Journal of Toxicology and Environmental Health
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Inhibition of metabolic coupling by metals Rita Loch‐Caruso , Isabel A. Corcos & James E. Trosko To cite this article: Rita Loch‐Caruso , Isabel A. Corcos & James E. Trosko (1991) Inhibition of metabolic coupling by metals, Journal of Toxicology and Environmental Health, 32:1, 33-48, DOI: 10.1080/15287399109531463 To link to this article: http://dx.doi.org/10.1080/15287399109531463
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INHIBITION OF METABOLIC COUPLING BY METALS Rita Loch-Caruso Toxicology Program, Department of Environmental and Industrial Health, School of Public Health, University of Michigan, Ann Arbor, Michigan
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Isabel A. Corcos Cellular and Molecular Biology Program, Department of Biology, University of Michigan, Ann Arbor, Michigan James E. Trosko Department of Pediatrics and Human Development, College of Medicine, Michigan State University, East Lansing, Michigan
Several metals were evaluated in cell cultures for their ability to inhibit metabolic coupling, the intercellular transfer of low-molecular-weight metabolites by directly connecting gap junctions. To monitor inhibition of metabolic coupling, wild-type Chinese hamster V79 cells proficient in metabolism of 6-thioguanine (6-TG) were cocultured with mutant V79 cells unable to metabolize 6-TG to its toxic metabolite (6-TGresistant cells). In the presence of 6-TG, inhibition of metabolic coupling by the metals was manifested as increased recovery of 6-TG-resistant cells compared to recovery in untreated cocultures. The toxic metal compounds, arsenic(V) acid, mercury(II) chloride, lead(II) acetate, and nickel(II)chloride, significantly (p < .05) increased recovery of 6-TG-resistant cells at concentrations that did not alter cell survival. However, because the increased recovery observed with nickel showed no concentration dependence, its importance may be negligible. Cadmium chloride increased 6-TGresistant cell recovery only at a toxic concentration, and zinc sulfate failed to increase
A portion of this work was presented at the 25th Annual Meeting of the Society of Toxicology, New Orleans, La., March 1986 {Toxicologist 6:267,1986). This project was supported by grants from the National Institute for Occupational Safety and Health (K01-OH00059) to R. Loch-Caruso and from the United States Air Force (AFOSR-89-0325) to J. E. Trosko. The United States Government is authorized to produce and distribute reprints for governmental purposes not withstanding any copyright notation therein. We thank Ms. Beth Lockwood for laboratory assistance, Ms. Suzan Cull for typing assistance, and the Audio-Visual Services staff of the University of Michigan's School of Public Health for production of graphs and photos. Requests for reprints should be sent to R. Loch-Caruso, Toxicology Program, Department of Environmental and Industrial Health, School of Public Health, The University of Michigan, Ann Arbor, Ml 48109-2029. 33 Journal of Toxicology and Environmental Health, 32:33-48, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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recovery. These data demonstrate that some metal compounds inhibit metabolic coupling, and suggest that inhibition of junctional communication should be further evaluated as a potential mechanism of toxicity of some metals.
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INTRODUCTION Gap junctions are membrane channels that allow direct intercellular transfer of low-molecular-weight substances (Larsen, 1983). Gap junctions may be important in the regulation of cell growth and differentiation (Loewenstein, 1979), embryogenesis (Sheridan, 1976; Fraser, 1985), germ-cell maturation (Dekel et al., 1981; Herlands and Schultz, 1984), and normal tissue function in the adult (Sheridan and Atkinson, 1985). Many toxicants have been shown to alter gap junctional communication, including tumor promoters (Murray and Fitzgerald, 1979; Yotti et al., 1979; Williams et al., 1981,1984; Trosko et al., 1982b; Slaga et al., 1981; Jone et al., 1982; Malcolm and Mills, 1983; Lawrence et al., 1984; Aylsworth et al., 1984), neurotoxicants (Tsushimoto et al., 1983a, 1983b; Williams et al., 1981; Wade et al., 1986; Zhong-Xiang et al., 1986, Trosko et al., 1987), and teratogens (Welsch and Stedman, 1984; Jone et al., 1985; Loch-Caruso et al., 1984; Loch-Caruso and Trosko, 1985). The present study examined whether certain metal compounds could alter junctional communication. Metabolic coupling, the gap junction-mediated transfer of lowmolecular-weight substances, was used to assay cell-to-cell communication in cultured V79 cells (Trosko et al., 1985). METHODS Chemicals All metal solutions were made with ultrapure water (Millipore MilliQ). Nickel(ll) chloride Q.T. Baker Inc., Phillipsburg, N.J.), cadmium acetate (Fisher Scientific, Pittsburgh, Pa.), arsenic(V) acid sodium salt (Sigma Chemical Co., St. Louis, Mo.), and zinc sulfate (Malinckrodt, St. Louis, Mo.) were dissolved directly in water. Lead(ll) acetate (Aldrich Chemical Co., Milwaukee, Wis.) was dissolved in a 0.06 N HCI solution and mercury(II) chloride (Fisher Scientific, Pittsburgh, Pa.) was dissolved in a 0.1 N HCI solution. All stock metal solutions were made to concentrations of 50 miW. The 12-tetradecanoylphorbol 13-acetate (TPA) was obtained from Consolidated Midland Corporation (Brewster, N.Y.) and was dissolved in absolute ethanol to a stock solution concentration of 1 mg/ml. Cell Culture Wild-type Chinese hamster V79 cells sensitive to 6-thioguanine (6-TG) (6-TGs cells) and a mutant line of V79 cells resistant to 6-TG (6-TGr cells)
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were used in these experiments. The cells were grown in modified Eagle's medium (Eagle's balanced salt solution; Eagle, 1959) with a 50% increase of all essential amino acids and vitamins except glutamine supplemented with a 100% increase of nonessential amino acids, 1 mM sodium pyruvate, and 3% fetal bovine serum (FBS). Stock cell cultures were maintained in medium without antibiotics, but gentamicin (50 /*g/ml) was added to the medium during experiments. The cells were incubated in a humidified air atmosphere with 5% CO2 at 37°C. Both cell lines were routinely checked for the presence of mycoplasma (Chen, 1977) and were found to be free of contamination.
