Possible role of ca2+ in heavy metal cytotoxicity

0306-4492/91 $3.00 + 0.00 0 1991 Pergamon Press plc

Camp. Eiochem. Physiol. Vol. IOOC, No. l/2, pp. 81-84, 1991 Printed in Great Britain

MINI-REVIEW

POSSIBLE

ROLE OF Ca2+ IN HEAVY METAL CYTOTOXICITY A.

VIARENGO*

and P.

NICoTERAt

Istituto

di Fisiologia Generale, Universitl di Genova, Corso Europa 26, I-16132, Genova, Italy and tDepartment of Toxicology, Karolinska Institutet, Box 60 400, S-104 01, Stockholm, Sweden (Received 1 October 1990)

Abstract-l. Organic xenobiotic metabolism often results in oxidative stress, involving GSH depletion, alteration of thiol/disulphide balance and peroxidation of membrane lipids. These events can lead to the disruption of Ca 2+ homeostasis, through impairment of the Ca 2+ translocases present in cellular membranes. Inhibition of the activity of Ca,Mg-ATPases due to oxidation of their SH groups would lead to uncontrolled rises in cytosolic Ca ‘+ levels resulting in loss of cell viability. 2. These observations seem to be of interest when interpreting the biochemical mechanisms of heavy metal cytotoxicity. Since these cations (such as HgZ+, Cu2+, Cd2+ and Zn2+ ) have an extremely high affinity for SH groups, they may affect the function of SH containing proteins, such as the Ca,Mg-ATPases, as in the case of oxidative stress. 3. Results are reported indicating that Hgr+ may stimulate Ca2+ Influx through voltage-dependent channels in different experimental systems. Moreover, evidence is presented that heavy metals can inhibit Ca,Mg-ATPase activity and affect mitochondrial functions in the cells of different organisms. 4. The possibility that heavy metal cytotoxicity is mediated through disruption of Ca*+ homeostasis is

INTRODUCTION

are typically sustained, and seem to differ from the rapid, physiological transients observed in response to hormones (Nicotera et al., 1988). Different mechanisms may be responsible for a disruption of Ca*+ homeostasis during toxic injury. It has been demonstrated that during oxidative stress, the increased rate of free radical production due to redox cycling of organic xenobiotics, such as menadione, results in a net decrease in the GSH content (Di Monte et al., 1984). This leads to an increased oxidation of protein sulphydryl groups, to the formation of mixed disulphides and is associated with an enhancement of the rate of peroxidation of membrane lipids (Younes and Siegers, 1984; Nicotera et al., 1988). It has been demonstrated that these events can affect the function of SH-containing enzymes, including the Ca*+ transport- ATPases present in liver microsomal fraction (Di Monte et al., 1984; Bellomo et al., 1985) and in hepatocyte plasma membranes (Bellomo et al., 1983; Nicotera et al., 1988). This can result in a rise in the level of cytosolic free Ca*+, which is closely associated with the onset of cell death, possibly due to an irreversible activation of phospholipases, endonucleases, proteases, and to cytoskeletal changes due to actin and tubulin depolymerization (Nicotera et al., 1988; Bellomo et al., 1989). The observation that Ca*+-mediated cytotoxicity is associated with an alteration of the cellular thiol/disulphide status appears to be of particular importance when interpreting the biochemical effects of heavy metals such as Hg*+, Cd*+, Cu*+, etc. In

The general involvement of calcium ions in the regulation of physiological processes is now wellestablished (Rasmussen and Barrett, 1984). Along with this knowledge has come the understanding that Caz+ can also play a critical role in a variety of pathological and toxicological processes (Orrenius et al., 1989). Ca’+-dependent cell killing does not seem to be restricted to a specific cell system or organ. Ca*+ appears to mediate neurotoxicity due to cyanide, lead, chlordecone and organotin compounds (Komulainen and Bondy, 1988) and liver toxicity in response to oxidative stress (Jewel1 et al., 1982), cyanide (Nicotera et al., 1989a), alkylating toxins (Long and Moore, 1986) and also thymic atrophy caused by thedioxin, 2,3,7,8-tetrachlorodibenzo-pdioxin (McConkey et al., 1990) and by organotin compounds (Aw et al., 1990). Normally, intracellular Ca*+ homeostasis is maintained by the concerted operation of extrusion and compartmentation systems (Carafoli, 1987). Alteration of these processes during cell injury can result in inhibition of Ca*+ extrusion or intracellular compartmentation mechanisms as well as in enhanced Ca*+ influx and release of Ca*+ from intracellular stores such as endoplasmic reticulum and mitochondria (Nicotera et al., 1989b). This can lead to the uncontrolled rises in cytosolic Ca*+ concentration which are usually associated with loss of cell viability. Toxic increases in cytosolic Ca’+ concentration *To whom all the correspondence

