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The effects of sulfhydryl blockers and metal ions on nickel accumulation by rat primary hepatocyte cultures
Toxicology Letters 118 (2000) 87 – 92 www.elsevier.com/locate/toxlet
The effects of sulfhydryl blockers and metal ions on nickel accumulation by rat primary hepatocyte cultures Hideaki Shimada a,*, Takayuki Funakoshi b, Takeshi Inoue a, Shoji Kojima a a
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto Uni6ersity, 5 -1 Oe-honmachi, Kumamoto 862 -0973, Japan b Kyushu Uni6ersity of Nursing and Social Welfare, 888 Tomio, Tamana, Kumamoto 865 -0062, Japan Received 27 July 2000; received in revised form 8 September 2000; accepted 11 September 2000
Abstract Previously, we found that nickel (Ni) accumulation by rat hepatocytes involves Ca channel transport processes. However, other mechanisms responsible for Ni accumulation are still unclear. Therefore, in the present study we examined the effects of sulfhydryl (SH) blockers on Ni accumulation by hepatocytes. Hepatocytes were exposed to various concentrations of N-ethylmaleimide (NEM) (0.5, 1 or 2 mM) or monoiodoacetic acid (MIA) (0.1, 0.25 or 0.5 mM), potent blockers of SH ligands, for 30 min and subsequently exposed to 10 mM NiCl2 for an additional 60 min. Pretreatment with NEM and MIA enhanced the Ni accumulation by hepatocytes to maximum of 156 and 73%, respectively. The effects of essential and nonessential metal ions on Ni accumulation were also investigated. Pretreatment with 10 mM of Cu, Zn, Co, Cd and Mn, decreased Ni accumulation by 46, 30, 20, 18 and 11%, respectively. In contrast, pretreatment with Hg (10 and 20 mM) enhanced the Ni accumulation by almost 81 and 140%, respectively. Furthermore, significant decrease in SH concentration in the hepatocyte membrane was observed by the treatment with NEM, MIA and Hg, but not with Cu, Zn and Cd. These results suggest that Ni accumulation by hepatocytes does not appear to be dependent on the SH carrier-mediated transport processes, and that to block the SH ligands in the plasma membrane may facilitate the Ni crossing of the cell membrane. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nickel accumulation; SH blockers; Hepatocytes; Metal ions
1. Introduction Nickel (Ni) is a very important environmental and industrial pollutant. The toxicity and carcino* Corresponding author. Tel.: +81-96-3714335; fax: + 8196-3714639. E-mail address: [email protected] (H. Shimada).
genicity of Ni compounds in experimental animals and humans have been well documented (Coogan et al., 1989; Sunderman, 1989). Exposure to Ni can result in damage to various tissues, including liver, kidney, lung and testes (Sunderman et al., 1985; Coogan et al., 1989; Xie et al., 1995, 1996). Parenteral administration of Ni in rats causes acute hepatotoxicity, including microvesicular
0378-4274/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 0 ) 0 0 2 6 8 - X
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fatty metamorphosis, hydropic degeneration, and foci inflammation (Donskoy et al., 1986). The ability of Ni to enter target cells appears to be a major determinant of the toxic effects of the metal (Costa et al., 1981; Costa and Heck, 1984). It is therefore of importance to define the mechanism of Ni accumulation by the cells. The available information indicates that in bullfrog ventricle strips and molluscan smooth muscles, Ni can cross the membrane through Ca channels and competes with Ca for specific receptors (Wang et al., 1984; Brommundt and Kavaler, 1987; Kavaler and Brommundt, 1987; Raffa et al., 1987). Abbracchio et al. (1982) reported that treatment of Chinese hamster ovary cells with Ni at 4°C resulted in 50% decrease of Ni accumulation compared to the control cells treated at 37°C, suggesting Ni accumulation occurs through energy-dependent processes. Previously, we reported that Ni accumulation by rat hepatocytes was time- and concentration-dependent processes, and involves Ca channel transport processes (Funakoshi et al., 1997). It has been reported that accumulation of Cd and Zn by rat hepatocytes occurs through SH carrier-mediated transport processes since SH blockers reduced the accumulation of both the metals (Failla and Cousins, 1978; Failla et al., 1979; Gerson and Shaikh, 1984). In contrast to Cd and Zn, Hg accumulation by the hepatocytes did not inhibited by the SH blocker, suggesting the accumulation mechanism for these metals might be different in the hepatocytes (Gerson and Shaikh, 1984). Therefore, in the present study we examined the effects of SH-blockers, N-ethylmaleimide (NEM) and monoiodoacetic acid (MIA), on Ni accumulation by rat primary hepatocyte cultures. The effects of essential metals such as Cu, Zn, Mn and Co, and non-essential toxic metals such as Cd and Hg, on Ni accumulation were also investigated.
2. Materials and methods
2.1. Chemicals Nickel chloride (NiCl2·6H2O), N-ethylmale-
imide (NEM), and monoiodoacetic acid (MIA) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Radioisotopic nickel (63Ni) was purchased from New England Nuclear Co. (Boston, MA). Other chemicals were of reagent grade.
