The mechanism of chloroform toxicity in isolated rat hepatocytes

Toxicology Letters, 69 (1993) 77-85 0

77

1993 Elsevier Science Publishers B.V. All rights reserved 0378-4274/93/$06.00

TOXLET 02928

The mechanism of chloroform toxicity in isolated rat hepatocytes

Nahla S. El-shenawy and Mohamed S. Abdel-Rahman Pharmacology and Toxicology Department, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ (USA)

(Received 6 October 1992) (Accepted 20 January 1993) Key words: Chloroform; Isolated hepatocyte; Viability; Glutathione

SUMMARY Chloroform (CHCI,) is widely used in the manufacture of drugs, cosmetics, plastics and cleaning agents. It is also found in chlorinated drinking water. This study was designed to investigate the toxic effect of CHCl, on isolated male rat hepatocytes using several toxicity parameters. The hepatocytes were isolated by a collagenase perfusion technique and the cell viability was determined by Trypan blue exclusion. The leakage of cytosolic enzymes such as aspartate transaminase (AST) and alanine transaminase (ALT) after treatment with CHCl, was measured. Reduced glutathione content (GSH) and its related enzymes, glutathione reductase (GSH-Rx) and glutathione peroxidase (GSH-Px), were also evaluated to study the effect of CHCl, on hepatocytes. Exposure to 100 and 1000 ppm CHCl, results in a significant decrease in cell after 30 min incubation. However, the effect of 1 and 10 ppm concentrations was observed at 60 min incubation. AST leakage was significantly increased in all treatment groups, while ALT was significantly increased at 100 and 1000 ppm CHCl, after 60 and 30 min, respectively. As early as 15 min, GSH was decreased significantly at 1000 ppm, but at 100 and 10 ppm CHCl, the decrease in GSH began after 30 and 120 min, respectively. GSH-Px activity did not changed. However, the activity of GSH-Rx was significantly decreased at 1000 ppm CHCl, and at the same time GSH content was decreased. The data indicate that the toxic effect of CHCl, was dose- and time-dependent. The degree of GSH depletion correlated with increased cytotoxicity and decreased GSH-Rx activity due to CHCl,.

INTRODUCTION

Chloroform is used in the manufacturing of lacquer and artificial silk. It has been used for over 100 years as a general anaesthetic [l]. In recent years there has been

Correspondence to: Dr. M.S. Abdel-Rahman, Department of Pharmacology and Toxicology, UMDNJNew Jersey Medical School, Newark, NJ 07103-2714, USA.

78

great speculation and concern about the effect of chlorination upon organic materials contained in natural water and waste water. A maximum concentration level of 100 ,@liter has been proposed for total trihalomethanes (THMs) of which CHCl, is a major component in finished drinking water [2]. This level recommended by United States Environmental Protection Agency (EPA) is based on a risk estimation that suggests the excess cancer risk in populations exposed to this level of CHCl, may be as high as 100 person per million [2]. Consequently, the EPA has proposed that all municipal water system serving at least 75 000 people would have to use granular, activated carbon filters to reduce CHCI, to 100 ppb [3]. Epidemiology studies have shown a correlation between human exposure to CHCl, in drinking water and colon cancer [4]. In addition, the National Institute for Occupational Safty and Health has recommended that no worker be exposed to atmospheric CHCl, in excess of 10 ppm during a 10-h workday or to more than 50 ppm for any IO-min period [5]. Glutathione has been implicated in the maintenance of cellular integrity and decreased levels of GSH in the liver may be either directly or indirectly responsible for cellular damage. In particular, GSH protects cells from potentially toxic electrophiles via the metabolism of xenobiotics [6]. CHCl, hepatotoxicity is due to the formation of phosgene which is produced during the metabolism by cytochrome P-450-dependent oxidation of CHCl, rather than to CHCl, per se [779]. Phosgene thus formed binds irreversibly to cellular GSH or tissue macromolecules. The availability of GSH in the liver during CHCl, metabolism seems to be of importance in preventing necrosis in vivo [lo]. GSH may protect the liver cell in two ways: it can prevent the alkylation of vitally important cellular structures by activated CHCl, metabolites [l 11,and it may act as an antioxidant [lo]. This effect is dependent not only on GSH but also on its related enzymes; GSH-Rx and GSH-Px. These enzymes are essential to maintain a normal level of GSH in the system. Recently, isolated hepatocytes have received increased attention as potentially useful screens for identifying hepatotoxic agents. Various indices have been used to investigate membrane integrity and metabolic function of hepatocyte [12]. Isolated hepatocytes have the ability to maintain many of the essential properties of the intact tissue; including similar permeability characteristics and the same concentration of cytochrome P-450 that plays an important role in CHCl, biotransformation