should be addressed. 81

A. VIARENGO and P. NICOTERA

82

fact, these cations have a high affinity for nucleophilic groups (Eichorn, 1973), and in particular for SH residues of amino acids and proteins (S > 0 > N). Therefore, the interactions between heavy metals and SH-containing proteins can affect their function, thus compromising several enzyme activities, similarly to that which has been observed during oxidative stress. Moreover, as recently demonstrated in marine invertebrates, and, in particularly, in the case of Cu2+-exposed mussels, the metal can decrease the cellular GSH content and increase the rate of membrane lipid peroxidation (Viarengo et al., 1990a). The role of Ca2+ in the cytotoxicity of heavy metals has recently been investigated in different biological systems. Here follows a brief review on this topic, including some of our more recent results. Cd+ UPTAKE

Ca2+ uptake into the cell is primarily dependent on a passive process of ion flux through transmembrane channels (Hille, 1984). Voltage-dependent Ca2+ channels can be blocked by La3+ but also by transition metals such as Ni2+ and Cd’+ (Hille, 1984; Sauer and Watabe, 1988). These data have led researchers to consider cadmium and, more generally, heavy metals able to reduce Ca2+ uptake into the cell. It was recently shown that, in fertilized sea urchin eggs, Hg2+ increases the Ca2+ influx through voltagedependent channels (Walter et al., 1989). It seems that in the Hg2+-exposed eggs the excess of Ca2+ that passively penetrates into the cells is, at least initially, accumulated in mitochondria. Although interesting, these data have been obtained by exposing sea urchin fertilized eggs to 100pM Hg’+, a concentration that is high. Therefore, extrapolation of these results to the interpretation of the Hg2+ effects on marine organisms in field conditions seems to be unrealistic. More recently, Nicotera, Rossi and coworkers have demonstrated that exposure of mammalian neuroadrenergic PC-12 cells to micromolar Hg2+ con-

0 -

I/J

d

100

?-

0

timi

hin;”

centrations causes a rapid increase in cytosolic free Ca2+, as evaluated utilizing the fluorescent probe FuraZ-AN (Fig. 1A). At lower concentrations Hg2+ did not seem to affect cytosolic Ca2+ directly. However, changes in Ca2+ concentration induced either by neurotransmitters or by hormones such as bradykinin and ATP (Fig. 1B and C) or KCl-stimulated depolarization were inhibited in cells pretreated with Hg2+. The effect of mercury was partially related to its ability to prevent Ca2+ influx through voltage-dependent channels and to depress the inositol phosphate production following hormone stimulation. Although the latter findings require more detailed study in order to understand the mechanism involved in the site of the inhibition, it appears clear that such modifications may compromise cell survival. Since the concentrations of mercury at which these effects were observed are low, and only marginally above the levels observed in normal subjects, these mechanisms may have relevance to the in vivo situation.

EFFECTS ON Ca,Mg-ATPASES

Concerning the possible effects of heavy metals on the Ca,Mg-ATPase activity, it was observed that in vitro Hg’+, Cu2+ and Zn2+ inhibit the enzyme activity present in the “crude” microsomal membrane fraction from fish gills (Shepard and Simkiss, 1978). However, it has been found that in vivo exposure of fish to 0.2pM Cu2+ for two months, rather than drastically inhibiting the enzyme activity, seems to induce the synthesis of additional enzyme units in gill cells (Shepard and Simkiss, 1978). These results do not enable us to understand the molecular mechanisms by which heavy metals affect the Ca2+ pump. Moreover, these data, obtained by utilizing a microsomal preparation, do not give information about the effects of heavy metals on the different cellular membranes, and in particular, on the Ca,Mg-ATPase present in the plasma membrane, which should represent one of the first targets of the metal cations

E J

j&k___ 100 6

L

I

tinZ

hin15

O

t, [$j

(LMf5

PC-12 cells. A: PC-12 Fig. 1. Effect of HgCl, on cytosolic Ca r+ in resting and stimulated neuroadrenergic cells were exposed to 2 PM HgCl, (trace a) or preincubated with the Ca2+-channel blocker verapamil (20 PM) and then exposed to HgCl, (trace b). B: PC-12 cells were stimulated with 20 PM bradykinin (trace a) or (trace b) preincubated for 10 min with 0.5 PM HgCl, and then stimulated by bradykinin as in trace (i.e. a. C: effect of HgCl, on ATP-stimulated cytosolic Ca2+ increase. At low HgCl, concentrations ~0.3 PM), a stimulation of the normal ATP-triggered response was observed. At concentrations above 0.5 PM a clear inhibition took place.