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Metabolic Coupling Assay The assay for metabolic coupling is based on the transfer of the toxic metabolite of 6-TG from the metabolically competent, wild-type 6-TGs cells to the mutant 6-TGr cells (Yotti et al., 1979). The 6-TG is not lethal to cells unless phosphorylated by hypoxanthine guanine phosphoribosyl transferase (HGPRT). The phosphorylated metabolite of 6-TG does not transfer across the plasma membrane of V79 cells to any measurable extent, presumably due to its charge, but is sufficiently small to pass through gap junctions. The 6-TGr cells lack HGPRT and survive in medium that includes 6-TG, whereas the 6-TGs cells have HGPRT activity and die in 6-TC-containing media. When 6-TGr and 6-TGs cells are cocultured at sufficiently high density, survival of 6-TGr cells declines due to transfer of the toxic 6-TG metabolite from the 6-TGs cells via gap junctions. If a test substance interrupts gap junction-mediated transfer of the toxic metabolite, increased numbers of 6-TGr cells survive in the coculture, evidenced by increased colony formation after 8 d of growth. The assay thus measures the recovery of 6-TGr cells in cocultures of 6-TGr and 6-TGs cells grown in media containing 6-TG. To perform the assay, a mixture of 100 6-TGr cells was seeded into 60mm dishes with 4 x 105 6-TGs cells, a density that allowed ample cell contact and opportunity for metabolic coupling (Fig. 1). As a measure of cytotoxicity, cell survival was determined in cocultures of 100 6-TGr cells and 104 6-TGs cells in 60-mm dishes, a lower cell density that allowed negligible cell contact and limited metabolic coupling (Fig. 1). For each experiment, plating efficiencies were obtained by growing 100 6-TGr cells per 60-mm dish, and these data were compared to a group in which 100 6-TGr cells were grown with 10" 6-TGs cells per 60-mm dish. Only slight, inconsistent differences were observed between these two groups. The latter group was thus taken as the unexposed control group for the cell survival experiments and was used for calculating the relative cell survivals of the remaining groups. The control group for the metabolic coupling experiments consisted of unexposed cocultures of 100 6TGr and 4 x 105 6-TGs cells. A positive control group consisted of 100 6TGr cells cocultured with 4 x 105 6-TGs cells with TPA (1 /xg/ml) added
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FIGURE 1. Metabolic cooperation between V79 wild-type 6-TGs cells and mutant 6-TGr cells. In 60mm tissue culture dishes containing 6-TG (10 /jg/ml), 100 6-TGr cells were cocultured with 6-TGs cells varying in number from 5 x 10 3 to8 x 10s cells per dish. The cultures were changed to fresh 6-TG medium on d 3, and stained for colony counting on d 8. Increased metabolic cooperation was observed with increased numbers of 6-TGs cells per dish, as reflected by decreased colony formation at the higher densities of 6-TGs cells.
the test compound, since previous reports noted strong inhibition of metabolic coupling in V79 cells treated with TPA (Yotti et al., 1979). The 6TGS cell population was checked for spontaneous 6-TGr mutants by plating 4 x 1 0 6-TGscells per 60-mm dish in the presence of 6-TG, and negligible colony formation was observed. The growth media of all treatment groups contained 6-TG (10 pg/ml). There were 10 plates per treatment group. The test substances were added in 1 ml of medium 3-4 h after the cells were seeded. The concentrations of the metals were selected from pilot studies and included noncytotoxic concentrations that effectively inhibited metabolic coupling, up to a toxic concentration, if possible. The pH of the medium was not significantly altered by addition of the test substances; any change was less than 0.1 pH unit. One hour after the test substances were added, 50 pi of a 1 mg/ml solution of 6-TG were added to each dish for a final concentration of 10 /xg 6-TG/ml medium. The plates were changed to fresh medium containing 6-TG (10 pg/ml) after 3 d of incubation. After a total of 8 d, the plates were stained with crystal violet and the number of colonies per plate was scored using a
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semiautomated colony counting system that allowed the experimenter to select the colonies to be included (VPC Systems, Ann Arbor, Mich.). Statistical Analysis
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Cell survivals and recoveries were expressed as the percent plating efficiency (using the unexposed cocultures of 100 6-TGr and 10 6-TCs cells), and these data were analyzed with the Kruskal-Wallis statistic (Gad and Weil, 1982). When a significant (p < .05) effect was observed, each treatment group was compared to the appropriate unexposed control group using distribution-free multiple comparisons tested at the p < .05 level for significance (Hollander and Wolfe, 1973). RESULTS Cytotoxicity Cytotoxicity was indicated by reduced cell survival in plates seeded with 100 6-TGr cells and 104 6-TGs cells. Cytotoxicity was observed within the concentration ranges employed for arsenic(V) acid (Fig. 2, p < .0001), mercury(ll) chloride (Fig. 3, p < .0001), nickel(ll) chloride (Fig. 4, p < .05), cadmium acetate (Fig. 5, p < .001), and zinc sulfate (Fig. 6, p < .0001). Cytotoxicity was not demonstrated for lead(ll) acetate (Fig. 7), since it precipitated in the medium at concentrations higher than those used in this experiment. The lowest concentrations at which significant (p < .05) cytotoxicity was observed were 20 pM arsenic(V) acid, 5 [xM mercury(ll) chloride, 4 fiM cadmium acetate, and 200 nM zinc sulfate. While the Kruskal-Wallis statistic for the nickel(ll) chloride experiment was significant at p < .05, pairwise comparison of the nickel-treated groups to the unexposed control group failed to detect differences significant at this level. This may be due to the conservative nature of the multiple comparison test used (Hollander and Wolfe, 1973). The largest differences observed with nickel were at the two highest concentrations, 100 and 150 (p < .15, not significant). Metabolic Coupling Arsenic(V) acid (Fig. 2, p < .0001), mercury(ll) chloride (Fig. 3, p < .0001), and lead(ll) acetate (Fig. 7, p < .0002) inhibited metabolic coupling, since recovery of 6-TGr cells increased at metal concentrations that did not alter cell survival. The concentrations that inhibited metabolic coupling were 5 and 10 fiM arsenic(V) acid, 2 and 3 fiM mercury(ll) chloride, and all concentrations of lead(ll) acetate, 50-500 fiM (p < .05). The Kruskal-Wallis statistic indicated a significant concentration effect for nickel(ll) chloride (Fig. 4, p < .0001), and post hoc comparisons indicated that 75 fiM nickel(ll) chloride was statistically different from controls (p < .05). However, the response was relatively small and a significant differ-
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FIGURE 2. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to arsenic(V) acid. Decreased cell survival was observed at 20, 30, and 50 pM arsenic(V) acid (stars, p < .05). Inhibition of metabolic cooperation was determined by increased recovery of 6-TC cells at concentrations that did not decrease cell survival, and was observed at 5 and 10 /xM arsenic(V) acid (stars, p < .05). Values were adjusted to plating efficiency, which was 77%. Each value represents the mean ± SEM of 10 dishes.
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FIGURE 3. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to mercury(ll) chloride. Decreased cell survival was observed at 5 \iM mercury(ll) chloride (asterisk) and significant inhibition of metabolic cooperation was observed at 2 and 3 iiM mercury(ll) chloride (stars) (p < .05). The plating efficiency was 61%. Each value represents the mean ± SEM of 10 dishes.
ence compared to unexposed controls was detected at only one dose. There was also no obvious pattern to the data. Thus, nickel(ll) chloride is apparently only a weak, perhaps negligible, inhibitor of metabolic coupling in this test system. Although lead(ll) acetate was an effective inhibitor of metabolic coupling, there was no evidence of a relationship between concentration and inhibition of metabolic coupling.