v31. Stacey et al. reported that 5-20 ~1 of CHCl,/2 ml isolated hepatocytes produced dose-dependent losses of K’ and release of ALT after 20 min incubation [14]. Exposure of isolated hepatocytes to 20 mM of CHCl, produced rapid AST leakage that plateaued between 10 and 60 min following exposure [15]. Ekstrom and Hogberg reported that 6.2 pmol/ml 14CHC13 decreased the intracellular level of GSH to about 15% within 60 min in hepatocytes isolated from phenobarbital-treated rats [8]. The present study was designed to evaluate the influence of CHCl, on membrane integrity and the leakage of cytosolic enzymes. Also, GSH content and the activities of GSH-Px and GSH-Rx were investigated.

79

MATERIALS AND METHODS

Chloroform was purchased from EM Industries, Inc, Gibbstown NJ with a chemical purity of 99.8%. Chloroform in dimethylsulphoxide (DMSO) (Sigma Chemical Co., St. Louis, MO) was prepared in different concentrations. Male Sprague-Dawley rats (225 & 25 g) were purchased from Taconic Farms (Germantown, NY). The animals were housed in an environmentally controlled room in the animal facilities of the university. After 1 week of quarantine, they were housed three per cage in a room with constant temperature (25X), humidity (50%) and a 12-h light/dark cycle. The animals received Purina laboratory chow and water ad libitum [ 16). ~epatocytes were isolated by a collagenase perfusion technique [17], with slight modi~cation as described by Farghali et al. [18]. The cells were counted in a hemocytometer. The viability of cells was measured by the Trypan blue exclusion technique [12]. Over 99% viable cells was obtained by this procedure in this study. Treatment and incubation of hepatocytes

Hepatocytes were resuspended in K-H buffer containing bovine serum albumin (BSA) in a final concentration 5 x lo6 cells/ml for Trypan blue exclusion technique and enzyme leakage. However, for determination of GSH content and its enzymes a concentration 65-70 x 1O6cells/ml was prepared. The cell suspension was poured into an Erlenmeyer flask. Hepatocytes were treated with CHCI, in DMSO at final concentrations of 1, 10, 100, 1000 ppm. DMSO (1 @/ml of cell suspension) was added to control flasks under the same conditions. Five to six animals were used for each treatment. Sample preparation for enzyme leakage

All samples were prepared in accordance with the established procedure in this laboratory [19]. AST and ALT leakage were measured according to Sigma Kits No. 59-50 and 58-50 UV, respectively. Enzyme activity was monitored in an aliquot of cell-free medium and compared to the total activity achieved after lysis of the cells [ 171. Glutathione assay

GSH content was determined using the method previously described by Beutler et al. [20]. GSH in the protein-free su~~atant was dete~ined at 412 nm, and it was expressed as nmollmg protein. Glutathione peroxidase and glutathione reductase assays

The activity of GSH-Px was determined by the assay described by Hafeman et al. [21]. An enzyme unit of activity was defined as a decrease in the log (GSH) of 0.001 per min. The activity of GSH-Rx was determined by method of Couri and Abdel-Rahman [22]. The unit of GSH-Rx activity was defined as an increase in the log (GSH) of 0.001 per min. Hepatocyte protein was measured by the Bio-Rad protein assay.