2.2. Rat hepatocyte cultures The hepatocytes were isolated from male Wistar rats (Kyudo Co., Ltd, Kumamoto, Japan), weighing 150–200 g, by the method of Berry and Friend (1969). Cell viability, determined by the trypan blue exclusion method, was greater than 95%. Primary cultures of hepatocytes were established by seeding 7× 105 cells onto six well plates. The cells were cultured in Eagle’s minimum essential medium (MEM) (Nissui Phamaceutical Co., Ltd, Tokyo, Japan) supplemented with 5% fetal bovine serum and 0.03% glutamine for 24 h before treatment. Cultures were maintained in a humidified atmosphere of 5% CO2/95% air at 37°C.
2.3. Determination of Ni accumulation A total of 24 h after hepatocyte isolation, cells were washed with Hank’s balanced salt solution (HBSS) (Sigma Chemical Co., St. Louis, MO) and then treated with 10 mM NiCl2 (4 kBq 63Ni/well) in Ca- and Mg-free HBSS for up to 60 min. After the treatment, the cells were washed three times with PBS containing 2 mM O,O%-bis (2aminoethyl) ethyleneglycol-N,N,N%,N%-tetraacetic acid (EGTA) to remove free and loosely bound metal. The washed cells were scraped to remove them from the plate and suspended in 1 ml of 1 N NaOH to dissolve the cells. Then an aliquot (100 ml) of the solution was added into 1 ml of liquid scintillator (ACS II, Amersham International plc, England). Cellular 63Ni radioactivity were measured with an Aloka liquid scintillation counter (model LCS-3500). Results were normalized to protein content by the method of Lowry et al. (1951).
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2.4. Effects of SH blockers on Ni accumulation The cells were pretreated with NEM (0.5, 1 and 2 mM) or MIA (0.1, 0.25 and 0.5 mM) for 30 min. The amount of Ni accumulation was determined after 60 min incubation with 10 mM 63 NiCl2.
2.5. Effects of metal ions on Ni accumulation The cells were pretreated with 10 mM CuCl2, ZnCl2, MnCl2 or CoCl2, or 3 and 10 mM CdCl2, or 3, 10 and 20 mM HgCl2 for 30 min. The amount of Ni accumulation was determined after 60 min incubation with 10 mM 63NiCl2.
2.6. Determination of SH concentration in hepatocyte membrane The cells were harvested as described above. The cell membrane was obtained by the method of Blazka and Shaikh (1992). Protein-bound and nonprotein-bound SH concentrations were determined by the method of Sedlak and Lindsay (1968).
2.7. Statistical analysis Data were analyzed by a one-way analysis of variance. When the analysis indicated significant difference, the treated groups were compared to the controls by Duncan’s new multiple range test (P B 0.05).
Fig. 1. Time course of Ni accumulation by hepatocytes. Hepatocytes were treated with 10 mM NiCl2 for up to 60 min. The data represent the mean 9S.D. for three independent cell preparations.
3.2. Effects of SH blockers on Ni accumulation To determine the role of SH ligands in the hepatocytes on Ni accumulation, the effects of SH blockers were studied (Fig. 2). Pretreatment with either NEM or MIA, potent blocker of SH ligands, significantly increased Ni accumulation in a dose-dependent manner. A maximal increase of Ni accumulation with NEM (156%) was observed at the 2 mM and with MIA (73%) at the 0.5 mM.
3. Results
3.1. Time course of Ni accumulation The Ni accumulation by hepatocytes during the course of 60 min was studied in serum-free medium containing 10 mM Ni (Fig. 1). The Ni accumulation was rapid during first 15 min and continued at a slower rate for the remainder of the 45 min observation period. The Ni was not cytotoxic in hepatocytes even at doses up to 50 mM, as assessed by the tetrazolium-based dye (MTT) assay (data not shown).
Fig. 2. Effects of NEM and MIA on Ni accumulation by hepatocytes. Hepatocytes were pretreated with NEM (0.5, 1 and 2 mM) or MIA (0.1, 0.25 and 0.5 mM) for 30 min, then treated with NiCl2 (10 mM) for an additional 60 min. The data represent the mean 9S.D. (n =3) and asterisk indicates a significant difference from control (P B0.05).
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Fig. 3. Effects of Cu, Zn, Co and Mn on Ni accumulation by hepatocytes. Hepatocytes were pretreated with 10 mM CuCl2, ZnCl2, CoCl2 or MnCl2 for 30 min, then treated with NiCl2 (10 mM) for additional 60 min. The data represent the mean9 S.D. (n =3) and asterisk indicates a significant difference from control (P B 0.05).
Fig. 4. Effects of Cd and Hg on Ni accumulation by hepatocytes. Hepatocytes were pretreated with 3 and 10 mM CdCl2 or 3, 10 and 20 mM HgCl2 for 30 min, then treated with NiCl2 (10 mM) for an additional 60 min. The data represent the mean9 S.D. (n = 3) and asterisk indicates a significant difference from control (PB0.05).