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Nlckelous Chloride
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FIGURE 4. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to nickel(ll) chloride. A statistically significant difference in metabolic cooperation was observed at 75 \iM nickel(ll) chloride (star, p < .05). The plating efficiency was 86%. Each value represents the mean ± SEM of 10 dishes.
Cadmium acetate increased 6-TGr cell recovery in an apparent concentration-related fashion (Fig. 5, p < .03), but a significant increase relative to unexposed controls was detected only at the highest and toxic concentration of 6 pM. Since cell survival also decreased in a concentration-related fashion, these data suggest that the primary effect of cadmium acetate was cytotoxicity. No increased recovery was observed with zinc sulfate (Fig. 6). DISCUSSION In the present study, metabolic coupling was inhibited by the metals arsenic(V), lead(ll), and mercury(ll), as indicated by increased cell recovery in these experiments. Inhibited metabolic coupling occurred at con-
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FIGURES. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to cadmium acetate. Decreased cell survival was observed at 4, 5, and 6 pM cadmium acetate (stars, p < .05) but statistically significant differences in the metabolic cooperation experiment occurred only at the cytotoxic concentration of 6 pM cadmium acetate (stars, p < .05). Because of the cytotoxicity, this effect is not interpreted as inhibition of metabolic cooperation (see text for discussion). The plating efficiency was 58%. Each value represents the mean ± SEM of 10 dishes.
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Zinc Sulfate
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CONCENTRATION FIGURE 6. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to zinc sulfate. Decreased cell survival was observed at 200 fiM zinc sulfate (star, p < .05), and no inhibition of metabolic cooperation was observed. The plating efficiency was 62%. Each value represents the mean + SEM of 10 dishes.
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Lead Acetate 100
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FIGURE 7. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to lead(ll) acetate. Cell survival was not significantly affected, while inhibition of metabolic cooperation was noted at all concentrations tested, 50-500 \iM lead(ll) acetate (stars, p < .05). Plating efficiency was 75%. Each value represents the mean ± SEM of 10 dishes.
centrations of these metals that were noncytotoxic as determined by cell survival. It is not likely that the increased cell recoveries observed in the metabolic coupling experiments were artifacts due to inhibition of HGPRT by the metals, since this would have allowed survival of the 6-TGs cells in addition to increased survival of the 6-TGr cells. The consequence of this would have been thousands of surviving cells and colonies, which was not observed. The responses to the metals arsenic, mercury, and lead were not as dramatic as those seen with TPA, the positive control used in these experiments. However, TPA inhibition of metabolic coupling is typically a near-maximal response for these cells in this assay (Trosko et al., 1985), while other cells and other test procedures have yielded weaker responses to TPA (Kavanagh et al., 1986; Zhong-Xiang et al., 1986). Similarly,
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the V79/6-TG metabolic coupling system is relatively insensitive to some substances, such as dieldrin, while similar exposure elicits high levels of inhibition in other cells and test systems (Wade et al., 1986; Zhong-Xiang et al., 1986). Additionally, recent observations suggest that assays for junctional communication may vary in their sensitivity (Loch-Caruso et al., 1990). It is possible that the culture and testing conditions employed in the present experiments are optimal for the TPA response but suboptimal for the detection of metal effects. Currently, we are investigating metal effects on gap junctional communication in other cell lines using different assays. The results presented here are consistent with those of OliveiraCastro and Loewenstein (1971), who reported that certain divalent cations, including magnesium, barium, and strontium, also inhibited junctional communication in salivary gland cells of Chironomous as measured by electrical coupling. In a report by Miki et al. (1987), millimolar concentrations of nickel(ll) sulfate were found to inhibit gap junctional communication in NIH 3T3 cells using a radioisotope transfer technique. Magnesium sulfate had no effect in their system by itself, but was able to antagonize the inhibition observed with nickel. In the present experiment, nickel's effect was unremarkable at concentrations up to 150 ixM, the highest concentration tested. The differences between the results of the two studies may be due, at least in part, to the different cells, assays, or concentrations used. Inhibition of gap junctional communication has been associated with tumor promotion (Trosko et al., 1983; Tong and Williams, 1987), and the V79/6-TG assay has been used to study a large number of tumor promoters. The majority of tumor promoters evaluated with this test system inhibit metabolic coupling (Elmore et al., 1987). In the present experiment, each of the metals associated with decreased metabolic couplingarsenic, lead, and mercury—also has been associated with tumorigenesis (summarized in Leonard et al., 1984), and lead has been specifically identified as a tumor promoter (Hiasa et al., 1983; Shirai et al., 1984). Zinc, which is nontumorigenic (Leonard et al., 1984), had no effect on metabolic coupling. The lack of a significant effect on metabolic coupling by the tumorigenic metals nickel and cadmium may indicate that the assay was not sensitive enough to detect metal effects, or that gap junctions may not be relevant to their tumorigenicity. The mechanism(s) whereby these metals inhibit metabolic coupling is unknown. Treatments that elevate intracellular levels of calcium or decrease intracellular pH have been shown to reduce junctional communication in a variety of cells (DeMello, 1984; Flagg-Newton and Loewenstein, 1979; Obaid et al., 1983; Rose et al., 1977; Spray et al., 1982). If there is a cationic regulatory mechanism for gap junctions, this may provide an explanation for the metal effects observed in the present experiment. Reports also link the calcium-binding protein calmodulin with regulation
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of gap junctions (Cole and Garfield, 1985; Peracchia et al., 1983; Peracchia and Girsch, 1985; Welsh et al., 1982). Since many toxic metals bind to calmodulin, and this has been proposed as a mechanism of toxicity (Chao et al., 1984; Cox and Harrison, 1983; Habermann et al., 1983; Mills and Johnson, 1985), another possibility is that metals interfere with junctional communication through their interaction with calmodulin. Alternatively, Davidson et al. (1984) showed that metabolic coupling requires extracellular calcium and magnesium, suggesting that certain metals may inhibit metabolic coupling by altering the extracellular matrix necessary to form cell contacts. Metals represent a significant toxicological concern, yet their mechanisms of action remain controversial. This report describes a cellular response to the toxic metals arsenic, mercury, and lead that has not been previously reported, inhibition of junction-mediated intercellular communication. While the responses observed here are weak relative to TPA, they are nonetheless significant at micromolar concentrations, and suggest that further work is warranted. Notably, evaluation of specific target cells of metal toxicity may reveal differences that offer further clues to their mechanism of action. Gap junctional communication has been associated with pituitary function, hormone responses, and parturition, to cite a few examples, all functions that may be adversely affected by metals. Determination of gap junctional responses in cells from specific, targeted tissues, such as pituitary, reproductive, and kidney, may allow more specific conclusions to be drawn concerning the importance of this cell response to the toxicity of metals. REFERENCES Aylsworth, C. F., Jone, C., Trosko, J. E., Meites, J., and Welsch, C. W. 1984. Promotion of 7,12dimethylbenz[a]anthracene-induced mammary tumorigenesis by high dietary fat in the rat: Possible role of intercellular communication. JNCI 72:637-645. Chao, S. H., Suzuki, Y., Zysk, J. R., and Cheung, W. Y. 1984. Activation of calmodulin by various metal cations as a function of ionic radius. Mol. Pharmacol. 26:75-82. Chen, T. R. 1977. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104:255-262. Cole, W. C., and Garfield, R. E. 1985. Alterations in coupling in uterine smooth muscle. In Cap Junctions, eds. M. L. V. Bennett and D. Spray, pp. 215-230. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Cox, J. A., and Harrison, S. D., Jr. 1983. Correlation of metal toxicity with in vitro calmodulin inhibition. Biochem. Biophys. Res. Commun. 115:106-111. Davidson, J. S., Baumgarten, I. M., and Harley, E. H. 1984. Effects of extracellular calcium and magnesium on junctional intercellular communication in human fibroblasts. Exp. Cell Res. 155:406-412. Dekel, N., Lawrence, T. S., Gilula, N. B., and Beers, W. H. 1981. Modulation of cell-to-cell communication in the cumulus-oocyte complex and the regulation of oocyte maturation by LH. Dev. Biol. 86:356-362. DeMello, W. C. 1984. Modulation of junctional permeability. Fed. Proc. 43:2692-2696.