80

Buta analyses

Data are presented as mean + standard error of the mean. For evaluation of significant differences between the treatments, one-way analysis of variance (ANOVA) with Scheffe’s test and Student’s t-test were applied [23]. RESULTS

Table I shows the time course of Trypan blue exclusion (percent viability) up to 2 h of incubation with different concentrations (I-1000 ppm) of @He&. The two highest CHCl, concentrations (100 and 1000 ppm) produced a significant decrease in hepatocyte viability at 30 min incubation. While, a significant decrease in cell viability in all treatment groups was observed after 60 and 120 incubation. The toxicity of CHCl, as evaluated by AST and ALT leakage, was summarized in Tables 11 and III, respectively. After 15 min of incubation, there was no change in AST leakage at 1 and 10 ppm CHCl, at3compared to the respective control. However, at the same time 100 and 1000 ppm CHCl, resulted in a significant elevation in AST. A significant increase in AST leakage after treatment with 10, 100 and 1000 ppm was observed after 30 min of incubation, but AST leakage in the 1 ppm treatment was similar to the control value. The concentration of 1 ppm of CHCl, released a significant amount of AST enzyme, as well as the other doses after 60 and 120 min (Table II). ALT leakage from the hepatocytes after the treatment with 1, 10 and 100 ppm was unaffected at 30 min. However, 1000 ppm CHC& produced a significant increase of the ALT leakage after 30 min incubation. A further increase in the release of the ALT enzyme was noted at 60 min with 100 and 1000 ppm treatments (Table III). Effect of CHCl, on glutathione content in isolated rat hepatocytes is described in Table IV. GSH content decreased gradually during the entire time course in the case of 10 ppm concentration. This decrease became significantly different from the conTABLE 1 EFFECT OF C~L~R~F~R~

UN VIABILITY OF ISOLATED RAT HEPATUCYTES Time @nmin)

Concentration(ppm)

0

30

60

120

Control” Chloroform

99.9 + O.Ob

99.6 zk0. I

99.3 z!z0.1

97.1 + 0.5

98.7 r 0.4 97.0 k. 0.7 91.9 I LO’ 84.6 zk0.7’

91.9 f 86.6 f 83.1 ? 69.6 f

82.7 rt 76.2 4 60.5 k 43.0 It

I 10 100 1000

1.0 1.0’ 1.4” 1.5’

1.3’ 2.0’ 1.3” lsc

a ~imethy~su~foxide was used as vehicle. bVal~es represent the mean + SE of percent viability from six rats (three to four incubations from each rat). ’ ~igni~~~y difkent from the respective control (ANOVA with Scheffe’s test) P < 0.05.

81

TABLE II EFFECT OF CHLOROFORM RAT HEPATOCYTES

ON ASPARTATE TRANSFERASE

LEAKAGE FROM ISOLATED

Time (mm) Concentration (ppm)

0

15

Control Chloroform 1 10 100 1000

4.7 f 0.1”

30

5.5 f 0.3 5.9 f 6.9 f 14.2 f 31.9 f

60

6.5 f 0.3

0.3 0.4 0.9b l.Ob

8.9 f 13.8 f 15.0 f 37.5 f

0.7 0.7b l.Ob 0.9b

120

8.6 f 0.3 13.9 f 20.5 f 24.7 f 41.5 f

9.6 + 0.3

1.3b l.Ob 1.4b l.Ob

24.4 f 1.6b 28.4 It l.lb 33.5 + 1.3b 54.7 ?r 1.3b

a Values represent the mean _+SE of the percent leakage from six rats (three to four incubations from each rat). bSignificantly different from the respective control (ANOVA with Scheffe’s test) P < 0.05.

trol after 2 h incubation. On the contrary, the GSH content was significantly reduced at 100 ppm CHCl, at 30 min and up to 120 min. As early as 15 min, the highest CHCl, concentration (1000 ppm) produced a significant reduction in GSH content which remained until the end of the study. In order to determine the effect of GSH depletion on GSH-Px activity, the enzyme activity was measured as a function of time with 1000 ppm CHCl,. As shown in Table V, the 1000 ppm treatment did not cause any significant increase in GSH-Px activity after 15 and 30 min. The results of this experiment indicated that the GSH depletion

