European Journal of Pharmacology - Environmental Toxicology and Pharmacology Section, 248 (1993) 337-340 0 1993 Elsevier Science Publishers B.V. All rights reserved 0926-6917/93/$06.00

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EJPTOX 40098

Short communication

Inhibition of hepatocyte gap junctional communication by 25hydroxycholesterol may be mediated through free radicals

Xinbiao Guo ‘, Yasuo Ohno * and Akira Takanaka

Division of Pharmacology, National Institute of Hygienic Sciences, 18-1, Kamiyoga, I-chome, Setagaya-ku, Tokyo 158, Japan

Received 18 August 1993, accepted 6 September 1993

25Hydroxycholestero1, an autoxidation product of cholesterol, has been shown to inhibit gap junctional communication between rat hepatocytes. In this study, we investigated whether free radicals are responsible for the effect of 25hydroxy- cholesterol on hepatocytes. Addition of superoxide dismutase, hydroxyl radical scavenger mannitol, or the antioxidants diphenylphenylenediamine and a-tocopherol markedly decreased the inhibitory effect of 25hydroxycholesterol. However, addition of catalase or the catalase inhibitor aminotriazole did not influence the inhibition of gap junctional communication by 25hydroxycholesterol. Data from this study suggest that free radicals other than hydrogen peroxide are involved in the mechanism of 25hydroxycholesterol-induced inhibition of gap junctional communication.

25Hydroxycholesterol; Gap junction; Hepatocytes; Free radical

1. Introduction

Gap junctional communication is a fundamental bio- logical process found in most animal tissues. The cell- cell communication through gap junction is said to be important in the regulation of cellular growth and differentiation, and in the homeostasis of tissues (Loewenstein, 1979). It has been suggested that inhibi- tion of gap junctional communication is a component of a variety of toxic responses (Elmore et al., 1987; Guo et al., 1992). The mechanism of gap junctional communication inhibition was still poorly understood. The production of free radicals, and the activation of protein kinase C activity have been proposed to explain the inhibitory effects of some chemicals on gap junc- tional communication (Enomoto and Yamasaki, 1985; Spray et al., 1988).

Oxysterols are derived from autoxidation of choles- terol in air or enzymatic metabolism of cholesterol in vivo, and have a wide spectrum of biological activities, including cytotoxicity against a variety of cells in vitro and in vivo, inhibition of cholesterol synthesis, inhibi- tion of DNA synthesis and modification of some im- munological responses (Smith and Johnson, 1989). Re-

* Corresponding author. Tel.: 03 (3700) 1141, ext. 349; Fax: 03 (3707) 6950.

1 Present address: Department of Environmental Health, Beijing Medical University, Beijing 100083, China.

cently, we found that some oxysterols, such as 7p-hy- droxycholesterol, 22S-hydroxycholesterol and 25hy- droxycholesterol inhibited gap junctional communica- tion between rat hepatocytes, and 25-hydroxy- cholesterol was the most potent (Guo et al., 1993). Zwijsen et al. (1992) also reported that 25hydroxy- cholesterol caused a significant decrease in gap junc- tional communication between human smooth muscle cells. Our previous study showed that inhibition of gap junctional communication by oxysterols was not a con- sequence of changes in cell viability, as measured by lactate dehydrogenase leakage and 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity. Furthermore, the effect of 25-hy- droxycholesterol on gap junctional communication seems to be independent of its effect on cholesterol synthesis function of cells. In this report, we investi- gated whether free radicals are involved in 25-hydroxy- cholesterol-induced inhibition of gap junctional com- munication between rat hepatocytes.

2. Materials and methods

2.1. Materials

25-Hydroxycholesterol, catalase and Lucifer Yellow CH were obtained from Sigma Chemical Co. (St. Louis. MO, USA). Superoxide dismutase was from Funakoshi Chemical Co. (Tokyo, Japan). Mannitol and cy-

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tocopherol were from Wako Chemical Co. (Tokyo, Japan). Aminotriazole and diphenylphenylenediamine were from Tokyo Kasei Chemical Co. (Tokyo, Japan). SKF-525A was from Smith Kline & French Labs. (Philadelphia, PA, USA). Leibovitz’s L-15 medium was from Flow Laboratories (Irvine, Scotland). Newborn calf serum was from Cell Culture Laboratories (Cleve- land, OH, USA). Collagenase was from Boehringer Mannheim (Germany). All other chemicals were of the reagent grade and were from Wako or Sigma.

