Toxicology Letters, 60 (1992) 99-106 0 1992 Elsevier Science Publishers B.V. All rights reserved 0378-4274/92/$ 5.00
99
TOXLET 2682
Indium inhibits gap junctional communication between rat hepatocytes in primary culture
Xinbiao Guo, Yasuo Ohno, Toru Kawanishi, Momoko Sunouchi and Akira Takanaka Division of Pharmacology, National Institute of Hygienic Sciences, Tokyo (Japan)
(Received 5 August 1991) (Accepted 10 October 1991) Key words: Indium; Gap junction; Hepatocyte; Primary culture
SUMMARY The effect of indium on gap junctional communication was investigated in primary cultured rat hepatocytes. Treatment of hepatocytes with indium chloride at concentrations of 100,uM to 1 mM for 2 h resulted in dose-dependent inhibition of gap junctional communication between hepatocytes. The effect of indium on hepatocytes was also evaluated using two indices for cell viability: lactate dehydrogenase (LDH) leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetr~olium bromide (MTT) reduction. Indium did not cause any increase in LDH leakage from hepatocytes at the above concentrations, but inhibition of MTT reduction was observed at concentrations above 500 PM. These results suggest that the gap junctions between hepatocytes may be vulnerable sites to indium toxicity.
INTRODUCTION
Iridium, a group IIIa metal in the periodic table, is widely distributed in nature in very minute quantities. In industry, it has been used for surface protection of metals or alloys, and for the manufacture of graphite, glass, motion picture screens and cathode oscillographs [l]. In particular, new uses have been found in recent years for indium as a dopant material in the microelectronics industry [2]. In medicine, indium (i.e. “‘In) is administered complexed with diethylenetriamine pentaacetic acid (DTPA), oxine, tropolone or bleomycin to locate and identify malignant tumors [3]. Although there have been no reported cases of systemic effects in humans exposed to indium, studies in animals have shown that indium is one of the most toxic metals [l].
Addressfor correspondence: Yasuo Ohno, Ph.D., Division of Pharmacology, National Institute of Hygienic
Sciences, 18-1, Kamiyoga, 1-chome, Setagaya-Ku, Tokyo 158, Japan.
100
Furthermore, the results from these studies have suggested that the liver is the target organ of indium. It was reported that indium administration produced multiple areas of necrosis in liver parenchymal cells of mice, with no changes in the Kupffer cells or the biliary system [4]. In addition, the marked reduction in cytochrome P-450 levels and its dependent drug-metabolizing enzyme activities, the inhibition of heme synthesis and the acceleration of heme catabolism have been demonstrated in the livers of indium-treated rats [5,6]. In the present study, we used primary cultured rat hepatocytes to investigate whether indium could alter gap junctional communication, a membrane channelmediated communication which is important in the regulation of cell growth and differentiation, and in the homeostasis of tissues [7]. Gap junctional communication has been reported to be altered by many toxicants, including tumor promoters, hepatotoxic chemicals, and teratogens [8811]. Our study demonstrated that indium inhibited gap junctional communication between rat hepatocytes in a dose-dependent manner. In this study, we also examined the effects of indium on membrane integrity and mitochondrial function of hepatocytes using lactate dehydrogenase (LDD, EC 1.1.1 27) leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction as indices, respectively. Our result suggests that gap junctions between hepatocytes may be vulnerable sites to indium toxicity. MATERIALS
AND METHODS
Materials Leibovitz’s L-15 medium, penicillin and streptomycin were obtained from Flow Laboratories (Irvine, U.K.). Newborn calf serum was obtained from Cell Culture Laboratories (Cleveland, OH, U.S.A.). Collagenase was obtained from Boehringer Mannheim, GmbH (F.R.G.). Indium chloride was obtained from Wako Chemical Co. (Tokyo, Japan). Lucifer Yellow CH and MTT were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All other chemicals were of reagent grade and were obtained from Wako Chemical Co. or Sigma Chemical Co.. Animals Male Sprague-Dawley rats (200-250 g weight range) were purchased from Japan SLC, and maintained on a 12-h light/dark cycle in a controlled-environment animal facility. Animals were fed rat chow and tap water ad libitum. Preparation of hepatocyte cultures Hepatocytes were prepared by the method of Moldeus et al. [12]. Cells were used only when the viability was higher than 85% at the time of plating. Hepatocytes were suspended in Leibovitz’s L- 15 medium supplemented with 2 nM insulin, 1 PM dexamethasone, 8.3 mM glucose, 2 mM nicotinamide, 10 mM HEPES, 50 IU/ml penicillin, 50 pg/ml streptomycin and 10% newborn calf serum. Approximately 2 x lo5 cells were plated in 2 ml of medium into 35-mm collagen-coated tissue culture dishes