Ca2+ in heavy metal cytotoxicity

penetrating into the cell (Webb et al., 1979). With regard to this, we have recently investigated the effects of heavy metals on the plasma membrane preparation obtained from mussel gills after previous biochemical characterization of the Ca,Mg-ATPase activity (Viarengo et al., 1990b). It appears that, in vitro, heavy metals inhibit Ca,Mg-ATPase. When 50 PM H$+ was present in the reaction mixture, a significant decrease of the ATPase activity was observed. Cd2+, Cu*+ and Zn2+ also decreased the enzyme activity, but only at higher concentrations. Figure 2 shows a polyacrylamide gel electrophoresis carried out to evaluate the formation of the phosphorylated intermediate (E - P) that represents a crucial step in the cycle of activity of the CaATPase, in the Ca2+ transmembrane transport (Inesi, 1985). It is shown that addition of 50/1M Hg2+ suffices to inhibit formation of the phosphorylated enzyme. In this case, too, Cu*+, Cd2+ and Zn2+ had similar effects only at higher concentrations. These data indicate that heavy metals inhibit the Ca,Mg-ATPase activity by affecting the formation of the phosphorylated enzyme. It is worth noting that preincubation of mussel gill plasma membranes with heavy metals (for 30 min at 19°C) increased the inhibitory effect, so that 10 PM Hg*+ significantly decreased both the catalytic activity and the formation of the phosphorylated intermediate. Data obtained from rat corpus luteum plasma membranes also indicate that, at least in oitro, Hg2+ may inhibit Ca*+ transport by affecting the E-P formation (Minami and Penniston, 1987). The in vitro effects of heavy metals on mussel gill plasma membrane Ca,Mg-ATPase seem to be confirmed by preliminary data from in viva exper-

KDa 136

L I

.

6-l

i Fig. 2. Effect of Hg*+ on the formation of phosphorylated Ca,Mg-ATPase intermediate (E - P). The plasma membrane preparation from mussel gill cells was phosphorylated utilizing Y-~‘P-ATP. La)+ was added to the reaction mixture to stabilize the E-P complex (Viarengo et al., 1390b). Separation of phosphorylated plasma membrane proteins was carried out by efectrophoresis on a 5-10% polyacrylamide gel and the formation of the E - P was evidentiated by autoradiography as described by Sarkadi er al. (1986). Autoradiography (A) and densitometric analysis at 530 nm (B) show that Hg2+, when present in the reaction mixture at a concentration of SOpM, significantly decreased the formation of the E -P, that was completely inhibited in presence of 200pM H$+; 1: control; 2: 50pM Hg2+; 3: 200pM Hg2+.

83

iments. In fact, when mussels were exposed for 3 days to Cu2+ (40 ~g/l/animal) the activity of the Ca2+ pump present in the plasma membrane of gill cells was significantly inhibited. Concerning the possible effects of heavy metals on the endoplasmic reticulum, it has been demonstrated that HgZf inhibits the Ca,Mg-ATPase present in the sarcoplasmic reticulum of rabbit muscle (Shamoo and Ryan, 1975). Overall, these data indicate that the Ca,MgATPases involved in calcium transport in different cell types of different organisms can be considered a heavy metal target. EFFECTS