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Eagle, H. 1959. Amino acid metabolism in mammalian cell cultures. Science 130:432-437. Elmore, E., Milman, H. A., and Wyatt, G. P. 1987. Applications of the Chinese hamster V79 metabolic cooperation assay in toxicology. In Biochemical Mechanisms and Regulation of Intercellular Communication, eds. H. A. Milman and E. Elmore, pp. 265-292. Princeton, N.J.: Princeton Scientific Publishing. Flagg-Newton, J., and Loewenstein, W. R. 1979. Experimental depression of junctional membrane permeability in mammalian cell culture. A study with tracer molecules in the 300 to 800 dalton range. J. Membrane Biol. 50:65-100. Fraser, S. E. 1985. Gap junctions and cell interactions during development. Trends Neurosci. 8:3-4. Friberg, L, Nordberg, G. F., and Vouk, V., eds. 1986. Handbook on the Toxicology of Metals, 2nd ed. New York: Elsevier. Gad, S. C., and Weil, C. S. 1982. Statistics for toxicologists. In Principles and Methods of Toxicology, ed. A. W. Hayes, pp. 273-320. New York: Raven Press. Habermann, E., Crowell, K., and Janicki, P. 1983. Lead and other metals can substitute for Ca 2+ in calmodulin. Arch. Toxicol. 54:61-70. Herlands, R. L., and Schultz, R. M. 1984. Regulation of mouse oocyte growth: Probable nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth. J. Exp. Zool. 229:317-325. Hiasa, Y., Ohshima, M., Kitahori, Y., Fujita, T., Yuasa, T., and Miyashiro, A. 1983. Basic lead acetate: Promoting effect on the development of renal tubular cell tumor in rats treated with N-ethylN-hydroxyethylnitrosamine. JNCI 70:761-765. Hollander, M., and Wolfe, D. A. 1973. Nonparametric Statistical Methods, pp. 114-132. New York: John Wiley and Sons. Jone, C. M., Trosko, J. E., Chang, C. C., Fujiki, H., and Sugimura, T. 1982. Inhibition of intercellular communication in Chinese hamster cells by teleocidin. Gann 73:874-878. Jone, C. M., Parker, L., Trosko, J. E., Netzloff, M., and Chang, C. C. 1985. Inhibition of metabolic cooperation by the anticonvulsants, diphenylhydantoin and phenobarbital. Teratogen. Carcinogen. Mutagen. 5:379-391. Kavanagh, T., Chang, C. C., and Trosko, J. E. 1986. Characterization of a human teratocarcinoma cell assay for inhibitors of metabolic cooperation. Cancer Res. 46:1359-1366. Larsen, W. J. 1983. Biological implications of gap junction structure, distribution and composition: A review. Tissue Cell 9:373-399. Lawrence, N. J., Parkinson, E. K., and Emmerson, A. 1984. Benzoyl peroxide interferes with metabolic cooperation between cultured human epidermal keratinocytes. Carcinogenesis 5:419421. Leonard, A., Gerber, C. B., Jacquet, P., and Lauwerys, R. R. 1984. Mutagenicity, carcinogenicity, and teratogenicity of industrially used metals. In Mutagenicity, Carcinogenicity, and Teratogenicity of Industrial Pollutants, ed. M. Kirsch-Volders, pp. 59-126. New York: Plenum Press. Loch-Caruso, R., and Trosko, J. E. 1985. Inhibited intercellular communication as a mechanistic link between teratogenesis and carcinogenesis. CRC Crit. Rev. Toxicol. 16:157-183. Loch-Caruso, R., Trosko, J. E., and Corcos, I. A. 1984. Interruption of cell-cell communication in Chinese hamster V79 cells by various alkyl glycol ethers: Implications for teratogenicity. Environ. Health Perspect. 57:119-123. Loch-Caruso, R., Caldwell, V., Cimini, M., and Juberg, D. 1990. Comparison of assays for gap junctional communication using human embryocarcinoma cells exposed to dieldrin. Fundam. Appl. Toxicol. 15:63-74. Loewenstein, W. R. 1979. Junctional intercellular communication and the control of growth. Biochim. Biophys. Acta 560:1-65. Malcolm, R., and Mills, L. J. 1983. Inhibition of metabolic cooperation between Chinese hamster V79 cells by tumor promoters and other chemicals. Ann. NY Acad. Sci. 407:448-450. Miki, H., Kasprzak, K. S., Kenney, S., and Heine, U. I. 1987. Inhibition of intercellular communication by nickel(II): Antagonistic effect of magnesium. Carcinogenesis 8:1757-1760.