101
(Corning). Cultures were incubated at 37°C in a humidified atmosphere of air. At 3 h after plating, the medium was changed to remove the unattached cells and was renewed every 24 h thereafter. Assay of gap junctional communication between hepatocytes Gap junctional communication between rat hepatocytes was evaluated by microinjection of the fluorescent dye, Lucifer Yellow CH, into one hepatocyte and observation of dye spread to adjacent cells. Lucifer Yellow CH 5% (w/v) in 0.3 M lithium chloride was backfilled into a microelectrode, and microinjected into individual cells using a Narishige IM-1 8gTMmicroinjector. Five minutes later, dye coupling was observed at room temperature with a Nikon Diaphot microscope with xenon arc lamp illumination using a Nikon B filter block (excitation at 450490 nm; emission > 520 nm). Photographs were taken on FUJI HR 400 film. The extent of dye coupling was expressed as the number of dye-coupled cells as a percentage of total adjacent cells. To investigate the effect of indium on gap junctional communication between hepatocytes, 24-h-old cultures were refed with medium (2 ml/dish) and were treated for 2 h with indium chloride at different concentrations. Cultures receiving vehicle (saline) alone were used as controls. Assay for viability of hepatocytes After treatment of hepatocytes with indium chloride as described above, two assays for cellular viability were performed: (1) LDH leakage was determined as described previously [ 131.Viability was expressed as the amount of LDH activity released from cells as a percentage of total LDH activity in the cells. (2) MTT reduction was assayed using the method described by Carmichael et al. [14]. The assay is dependent on the cellular reduction of MTT by the mitochondrial dehydrogenase of viable cells to a blue formazan product. The amount of the reduced product from MTT was expressed as the absorbance at 560 nm. Statistical analyses ANOVA was used for comparison of variance among groups. When a significant (P < 0.05) effect was observed, each treatment group was compared with the untreated control group using Scheffe’s test [15]. RESULTS
The incidence of dye coupling in untreated rat hepatocytes during the period of culture is shown in Figure 1. The incidence of dye coupling at 4 h of culture was 79 + 8%. A marked decrease in dye coupling was observed over the first 24 h of culture. After that, the decrease became less, and the incidence of dye coupling changed from 47 f 3% to 39 * 4% over the next 24 h of culture. Figure 2 is a fluorescence micrograph which shows dye coupling between a rat hepatocyte microinjected with Lucifer Yellow CH and adjacent hepatocytes in 24-h-old cultures. In this experiment, the effect of indium on gap junctional communication between
102
I
0'
0
48
24
Time
in
Culture
(hours)
Fig. 1.The timeecourse of changes in dye coupling between untreated rat hepatocytes in primary culture. 45-453 adjacent hepatocytes were observed for each value. Results are expressed as the mean k SE.
hepatocytes was determined after treatment of 24-h-old hepatocytes with indium chloride for 2 h. Dye coupling between hepatocytes exposed to 10 ,uM indium chloride remained at the same levels as those in untreated cells. When hepatocytes were exposed to indium chloride at concentrations above 100 ,uM, a significant inhibition of dye coupling was observed. Dye coupling between hepatocytes exposed to 100 ,uM indium was decreased to 67% of that in untreated cells. Moreover, indium inhibited the dye coupling in a dose-dependent manner. After treatment with 1 mM indium, the dye coupling between hepatocytes was almost completely inhibited (Fig. 3).
Fig. 2. A fluorescence micrograph showing dye coupling between a rat hepatocyte (A) microinjected with Lucifer Yellow CH and adjacent hepatocytes in 24-h-old cultures.
