conjugates are nephrotoxic in vivo and cytotoxic in kidney cells in culture; moreover, they are mutagenic in bacteria [lo], bind to DNA in isolated renal tubular cells [l 11,and induce unscheduled DNA synthesis in a renal cell line [12]. However, genotoxic events are observed over a very narrow concentration range, and acute cell death dominates the picture [12]. In accordance with these in vitro observations the parent haloalkenes produce renal adenocarcinomas in vivo only with treatment regimens that concomi-
118
tantly cause severe nephrotoxicity [ 131. The recent fluorescence digital imaging microscopy studies mentioned above on the spatial and temporal changes in [Ca*+& induced by DCVC in kidney cells [6] have provided interesting new perspectives about the possible role of ion deregulation in the long-term e&cts of these halogenated alkenes. DCVC, which promotes N,N-dimethylnitrosamine-initiated renal tumors [14], impairs the ability of mitochondria to sequester cytosolic Ca2+ without affecting the mitochondrial membrane potential. This effect is common to some tumor promo~rs, such as organic hydroperoxides, that diminish the concentration of reduced pyridine nucleotides in the mitochondria [l&16] and has also been demonstrated with compounds that inhibit sul~ydrylsensitive dehydrogenases in mitochondria [17]. The specific interference with the Ca2*-buffering capacity of the mitochondria results in increased [Ca2+]i at an early point in the toxicity of DCVC. Activation of Ca 2+-dependent endonucleases and production of DNA strand breaks may induce poly-ADP-ribosylation of nuclear proteins [3]; DNA strand breaks have been demonstrated with DCVC both in vivo and in vitro [ 181.High levels of poly-ASP-ribosylation may cause cell death by depletion of NAD+ and ATP, whereas moderate or low levels that do not cause cell death may alter chromatin conformation and function and induce long-term changes [3f. On the other hand, an increase of [Ca2+k is associated with overexpression of C--X and c-myc [ 19,201, effects that may dominate the picture if the changes in [Ca2+]i take place in the early, reversible phase of toxicity. Overexp~ssion of these genes may provide a constant stimulus for cell proliferation and contribute to tumor formation. Although our understanding of the importance of these different events, which are summarized in Figure 1, is incomplete, experimental evidence indicates that changes in [Ca2”]i may function as a link between extranuclear and nuclear events. Quin-2, a Ca2+ chelator, inhibits the formation of DNA strand breaks induced by active oxygen species 131.An important issue in this context is that treatment of cells with tumor promoters may alter their response to changes in [Ca2+ ]i. Phorbol esters inhibit DNA fragmentation caused by increased [Ca*+]i induced by glucocorticoids or by Ca2+ ionophores [2 l]. Ca2+ -mediated events may abo be up-regulated indirectly by alkalinization of the cytoplasm via protein kinase C and via the phosphatidylinositol pathway, which is central to the effects induced by several tumor promoters [19]. Recently the tumor promoter thapsigargin has been shown to increase [Ca2+]i by a different pathway; it is a specific and potent inhibitor of the microsomal Ca*+-ATPase [22]. Moreover, neoplastic cells have a different ion composition and show a different response to changes in [Ca2+], compared with their normal counterparts 1231;finally, increased [Ca”]i is probably involved in the decrease of the gap junction areas observed during the course of neoplastic transformation [19] and Ca2+ ionophores can produce preneoplastic lesions [Sj. Attempts to establish the molecular mechanisms that function as the link between acute toxicity and persistent chronic changes in the kidney are important. In addi-