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Mills, J. S., and Johnson, J. D. 1985. Metal ions as allosteric regulators of calmodulin. J. Biol. Chew. 260:15100-15105. Murray, A. W., and Fitzgerald, D. J. 1979. Tumor promoters inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells. Biochem. Biophys. Res. Commun. 91:395-401. Obaid, A. L., Socolar, S. J., and Rose, B. 1983. Cell-to-cell channels with two independently regulated gates in series: Analysis of junctional conductance modulation by membrane potential, calcium, and pH. J. Membrane Biol. 73:69-89. Oliveira-Castro, G., and Loewenstein, W. R. 1971. Junctional membrane permeability: Effects of divalent cations. J. Membrane Biol. 5:51-77. Peracchia, C., and Girsch, S. J. 1985. Functional modulation of cell coupling: Evidence for a calmodulin-driven channel gate. Am. J. Physiol. 248:H765-H782. Peracchia, C., Bernardini, G., and Peracchia, L. L. 1983. Is calmodulin involved in the regulation of gap junction permeability? Pflugers Arch. 399:152-154. Peracchia, C., Bernardini, G., and Peracchia, L. L. 1983. Is calmodulin involved in the regulation of gap junction permeability? Pflugers Arch. 399:152-154. Rose, B., Simpson, I., and Loewenstein, W. R. 1977. Calcium ion produces graded changes in permeability of membrane channels in cell junction. Nature (Lond.) 267:625-627. Sheridan, J. D. 1976. Cell coupling and cell communication during embryogenesis. In The Cell Surface in Animal Embryogenesis and Development, eds. G. Poste and G. L. Nicolson, pp. 409-447. New York: Elsevier/North-Holland Biomedical Press. Sheridan, J. D., and Atkinson, M. M. 1985. Physiological roles of permeable junctions: Some possibilities. Annu. Rev. Physiol. 47:337-353. Shirai, T., Ohshima, M., Masuda, A., Tamano, S., and Ito, N. 1984. Promotion of 2(ethylnitrosoamino)ethanol-induced renal carcinogenesis by nephrotoxic compounds: Positive responses with folic acid, basic lead acetate, and N-(3,5-dichlorophenyl) succinimide but not with 2,3-dibromo-1-propanol phosphate. JNCI 72:477-482. Slaga, T. J., Klein-Szanto, A. S. P., Triplett, L. C., Yotti, L. P., and Trosko, J. E. 1981. Skin tumor promoting activity of benzoyl peroxide: A widely used free radical generating compound. Science 213:1023-1025. Spray, D. C., Stern, J. H., Harris, A. L., and Bennett, M. V. L. 1982. Gap junctional conductance: Comparison of sensitivities to H and Ca ions. Proc. Natl. Acad. Sci. USA 79:441-445. Tong, C. C., and Williams, G. M. 1987. The effect of tumor promoters on cell-cell communication in liver epithelial cell systems. In Biochemical Mechanisms and Regulation of Intercellular Communication, eds. H. A. Milman and E. Elmore, pp. 251-263. Princeton, N.J.: Princeton Scientific Publishing. Trosko, J. E., Chang, C. C., and Netzloff, M. 1982a. The role of inhibited cell-cell communication in teratogenesis. Teratogen. Carcinogen. Mutagen. 2:31-45. Trosko, J. E., Jone, C., Aylsworth, C., and Tsushimoto, G. 1982b. Elimination of metabolic cooperation is associated with the tumor promoters, oleic acid and anthralin. Carcinogenesis 3:11011103. Trosko, J. E., Chang, C. C., and Medcalf, A. 1983. Mechanisms of tumor promotion: Potential role of intercellular communication. Cancer Invest. 1:511-526. Trosko, J. E., Jone, C., Aylsworth, C., and Chang, C. C. 1985. In vitro assay to detect inhibitors of intercellular communication. In Handbook of Carcinogen Testing, eds. H. A. Milman and E. K. Weisburger, pp. 422-437. Park Ridge, NJ.: Noyes Publishing. Trosko, J. E., Jone, C., and Chang, C. C. 1987. Inhibition of gap junctional-mediated intercellular communication in vitro by aldrin, dieldrin and toxaphene: A possible cellular mechanism for their tumor promoting and neurotoxic effects. Molec. Toxicol. 1:83-93. Tsushimoto, G., Asano, S., Trosko, J. E., and Chang, C. C. 1983a. Inhibition of intercellular communication by various congeners of polybrominated biphenyl and polychlorinated biphenyl. In PCB's: Human and Environmental Hazards, eds. F. D'lmitri and M. Kamrin, pp. 241-252. Ann Arbor, Mich.: Ann Arbor Science Publishing. Tsushimoto, G., Chang, C. C., Trosko, J. E., and Matsumura, F. 1983b. Cytotoxic, mutagenic, and cell-cell communication inhibitory properties of DDT, lindane, and chlordane on Chinese hamster cells in vitro. Arch. Environ. Contam. Toxicol. 12:721-730.
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Wade, M. H., Trosko, J. E., and Schindler, M. 1986. A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science 232:525-528. Welsch, F., and Stedman, D. B. 1984. Inhibition of metabolic cooperation between Chinese hamster V79 cells by structurally diverse teratogens. Teratogen. Carcinogen. Mutagen. 4:285-301. Welsh, M. J., Aster, J. .C, Ireland, M., Alcala, J., and Maisel, H. 1982. Calmodulin binds to chick lens gap junction protein in a calcium-independent manner. Science 216:642-644. Williams, G. M., Telang, S., and Tong, C. 1981. Inhibition of intercellular communication between liver cells by the liver promoter 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane. Cancer Lett. 11:339-344. Williams, G. M., Tong, C., and Telang, S. 1984. Polybrominated biphenyls are non-genotoxic and produce an epigenetic membrane effect in cultured liver cells. Environ. Res. 34:310-320. Yotti, L. P., Chang, C. C., and Trosko, J. E. 1979. Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science 206:1089-1091. Zhong-Xiang, L., Kavanagh, T., Trosko, J. E., and Chang, C. C. 1986. Inhibition of gap junctional intercellular communication in human teratocarcinoma cells by organochlorine pesticides. Toxicol. Appl. Pharmacol. 83:10-19. Received July 3, 1987 Accepted June 5, 1990
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Inhibition of metabolic coupling by metals Rita Loch‐Caruso , Isabel A. Corcos & James E. Trosko To cite this article: Rita Loch‐Caruso , Isabel A. Corcos & James E. Trosko (1991) Inhibition of metabolic coupling by metals, Journal of Toxicology and Environmental Health, 32:1, 33-48, DOI: 10.1080/15287399109531463 To link to this article: http://dx.doi.org/10.1080/15287399109531463
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INHIBITION OF METABOLIC COUPLING BY METALS Rita Loch-Caruso Toxicology Program, Department of Environmental and Industrial Health, School of Public Health, University of Michigan, Ann Arbor, Michigan
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Isabel A. Corcos Cellular and Molecular Biology Program, Department of Biology, University of Michigan, Ann Arbor, Michigan James E. Trosko Department of Pediatrics and Human Development, College of Medicine, Michigan State University, East Lansing, Michigan
Several metals were evaluated in cell cultures for their ability to inhibit metabolic coupling, the intercellular transfer of low-molecular-weight metabolites by directly connecting gap junctions. To monitor inhibition of metabolic coupling, wild-type Chinese hamster V79 cells proficient in metabolism of 6-thioguanine (6-TG) were cocultured with mutant V79 cells unable to metabolize 6-TG to its toxic metabolite (6-TGresistant cells). In the presence of 6-TG, inhibition of metabolic coupling by the metals was manifested as increased recovery of 6-TG-resistant cells compared to recovery in untreated cocultures. The toxic metal compounds, arsenic(V) acid, mercury(II) chloride, lead(II) acetate, and nickel(II)chloride, significantly (p < .05) increased recovery of 6-TG-resistant cells at concentrations that did not alter cell survival. However, because the increased recovery observed with nickel showed no concentration dependence, its importance may be negligible. Cadmium chloride increased 6-TGresistant cell recovery only at a toxic concentration, and zinc sulfate failed to increase
A portion of this work was presented at the 25th Annual Meeting of the Society of Toxicology, New Orleans, La., March 1986 {Toxicologist 6:267,1986). This project was supported by grants from the National Institute for Occupational Safety and Health (K01-OH00059) to R. Loch-Caruso and from the United States Air Force (AFOSR-89-0325) to J. E. Trosko. The United States Government is authorized to produce and distribute reprints for governmental purposes not withstanding any copyright notation therein. We thank Ms. Beth Lockwood for laboratory assistance, Ms. Suzan Cull for typing assistance, and the Audio-Visual Services staff of the University of Michigan's School of Public Health for production of graphs and photos. Requests for reprints should be sent to R. Loch-Caruso, Toxicology Program, Department of Environmental and Industrial Health, School of Public Health, The University of Michigan, Ann Arbor, Ml 48109-2029. 33 Journal of Toxicology and Environmental Health, 32:33-48, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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recovery. These data demonstrate that some metal compounds inhibit metabolic coupling, and suggest that inhibition of junctional communication should be further evaluated as a potential mechanism of toxicity of some metals.