103
=‘
120
kt 100 c
60
p
60
‘-
0" Ef
40
": CI
20 0 10
100
1000
500
lndium
(FM)
Fig. 3. The dose-depedent inhibition of dye coupling between rat hepatocytes by indium chloride. 24-h-old hepatocyte cultures were treated with indium chloride for 2 h. 3@48 adjacent recipient hepatocytes were observed for each value. Results are expressed as the mean ? SE.
The effect of indium on LDH leakage from hepatocytes is shown in Figure 4. Treatment of hepatocytes for 2 h with indium chloride at concentrations of 100 ,uM to 1 mM did not cause any increase in LDH leakage from cells. On the other hand, as shown in Figure 5, MTT reduction activity of hepatocytes showed no significant changes when indium was added at concentrations below 250 ,uM. However, a significant inhibition of MTT reduction activity was observed when hepatocytes were exposed to indium chloride at both 500 PM and 1 mM. DISCUSSION
Gap junctional communication has been said to be important in the regulation of cell growth and differentiation. The coordinated function of the liver as an organ also depends significantly on gap junctional communication between hepatocytes. The gap junction between hepatocytes may function to link together the volume of coupled cells, and thus to increase the sink or buffer capacity of the cytoplasm [16]. In 100
3
L
60
ab p
60
x ii
40
4
20
0 0
.I 00
250
500
lndium
(PM)
1000
Fig. 4. The effect of indium chloride on LDH leakage from rat hepatocytes. 24-h-old hepatocyte cultures were treated with indium chloride for 2 h. LDH leakage was measured as described in ‘Materials and Methods’. Values are expressed as the mean +_SE (n = 3).
104
0
10
50
lndium
100
500
lODO
(JIM)
Fig. 5. The effect of indium chloride on the MTT reduction activity of rat hepatocytes. 24-h-old hepatocyte cultures were treated with indium chloride for 2 h. MTT assay was performed as described in ‘Materials and Methods’. Values are expressed as the mean It SE (n = 9). *Significantly different from control: P < 0.05.
addition, the gap junction has been shown to be permeable to second messengers, including CAMP, inositol 1,4,Striphosphate (Imp,), and calcium, and many essential substances, such as sugars, glycolytic substances, amino acids and ions [17]. The dysfunction of gap junctional communication has been implicated in mechanisms of tumor promotion, teratogenesis and toxicity caused by chemicals. Primary cultures of hepatocytes represent a potentially useful model for studying the highly differentiated liver cell functions and the mechanism of hepatotoxicity under strictly controlled conditions. However, these cultures undergo a number of biochemical and morphological changes which partly limit the results obtained to be comparable with the in vivo system [lx]. The rapid loss of gap junctions between hepatocytes is one of the above changes occurring in primary culture. It was reported that after 10 h of culture, dye coupling between rat hepatocytes decreased to undetectable levels [19]. We also found in this study that dye coupling between hepatocytes decreased markedly during culture. Nevertheless, the incidence of dye coupling between hepatocytes cultured in our system remained at 39% even after 48 h of culture. This indicates that the culture system used in this study is favorable to the maintenance of gap junctional communication between rat hepatocytes in primary culture. The results from the present study have demonstrated that indium causes pronounced inhibition of gap junctional communication between rat hepatocytes in a dose-dependent manner. This effect of indium was observed at 100 FM, a dose that did not decrease the viability of hepatocytes. In this study, we measured the viability of cells using two indices: LDH leakage and MTT reduction. LDH leakage is a widely used index for viability of cells, the increase of which usually reflects, membrane damage of cells. MTT is a substance which can be reduced by mitochondrial succinate dehydrogenase, an enzyme involved in the production of ATP, to strongly absorbing formazan products. MTT reduction is therefore considered to reflect the mitochondrial function of cells [20]. As shown by our results, the increase in LDH leakage was not detected even when hepatocytes were exposed to 1 mM indium chloride, the concentration at which gap junctional co~uni~ation between hepatocytes was al-