3.3. Effects of metal ions on Ni accumulation
3.4. Effects of SH blockers and metal ions on SH concentration in hepatocyte membrane
The effects of essential metals, Cu, Zn, Co and Mn, on Ni accumulation are shown in Fig. 3. No toxicity was observed in the presence of 10 mM each metal after incubation for 30 min as determined by the MTT assay (data not shown). Significant decrease in the Ni accumulation was observed in the cells pretreated with all the metals. Among the metals, Cu was the most effective inhibitor, causing about 46% inhibition. The effects of nonessential toxic metals, Cd and Hg, on Ni accumulation were also investigated (Fig. 4). No toxicity was observed in the presence of 20 mM Hg after incubation for 30 min, however, Cd concentration greater than 10 mM caused cytotoxic (data not shown). Pretreatment with Cd at the concentration of 10 mM significantly decreased Ni accumulation by approximately 15%. In contrast, pretreatment with Hg at the concentrations of 10 and 20 mM resulted in marked increase in Ni accumulation by almost 81 and 140%, respectively.
Table 1 shows the effects of NEM and MIA on SH concentration in hepatocyte membrane. Treatment of NEM (2 mM) significantly decreased the protein-bound SH concentration by approxiTable 1 Effects of SH blockers on SH concentration in hepatocyte membrane Treatmenta
Control NEM MIA a
SH concentration (nmol/mg protein)b Total SH
Protein-bound Nonprotein SH SH
32.06 9 3.25 15.79 90.62c 19.33 90.03c
27.48 9 2.70 11.29 90.42c 16.37 9 0.06c
4.57 9 0.12 4.51 9 0.21 2.96 9 0.07c
Hepatocytes were treated with NEM (2 mM) or MIA (0.5 mM) for 30 min. b The data represent the mean 9 S.D. for three independent cell preparations. c Significantly different from control (PB0.05).
H. Shimada et al. / Toxicology Letters 118 (2000) 87–92 Table 2 Effects of heavy metals on SH concentration in hepatocyte membrane Treatmenta
Control Cu Zn Cd Hg
SH concentration (nmol/mg protein)b Total SH
Protein-bound Nonprotein SH SH
30.87 9 0.41 29.07 9 0.45 31.64 90.76 30.28 90.99 18.53 90.11c
26.759 0.14 24.269 0.27 27.94 90.52 25.88 90.47 13.909 0.14c
4.129 0.55 4.81 90.18 3.7090.25 4.409 0.52 4.6390.14
a Hepatocytes were treated with 10 mM Cu, Zn, Cd or Hg for 30 min. b The data represent the mean 9 S.D. for three independent cell preparations. c Significantly different from control (PB0.05).
mately 59%, while MIA (0.5 mM) significantly decreased both the protein-bound and nonprotein SH concentrations by almost 40 and 35%, respectively. The effects of Cu, Zn, Cd and Hg on the SH concentration are shown in Table 2. Treatment of Hg significantly decreased the proteinbound SH concentration by almost 48%. However, other metal ions did not cause decrease in SH concentration in hepatocyte membrane.
4. Discussion The results of the present study demonstrate that Ni accumulation by hepatocytes does not appear to be dependent on the SH-mediated transport processes. It has been reported that accumulations of divalent heavy metals such as Cd and Zn, by rat hepatocytes were involved in carrier-mediated processes (Stacey and Klaassen, 1980, 1981). In fact, Cd and Zn accumulations by hepatocytes were significantly decreased by the treatment of SH blockers, indicating SH-mediated transport processes involved the accumulation (Failla and Cousins, 1978; Blazka and Shaikh, 1992). Contrary to these metals, in the present study the treatment of SH blockers did not decrease the Ni accumulation by hepatocytes. These findings suggest that Ni accumulation by hepatocytes does not involve the SH carrier-mediated
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transport processes unlike other heavy metals such as Cd and Zn. In the present study, the treatment of SH blockers, NEM and MIA, enhanced unexpectedly the Ni accumulation to maximum of 156 and 73%, respectively. Similarly, the treatment of Hg enhanced the Ni accumulation to a maximum of 140%. It is well known that Hg can bind tightly to SH ligands (Jacobson and Turner, 1980). In fact, our present data showed the treatment of Hg decreased the SH concentrations in the plasma membrane as well as the SH blockers. These results suggest that both Hg and SH blockers might enhance the Ni accumulation by the same mechanism. For the possible mechanism, it can be speculated that SH ligands in the plasma membrane may interfere in the Ni crossing of the cell membrane. However, the precise mechanisms involved this enhanced