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INTRODUCTION Gap junctions are membrane channels that allow direct intercellular transfer of low-molecular-weight substances (Larsen, 1983). Gap junctions may be important in the regulation of cell growth and differentiation (Loewenstein, 1979), embryogenesis (Sheridan, 1976; Fraser, 1985), germ-cell maturation (Dekel et al., 1981; Herlands and Schultz, 1984), and normal tissue function in the adult (Sheridan and Atkinson, 1985). Many toxicants have been shown to alter gap junctional communication, including tumor promoters (Murray and Fitzgerald, 1979; Yotti et al., 1979; Williams et al., 1981,1984; Trosko et al., 1982b; Slaga et al., 1981; Jone et al., 1982; Malcolm and Mills, 1983; Lawrence et al., 1984; Aylsworth et al., 1984), neurotoxicants (Tsushimoto et al., 1983a, 1983b; Williams et al., 1981; Wade et al., 1986; Zhong-Xiang et al., 1986, Trosko et al., 1987), and teratogens (Welsch and Stedman, 1984; Jone et al., 1985; Loch-Caruso et al., 1984; Loch-Caruso and Trosko, 1985). The present study examined whether certain metal compounds could alter junctional communication. Metabolic coupling, the gap junction-mediated transfer of lowmolecular-weight substances, was used to assay cell-to-cell communication in cultured V79 cells (Trosko et al., 1985). METHODS Chemicals All metal solutions were made with ultrapure water (Millipore MilliQ). Nickel(ll) chloride Q.T. Baker Inc., Phillipsburg, N.J.), cadmium acetate (Fisher Scientific, Pittsburgh, Pa.), arsenic(V) acid sodium salt (Sigma Chemical Co., St. Louis, Mo.), and zinc sulfate (Malinckrodt, St. Louis, Mo.) were dissolved directly in water. Lead(ll) acetate (Aldrich Chemical Co., Milwaukee, Wis.) was dissolved in a 0.06 N HCI solution and mercury(II) chloride (Fisher Scientific, Pittsburgh, Pa.) was dissolved in a 0.1 N HCI solution. All stock metal solutions were made to concentrations of 50 miW. The 12-tetradecanoylphorbol 13-acetate (TPA) was obtained from Consolidated Midland Corporation (Brewster, N.Y.) and was dissolved in absolute ethanol to a stock solution concentration of 1 mg/ml. Cell Culture Wild-type Chinese hamster V79 cells sensitive to 6-thioguanine (6-TG) (6-TGs cells) and a mutant line of V79 cells resistant to 6-TG (6-TGr cells)
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were used in these experiments. The cells were grown in modified Eagle's medium (Eagle's balanced salt solution; Eagle, 1959) with a 50% increase of all essential amino acids and vitamins except glutamine supplemented with a 100% increase of nonessential amino acids, 1 mM sodium pyruvate, and 3% fetal bovine serum (FBS). Stock cell cultures were maintained in medium without antibiotics, but gentamicin (50 /*g/ml) was added to the medium during experiments. The cells were incubated in a humidified air atmosphere with 5% CO2 at 37°C. Both cell lines were routinely checked for the presence of mycoplasma (Chen, 1977) and were found to be free of contamination.
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Metabolic Coupling Assay The assay for metabolic coupling is based on the transfer of the toxic metabolite of 6-TG from the metabolically competent, wild-type 6-TGs cells to the mutant 6-TGr cells (Yotti et al., 1979). The 6-TG is not lethal to cells unless phosphorylated by hypoxanthine guanine phosphoribosyl transferase (HGPRT). The phosphorylated metabolite of 6-TG does not transfer across the plasma membrane of V79 cells to any measurable extent, presumably due to its charge, but is sufficiently small to pass through gap junctions. The 6-TGr cells lack HGPRT and survive in medium that includes 6-TG, whereas the 6-TGs cells have HGPRT activity and die in 6-TC-containing media. When 6-TGr and 6-TGs cells are cocultured at sufficiently high density, survival of 6-TGr cells declines due to transfer of the toxic 6-TG metabolite from the 6-TGs cells via gap junctions. If a test substance interrupts gap junction-mediated transfer of the toxic metabolite, increased numbers of 6-TGr cells survive in the coculture, evidenced by increased colony formation after 8 d of growth. The assay thus measures the recovery of 6-TGr cells in cocultures of 6-TGr and 6-TGs cells grown in media containing 6-TG. To perform the assay, a mixture of 100 6-TGr cells was seeded into 60mm dishes with 4 x 105 6-TGs cells, a density that allowed ample cell contact and opportunity for metabolic coupling (Fig. 1). As a measure of cytotoxicity, cell survival was determined in cocultures of 100 6-TGr cells and 104 6-TGs cells in 60-mm dishes, a lower cell density that allowed negligible cell contact and limited metabolic coupling (Fig. 1). For each experiment, plating efficiencies were obtained by growing 100 6-TGr cells per 60-mm dish, and these data were compared to a group in which 100 6-TGr cells were grown with 10" 6-TGs cells per 60-mm dish. Only slight, inconsistent differences were observed between these two groups. The latter group was thus taken as the unexposed control group for the cell survival experiments and was used for calculating the relative cell survivals of the remaining groups. The control group for the metabolic coupling experiments consisted of unexposed cocultures of 100 6TGr and 4 x 105 6-TGs cells. A positive control group consisted of 100 6TGr cells cocultured with 4 x 105 6-TGs cells with TPA (1 /xg/ml) added
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FIGURE 1. Metabolic cooperation between V79 wild-type 6-TGs cells and mutant 6-TGr cells. In 60mm tissue culture dishes containing 6-TG (10 /jg/ml), 100 6-TGr cells were cocultured with 6-TGs cells varying in number from 5 x 10 3 to8 x 10s cells per dish. The cultures were changed to fresh 6-TG medium on d 3, and stained for colony counting on d 8. Increased metabolic cooperation was observed with increased numbers of 6-TGs cells per dish, as reflected by decreased colony formation at the higher densities of 6-TGs cells.