2.2. Preparation of hepatocyte cultures

Hepatocytes were prepared from male Sprague- Dawley rats (200-250 g weight range, 7 weeks of age) by the method of Mold&s et al. (1978). Cells were used only when the viability was higher than 90% just before plating. Hepatocytes were suspended in Lei- bovitz’s L-15 medium supplemented with 100 nM in- sulin, 1 PM dexamethasone, 8.3 mM glucose, 2 mM nicotinamide, 10 mM HEPES, 50 III/ml penicillin, 50 pg/ml streptomycin and 5% newborn calf serum. 2 x

lo5 cells in 2 ml of medium were plated onto 35-mm collagen-coated tissue culture dishes. 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 with 2 ml of the same culture medium except for omission of serum. Hepatocytes cultured for 12 h were used for experiments. 25-Hydroxycholesterol and cr-tocopherol were dissolved in ethanol. Superoxide dismutase, cata- lase, aminotriazole and mannitol were dissolved in saline. Diphenylphenylenediamine was dissolved in dimethyl sulfoxide. Control cultures received the same volume of vehicle only, and the final concentrations of ethanol or dimethyl sulfoxide in all cultures were less than 0.25% (v/v) which did not affect hepatocyte dye coupling or viability. If not mentioned otherwise, the treatment period was 12 h.

2.3. Assay of gap junctional communication between hepa tocytes

Gap junctional communication between rat hepato- cytes was evaluated as described previously (Guo et al., 1992). Briefly, 5% (w/v) Lucifer Yellow CH in 0.3 M lithium chloride was backfilled into a glass capillary needle, and microinjected into individual ‘donor’ hepa- tocytes using a Narishige IM-188 microinjector. 5 min later, ‘recipient’ cells directly adjacent to the microin- jetted dye-loaded cell were evaluated for the evidence of dye coupling at room temperature under a Nikon phase-contrast fluorescent microscope. All cells di- rectly adjacent to the donor cell were observed, and the extent of dye coupling was expressed as the per- centage of dye-coupled recipients.

The viability of cells was determined by the leakage of lactate dehydrogenase into the culture medium as described previously (Guo et al., 1992).

Data were compared by ANOVA followed by Scheffe’s multiple comparison test.

3. Results

Table 1 shows the effects of several substances on 25-hydroxycholesterol-induced inhibition of dye cou- pling. Treatment of rat hepatocytes with 10 PM 25hy- droxycholesterol for 12 h significantly inhibited gap junctional communication. The incidence of dye cou- pling in 25-hydroxycholesterol-treated cultures de- creased to 47% of that in control ones. The addition of superoxide dismutase to 25-hydoxycholesterol-treated hepatocytes completely prevented the inhibitory effect of 25-hydroxycholesterol on dye coupling. Catalase, however, did not protect cells from the inhibition of dye coupling by 25-hydroxycholesterol. Moreover, the inhibitory effect of 25-hydroxycholesterol on dye cou- pling was not significantly influenced by addition of aminotriazole, a catalase inhibitor. The possible in- volvement of free radicals in 25-hydroxycholesterol-in- duced inhibition of dye coupling was further investi- gated using a nonenzymatic free radical scavenger mannitol, and the antioxidants diphenylphenylenedi- amine and a-tocopherol. Mannitol significantly pro- tected cells from 25-hydroxycholesterol. Diphenylphen- ylenediamine and a-tocopherol nearly completely re- versed the inhibition of dye coupling by 25-hydroxy-

TABLE 1

Effects of various substances on the inhibition of dye coupling between rat hepatocytes by 25hydroxycholesterol

Rat hepatocytes were treated for 12 h with 10 FM 25hydroxy- cholesterol (25OH) alone, or 25-OH plus the following substances added simutaneously: 200 U/ml superoxide dismutase, 2000 U/ml catalase, 20 mM mannitol, 10 PM diphenylphenylenediamine, or 100 PM a-tocopherol. Aminotriazole (1 mM) was added to the culture 2 h prior to the addition of 25OH. Data were obtained from at least three separate experiments and 3-5 dishes were used for each group of experiments. Results are expressed as means of dye couplingrt S.E., and the number of injections are given in parenthesis (N).

Treatment (N) Dye coupling (%b)

Control 25-OH 25-OH + superoxide dismutase 25-OH + catalase 25-OH + aminotriazole 25-OH + mannitol 25-OH + diphenylphenylenediamine 25-OH + a-tocopherol

105 70+5 24 33+5 a 20 72+6 b 20 39+6 a 29 42+4 a 28 51*5 a*b 53 61+4h 36 61+5 b

a Significantly different from control cultures, P < 0.05. b Signifi- cantly different from cultures treated with 25-OH alone, P < 0.05.

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80 , I

COfltrOl %-OH 25.OH+SKF-525A SKF-525A

Fig. 1. The effect of SKF-525A on 25hydroxycholesterol-induced inhibition of dye coupling.

cholesterol. Mannitol, diphenylphenylenediamine and a-tocopherol on their own had no effect on hepatocyte dye coupling (data not shown).