TABLE III EFFECT OF CHLOROFORM RAT HEPATOCYTES

ON ALANINE

TRANSFERASE

LEAKAGE

FROM ISOLATED

Time (min) Concentration @pm)

0

30

60

Control Chloroform 1 10 100 1000

31.4 f. 0.8”

37.5 + 1.1

44.9 + 1.3

37.7 f 38.1 f 40.6 f 53.1 f

47.9 f 46.7 f 51.1 + 65.1 f

1.2 0.8 0.7 0.9b

1.0 1.0 0.8b 1.6b

‘Values represent the mean + SE of the percent leakage from five rats (three to four incubations from each rat). bSignificantly different from the respective control (ANOVA with Scheffe’s test) P < 0.05.

82

TABLE IV EFFECT OF CHLOROFORM CYTES

ON GLUTATHIONE

CONTENT IN ISOLATED RAT HEPATO-

Treatment Time (min) 0

I5 30 60 90 120

Control

10 ppm

100 ppm

1000 ppm

13.5 + 0.9 11.2 zk 1.3 8.1 + 0.9 7.3 + 0.7 5.4 + 0.Q

12.3 k 1.0 6.8 T 0.3b 6.3 * 0.7b 5.0 + W 3.8 ?. 0.6”

10.3 f 6.4 k 4.5 k 4.0 i 3.1 +

15.5 It 0.8”

15.0rt 0.8 12.0 + 10.0 t 8.9 I 7.8 rt

i.2 0.8 0.8 0.6

l.lh O.Sh OZb O.Zb O.?

a Values represent the mean ?c SE of the glutathione content (nmol/mg protein) from six rats bSignificantly different from the respective control (ANOVA with Scheffe’s test) P c 0.05.

was not due to oxidation of GSH by CHCI,. The GSH-Rx activity was significantly decreased at 1000 ppm CHClj after 15 and 30 min treatments (Table VI). DISCUSSION

The data represented here reflect the utilization of isolated liver cells to investigate the toxic effect of CHCI, using three different parameters. In this study, Trypan blue exclusion was used to determine cell viability. Staining of the cells by Trypan blue indicated severe irreversible damage and reflected the endpoint to evaluate the effect of CHCl, [12]. Release of ALT into the medium from isolated hepatocyte suspensions may indicate the disruption of the cell membrane, while mitochondrial damage is responsible for the major portion of AST leakage [24]. Our results suggest that the

TABLE V EFFECT OF CHLOROFORM RAT HEPATOCYTES

ON GLUTATHIONE

PEROXIDASE

ACTIVITY IN ISOLATED

Time (min) Concentration (ppm)

15

30

Control Chloroform (1000)

11.4 + I.ti” 8.5 i 2.9

8.6 rt 1.6 6.6 + 1.7

a Values represent the mean + SE in Ulmg protein from five rats. The unit is defined as a decrease in the log (GSH) of 0.001 per min.

83

major effect of CHCl, is on the mitochondria, as the effect on ALT leakage was lower in comparison with the AST leakage. It is worth noting that the 1000 ppm dose increased ALT leakage approx. 2-fold, while the same concentration increased AST leakage 6-fold compared to the respective controls. This observed effect is in agreement with the reports of other investigators. They reported that the mitochondria is the target organelle for the toxic effect of CHCl, by measuring mitochondrial succinate oxidation in isolated rat hepatocytes [ 151 and Tetrazolum salt in primary cultured rat hepatocytes [25]. The results from this investigation revealed a relationship between the loss of GSH and cytotoxicity of CHCl,. A protective role for mitochondrial GSH in cytotoxicity was first proposed by Meredith and Reed [26]. They showed that the onset of cell injury in isolated rat hepatocytes by ethacrynic acid correlated with depletion of mitochondrial GSH, whereas the cytosolic pool could be depleted without affecting cell viability. Previously, several reports had demonstrated that the cytotoxicity, as measured by lipid peroxidation, liver necrosis, and loss of intracellular enzymes in vivo and in vitro, occurred only if the intracellular concentration of GSH fell below lo-15% of the initial value, which is the amount associated with mitochondria [8,27,28]. Mitochondrial GSH may be important in regulating inner membrane permeability by maintaining mitochondrial sulfhydryl groups in the reduced state [29]. The effect of CHCl, on isolated hepatocytes in this study is most likely related to its metabolites. Ekstrom and Hogberg [8] and also Stacy et al. [14] reported that a metabolite of CHCl, is responsible for its hepatotoxicity. The GSH depletion in this study can not be explained as an oxidation of GSH to GSSG because there is no increase in the activity of GSH-Px (Table V). This observation is similar to that described by Ekstrom and Hbgberg, in which 6.2 mM 14CHCl, produced a rapid loss in hepatocyte GSH level from 35 to 5 nmol/106 cells within an hour and there was no significant increase in the concentration of GSSG. Furthermore, they concluded that the period of maximal CHCl, metabolism in the hepatocyte model system was less than 20 min when 6.2 mM was used [8].