105
most completely inhibited. On the other hand, treatment of hepatocytes with indium chloride at both 500 ,uM and 1 mM caused a significant decrease in MTT reduction activity of hepatocytes, though the two doses of indium chloride used were much higher than that required to inhibit gap junctional communication. Thus, our results suggested that inhibition of gap junctional communication was an early effect of indium on hepatocytes. The meaning of this inhibitory effect of indium on gap junctional communication between hepatocytes is still unknown. It is conceivable that the effect may be an early sign of indium toxicity. Inhibition of gap junctional communication could weaken the buffering ability of hepatocytes to toxic insults, thus preventing healthy cells from rescuing unhealthy ones, and accelerating the process of cell death. However, we cannot exclude the possibility that the closure of gap junctional communication caused by indium may be a protective response of hepatocytes. This mechanism may prevent the spread of toxic substances from damaged cells to healthy ones, and the loss of necessary metabolites from healthy cells at the same time [ 151. In conclusion, our study has demonstrated that indium inhibits gap junctional communication between rat hepatocytes in a dose-dependent manner. The effect of indium was observed at a concentration that did not result in a decrease in the viability of hepatocytes as assayed by LDH leakage and MTT reduction. Our results suggest that gap junctions between hepatocytes may be vulnerable sites to indium toxicity. REFERENCES 1 Venugopal, B. and Luckey, T.D. (1978) Metal Toxicity in Mammals, Vol. II. Plenum Press, New York, pp. 116-121. 2 Lewis, D.R. (1986) Dopant materials used in the microelectronics industry. Occup. Med. 1, 35-47. 3 Signore, A., Sensi, M., Pozzilli, C., Negri, M., Lenzi, G.L. and Pozzilli, P. (1985) Effect of unlabeled indium oxine and indium tropolone on the function of isolated human lymphocytes. J. Nucl. Med. 26, 612-615. 4 Castronovo, F.P. and Wagner, H.N. (1971) Factors affecting the toxicity of the element indium. Br. J. Exp. Pathol. 52, 543-559. 5 Fowler, B.A., Kardish, R.M. and Woods, J.S. (1983) Alteration of hepatic microsomal structure and function by indium chloride: u~trastructural, mo~hometric, and bi~hemi~l studies. Lab. Invest. 48, 471478. 6 Woods, J.S., Carver, G.T. and Fowler, B.A. (1979) Altered regulation of hepatic heme metabolism by indium chloride. Toxicol. Appl. Pharmacol. 49,455461, 7 Loewenstein, W.R. (1979) Junctional intercellular communication and the control of growth. Biochim. Biophys. Acta 560, I-65. 8 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. 9 Ruth, R.J., Klaunig, J.E. and Pereira, M.A. (1987) Inhibition of intercellular communication between mouse hepatocytes by tumor promoters. Toxicol. Appl. Pharmacol. 87, 11l-120. 10 Saez, J.C., Bennett, M.V.L. and Spray, DC. (1987) Carbon tetrachloride at hepatotoxic levels blocks reversibly gap junctions between rat hepatocytes. Science 236,967-969. 11 Welsch, F. and Stedman, D.B. (1984) Inhibition of metabolic cooperation between Chinese hamster V79 cells by structurally diverse teratogens. Teratog. Carcinog. Mutag. 4, 285-301.