the test compound, since previous reports noted strong inhibition of metabolic coupling in V79 cells treated with TPA (Yotti et al., 1979). The 6TGS cell population was checked for spontaneous 6-TGr mutants by plating 4 x 1 0 6-TGscells per 60-mm dish in the presence of 6-TG, and negligible colony formation was observed. The growth media of all treatment groups contained 6-TG (10 pg/ml). There were 10 plates per treatment group. The test substances were added in 1 ml of medium 3-4 h after the cells were seeded. The concentrations of the metals were selected from pilot studies and included noncytotoxic concentrations that effectively inhibited metabolic coupling, up to a toxic concentration, if possible. The pH of the medium was not significantly altered by addition of the test substances; any change was less than 0.1 pH unit. One hour after the test substances were added, 50 pi of a 1 mg/ml solution of 6-TG were added to each dish for a final concentration of 10 /xg 6-TG/ml medium. The plates were changed to fresh medium containing 6-TG (10 pg/ml) after 3 d of incubation. After a total of 8 d, the plates were stained with crystal violet and the number of colonies per plate was scored using a
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semiautomated colony counting system that allowed the experimenter to select the colonies to be included (VPC Systems, Ann Arbor, Mich.). Statistical Analysis
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Cell survivals and recoveries were expressed as the percent plating efficiency (using the unexposed cocultures of 100 6-TGr and 10 6-TCs cells), and these data were analyzed with the Kruskal-Wallis statistic (Gad and Weil, 1982). When a significant (p < .05) effect was observed, each treatment group was compared to the appropriate unexposed control group using distribution-free multiple comparisons tested at the p < .05 level for significance (Hollander and Wolfe, 1973). RESULTS Cytotoxicity Cytotoxicity was indicated by reduced cell survival in plates seeded with 100 6-TGr cells and 104 6-TGs cells. Cytotoxicity was observed within the concentration ranges employed for arsenic(V) acid (Fig. 2, p < .0001), mercury(ll) chloride (Fig. 3, p < .0001), nickel(ll) chloride (Fig. 4, p < .05), cadmium acetate (Fig. 5, p < .001), and zinc sulfate (Fig. 6, p < .0001). Cytotoxicity was not demonstrated for lead(ll) acetate (Fig. 7), since it precipitated in the medium at concentrations higher than those used in this experiment. The lowest concentrations at which significant (p < .05) cytotoxicity was observed were 20 pM arsenic(V) acid, 5 [xM mercury(ll) chloride, 4 fiM cadmium acetate, and 200 nM zinc sulfate. While the Kruskal-Wallis statistic for the nickel(ll) chloride experiment was significant at p < .05, pairwise comparison of the nickel-treated groups to the unexposed control group failed to detect differences significant at this level. This may be due to the conservative nature of the multiple comparison test used (Hollander and Wolfe, 1973). The largest differences observed with nickel were at the two highest concentrations, 100 and 150 (p < .15, not significant). Metabolic Coupling Arsenic(V) acid (Fig. 2, p < .0001), mercury(ll) chloride (Fig. 3, p < .0001), and lead(ll) acetate (Fig. 7, p < .0002) inhibited metabolic coupling, since recovery of 6-TGr cells increased at metal concentrations that did not alter cell survival. The concentrations that inhibited metabolic coupling were 5 and 10 fiM arsenic(V) acid, 2 and 3 fiM mercury(ll) chloride, and all concentrations of lead(ll) acetate, 50-500 fiM (p < .05). The Kruskal-Wallis statistic indicated a significant concentration effect for nickel(ll) chloride (Fig. 4, p < .0001), and post hoc comparisons indicated that 75 fiM nickel(ll) chloride was statistically different from controls (p < .05). However, the response was relatively small and a significant differ-
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FIGURE 2. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to arsenic(V) acid. Decreased cell survival was observed at 20, 30, and 50 pM arsenic(V) acid (stars, p < .05). Inhibition of metabolic cooperation was determined by increased recovery of 6-TC cells at concentrations that did not decrease cell survival, and was observed at 5 and 10 /xM arsenic(V) acid (stars, p < .05). Values were adjusted to plating efficiency, which was 77%. Each value represents the mean ± SEM of 10 dishes.
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FIGURE 3. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to mercury(ll) chloride. Decreased cell survival was observed at 5 \iM mercury(ll) chloride (asterisk) and significant inhibition of metabolic cooperation was observed at 2 and 3 iiM mercury(ll) chloride (stars) (p < .05). The plating efficiency was 61%. Each value represents the mean ± SEM of 10 dishes.
ence compared to unexposed controls was detected at only one dose. There was also no obvious pattern to the data. Thus, nickel(ll) chloride is apparently only a weak, perhaps negligible, inhibitor of metabolic coupling in this test system. Although lead(ll) acetate was an effective inhibitor of metabolic coupling, there was no evidence of a relationship between concentration and inhibition of metabolic coupling.
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Nlckelous Chloride
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FIGURE 4. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to nickel(ll) chloride. A statistically significant difference in metabolic cooperation was observed at 75 \iM nickel(ll) chloride (star, p < .05). The plating efficiency was 86%. Each value represents the mean ± SEM of 10 dishes.
Cadmium acetate increased 6-TGr cell recovery in an apparent concentration-related fashion (Fig. 5, p < .03), but a significant increase relative to unexposed controls was detected only at the highest and toxic concentration of 6 pM. Since cell survival also decreased in a concentration-related fashion, these data suggest that the primary effect of cadmium acetate was cytotoxicity. No increased recovery was observed with zinc sulfate (Fig. 6). DISCUSSION In the present study, metabolic coupling was inhibited by the metals arsenic(V), lead(ll), and mercury(ll), as indicated by increased cell recovery in these experiments. Inhibited metabolic coupling occurred at con-
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Cadmium Acetate 100 9O j
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FIGURES. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to cadmium acetate. Decreased cell survival was observed at 4, 5, and 6 pM cadmium acetate (stars, p < .05) but statistically significant differences in the metabolic cooperation experiment occurred only at the cytotoxic concentration of 6 pM cadmium acetate (stars, p < .05). Because of the cytotoxicity, this effect is not interpreted as inhibition of metabolic cooperation (see text for discussion). The plating efficiency was 58%. Each value represents the mean ± SEM of 10 dishes.