response will require further study to define. Several studies have shown that Cd competes with Ca, Cu and Zn for common uptake pathways (Stacey and Klaassen, 1980, 1981; Ettinger et al., 1986; Blazka and Shaikh, 1991, 1992). Recently, our laboratory has shown that Ca channels are involved in the Ni accumulation by hepatocytes (Funakoshi et al., 1997). Hughes and Barritt (1989) reported that Ca channels are inhibited by many metal ions such as Zn, Cd, Mn and Ni. This is supported by our findings that Ni accumulation by hepatocytes was decreased by Cu, Zn, Co, Mn and Cd. Thus, reduced Ni accumulation in the presence of Cu, Zn, Co, Mn and Cd may be due to competition or inhibition for Ca channels. Among the divalent metals tested, Cu was the most effective at decreasing in the Ni accumulation (46% decrease), suggesting that Ni accumulation may occur by a processes associated with Cu uptake. It has been reported that transport processes involving membrane carriers are temperature-dependent (West, 1983). In our previous study, Ni accumulation by hepatocytes was decreased by low temperature with a 20% decrease (Funakoshi et al., 1997). Furthermore, Ca channel blockers decreased the Ni accumulation with a maximum inhibition of 20% (Funakoshi et al., 1997). In this case, the percentage of the inhibitory effects of Ca
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channel blockers on Ni accumulation was actually the same as that of low temperature, indicating that inhibition by low temperature may correspond to inhibition of the Ca channels by the blockers (Funakoshi et al., 1997). In the present study, the treatment of SH blockers did not inhibit the Ni accumulation. In combination with present results, these studies suggest that a majority of Ni accumulation by hepatocytes might occur through passive diffusion. In conclusion, the results of the present study indicate that SH carrier-mediated transport processes may be unimportant in Ni accumulation by hepatocytes. Further study will be required to assess what other mechanisms are involved. References Abbracchio, M.P., Evans, R.M., Heck, J.D., Cantoni, O., Costa, M., 1982. The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components. Biol. Trace Element Res. 4, 289–301. Berry, M.N., Friend, D.S., 1969. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell Biol. 43, 506–520. Blazka, M.E., Shaikh, Z.A., 1991. Differences in the uptake of cadmium and mercury by rat hepatocytes primary cultures: role of calcium channels. Toxicol. Appl. Pharmacol. 110, 355 – 363. Blazka, M.E., Shaikh, Z.A., 1992. Cadmium and mercury accumulation in rat hepatocytes: interactions with other metal ions. Toxicol. Appl. Pharmacol. 113, 118–125. Brommundt, G., Kavaler, F., 1987. La3 + , Mn2 + and Ni2 + effects on Ca2 + pump and on Na+-Ca2 + exchange in bullfrog ventricle. Am. J. Physiol. 253, C45–C51. Coogan, T.P., Latta, D.M., Snow, E.T., Costa, M., 1989. Toxicity and carcinogenicity of nickel compounds. CRC Crit. Rev. Toxicol. 19 (4), 341–384. Costa, M., Heck, J.D., 1984. Perspectives on the mechanism of nickel carcinogenesis. In: Eichorn, G.L., Marzilli, L.G. (Eds.), Advances in Inorganic Biochemistry, vol. 6. Elsevier, New York, pp. 285–309. Costa, M., Simmons-Hansen, J., Bedrossian, C.W.M., Bonura, J., Caprioli, R.M., 1981. Phagocytosis, cellular distribution and carcinogenic activity of particulate nickel compounds in tissue culture. Cancer Res. 41, 2868–2876. Donskoy, E., Donskoy, M., Faripour, F., Gillies, C.G., Marzouk, A., Reid, M.C., Zaharia, O., Sunderman, F.W., Jr, 1986. Hepatic toxicity of nickel chloride in rats. Ann. Clin. Lab. Sci. 16, 108 – 117. Ettinger, M.J., Darwish, H.M., Schmitt, R.C., 1986. Mechanisms of copper transport from plasma to hepatocytes. Fed. Proc. 45, 2800 – 2804.