119
tion, efforts should be made to establish a step-by-step in vivo model for renal adenocarcinomas to correlate ion deregulation with the sequential cellular changes in the course of chemically induced renal neoplasia. ACKNOWLEDGEMENTS
This research was supported by NIEHS grant ES03127 (M.W.A.) and by the Boehringer Ingelheim Foundation (S.V.). REFERENCES 1 Orrenius, S.. McConkey, D.J., Jones, D.P. and Nicotera, P. (1988) Ca2+-activated mechanisms in toxicity and programmed cell death. ISI Atlas of Science: Pha~a~ology, 319-324. 2 Orrenius, S., McConkey, D.J., Bellomo, G. and Nicotera, P. (1989) Role of Ca2+ in toxic cell killing. Trends Pharmacol. Sci. 10.281-285. 3 Muehlematter, D., Larsson. R. and Cerutti, P. (1988) Active oxygen induced DNA strand breakage and poly ADP-ribosylation in promotable and non-promotable JB6 mouse epide~al cells. Carcinogenesis 9, 239-245. 4 Brown, P.C., Chen, J.C. and Jones, T.W. (1990) The role of phospholipases, proteases and endonucleases in the isolated rat renal epithelial cell toxicity of ~-(1,~,3,4,4-pentachlorobutadienyl)glutathione, Toxicologist 10, 265. 5 Trump, B.F., Berezesky, I.K., Smith, M.W., Phelps, P.C. and Elliget, K.A. (1989) The relationship between ion deregulation and acute and chronic toxicity. Toxicol. Appt. Pharmacol. 97,622. 6 Vamvakas, S., Sharma. V.K., Sheu, S.-S. and Anders, M.W. ((990) Perturbation of intracellular calcium distribution in kidney cells by nephrotoxic haloalkenyl cysteine S-conjugates. Mol. Pharmacol. (in press). 7 Lipsky, M.M. and Trump, B.F. (1987) Chemically induced renal epithelial neoplasia in experimental animals. In: G.W. Richter (Ed.), International Review of Experimental Pathology, Kidney Diseases (Vol. 30). Academic Press, San Diego, pp. 357-383. 8 Dekant, W., Vamvakas, S. and Anders, M.W. (1989) Bioactivation of nephrotoxic haloalkenes but glutathione conjugation: formation of toxic and mutagenic intermediates by cysteine conjugate/?-lyase. Drug Metab. Rev. 20,43383. 9 Elfarra, A.A. and Anders, M.W. (1984) Renal processing of glutathione conjugates: role in nephrotoxicity. Biochem. Pharmacol. 33, 3729~-3732. 10 Dekant, W., Vamvakas, S., Berthold, K., Schmidt, S., Wild, D. and Henschler, D. (1986) Bacterial /?-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene. Chem.-Biol. Interact. 60, 3145. 1I Vamvakas, S., Miiller, D.A., Dekant, W. and Henschler, D. (1988) DNA-binding of sulfur”containing metabolites from “5S-(pentachlorobutadienyl)-L-cysteine in bacteria and isolated renal tubular cells. Rev. Drug. Metab. Drug Interact. 6, 349-358. 12 Vamvakas, S., Dekant, W. and Henschler, D. (1989) Assessment of unscheduled DNA synthesis in a cultured line of renal epithelial cells exposed to cysteine S-conjugates of haloalkenes and haloalkanes. Mutat. Res. 222,329-335. 13 Kociba, R.J., Keyes. D.G.. Jersey, G.C., Ballard, J.J., Dittenber, D.A., Quast, J.F., Wade, L.E., Humiston. C.G. and Schwetz, B.A. (1977) Results of a two year chronic toxicity study with hexachlorobutadiene in rats. Am. Ind. Hyg. Assoc. J. 38. 589-602. 14 Meadows, SD., Gandolfi. A.J., Nagle. R.B. and Shively, J.W. (1988) Enhancement of DMN-induced