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Zinc Sulfate
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CONCENTRATION FIGURE 6. Cell survival (top) and metabolic cooperation (bottom) among V79 cells exposed to zinc sulfate. Decreased cell survival was observed at 200 fiM zinc sulfate (star, p < .05), and no inhibition of metabolic cooperation was observed. The plating efficiency was 62%. Each value represents the mean + SEM of 10 dishes.
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Lead Acetate 100
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centrations of these metals that were noncytotoxic as determined by cell survival. It is not likely that the increased cell recoveries observed in the metabolic coupling experiments were artifacts due to inhibition of HGPRT by the metals, since this would have allowed survival of the 6-TGs cells in addition to increased survival of the 6-TGr cells. The consequence of this would have been thousands of surviving cells and colonies, which was not observed. The responses to the metals arsenic, mercury, and lead were not as dramatic as those seen with TPA, the positive control used in these experiments. However, TPA inhibition of metabolic coupling is typically a near-maximal response for these cells in this assay (Trosko et al., 1985), while other cells and other test procedures have yielded weaker responses to TPA (Kavanagh et al., 1986; Zhong-Xiang et al., 1986). Similarly,
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the V79/6-TG metabolic coupling system is relatively insensitive to some substances, such as dieldrin, while similar exposure elicits high levels of inhibition in other cells and test systems (Wade et al., 1986; Zhong-Xiang et al., 1986). Additionally, recent observations suggest that assays for junctional communication may vary in their sensitivity (Loch-Caruso et al., 1990). It is possible that the culture and testing conditions employed in the present experiments are optimal for the TPA response but suboptimal for the detection of metal effects. Currently, we are investigating metal effects on gap junctional communication in other cell lines using different assays. The results presented here are consistent with those of OliveiraCastro and Loewenstein (1971), who reported that certain divalent cations, including magnesium, barium, and strontium, also inhibited junctional communication in salivary gland cells of Chironomous as measured by electrical coupling. In a report by Miki et al. (1987), millimolar concentrations of nickel(ll) sulfate were found to inhibit gap junctional communication in NIH 3T3 cells using a radioisotope transfer technique. Magnesium sulfate had no effect in their system by itself, but was able to antagonize the inhibition observed with nickel. In the present experiment, nickel's effect was unremarkable at concentrations up to 150 ixM, the highest concentration tested. The differences between the results of the two studies may be due, at least in part, to the different cells, assays, or concentrations used. Inhibition of gap junctional communication has been associated with tumor promotion (Trosko et al., 1983; Tong and Williams, 1987), and the V79/6-TG assay has been used to study a large number of tumor promoters. The majority of tumor promoters evaluated with this test system inhibit metabolic coupling (Elmore et al., 1987). In the present experiment, each of the metals associated with decreased metabolic couplingarsenic, lead, and mercury—also has been associated with tumorigenesis (summarized in Leonard et al., 1984), and lead has been specifically identified as a tumor promoter (Hiasa et al., 1983; Shirai et al., 1984). Zinc, which is nontumorigenic (Leonard et al., 1984), had no effect on metabolic coupling. The lack of a significant effect on metabolic coupling by the tumorigenic metals nickel and cadmium may indicate that the assay was not sensitive enough to detect metal effects, or that gap junctions may not be relevant to their tumorigenicity. The mechanism(s) whereby these metals inhibit metabolic coupling is unknown. Treatments that elevate intracellular levels of calcium or decrease intracellular pH have been shown to reduce junctional communication in a variety of cells (DeMello, 1984; Flagg-Newton and Loewenstein, 1979; Obaid et al., 1983; Rose et al., 1977; Spray et al., 1982). If there is a cationic regulatory mechanism for gap junctions, this may provide an explanation for the metal effects observed in the present experiment. Reports also link the calcium-binding protein calmodulin with regulation
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of gap junctions (Cole and Garfield, 1985; Peracchia et al., 1983; Peracchia and Girsch, 1985; Welsh et al., 1982). Since many toxic metals bind to calmodulin, and this has been proposed as a mechanism of toxicity (Chao et al., 1984; Cox and Harrison, 1983; Habermann et al., 1983; Mills and Johnson, 1985), another possibility is that metals interfere with junctional communication through their interaction with calmodulin. Alternatively, Davidson et al. (1984) showed that metabolic coupling requires extracellular calcium and magnesium, suggesting that certain metals may inhibit metabolic coupling by altering the extracellular matrix necessary to form cell contacts. Metals represent a significant toxicological concern, yet their mechanisms of action remain controversial. This report describes a cellular response to the toxic metals arsenic, mercury, and lead that has not been previously reported, inhibition of junction-mediated intercellular communication. While the responses observed here are weak relative to TPA, they are nonetheless significant at micromolar concentrations, and suggest that further work is warranted. Notably, evaluation of specific target cells of metal toxicity may reveal differences that offer further clues to their mechanism of action. Gap junctional communication has been associated with pituitary function, hormone responses, and parturition, to cite a few examples, all functions that may be adversely affected by metals. Determination of gap junctional responses in cells from specific, targeted tissues, such as pituitary, reproductive, and kidney, may allow more specific conclusions to be drawn concerning the importance of this cell response to the toxicity of metals. REFERENCES Aylsworth, C. F., Jone, C., Trosko, J. E., Meites, J., and Welsch, C. W. 1984. Promotion of 7,12dimethylbenz[a]anthracene-induced mammary tumorigenesis by high dietary fat in the rat: Possible role of intercellular communication. JNCI 72:637-645. Chao, S. H., Suzuki, Y., Zysk, J. R., and Cheung, W. Y. 1984. Activation of calmodulin by various metal cations as a function of ionic radius. Mol. Pharmacol. 26:75-82. Chen, T. R. 1977. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104:255-262. Cole, W. C., and Garfield, R. E. 1985. Alterations in coupling in uterine smooth muscle. In Cap Junctions, eds. M. L. V. Bennett and D. Spray, pp. 215-230. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Cox, J. A., and Harrison, S. D., Jr. 1983. Correlation of metal toxicity with in vitro calmodulin inhibition. Biochem. Biophys. Res. Commun. 115:106-111. Davidson, J. S., Baumgarten, I. M., and Harley, E. H. 1984. Effects of extracellular calcium and magnesium on junctional intercellular communication in human fibroblasts. Exp. Cell Res. 155:406-412. Dekel, N., Lawrence, T. S., Gilula, N. B., and Beers, W. H. 1981. Modulation of cell-to-cell communication in the cumulus-oocyte complex and the regulation of oocyte maturation by LH. Dev. Biol. 86:356-362. DeMello, W. C. 1984. Modulation of junctional permeability. Fed. Proc. 43:2692-2696.
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