Failla, M.L., Cousins, R.J., 1978. Zinc uptake by isolated rat liver parenchymal cells. Biochim. Biophys. Acta 538, 435 – 444. Failla, M.L., Cousins, R.J., Mascenik, M.J., 1979. Cadmium accumulation and metabolism by rat liver parenchymal cells in primary monolayer culture. Biochim. Biophys. Acta 583, 63 – 72. Funakoshi, T., Inoue, T., Shimada, H., Kojima, S., 1997. The mechanisms of nickel uptake by rat primary hepatocyte cultures: role of calcium channels. Toxicology 124, 21 – 26. Gerson, R.J., Shaikh, Z.A., 1984. Differences in the uptake of cadmium and mercury by rat hepatocyte primary cultures. Biochem. Pharmacol. 33, 199 – 203. Hughes, B.P., Barritt, G.J., 1989. Inhibition of the liver cell receptor-activated Ca2 + inflow system by metal ion inhibitors of voltage-operated Ca2 + channels but not other inhibitors of Ca2 + inflow. Biochim. Biophys. Acta 1013, 197 – 205. Jacobson, K.B., Turner, J.E., 1980. The international of cadmium and certain other metal ions with proteins and nucleic acids. Toxicology 16, 1 – 37. Kavaler, F., Brommundt, G., 1987. Potentiation of contraction in bullfrog ventricle strips by manganese and nickel. Am. J. Physiol. 253, C52 – C59. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 19, 265 – 275. Raffa, R.B., Bianchi, P., Narayan, S.R., 1987. Reversible inhibition of acetylcholine contracture of molluscan smooth muscle by heavy metals: correlation to Ca++ and metal content. J. Pharmacol. Exp. Ther. 243, 200 – 204. Sedlak, J., Lindsay, R.H., 1968. Estimation of total, proteinbound, and nonprotein sulfhydril groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192 – 205. Stacey, N.H., Klaassen, C.D., 1980. Cadmium uptake by isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 55, 448 – 455. Stacey, N.H., Klaassen, C.D., 1981. Zinc uptake by isolated rat hepatocytes. Biochim. Biophys. Acta 640, 693 – 697. Sunderman, F.W., Jr, Zaharia, O., Marzouk, A., Reid, M.C., 1985. Increased lipid peroxidation in liver, kidney and lung of NiCl2-treated rats. In: Brown, S.S., Sunderman, F.W., Jr (Eds.), Progress in Nickel Toxicology. Blackwell, Oxford, pp. 93 – 96. Sunderman, F.W., Jr., 1989. Mechanisms of nickel carcinogenesis. Scand. J. Work Environ. Health 15, 1 – 12. Wang, Z., Bianchi, C.P., Narayan, S.R., 1984. Nickel inhibition of calcium release from subsarcolemmal calcium stores of molluscan smooth muscle. J. Pharmacol. Exp. Ther. 229, 696 – 701. West, I.C., 1983. The Biochemistry of Membrane Transport. Chapman & Hall, London. Xie, J., Funakoshi, T., Shimada, H., Kojima, S., 1995. Effects of chelating agents on testicular toxicity in mice caused by acute exposure to nickel. Toxicology 103, 147 – 155. Xie, J., Funakoshi, T., Shimada, H., Kojima, S., 1996. Comparative effects of chelating agents on pulmonary toxicity of systemic nickel in mice. J. Appl. Toxicol. 16, 317 – 324.
The effects of sulfhydryl blockers and metal ions on nickel accumulation by rat primary hepatocyte cultures Hideaki Shimada a,*, Takayuki Funakoshi b, Takeshi Inoue a, Shoji Kojima a a
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Kumamoto Uni6ersity, 5 -1 Oe-honmachi, Kumamoto 862 -0973, Japan b Kyushu Uni6ersity of Nursing and Social Welfare, 888 Tomio, Tamana, Kumamoto 865 -0062, Japan Received 27 July 2000; received in revised form 8 September 2000; accepted 11 September 2000
Abstract Previously, we found that nickel (Ni) accumulation by rat hepatocytes involves Ca channel transport processes. However, other mechanisms responsible for Ni accumulation are still unclear. Therefore, in the present study we examined the effects of sulfhydryl (SH) blockers on Ni accumulation by hepatocytes. Hepatocytes were exposed to various concentrations of N-ethylmaleimide (NEM) (0.5, 1 or 2 mM) or monoiodoacetic acid (MIA) (0.1, 0.25 or 0.5 mM), potent blockers of SH ligands, for 30 min and subsequently exposed to 10 mM NiCl2 for an additional 60 min. Pretreatment with NEM and MIA enhanced the Ni accumulation by hepatocytes to maximum of 156 and 73%, respectively. The effects of essential and nonessential metal ions on Ni accumulation were also investigated. Pretreatment with 10 mM of Cu, Zn, Co, Cd and Mn, decreased Ni accumulation by 46, 30, 20, 18 and 11%, respectively. In contrast, pretreatment with Hg (10 and 20 mM) enhanced the Ni accumulation by almost 81 and 140%, respectively. Furthermore, significant decrease in SH concentration in the hepatocyte membrane was observed by the treatment with NEM, MIA and Hg, but not with Cu, Zn and Cd. These results suggest that Ni accumulation by hepatocytes does not appear to be dependent on the SH carrier-mediated transport processes, and that to block the SH ligands in the plasma membrane may facilitate the Ni crossing of the cell membrane. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nickel accumulation; SH blockers; Hepatocytes; Metal ions
1. Introduction Nickel (Ni) is a very important environmental and industrial pollutant. The toxicity and carcino* Corresponding author. Tel.: +81-96-3714335; fax: + 8196-3714639. E-mail address: [email protected] (H. Shimada).