106 12 Moldeus, P., Hegberg, J. and Orrenius, S. (1978) Isolation and use of liver cells. In: S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. 52, Dekker, New York, pp. 6&71. 13 Ohno, Y., Hirota, K., Kawanishi T. and Takanaka, A. (1990) Loss of viability after disulfiram treatment without preceding depletion of intracellular GSH. J. Toxicol. Sci. 15, 63-73. 14 Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D. and Mitchell, J.B. (1987) Evaluation of a tetrazolium-based semiautomated calorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47, 936942. 15 Gad, S.C. and Weil, C.S. (1989) Statistics for toxicologists. In: A.W. Hayes (Ed.), Principles and Methods of Toxicology, 2nd Edit., Raven Press, New York, pp. 435483. 16 Spray, D.C., Saez, J.C. and Hertzberg, E.L. (1988) Gap junctions between hepatocytes: structural and regulatory features. In: I.M. Arias, W.B. Jakoby, H. Popper, D. Schachter and D.A. Shafritz (Eds.), The Liver: Biology and Pathobiology, 2nd Edit., Raven Press, New York, pp. 851-866. 17 Saez, J.C., Connor, J.A., Spray, DC. and Bennett, M.V.L. (1989) Hepatocyte gapjunctions are permeable to the second messenger, inositol 1,4,5-triphosphate, and to calcium ions. Proc. Natl. Acad. Sci. USA 86,2708-2712. 18 Guguen-Guillouzo, C., Gripon, P., Vandenberghe, Y., Lamballe, F., Ratanasavanh, D. and Guillouzo, A. (1988) Hepatotoxicity and molecular aspects of hepatocyte function in primary culture. Xenobiotica 18, 7733783. 19 Spray, D.C., Fujita, M., Saez, J.C., Choi, H., Watanabe, T., Hertzberg, E., Rosenberg, L.C. and Reid, L.M. (1987) Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures. J. Cell. Biol. 105, 541-551. 20 Cook, J.A. and Mitchell, J.B. (1989) Viability measurements in mammalian cell systems. Anal. Biothem. 179, I-7.
99
TOXLET 2682
Indium inhibits gap junctional communication between rat hepatocytes in primary culture
Xinbiao Guo, Yasuo Ohno, Toru Kawanishi, Momoko Sunouchi and Akira Takanaka Division of Pharmacology, National Institute of Hygienic Sciences, Tokyo (Japan)
(Received 5 August 1991) (Accepted 10 October 1991) Key words: Indium; Gap junction; Hepatocyte; Primary culture
SUMMARY The effect of indium on gap junctional communication was investigated in primary cultured rat hepatocytes. Treatment of hepatocytes with indium chloride at concentrations of 100,uM to 1 mM for 2 h resulted in dose-dependent inhibition of gap junctional communication between hepatocytes. The effect of indium on hepatocytes was also evaluated using two indices for cell viability: lactate dehydrogenase (LDH) leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetr~olium bromide (MTT) reduction. Indium did not cause any increase in LDH leakage from hepatocytes at the above concentrations, but inhibition of MTT reduction was observed at concentrations above 500 PM. These results suggest that the gap junctions between hepatocytes may be vulnerable sites to indium toxicity.
INTRODUCTION
Iridium, a group IIIa metal in the periodic table, is widely distributed in nature in very minute quantities. In industry, it has been used for surface protection of metals or alloys, and for the manufacture of graphite, glass, motion picture screens and cathode oscillographs [l]. In particular, new uses have been found in recent years for indium as a dopant material in the microelectronics industry [2]. In medicine, indium (i.e. “‘In) is administered complexed with diethylenetriamine pentaacetic acid (DTPA), oxine, tropolone or bleomycin to locate and identify malignant tumors [3]. Although there have been no reported cases of systemic effects in humans exposed to indium, studies in animals have shown that indium is one of the most toxic metals [l].
Addressfor correspondence: Yasuo Ohno, Ph.D., Division of Pharmacology, National Institute of Hygienic
Sciences, 18-1, Kamiyoga, 1-chome, Setagaya-Ku, Tokyo 158, Japan.