120 kidney tumors by 1,2-dichlorovinyl-cysteine in Swiss-Webster mice. Drug Chem. Toxicol. 11,307-3 18. 15 Richter, C. and Balz, F. (1985) Ca2+ movements induced by hydroperoxide in mitochondria. In: H. Sies (Ed.), Oxidative Stress. Academic Press, London, pp. 221-241. 16 Cerutti, P.A. (1985) Prooxidant states and tumor promotion. Science 227,375-381. 17 Moore, G.A., Weis, M., Orrenius, S. and O’Brien, P.J. (1988) Role of sulfhydryl groups in benzoquinone-induced Ca*+ release by rat liver mitochondria. Arch. B&hem. Biophys. 267,539-550. 18 Jaffe, D.R., Hassal, D., Gandolfi, A.J. and Brendel, K. (1985) Production of DNA single strand breaks in rabbit renal tissue after exposure to 1,2-dichlorovinylcysteine. Toxicology 35,25-33. 19 Trump, B.G. and Berezesky, I. (1987) Ion regulation, cell injury and carcinogenesis. Carcinogenesis 8,1027-1031. 20 Moore, J.P., Todd, J.A., Hesketh, T.R. and Metcalfe, J.C. (1986) c-fos and c-myc gene activation, ionic signals and DNA synthesis in thymocytes. J. Biol. Chem. 261,8158-8162. 21 McConkey, D.J., Hartell, P., Jordal, M. and Orrenius, S. (1989) Inhibitions of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C. J. Biol. Chem. 264, 13399-13402. 22 Thastrup, O., Cullen, P.J., Drobak, B.K., Hanley, P.M. and Dawson, A.P. (1990) Thapsigargin, a tumor promoter, discharges intra~llular Ca 2+ stores by specific inhibition of the endoplasmic reticulum CaZ+-ATPase. Proc. Natl. Acad. Sci. USA 87,2466-2470. 23 Banyar, M.R.C. and Tellam, R.L. (1985) The free cytoplasmic calcium concentration of tumorigenic and non-tumorigenic human somatic cell hybrids. Br. J. Cancer 51,761-766.
115
Elsevier
TOXLET
02395
Perturbation of calcium homeostasis as a link between acute cell injury and carcinogenesis in the kidney
Spyridon Vamvakas and M.W. Anders Department of Pharmacology, University of Rochester, Rochester, NY (U.S.A.)
Key words: Calcium microscopy;
homeostasis;
ADP-ribosylation;
9( 1,2-Dichlorovinyl)-L-cysteine; Carcinogenesis;
Fluorescence
digital imaging
Cell death
Transport systems on the plasma membrane, the mitochondria, and the endoplasmic reticulum (ER) maintain cytosolic Ca*+ ([Ca*+]J at a very low concentration (x 100 nM) relative to the extracellular Ca*+ concentration ([Ca*+],), which is x 1 mM under physiological conditions. Uncontrolled and sustained increases in [Ca*+]i in chemical-induced toxicity can occur via an enhanced influx of [Ca*+], or by impairment of the intracellular buffering systems, or both. The role of Ca*+-mediated events in acute cell injury and cell death has been studied extensively. Although there are still questions, especially in distinguishing causal effects from epiphenomena, our understanding of this field has improved considerably. Activation of Ca*+ -dependent p hospholipases, proteases, and endonucleases as well as disruption of the cytoskeletal organization (Fig. 1) is involved in the acute injury and cell death associated with elevated [Ca*+]i [1,2]. Ca2+-activated phospholipases catalyze phospholipid breakdown to arachidonic acid and cytotoxic lysophosphohpids; moreover, deregulation of phospholipid turnover may disrupt the integrity of the cell membrane, further impairing the ability of the cell to maintain a normal ion homeostasis. Ca* + -dependent p roteases are involved in cytoskeletal organization, cleavage of receptors, regulation of mitosis, and proteolytic activation of protein kinase C. The use of intracellular Ca *+ chelators to buffer increases in [Ca*+]i delays bleb formation; similar effects can be achieved with selective inhibitors of
Address for correspondence: Elmwood
Avenue,
0378-4274/90/$3.50
Rochester,
S. Vamvakas,
Department
of Pharmacology,
University
NY 14642, U.S.A.
@ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
of Rochester,
601
116 CELL
DAMAGE I
YITOCHONDRIA
PLASMA MEUBRANE I
ENDOPLASYIC RETICULUM I
I
DIACYLGLYCEROL'j)
YEYBRANE
CYTO;KELETAL
DAYACE
ALTERATIONS
I
POLY
I-L
ADP-
RIBDSYLATION
CELL
DEATH
LONG-TERM IN CELLULAR
PERTURBATIONS HOMEOSTASIS
Fig. 1. Processes involved in Ca 2+-mediated acute and long-term cellular toxicity.
Ca2+-dependent proteases. Finally the Ca2+ ionophore A23 187 as well as glucocorticoids and phorbol esters, which facilitate the influx of [Ca2+], in immature thymocytes, cause chromatin condensation and DNA fragmentation. Induction of Ca2+dependent DNA strand-breaks has been demonstrated in mouse epidermal cells where inclusion of the Ca2+ chelator Quin-2 in the incubation system abolished the effect [3], and the toxicity of S-(pentachlorobutadienyl)glutathione and S-(dichlorovinyl)-L-cysteine (DCVC) in kidney cells is diminished by aurintricarboxylic acid, a selective inhibitor of Ca2+-dependent endonucleases [4; S. Vamvakas, unpublished data]. The acute effects of two well-established nephrotoxins on the Ca2+ homeostasis in kidney cells will be briefly described to illustrate two different pathways operating in the deregulation of this ion. Mercuric chloride (HgC12) produces acute necrosis in the proximal tubule in both rodents and humans_ [5]; 50 PM HgC12 induces an intial rise in [Ca2+]i to x 1000 nM, probably due to redistribution from the ER. After a rapid decrease, [Ca2+]i exhibits a second gradual increase due to an influx of [Ca2+], that is less marked but sustained. HgC12 does not interfere with the mitochondrial Ca2+ transport systems, as confirmed by the morphological picture of the injury. The necrotic phase is characterized by calcification in the proximal tubules consisting predominantly of calcium phosphate and hydroxylapatite deposits within the mitochondria, indicating that mitochondria are able to accumulate Ca2+ even at a very late stage in the course of HgClz toxicity.
117
In contrast, the key metabolite of the nephrocarcinogen trichloroethylene DCVC, which is nephrotoxic in vivo and cytotoxic to kidney cells in culture, appears to perturb primarily the mitochondrial Ca *+ homeostasis. The effects of DCVC on intracellular Ca*+ compartmentation have been studied with fluorescence digital imaging microscopy and the Ca*+ indicator fura- in single living kidney cells during the development of cell injury [6]. In the early stage of DCVC-induced toxicity in cultured kidney cells, the average [Ca*+]i increases 3-5-fold compared with control values. However the mitochondria, stained concomitantly with rhodamine 123, have significantly lower Ca*+ concentrations than the rest of the cell. This indicates impairment of the ability of the mitochondrial low-affinity, high-capacity Ca*+ buffering system to sequester Ca*+, although the mitochondrial membrane potential is still intact according to the staining with rhodamine 123. Plasma membrane blebs, collapse of the mitochondrial membrane potential, and loss of viability by propidium iodide staining are observed during later stages in the course of DCVC-induced toxicity. Although it has become increasingly evident in recent years that most renal carcinogens induce toxicity in proximal tubule epithelial cells and the toxic lesions precede and occur along with the development of neoplasia [7], it is still difficult to establish a causal link between chemically induced nephrotoxicity and the formation of kidney tumors and is even more difficult to characterize the involvement of Ca*+-dependent effects in these processes. A number of renal carcinogens, including nitrosamines, arylamines and amides, and polycyclic hydrocarbons, act via a direct genotoxic mechanism; either the parent compounds themselves or their metabolites interact with DNA, resulting in heritable alterations in target cells. On the other hand, non-mutagenic compounds, e.g. branched-chain hydrocarbons or the