ON MITOCHONDRIA

The data concerning the biochemical effects of heavy metals on mitochondrial calcium compartmentation appear to be of particular interest. It has been shown that in fertilized sea urchin eggs Hg2+ stimulates, at least initially, the oxygen consumption rate of mitochondria (Walter et al., 1989). This stimulation depends on uncoupling phenomena, since it is unaffected by oligomycin. It seems that the pathological increase of cellular calcium induced by the metal is buffered mainly by mitochondria, which results in uncoupling and consequent reduction of the mitochondrial ATP synthesis and probably in alteration of cell metabolism. It is important to mention, however, that in mussels exposed to Cu2* (40 pg/animal) for 3 days, copper ions are able to alter the mitochondrial physiology by permeabilizing the inner mitochondrial membrane (Viarengo et al., 1985). It is likely that Cu2+, probably by enhancing the oxyradical production, stimulates the lipid peroxidation of mitochondrial membranes, which consequently become permeable also to cations and high molecular weight molecules. Lipid peroxidation of mitochondrial membranes has also been demonstrated in Cu-exposed fish (Aloj Totaro et al., 1986). As mentioned before, mitochondria are a site of endocellular Ca*+ compartmentation. Therefore, alteration of mitochondrial membrane permeability may cause Ca*+ release into cytosol, thus affecting the physiology of the cells. The overall data presented in this paper indicate that heavy metals alter the physiological status of the cells by affecting, although in differing ways, the mechanisms involved in Ca2+ homeostasis. It seems clear that different heavy metals affect different aspects of the complex Ca-homeostasis process; therefore, at the moment, no generalization can be made on the way heavy metals act. It is worth noting, however, that important structural and enzymatic SH-containing proteins, such as tubulin (Viarengo et al., 1989), actin (M. N. Moore, personal communication) Na,K-ATPases (Bouquegneau and Gilles, 1979), DNA and RNA polymerases (Viarengo et al., 1982), could represent a critical target for heavy metal toxicity (Webb, 1979). Moreover, it must be pointed out that heavy metals may also affect other important cellular functions, such as lysosomal activity, protein synthesis, nuclear metabolism, etc. (Webb, 1979; Viarengo, 1989) and

A. VIARENGO and P. NICOTERA

84

it is not clear whether such cations can affect these processes directly or indirectly, through an impairment of Caz+ homeostasis mechanisms. Therefore, although it appears clear that heavy metals can affect homeostasis of the cytosolic free Ca*+, further research has to be carried out to clarify the possible role of calcium in mediating heavy metal cytotoxicity. Acknowledgements-This work was supported by a CNR Grant (Progetto Biotecnologie-cap. B 1205 7821), a SNV Grant (Contr. 5311320-5), CFN-Grant (Contr. L-90-08) e Fondazione Clinica de1 lavoro IRCCS. Universita di Pavia.

REFERENCES Aloj Totaro E., Pisanti F. A., Glees P. and Continillo A. (1986) The effect of copper pollution on mitochondrial degeneration. Mar. environ. Res. 18, 245-253. Aw T. Y., Nicotera P., Manzo L. and Orrenius S. (1990) Tributyltin stimulates apoptosis in rat thymocytes. Archs Biochem. Biophys. (In press). Bellomo G., Mirabelli F., Richelmi P. and Orrenius S. (1983) A critical role of sulnhvdrvl arouu(s) in ATP metacdependent Ca2+ sequestration - ’ _by -the plasma _. ’ membrane fraction from the rat liver. FEBS Letr. 163, 136141. Bellomo G., Richelmi P., Mirabelli F., Marinoni V. and Abbagnano A. (1985) Inhibition of liver microsomal calcium ion sequestration by oxidative stress: role of protein sulphydryl groups. In Free Radicals in Liver Injury (Edited by Poli G., Cheeseman K. H., Dianzani M. U. and Slater T. F.), pp. 139-142. IRL Press, Oxford. Bellomo G., Thor H., Mirabelli F., Richelmi P., Finardi G. and Orrenius S. (1989) Alteration in hepatocyte cytoskeleton during the metabolism of quinones. In Free Radicals in the Pathogenesis of Liver Injury. Advances in the Biosciences (Edited by Poli G., Cheeseman K. H.,

Dianzani M. U. and Slater T. F.), Vol. 76, pp. 21-35. Pergamon Press, Oxford. Bouquegneau J. M. and Gilles R. (1979) Osmoregulation and Pollution of the Aquatic Medium. In Metabolism and Osmoregulation in Animals (Edited by Gilles R.), pp. 563-587. John Wiley & Sons, New York. Carafoli E. (1987) Intracellular calcium homeostasis. A. Rev. Biochem. 56, 395-433.

Di Monte D., Bellomo G., Thor H., Nicotera P. and Orrenius S. (1984) Menadione-induced cytotoxicity is associated with protein thiol oxidation and alteration in intracellular Ca’+ homeostasis. Archs Biochem. Biophys. 235, 343-350.

Eichorn G. L. (1973) Inorganic Biochemistry (Edited by G. L. Eichorn), pp: 12101Elsevier, Amsterdam. Hille B. (1984) Calcium channels. In Ionic Channels and Excitable Membranes, pp. 76-98. Sinauer Associates, Sunderland, MA. Inesi G. (1985) Mechanism of calcium transport. A. Rev. Physiol. 41, 573601.