100
Furthermore, the results from these studies have suggested that the liver is the target organ of indium. It was reported that indium administration produced multiple areas of necrosis in liver parenchymal cells of mice, with no changes in the Kupffer cells or the biliary system [4]. In addition, the marked reduction in cytochrome P-450 levels and its dependent drug-metabolizing enzyme activities, the inhibition of heme synthesis and the acceleration of heme catabolism have been demonstrated in the livers of indium-treated rats [5,6]. In the present study, we used primary cultured rat hepatocytes to investigate whether indium could alter gap junctional communication, a membrane channelmediated communication which is important in the regulation of cell growth and differentiation, and in the homeostasis of tissues [7]. Gap junctional communication has been reported to be altered by many toxicants, including tumor promoters, hepatotoxic chemicals, and teratogens [8811]. Our study demonstrated that indium inhibited gap junctional communication between rat hepatocytes in a dose-dependent manner. In this study, we also examined the effects of indium on membrane integrity and mitochondrial function of hepatocytes using lactate dehydrogenase (LDD, EC 1.1.1 27) leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction as indices, respectively. Our result suggests that gap junctions between hepatocytes may be vulnerable sites to indium toxicity. MATERIALS
AND METHODS
Materials Leibovitz’s L-15 medium, penicillin and streptomycin were obtained from Flow Laboratories (Irvine, U.K.). Newborn calf serum was obtained from Cell Culture Laboratories (Cleveland, OH, U.S.A.). Collagenase was obtained from Boehringer Mannheim, GmbH (F.R.G.). Indium chloride was obtained from Wako Chemical Co. (Tokyo, Japan). Lucifer Yellow CH and MTT were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All other chemicals were of reagent grade and were obtained from Wako Chemical Co. or Sigma Chemical Co.. Animals Male Sprague-Dawley rats (200-250 g weight range) were purchased from Japan SLC, and maintained on a 12-h light/dark cycle in a controlled-environment animal facility. Animals were fed rat chow and tap water ad libitum. Preparation of hepatocyte cultures Hepatocytes were prepared by the method of Moldeus et al. [12]. Cells were used only when the viability was higher than 85% at the time of plating. Hepatocytes were suspended in Leibovitz’s L- 15 medium supplemented with 2 nM insulin, 1 PM dexamethasone, 8.3 mM glucose, 2 mM nicotinamide, 10 mM HEPES, 50 IU/ml penicillin, 50 pg/ml streptomycin and 10% newborn calf serum. Approximately 2 x lo5 cells were plated in 2 ml of medium into 35-mm collagen-coated tissue culture dishes
101
(Corning). Cultures were incubated at 37°C in a humidified atmosphere of air. At 3 h after plating, the medium was changed to remove the unattached cells and was renewed every 24 h thereafter. Assay of gap junctional communication between hepatocytes Gap junctional communication between rat hepatocytes was evaluated by microinjection of the fluorescent dye, Lucifer Yellow CH, into one hepatocyte and observation of dye spread to adjacent cells. Lucifer Yellow CH 5% (w/v) in 0.3 M lithium chloride was backfilled into a microelectrode, and microinjected into individual cells using a Narishige IM-1 8gTMmicroinjector. Five minutes later, dye coupling was observed at room temperature with a Nikon Diaphot microscope with xenon arc lamp illumination using a Nikon B filter block (excitation at 450490 nm; emission > 520 nm). Photographs were taken on FUJI HR 400 film. The extent of dye coupling was expressed as the number of dye-coupled cells as a percentage of total adjacent cells. To investigate the effect of indium on gap junctional communication between hepatocytes, 24-h-old cultures were refed with medium (2 ml/dish) and were treated for 2 h with indium chloride at different concentrations. Cultures receiving vehicle (saline) alone were used as controls. Assay for viability of hepatocytes After treatment of hepatocytes with indium chloride as described above, two assays for cellular viability were performed: (1) LDH leakage was determined as described previously [ 131.Viability was expressed as the amount of LDH activity released from cells as a percentage of total LDH activity in the cells. (2) MTT reduction was assayed using the method described by Carmichael et al. [14]. The assay is dependent on the cellular reduction of MTT by the mitochondrial dehydrogenase of viable cells to a blue formazan product. The amount of the reduced product from MTT was expressed as the absorbance at 560 nm. Statistical analyses ANOVA was used for comparison of variance among groups. When a significant (P < 0.05) effect was observed, each treatment group was compared with the untreated control group using Scheffe’s test [15]. RESULTS
The incidence of dye coupling in untreated rat hepatocytes during the period of culture is shown in Figure 1. The incidence of dye coupling at 4 h of culture was 79 + 8%. A marked decrease in dye coupling was observed over the first 24 h of culture. After that, the decrease became less, and the incidence of dye coupling changed from 47 f 3% to 39 * 4% over the next 24 h of culture. Figure 2 is a fluorescence micrograph which shows dye coupling between a rat hepatocyte microinjected with Lucifer Yellow CH and adjacent hepatocytes in 24-h-old cultures. In this experiment, the effect of indium on gap junctional communication between
102
I
0'
0
48
24
Time
in
Culture
(hours)
Fig. 1.The timeecourse of changes in dye coupling between untreated rat hepatocytes in primary culture. 45-453 adjacent hepatocytes were observed for each value. Results are expressed as the mean k SE.