TABLE VI EFFECT OF CHLOROFORM HEPATOCYTES

ON GLUTATHIONE

REDUCTASE ACTIVITY IN ISOLATED RAT

Time (min) Concentration (ppm)

15

30

Control Chloroform (1000)

6.7 f 1.1” 3.2 f 0.7b

5.5 f 0.8 3.0 f 0.3b

a Values represent the mean f SE in U/mg protein from six rats. The unit was defined as an increase in log (GSH) of 0.001 per min. bSignificantly different from the control (Student’s t-test) P < 0.02.

84

The GSH-Rx activity was decreased after hepatocytes were treated with 1000 ppm CHC& (Table VI). A similar result was reported on the effect of arsenite on GSH-Rx. Williams has demonstrated that GSH-Rx contains a ‘redox-active’ disulfide bond, which in the presence of NADPH has accepted an electron pair through bond cleavage to form a dithiol. The arsenite then combines with the dithiol[30]. Most likely the mechanism that is operative with arsenite is also occurring with CHCl, metabolite. In conclusion, this study demonstrates that CHCl, toxicity is a dose- and timeresponse relationship. The decrease of GSH content plays a major role in CHCl, toxicity. REFERENCES 1 Challen, P.J.R., Hichish, D.E. and Bedford, J. (1958) Chronic chloroform in toxication. Br. J. Industr. Med. 15,243-249. 2 Environmental Protection Agency (EPA) (1977) Statement of basis and purpose for the regulation of trihalomethanes. EPA, office of water supply, Cincinnati, OH. 3 Trihalomethane limits in water proposed (1978) Chem. Eng. News, January 30, 7. 4 Gottlieb, M.S. and Carr, J.K. (1981) Cancer and drinking water in Louisiana: colon and rectum. Int. J. Epidemiol. 10, 117-125. 5 Utidjian, H.M.D. (1976) Recommendations for a chloroform standard, J. Occup. Med. l&253-257. 6 Eldjarn, L. and Bremer, J. (1962) The inhibitor effect at hexokinase level of disulphides on glucose metabolism in human erythrocytes. Biochem. J. 84,286291. 7 Pohl, L.R., Bhooshan, B., Whitaker, N.F. and Krishna, G. (1977) Phosgene: A metabolite of chloroform. Biochem. Biophys. Res. Commun. 79,684691. 8 Ekstrom, T. and Hogberg, J. (1980) Chloroform-induced glutathione depletion and toxicity in freshly isolated hepatocytes. Biochem. Pharmacol. 29, 30.59-3065. 9 Mansuy, D., Beaune, P., Cresteil, T., Lange, M. and Leroux, J. (1977) Evidence for phosgene formation during liver microsomal oxidation of chloroform. Bioch. Biophy. Res. Commun. 79, 513-517. 10 Brown, B.R., Sipes, LG. and Sagalyn, A.M. (1974) Mechanism of acute hepatic toxicity: CHCl, halothane and glutathione. Anesthesiology 41, 554-561. 11 Pohl, L.R. (1979) Biochemical toxicology of CHCI,. In: E. Hodgson, J.R. Bend and R.M. Philpot (Eds.), Review in Biochemical Toxicology. Elsevier, Amsterdam, pp. 79-107. 12 Baur, H., Kasperek, S. and Pfaff, E. (1985) Criteria of viability of isolated live cells. Hoppe-Seyler’s 2 Physiol. Chem. 356, 827-838. 13 LaBrecque, D.R. and Howard, R.B. (1976) The preparation and characte~zation of intact isolated parenchymal cells from rat liver. Methods Cell Biol. 14,327-340. 14 Stacey, N.H., Priestly, B.G. and Hall, R.C. (1978) Toxicity of halogenated volatile anesthetics in isolated rat hepatocytes. Anesthesiology 48, 17-22. 15 Berger, M.L. and Sozeri, T. (1987) Rapid halogenated hydrocarbon toxicity in isolated hepatocytes is mediated by direct solvent effects. Toxicology 45, 319-330. 16 National Institutes of Health (1985) Guide for the Care and use of Laboratory animals. NIH contract No. Nol-RR-2-2135, Beathesda, MD, pp. 11-28. 17 Mold&us, P., Hogberg, J. and Orrenius, S. (1978) Isolation and use of liver cells. In: S. Fleisher and L. Packer (Eds.), Methods in Enzymology. Academic Press, New York, pp. 60-71. 18 Farghali, H., Machchkova, Z., Kamemikova, L., Jankn, I. and Masek, K, (1984) The protection from hepatotoxicity of some compounds by the synthetic immunomodulator muramyl dipeptide (MDP) in rat hepatocytes and in vivo. Methods Find. Exp. Clin. Pharmacoi. 6,4491154. 19 Abdel-Rahman, MS. and Saxena, S. (1988) Effect of benzyl chloride on rat hepatocytes. J. Toxicol. Environ. Health 25,453+459.