Jewel1 S. A., Bellomo G., Thor H., Orrenius S. and Smith M. T. (1982) Bleb formation in hepatocytes during drug metabolism is caused by disturbances in thiol and Ca2+ homeostasis. Science 217, 1257-1259. Komulainen H. and Bondy S. C. (1988) Increased free intracellular Ca *+ by toxic agents: an index of potential neurotoxicity. Trends Pharmac. Sci. 9, 154-156. Long R. M. and Moore L. (1986) Cytosolic Ca*+ after carbon tetrachloride, 1,l -dichloroethylene, and phenylephrine exposure. Biochem. Pharmac. 35, 3301-3307.

McConkey D. J., Hartzell P. and Orrenius S. (1990) 2,3,7,8-tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca*+-mediated endonuclease activation. Archs Biochem. Biophys. (In press). Minami J. and Penniston J. T. (1987) Ca2+ uptake by corpus luteum plasma membranes. Biochem. J. 242, 889-894. Nicotera P., McConkey D. J., Svensson S. A., Bellomo G. and Orrenius S. (1988) Correlation between cytosolic Ca2+ concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicology 52, 5563. Nicotera P., Thor H. and Orrenius S. (1989a) Cytosolic-free Ca*+ and cell killing in hepatoma lclc7 cells exposed to chemical anoxia. FASEB J. 3, 594. Nicotera P., McConkey D. J., Dypbukt J. M., Jones D. P. and Orrenius S. (1989b) Caz+-activated mechanisms in cell killing. Drug. Metab. Rev. 20, 193-201. Orrenius S., McConkey D. J., Bellomo G. and Nicotera P. (1989) Role of the Ca2+ in toxic cell killing. Trends Pharmac. Sci. 10, 281-285. Rasmussen H. and Barrett P. Q. (1984) Calcium messenger system: an integrated view. Physiol. Rev. 64, 938-984. Sarkadi B., Enyedi A., Foldes-Papp Z. and Gardos G. (1986) Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis. J. biol. Chem. 261, 9552-9557. Sauer G. R. and Watabe N. (1988) The effects of heavy metals and metabolic inhibitors on calcium uptake by gills and scales of Fundulus Heteroclitus in vitro. Comp. Biochem. Physiol. 91C, 473478.

Shepard K. and Simkiss K. (1978) The effects of heavy metal ions on Cal+ ATPase extracted from fish gills. Comp. Biochem. Physiol. 61B, 69-72.

Shamoo A. E. and Ryan T. E. (1975) Isolation of ionophores from ion-transport system. Ann. N.Y. Acad. Sci. 264, 83-97.

Viarengo A. (1989) Heavy metals in marine invertebrates: mechanisms of regulation and toxicity at the cellular level. CRC Crit. Rev. Aquat. Sci. 1, 295-317. Viarengo A., Pertica M., Mancinelli G., Palmer0 S. and Orunesu M. (1982) Effects of Cu2+ on nuclear RNA polymerase activities in mussel digestive gland. Mar. Biol. Lett. 3, 3455352.

Viarengo A., Secondini A., Scoppa P., Moore M. N. and Orunesu M. (1985) Effetti de1 Cu*+ e de1 Cd’+ sull’attivita mitocondriale e sulla carica energetica adenilica delle cellule della ghiandola digestiva di mitili. Atfi Riun. SZF, Ottobre. Pisa. Viarengo A., Arena N., Pertica M., Canesi L., Martin0 G., Gaspa L. and Orunesu M. (1989) Effect of copper on microtubule structure in the gill cells of metal-exposed mussels. Mar. environ. Res. 28, 453454. Viarengo A., Canesi L., Pertica M., Poli G., Moore M. N. and Orunesu M. (1990a) Heavy metal effects on lipid peroxidation in the tissues of Mytilus galloprovincialis Lam. Comp. Biochem. Physiol. 97C, 3742. Viarengo A.: Pertica M., .Mancinelli G., Damonte G. and Martin0 G. (1990b) Biochemical characterization of plasma membrane da,Mg-ATPase from the gills of Mytilus galloprovincialis Lam. Proc. 12th ESCPB Conference, Utrecht, August 1990. Walter P., Allemand D., De Renzis G. and Payan P. (1989) Mediating effect of calcium in HgCl, cytotoxicity in sea urchin egg: role of mitochondria in Ca*+-mediated cell death. Biochim. biophys. Acta 3, 3644. Webb M. (1979) Interaction of cadmium with cellular components. In The Chemistry, Biochemistry and Biology of Cadmium (Edited by Webb M.), pp. 285-340. Elsevier, Amsterdam. Younes M. and Siegers C. P. (1984) Interrelation between lipid peroxidation and other hepatotoxic events. Biochem. Pharmac. 33, 2001-2003.