cationic chelator nitrilotriacetic acid (NTA), produce tumors primarily via extranuclear, epigenetic pathways. NTA is not biotransformed and does not react with DNA; it does, however, produce proximal tubule cell toxicity and may exert its nephrocarcinogenic action via alterations in the homeostasis of divalent cations such as Zn*+ and Ca*+ [7]. The role of genotoxicity versus epigenetic effects in the nephrocarcinogenicity of compounds that are intermediates between the two categories mentioned above, namely compounds that are weak genotoxins and potent nephrotoxins, is still unclear. Several halogenated alkenes such as hexachlorobutadiene, trichloroethylene and tetrachloroethylene are potent nephrotoxins [8]. The corresponding cysteine conjugates, which are the penultimate metabolites formed from these chloroalkenes in vivo, are cleaved by P-lyase to reactive intermediates in the kidney [9]. These cysteine conjugates are nephrotoxic in vivo and cytotoxic in kidney cells in culture; moreover, they are mutagenic in bacteria [lo], bind to DNA in isolated renal tubular cells [l 11,and induce unscheduled DNA synthesis in a renal cell line [12]. However, genotoxic events are observed over a very narrow concentration range, and acute cell death dominates the picture [12]. In accordance with these in vitro observations the parent haloalkenes produce renal adenocarcinomas in vivo only with treatment regimens that concomi-
118
tantly cause severe nephrotoxicity [ 131. The recent fluorescence digital imaging microscopy studies mentioned above on the spatial and temporal changes in [Ca*+& induced by DCVC in kidney cells [6] have provided interesting new perspectives about the possible role of ion deregulation in the long-term e&cts of these halogenated alkenes. DCVC, which promotes N,N-dimethylnitrosamine-initiated renal tumors [14], impairs the ability of mitochondria to sequester cytosolic Ca2+ without affecting the mitochondrial membrane potential. This effect is common to some tumor promo~rs, such as organic hydroperoxides, that diminish the concentration of reduced pyridine nucleotides in the mitochondria [l&16] and has also been demonstrated with compounds that inhibit sul~ydrylsensitive dehydrogenases in mitochondria [17]. The specific interference with the Ca2*-buffering capacity of the mitochondria results in increased [Ca2+]i at an early point in the toxicity of DCVC. Activation of Ca 2+-dependent endonucleases and production of DNA strand breaks may induce poly-ADP-ribosylation of nuclear proteins [3]; DNA strand breaks have been demonstrated with DCVC both in vivo and in vitro [ 181.High levels of poly-ASP-ribosylation may cause cell death by depletion of NAD+ and ATP, whereas moderate or low levels that do not cause cell death may alter chromatin conformation and function and induce long-term changes [3f. On the other hand, an increase of [Ca2+k is associated with overexpression of C--X and c-myc [ 19,201, effects that may dominate the picture if the changes in [Ca2+]i take place in the early, reversible phase of toxicity. Overexp~ssion of these genes may provide a constant stimulus for cell proliferation and contribute to tumor formation. Although our understanding of the importance of these different events, which are summarized in Figure 1, is incomplete, experimental evidence indicates that changes in [Ca2”]i may function as a link between extranuclear and nuclear events. Quin-2, a Ca2+ chelator, inhibits the formation of DNA strand breaks induced by active oxygen species 131.An important issue in this context is that treatment of cells with tumor promoters may alter their response to changes in [Ca2+ ]i. Phorbol esters inhibit DNA fragmentation caused by increased [Ca*+]i induced by glucocorticoids or by Ca2+ ionophores [2 l]. Ca2+ -mediated events may abo be up-regulated indirectly by alkalinization of the cytoplasm via protein kinase C and via the phosphatidylinositol pathway, which is central