genicity of Ni compounds in experimental animals and humans have been well documented (Coogan et al., 1989; Sunderman, 1989). Exposure to Ni can result in damage to various tissues, including liver, kidney, lung and testes (Sunderman et al., 1985; Coogan et al., 1989; Xie et al., 1995, 1996). Parenteral administration of Ni in rats causes acute hepatotoxicity, including microvesicular
0378-4274/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 0 ) 0 0 2 6 8 - X
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fatty metamorphosis, hydropic degeneration, and foci inflammation (Donskoy et al., 1986). The ability of Ni to enter target cells appears to be a major determinant of the toxic effects of the metal (Costa et al., 1981; Costa and Heck, 1984). It is therefore of importance to define the mechanism of Ni accumulation by the cells. The available information indicates that in bullfrog ventricle strips and molluscan smooth muscles, Ni can cross the membrane through Ca channels and competes with Ca for specific receptors (Wang et al., 1984; Brommundt and Kavaler, 1987; Kavaler and Brommundt, 1987; Raffa et al., 1987). Abbracchio et al. (1982) reported that treatment of Chinese hamster ovary cells with Ni at 4°C resulted in 50% decrease of Ni accumulation compared to the control cells treated at 37°C, suggesting Ni accumulation occurs through energy-dependent processes. Previously, we reported that Ni accumulation by rat hepatocytes was time- and concentration-dependent processes, and involves Ca channel transport processes (Funakoshi et al., 1997). It has been reported that accumulation of Cd and Zn by rat hepatocytes occurs through SH carrier-mediated transport processes since SH blockers reduced the accumulation of both the metals (Failla and Cousins, 1978; Failla et al., 1979; Gerson and Shaikh, 1984). In contrast to Cd and Zn, Hg accumulation by the hepatocytes did not inhibited by the SH blocker, suggesting the accumulation mechanism for these metals might be different in the hepatocytes (Gerson and Shaikh, 1984). Therefore, in the present study we examined the effects of SH-blockers, N-ethylmaleimide (NEM) and monoiodoacetic acid (MIA), on Ni accumulation by rat primary hepatocyte cultures. The effects of essential metals such as Cu, Zn, Mn and Co, and non-essential toxic metals such as Cd and Hg, on Ni accumulation were also investigated.
2. Materials and methods
2.1. Chemicals Nickel chloride (NiCl2·6H2O), N-ethylmale-
imide (NEM), and monoiodoacetic acid (MIA) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Radioisotopic nickel (63Ni) was purchased from New England Nuclear Co. (Boston, MA). Other chemicals were of reagent grade.
2.2. Rat hepatocyte cultures The hepatocytes were isolated from male Wistar rats (Kyudo Co., Ltd, Kumamoto, Japan), weighing 150–200 g, by the method of Berry and Friend (1969). Cell viability, determined by the trypan blue exclusion method, was greater than 95%. Primary cultures of hepatocytes were established by seeding 7× 105 cells onto six well plates. The cells were cultured in Eagle’s minimum essential medium (MEM) (Nissui Phamaceutical Co., Ltd, Tokyo, Japan) supplemented with 5% fetal bovine serum and 0.03% glutamine for 24 h before treatment. Cultures were maintained in a humidified atmosphere of 5% CO2/95% air at 37°C.
2.3. Determination of Ni accumulation A total of 24 h after hepatocyte isolation, cells were washed with Hank’s balanced salt solution (HBSS) (Sigma Chemical Co., St. Louis, MO) and then treated with 10 mM NiCl2 (4 kBq 63Ni/well) in Ca- and Mg-free HBSS for up to 60 min. After the treatment, the cells were washed three times with PBS containing 2 mM O,O%-bis (2aminoethyl) ethyleneglycol-N,N,N%,N%-tetraacetic acid (EGTA) to remove free and loosely bound metal. The washed cells were scraped to remove them from the plate and suspended in 1 ml of 1 N NaOH to dissolve the cells. Then an aliquot (100 ml) of the solution was added into 1 ml of liquid scintillator (ACS II, Amersham International plc, England). Cellular 63Ni radioactivity were measured with an Aloka liquid scintillation counter (model LCS-3500). Results were normalized to protein content by the method of Lowry et al. (1951).
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2.4. Effects of SH blockers on Ni accumulation The cells were pretreated with NEM (0.5, 1 and 2 mM) or MIA (0.1, 0.25 and 0.5 mM) for 30 min. The amount of Ni accumulation was determined after 60 min incubation with 10 mM 63 NiCl2.
2.5. Effects of metal ions on Ni accumulation The cells were pretreated with 10 mM CuCl2, ZnCl2, MnCl2 or CoCl2, or 3 and 10 mM CdCl2, or 3, 10 and 20 mM HgCl2 for 30 min. The amount of Ni accumulation was determined after 60 min incubation with 10 mM 63NiCl2.
2.6. Determination of SH concentration in hepatocyte membrane The cells were harvested as described above. The cell membrane was obtained by the method of Blazka and Shaikh (1992). Protein-bound and nonprotein-bound SH concentrations were determined by the method of Sedlak and Lindsay (1968).
2.7. Statistical analysis Data were analyzed by a one-way analysis of variance. When the analysis indicated significant difference, the treated groups were compared to the controls by Duncan’s new multiple range test (P B 0.05).
Fig. 1. Time course of Ni accumulation by hepatocytes. Hepatocytes were treated with 10 mM NiCl2 for up to 60 min. The data represent the mean 9S.D. for three independent cell preparations.
3.2. Effects of SH blockers on Ni accumulation To determine the role of SH ligands in the hepatocytes on Ni accumulation, the effects of SH blockers were studied (Fig. 2). Pretreatment with either NEM or MIA, potent blocker of SH ligands, significantly increased Ni accumulation in a dose-dependent manner. A maximal increase of Ni accumulation with NEM (156%) was observed at the 2 mM and with MIA (73%) at the 0.5 mM.