20 Beutler, E., Duron, 0. and Kelly, B.M. (1963) Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 61,882-888. 21 Hafeman, D.G., Sunde, R.A. and Hoekstra, W.G. (1974) Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J. Nutr. 104, 580-587. 22 Couri, D. and Abdel-Rahman, MS. (1980) Effect of chlorine dioxide and metabolites on glutathione dependent system in rat and chicken blood. J. Environ. Pathol. Toxicol. 3,451-460. 23 Zar, J.H. (1984) Biostatistical analysis. 2nd Edn. Prentice-Hall, Englewood Cliffs. NJ, pp. 196198. 24 Story, D.L., Gees, S.J. and Tyson, CA. (1983) Response of isolated hepatocytes to organic and inorganic cytotoxines. J. Toxicol. Environ. Health 11,483-501. 25 Lamb, R.G., Borzelleca, J.F., Condie, L.W. and Gennings, C. (1989) Toxic interactions between carbon tetrachloride and chloroform in cultured rat hepatocytes. Toxicol. Appl. Pharmacol. 101, 106113. 26 Meredith, M.J. and Reed, D.J. (1982) Status of the mitochondrial pool of glutathione in the isolated hepatocytes. J. Biol. Chem. 257, 3747-3753. 27 Anundi, I., Hogberg, J. and Stead, A.H. (1979) Glutathione depletion in isolated hepatocytes: its relation to lipid peroxidation and cell damage. Acta Pharmacol. Toxicol. 45,45-50. 28 Casini, A.F., Pompella, A. and Comporti, M. (1985) Liver glutathione depletion induced by bromobenzene, iodobenzene, and diethylmalate poisoning and its relation to lipid peroxidation and necrosis. Am. J. Pathol. 118,225-237. 29 Reed, D.J. (1989) Role of glutathione. In: N. Taniguchi, T.H.Y. Sakamoto and A. Meister (Eds.), Glutathione Centennial Molecular Perspectives and Clinical Implications. Academic Press, Inc., New York. 30 Williams, C.H. (1976) Flavin-containing dehydrogenases. In: P.D. Boyer (Ed.), The Enzymes, Academic Press, New York, Vol. 13, pp. 89-137.