hepatocytes was determined after treatment of 24-h-old hepatocytes with indium chloride for 2 h. Dye coupling between hepatocytes exposed to 10 ,uM indium chloride remained at the same levels as those in untreated cells. When hepatocytes were exposed to indium chloride at concentrations above 100 ,uM, a significant inhibition of dye coupling was observed. Dye coupling between hepatocytes exposed to 100 ,uM indium was decreased to 67% of that in untreated cells. Moreover, indium inhibited the dye coupling in a dose-dependent manner. After treatment with 1 mM indium, the dye coupling between hepatocytes was almost completely inhibited (Fig. 3).
Fig. 2. A fluorescence micrograph showing dye coupling between a rat hepatocyte (A) microinjected with Lucifer Yellow CH and adjacent hepatocytes in 24-h-old cultures.
103
=‘
120
kt 100 c
60
p
60
‘-
0" Ef
40
": CI
20 0 10
100
1000
500
lndium
(FM)
Fig. 3. The dose-depedent inhibition of dye coupling between rat hepatocytes by indium chloride. 24-h-old hepatocyte cultures were treated with indium chloride for 2 h. 3@48 adjacent recipient hepatocytes were observed for each value. Results are expressed as the mean ? SE.
The effect of indium on LDH leakage from hepatocytes is shown in Figure 4. Treatment of hepatocytes for 2 h with indium chloride at concentrations of 100 ,uM to 1 mM did not cause any increase in LDH leakage from cells. On the other hand, as shown in Figure 5, MTT reduction activity of hepatocytes showed no significant changes when indium was added at concentrations below 250 ,uM. However, a significant inhibition of MTT reduction activity was observed when hepatocytes were exposed to indium chloride at both 500 PM and 1 mM. DISCUSSION
Gap junctional communication has been said to be important in the regulation of cell growth and differentiation. The coordinated function of the liver as an organ also depends significantly on gap junctional communication between hepatocytes. The gap junction between hepatocytes may function to link together the volume of coupled cells, and thus to increase the sink or buffer capacity of the cytoplasm [16]. In 100
3
L
60
ab p
60
x ii
40
4
20
0 0
.I 00
250
500
lndium
(PM)
1000
Fig. 4. The effect of indium chloride on LDH leakage from rat hepatocytes. 24-h-old hepatocyte cultures were treated with indium chloride for 2 h. LDH leakage was measured as described in ‘Materials and Methods’. Values are expressed as the mean +_SE (n = 3).
104
0
10
50
lndium
100
500
lODO
(JIM)
Fig. 5. The effect of indium chloride on the MTT reduction activity of rat hepatocytes. 24-h-old hepatocyte cultures were treated with indium chloride for 2 h. MTT assay was performed as described in ‘Materials and Methods’. Values are expressed as the mean It SE (n = 9). *Significantly different from control: P < 0.05.