3. Results
3.1. Time course of Ni accumulation The Ni accumulation by hepatocytes during the course of 60 min was studied in serum-free medium containing 10 mM Ni (Fig. 1). The Ni accumulation was rapid during first 15 min and continued at a slower rate for the remainder of the 45 min observation period. The Ni was not cytotoxic in hepatocytes even at doses up to 50 mM, as assessed by the tetrazolium-based dye (MTT) assay (data not shown).
Fig. 2. Effects of NEM and MIA on Ni accumulation by hepatocytes. Hepatocytes were pretreated with NEM (0.5, 1 and 2 mM) or MIA (0.1, 0.25 and 0.5 mM) for 30 min, then treated with NiCl2 (10 mM) for an additional 60 min. The data represent the mean 9S.D. (n =3) and asterisk indicates a significant difference from control (P B0.05).
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Fig. 3. Effects of Cu, Zn, Co and Mn on Ni accumulation by hepatocytes. Hepatocytes were pretreated with 10 mM CuCl2, ZnCl2, CoCl2 or MnCl2 for 30 min, then treated with NiCl2 (10 mM) for additional 60 min. The data represent the mean9 S.D. (n =3) and asterisk indicates a significant difference from control (P B 0.05).
Fig. 4. Effects of Cd and Hg on Ni accumulation by hepatocytes. Hepatocytes were pretreated with 3 and 10 mM CdCl2 or 3, 10 and 20 mM HgCl2 for 30 min, then treated with NiCl2 (10 mM) for an additional 60 min. The data represent the mean9 S.D. (n = 3) and asterisk indicates a significant difference from control (PB0.05).
3.3. Effects of metal ions on Ni accumulation
3.4. Effects of SH blockers and metal ions on SH concentration in hepatocyte membrane
The effects of essential metals, Cu, Zn, Co and Mn, on Ni accumulation are shown in Fig. 3. No toxicity was observed in the presence of 10 mM each metal after incubation for 30 min as determined by the MTT assay (data not shown). Significant decrease in the Ni accumulation was observed in the cells pretreated with all the metals. Among the metals, Cu was the most effective inhibitor, causing about 46% inhibition. The effects of nonessential toxic metals, Cd and Hg, on Ni accumulation were also investigated (Fig. 4). No toxicity was observed in the presence of 20 mM Hg after incubation for 30 min, however, Cd concentration greater than 10 mM caused cytotoxic (data not shown). Pretreatment with Cd at the concentration of 10 mM significantly decreased Ni accumulation by approximately 15%. In contrast, pretreatment with Hg at the concentrations of 10 and 20 mM resulted in marked increase in Ni accumulation by almost 81 and 140%, respectively.
Table 1 shows the effects of NEM and MIA on SH concentration in hepatocyte membrane. Treatment of NEM (2 mM) significantly decreased the protein-bound SH concentration by approxiTable 1 Effects of SH blockers on SH concentration in hepatocyte membrane Treatmenta
Control NEM MIA a
SH concentration (nmol/mg protein)b Total SH
Protein-bound Nonprotein SH SH
32.06 9 3.25 15.79 90.62c 19.33 90.03c
27.48 9 2.70 11.29 90.42c 16.37 9 0.06c
4.57 9 0.12 4.51 9 0.21 2.96 9 0.07c
Hepatocytes were treated with NEM (2 mM) or MIA (0.5 mM) for 30 min. b The data represent the mean 9 S.D. for three independent cell preparations. c Significantly different from control (PB0.05).
H. Shimada et al. / Toxicology Letters 118 (2000) 87–92 Table 2 Effects of heavy metals on SH concentration in hepatocyte membrane Treatmenta
Control Cu Zn Cd Hg
SH concentration (nmol/mg protein)b Total SH
Protein-bound Nonprotein SH SH
30.87 9 0.41 29.07 9 0.45 31.64 90.76 30.28 90.99 18.53 90.11c
26.759 0.14 24.269 0.27 27.94 90.52 25.88 90.47 13.909 0.14c
4.129 0.55 4.81 90.18 3.7090.25 4.409 0.52 4.6390.14
a Hepatocytes were treated with 10 mM Cu, Zn, Cd or Hg for 30 min. b The data represent the mean 9 S.D. for three independent cell preparations. c Significantly different from control (PB0.05).
mately 59%, while MIA (0.5 mM) significantly decreased both the protein-bound and nonprotein SH concentrations by almost 40 and 35%, respectively. The effects of Cu, Zn, Cd and Hg on the SH concentration are shown in Table 2. Treatment of Hg significantly decreased the proteinbound SH concentration by almost 48%. However, other metal ions did not cause decrease in SH concentration in hepatocyte membrane.