Effect of SKF-525A, an inhibitor of mixed function oxidase, on 25-hydroxycholesterol-induced inhibition of dye coupling is shown in fig. 1. The addition of SKF- 525A to the cultures substantially prevented the in- hibitory effect of 25-hydroxycholesterol on dye cou- pling between hepatocytes.

There were no changes in viability of hepatocytes after the treatment described above.

4. Discussion

While a variety of chemicals have been shown to inhibit gap junctional communication in hepatocytes, the mechanism by which these chemicals inhibit gap junctional communication remains uncertain. The acti- vation of protein kinase C has been proposed to ex- plain the inhibitory effects of some agents, like diacyl- glycerol and phorbol esters on gap junction in some type of cells. However, the uncoupling actions of these agents are not observed in primary cultures of hepato- cytes, although the activation of protein kinase C by phorbol esters in hepatocytes, and the phosphorylation of liver gap junction by protein kinase C have been reported (Spray et al., 1988). We also observed that 12-0-tetradecanoyl-phorbol-13-acetate did not inhibit dye coupling between rat hepatocytes in primary cul- ture (unpublished data). Recently, the role of free radicals in the inhibition of gap junctional communica- tion between hepatocytes was proposed in several stud- ies. Saez et al. (1987) found that the electrical coupling and dye coupling between pairs of rat hepatocytes were blocked reversibly by brief exposure to carbon tetra- chloride. They suggested that free radicals were media- tors of carbon tetrachloride-induced effect on gap junction functioning. Research by Ruth and Klaunig (1988) showed that antioxidants prevented the inhibi-

tion of gap junctional communication between mouse hepatocytes by paraquat, glucose oxidase, hydrogen peroxide, and several tumor promoters, such as pheno- barbital, lindane and dichlorodiphenyltrichloroethane.

In this study, we investigated the possible involve- ment of free radicals in the mechanism of 25-hydroxy- cholesterol-induced inhibition of gap junctional com- munication between rat hepatocytes. Superoxide dis- mutase is a free radical scavenging enzyme, and plays a role in removing of superoxide radical produced in cells. The complete protection by superoxide dismutase suggested the involvement of superoxide in the mecha- nism of 25-hyroxycholesterol-induced inhibition of gap junctional communication. Our results showed that hydrogen peroxide may not play a role in the mecha- nism responsible for the inhibition of gap junctional communication by 25-hydroxycholesterol. This was demonstrated by the addition of catalase, or using a catalase inhibitor aminotriazole. As superoxide radical is a poorly reactive oxygen species in biological system, the effect of superoxide on cells could involve the production of much more reactive hydroxyl radical, which is an initiator of lipid peroxidation (Halliwell and Gutteridge, 1992). The protective effects of the hydroxyl radical scavenger mannitol, and the anti- oxidants diphenylphenylenediamine and a-tocopherol on hepatocytes exposed to 25-hydroxycholesterol strongly suggested the involvement of hydroxyl radical and lipid peroxidation in the mechanism of 25-hydroxy- cholesterol-induced inhibition of gap junctional com- munication. Lipid peroxidation initiated by hydroxyl radical could change the microenvironment for the function of gap junction. Moreover, hydroxyl radical may also directly attack gap junction protein to cause the dysfunction of gap junctional communication.

The reason for the failure to prevent the effect of 25-hydroxycholesterol by catalase is not clear. The mechanism generally proposed to explain the hydroxyl radical production involves the presence of superoxide and hydrogen peroxide, which is called the iron-cata- lyzed Haber-Weiss reaction. Several recent studies in- dicated that hydroxyl radical can be formed by the interaction between superoxide and nitric oxide, a re- action which is not dependent on the presence of hydrogen peroxide (Halliwell and Gutteridge, 1992). It is conceivable that 25-hydroxycholesterol-induced inhi- bition of gap junctional communication may be medi- ated by a hydrogen peroxide-independent process.

Oxysterols, such as 25-hydroxycholesterol and 26-hy- droxycholesterol can be efficiently metabolized by mixed function oxidase system in the liver, and finally converted to bile acids (Swell et al., 1981; Toll et al., 1992). Our results showed that SKF-525A, an inhibitor of mixed function oxidase, significantly alleviated the inhibitory effect of 25-hydroxycholesterol on gap junc- tion functioning. This suggests that free radicals, which

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may mediate the inhibition of gap junctional communi- cation, could be produced via metabolism of 25-hy- droxycholesterol.

In conclusion, our study suggests that free radicals are involved in the mechanism of 25-hydroxy- cholesterol-induced inhibition of gap junctional com- munication. The sequence of events to generate hy- droxyl radical in 25-hydroxycholesterol-treated hepato- cytes may be a hydrogen peroxide-independent pro- cess.

Acknowledgement

This research was supported by a grant from Human Sciences Foundation, Japan.

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