addition, the gap junction has been shown to be permeable to second messengers, including CAMP, inositol 1,4,Striphosphate (Imp,), and calcium, and many essential substances, such as sugars, glycolytic substances, amino acids and ions [17]. The dysfunction of gap junctional communication has been implicated in mechanisms of tumor promotion, teratogenesis and toxicity caused by chemicals. Primary cultures of hepatocytes represent a potentially useful model for studying the highly differentiated liver cell functions and the mechanism of hepatotoxicity under strictly controlled conditions. However, these cultures undergo a number of biochemical and morphological changes which partly limit the results obtained to be comparable with the in vivo system [lx]. The rapid loss of gap junctions between hepatocytes is one of the above changes occurring in primary culture. It was reported that after 10 h of culture, dye coupling between rat hepatocytes decreased to undetectable levels [19]. We also found in this study that dye coupling between hepatocytes decreased markedly during culture. Nevertheless, the incidence of dye coupling between hepatocytes cultured in our system remained at 39% even after 48 h of culture. This indicates that the culture system used in this study is favorable to the maintenance of gap junctional communication between rat hepatocytes in primary culture. The results from the present study have demonstrated that indium causes pronounced inhibition of gap junctional communication between rat hepatocytes in a dose-dependent manner. This effect of indium was observed at 100 FM, a dose that did not decrease the viability of hepatocytes. In this study, we measured the viability of cells using two indices: LDH leakage and MTT reduction. LDH leakage is a widely used index for viability of cells, the increase of which usually reflects, membrane damage of cells. MTT is a substance which can be reduced by mitochondrial succinate dehydrogenase, an enzyme involved in the production of ATP, to strongly absorbing formazan products. MTT reduction is therefore considered to reflect the mitochondrial function of cells [20]. As shown by our results, the increase in LDH leakage was not detected even when hepatocytes were exposed to 1 mM indium chloride, the concentration at which gap junctional co~uni~ation between hepatocytes was al-
105
most completely inhibited. On the other hand, treatment of hepatocytes with indium chloride at both 500 ,uM and 1 mM caused a significant decrease in MTT reduction activity of hepatocytes, though the two doses of indium chloride used were much higher than that required to inhibit gap junctional communication. Thus, our results suggested that inhibition of gap junctional communication was an early effect of indium on hepatocytes. The meaning of this inhibitory effect of indium on gap junctional communication between hepatocytes is still unknown. It is conceivable that the effect may be an early sign of indium toxicity. Inhibition of gap junctional communication could weaken the buffering ability of hepatocytes to toxic insults, thus preventing healthy cells from rescuing unhealthy ones, and accelerating the process of cell death. However, we cannot exclude the possibility that the closure of gap junctional communication caused by indium may be a protective response of hepatocytes. This mechanism may prevent the spread of toxic substances from damaged cells to healthy ones, and the loss of necessary metabolites from healthy cells at the same time [ 151. In conclusion, our study has demonstrated that indium inhibits gap junctional communication between rat hepatocytes in a dose-dependent manner. The effect of indium was observed at a concentration that did not result in a decrease in the viability of hepatocytes as assayed by LDH leakage and MTT reduction. Our results suggest that gap junctions between hepatocytes may be vulnerable sites to indium toxicity. REFERENCES 1 Venugopal, B. and Luckey, T.D. (1978) Metal Toxicity in Mammals, Vol. II. Plenum Press, New York, pp. 116-121. 2 Lewis, D.R. (1986) Dopant materials used in the microelectronics industry. Occup. Med. 1, 35-47. 3 Signore, A., Sensi, M., Pozzilli, C., Negri, M., Lenzi, G.L. and Pozzilli, P. (1985) Effect of unlabeled indium oxine and indium tropolone on the function of isolated human lymphocytes. J. Nucl. Med. 26, 612-615. 4 Castronovo, F.P. and Wagner, H.N. (1971) Factors affecting the toxicity of the element indium. Br. J. Exp. Pathol. 52, 543-559. 5 Fowler, B.A., Kardish, R.M. and Woods, J.S. (1983) Alteration of hepatic microsomal structure and function by indium chloride: u~trastructural, mo~hometric, and bi~hemi~l studies. Lab. Invest. 48, 471478. 6 Woods, J.S., Carver, G.T. and Fowler, B.A. (1979) Altered regulation of hepatic heme metabolism by indium chloride. Toxicol. Appl. Pharmacol. 49,455461, 7 Loewenstein, W.R. (1979) Junctional intercellular communication and the control of growth. Biochim. Biophys. Acta 560, I-65. 8 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. 9 Ruth, R.J., Klaunig, J.E. and Pereira, M.A. (1987) Inhibition of intercellular communication between mouse hepatocytes by tumor promoters. Toxicol. Appl. Pharmacol. 87, 11l-120. 10 Saez, J.C., Bennett, M.V.L. and Spray, DC. (1987) Carbon tetrachloride at hepatotoxic levels blocks reversibly gap junctions between rat hepatocytes. Science 236,967-969. 11 Welsch, F. and Stedman, D.B. (1984) Inhibition of metabolic cooperation between Chinese hamster V79 cells by structurally diverse teratogens. Teratog. Carcinog. Mutag. 4, 285-301.
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