4. Discussion The results of the present study demonstrate that Ni accumulation by hepatocytes does not appear to be dependent on the SH-mediated transport processes. It has been reported that accumulations of divalent heavy metals such as Cd and Zn, by rat hepatocytes were involved in carrier-mediated processes (Stacey and Klaassen, 1980, 1981). In fact, Cd and Zn accumulations by hepatocytes were significantly decreased by the treatment of SH blockers, indicating SH-mediated transport processes involved the accumulation (Failla and Cousins, 1978; Blazka and Shaikh, 1992). Contrary to these metals, in the present study the treatment of SH blockers did not decrease the Ni accumulation by hepatocytes. These findings suggest that Ni accumulation by hepatocytes does not involve the SH carrier-mediated
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transport processes unlike other heavy metals such as Cd and Zn. In the present study, the treatment of SH blockers, NEM and MIA, enhanced unexpectedly the Ni accumulation to maximum of 156 and 73%, respectively. Similarly, the treatment of Hg enhanced the Ni accumulation to a maximum of 140%. It is well known that Hg can bind tightly to SH ligands (Jacobson and Turner, 1980). In fact, our present data showed the treatment of Hg decreased the SH concentrations in the plasma membrane as well as the SH blockers. These results suggest that both Hg and SH blockers might enhance the Ni accumulation by the same mechanism. For the possible mechanism, it can be speculated that SH ligands in the plasma membrane may interfere in the Ni crossing of the cell membrane. However, the precise mechanisms involved this enhanced response will require further study to define. Several studies have shown that Cd competes with Ca, Cu and Zn for common uptake pathways (Stacey and Klaassen, 1980, 1981; Ettinger et al., 1986; Blazka and Shaikh, 1991, 1992). Recently, our laboratory has shown that Ca channels are involved in the Ni accumulation by hepatocytes (Funakoshi et al., 1997). Hughes and Barritt (1989) reported that Ca channels are inhibited by many metal ions such as Zn, Cd, Mn and Ni. This is supported by our findings that Ni accumulation by hepatocytes was decreased by Cu, Zn, Co, Mn and Cd. Thus, reduced Ni accumulation in the presence of Cu, Zn, Co, Mn and Cd may be due to competition or inhibition for Ca channels. Among the divalent metals tested, Cu was the most effective at decreasing in the Ni accumulation (46% decrease), suggesting that Ni accumulation may occur by a processes associated with Cu uptake. It has been reported that transport processes involving membrane carriers are temperature-dependent (West, 1983). In our previous study, Ni accumulation by hepatocytes was decreased by low temperature with a 20% decrease (Funakoshi et al., 1997). Furthermore, Ca channel blockers decreased the Ni accumulation with a maximum inhibition of 20% (Funakoshi et al., 1997). In this case, the percentage of the inhibitory effects of Ca
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channel blockers on Ni accumulation was actually the same as that of low temperature, indicating that inhibition by low temperature may correspond to inhibition of the Ca channels by the blockers (Funakoshi et al., 1997). In the present study, the treatment of SH blockers did not inhibit the Ni accumulation. In combination with present results, these studies suggest that a majority of Ni accumulation by hepatocytes might occur through passive diffusion. In conclusion, the results of the present study indicate that SH carrier-mediated transport processes may be unimportant in Ni accumulation by hepatocytes. Further study will be required to assess what other mechanisms are involved. References Abbracchio, M.P., Evans, R.M., Heck, J.D., Cantoni, O., Costa, M., 1982. The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components. Biol. Trace Element Res. 4, 289–301. Berry, M.N., Friend, D.S., 1969. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J. Cell Biol. 43, 506–520. Blazka, M.E., Shaikh, Z.A., 1991. Differences in the uptake of cadmium and mercury by rat hepatocytes primary cultures: role of calcium channels. Toxicol. Appl. Pharmacol. 110, 355 – 363. Blazka, M.E., Shaikh, Z.A., 1992. Cadmium and mercury accumulation in rat hepatocytes: interactions with other metal ions. Toxicol. Appl. Pharmacol. 113, 118–125. Brommundt, G., Kavaler, F., 1987. La3 + , Mn2 + and Ni2 + effects on Ca2 + pump and on Na+-Ca2 + exchange in bullfrog ventricle. Am. J. Physiol. 253, C45–C51. Coogan, T.P., Latta, D.M., Snow, E.T., Costa, M., 1989. Toxicity and carcinogenicity of nickel compounds. CRC Crit. Rev. Toxicol. 19 (4), 341–384. Costa, M., Heck, J.D., 1984. Perspectives on the mechanism of nickel carcinogenesis. In: Eichorn, G.L., Marzilli, L.G. (Eds.), Advances in Inorganic Biochemistry, vol. 6. Elsevier, New York, pp. 285–309. Costa, M., Simmons-Hansen, J., Bedrossian, C.W.M., Bonura, J., Caprioli, R.M., 1981. Phagocytosis, cellular distribution and carcinogenic activity of particulate nickel compounds in tissue culture. Cancer Res. 41, 2868–2876. Donskoy, E., Donskoy, M., Faripour, F., Gillies, C.G., Marzouk, A., Reid, M.C., Zaharia, O., Sunderman, F.W., Jr, 1986. Hepatic toxicity of nickel chloride in rats. Ann. Clin. Lab. Sci. 16, 108 – 117. Ettinger, M.J., Darwish, H.M., Schmitt, R.C., 1986. Mechanisms of copper transport from plasma to hepatocytes. Fed. Proc. 45, 2800 – 2804.
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