to the effects induced by several tumor promoters [19]. Recently the tumor promoter thapsigargin has been shown to increase [Ca2+]i by a different pathway; it is a specific and potent inhibitor of the microsomal Ca*+-ATPase [22]. Moreover, neoplastic cells have a different ion composition and show a different response to changes in [Ca2+], compared with their normal counterparts 1231;finally, increased [Ca”]i is probably involved in the decrease of the gap junction areas observed during the course of neoplastic transformation [19] and Ca2+ ionophores can produce preneoplastic lesions [Sj. Attempts to establish the molecular mechanisms that function as the link between acute toxicity and persistent chronic changes in the kidney are important. In addi-
119
tion, efforts should be made to establish a step-by-step in vivo model for renal adenocarcinomas to correlate ion deregulation with the sequential cellular changes in the course of chemically induced renal neoplasia. ACKNOWLEDGEMENTS
This research was supported by NIEHS grant ES03127 (M.W.A.) and by the Boehringer Ingelheim Foundation (S.V.). REFERENCES 1 Orrenius, S.. McConkey, D.J., Jones, D.P. and Nicotera, P. (1988) Ca2+-activated mechanisms in toxicity and programmed cell death. ISI Atlas of Science: Pha~a~ology, 319-324. 2 Orrenius, S., McConkey, D.J., Bellomo, G. and Nicotera, P. (1989) Role of Ca2+ in toxic cell killing. Trends Pharmacol. Sci. 10.281-285. 3 Muehlematter, D., Larsson. R. and Cerutti, P. (1988) Active oxygen induced DNA strand breakage and poly ADP-ribosylation in promotable and non-promotable JB6 mouse epide~al cells. Carcinogenesis 9, 239-245. 4 Brown, P.C., Chen, J.C. and Jones, T.W. (1990) The role of phospholipases, proteases and endonucleases in the isolated rat renal epithelial cell toxicity of ~-(1,~,3,4,4-pentachlorobutadienyl)glutathione, Toxicologist 10, 265. 5 Trump, B.F., Berezesky, I.K., Smith, M.W., Phelps, P.C. and Elliget, K.A. (1989) The relationship between ion deregulation and acute and chronic toxicity. Toxicol. Appt. Pharmacol. 97,622. 6 Vamvakas, S., Sharma. V.K., Sheu, S.-S. and Anders, M.W. ((990) Perturbation of intracellular calcium distribution in kidney cells by nephrotoxic haloalkenyl cysteine S-conjugates. Mol. Pharmacol. (in press). 7 Lipsky, M.M. and Trump, B.F. (1987) Chemically induced renal epithelial neoplasia in experimental animals. In: G.W. Richter (Ed.), International Review of Experimental Pathology, Kidney Diseases (Vol. 30). Academic Press, San Diego, pp. 357-383. 8 Dekant, W., Vamvakas, S. and Anders, M.W. (1989) Bioactivation of nephrotoxic haloalkenes but glutathione conjugation: formation of toxic and mutagenic intermediates by cysteine conjugate/?-lyase. Drug Metab. Rev. 20,43383. 9 Elfarra, A.A. and Anders, M.W. (1984) Renal processing of glutathione conjugates: role in nephrotoxicity. Biochem. Pharmacol. 33, 3729~-3732. 10 Dekant, W., Vamvakas, S., Berthold, K., Schmidt, S., Wild, D. and Henschler, D. (1986) Bacterial /?-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene. Chem.-Biol. Interact. 60, 3145. 1I Vamvakas, S., Miiller, D.A., Dekant, W. and Henschler, D. (1988) DNA-binding of sulfur”containing metabolites from “5S-(pentachlorobutadienyl)-L-cysteine in bacteria and isolated renal tubular cells. Rev. Drug. Metab. Drug Interact. 6, 349-358. 12 Vamvakas, S., Dekant, W. and Henschler, D. (1989) Assessment of unscheduled DNA synthesis in a cultured line of renal epithelial cells exposed to cysteine S-conjugates of haloalkenes and haloalkanes. Mutat. Res. 222,329-335. 13 Kociba, R.J., Keyes. D.G.. Jersey, G.C., Ballard, J.J., Dittenber, D.A., Quast, J.F., Wade, L.E., Humiston. C.G. and Schwetz, B.A. (1977) Results of a two year chronic toxicity study with hexachlorobutadiene in rats. Am. Ind. Hyg. Assoc. J. 38. 589-602. 14 Meadows, SD., Gandolfi. A.J., Nagle. R.B. and Shively, J.W. (1988) Enhancement of DMN-induced
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