XENOBIOTICA,

1990, VOL. 20, NO. 9, 933-943

Molecular mechanisms for bromotrichloromethane cytotoxicity in isolated rat hepatocytes L. G. McGIRR, S. KHAN, V. LAURIAULT and P. J. O’BRIENT Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 1A1, Canada

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Received 16 February 1990; accepted 8 May 1990

1. Bromotrichloromethane added to isolated rat hepatocytes resulted in increased cell death as determined by trypan blue uptake. Toxicity increased in a concentrationdependent fashion between 2.0-50 M bromotrichloromethane. 2. Lipid peroxidation (malondialdehyde)increased in a time-dependent fashion but in contrast to toxicity reached a maximum level at 20 m M bromotrichloromethane. 3. Hypoxia increased the toxicity of bromotrichloromethane three-fold but only decreased the amount of lipid peroxidation to a small degree. 4. In spite of this poor correlation between toxicity and lipid peroxidation, the antioxidant butylated hydroxyanisoleand the iron chelator desferal protected the cells from toxicity under both aerobic and hypoxic conditions and prevented lipid peroxidation. 5. During treatment with bromotrichloromethane, cellular glutathione levels slowly decreased and oxidized glutathione appeared in the media. The addition of cystine to the incubation media prevented the formation of extracellular oxidized glutathione, indicating that cellular glutathione had leaked from the cell during treatment and was oxidized in the incubation media. Although this suggested that glutathione does not play a protective role against bromotrichloromethane toxicity, diethyl maleate-pretreatment of the cells to decrease glutathione levels markedly increased bromotrichloromethane toxicity.

6. The addition of ascorbic acid to the incubation media increased bromotrichloromethane toxicity. This was attributed to the reductive activation of bromotrichloromethane in an iron and oxygendependent reaction. 7. It was concluded that peroxidation of essential phospholipids contributes to bromotrichloromethane-inducedhepatocyte cytotoxicity.

Introduction The mechanism of liver toxicity induced by carbon tetrachloride is believed to be the result of reductive metabolism catalysed by the cytochrome P-450-dependent mixed function oxidase system. Metabolism results in covalent binding to protein and lipid as well as the induction of extensive microsomal lipid peroxidation prior to cell death (Recknagel and Glende 1973, Slater 1982). Both covalent binding and lipid peroxidation are the result of the formation of the trichloromethyl radical which has been spin-trapped using phenyl-N-tert-butyl nitrone in several in oitro (Poyer et al. 1978)and in vioo systems (Lai et al. 1979). Each of these two mechanisms can be demonstrated to play a role in the inactivation of several microsomal enzymes. Cytochrome P-450 appears to be inactivated via a covalent binding mechanism (Yamazoe et al. 1979, DeGroot and Haas 1981) whereas glucose 6-phosphate is inactivated by a lipid peroxidation mechanism (Masuda 1981, Poli et al. 1981). The degree to which these two mechanisms participate in cell death/necrosis is still controversial. An early cytotoxic effect is the increased cytosolic CaZ+levels arising from inhibition of the Ca2+pumps of the endoplasmic reticulum and mitochondria (Long and Moore 1986a, b). The inhibition of microsomal Ca2+ sequestration

t To whom correspondence should be addressed. 0049-8254/90 $3.00 @> 1990 Taylor & Francis I,td

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seems to involve both covalent binding and lipid peroxidation mechanisms (Waller et at. 1983). Increased cytosolic Ca2+levels could be responsible for the activation of phosphorylase a (Long and Moore 1986a, b), protein kinase C (Poli et al. 1988) and phospholipases (Glende and Pushpendran 1986). Several studies have shown that some antioxidants give partial protection against CC14-induced toxicity (Cawthorne et at. 1970, Torrielli and Ugazio 1975) but other studies have indicated that a poor correlation exists between lipid peroxidation and cell death (Shen et al. 1982, Dogterom et at. 1988). The CC1,-induced inhibition of lipoprotein secretion by isolated hepatocytes has been attributed to the covalent binding to protein rather than lipid peroxidation (Dianzani et al. 1981, Becker et al. 1987). The blockage of lipoprotein secretion is believed to contribute to the fatty liver induced by CCl,. Bromotrichloromethane (BrCCl,) is much more hepatotoxic than CCl, (Burdino et al. 1973) and is also more effective at inducing lipid peroxidation (Gavino et al. 1984) and covalent binding (Sipes et al. 1977). In this paper, the relationship between lipid peroxidation and BrCC1,-induced cell death was investigated in isolated hepatocytes by attenuation of lipid peroxidation, and activation mechanisms under aerobic conditions compared to hypoxic conditions.

Materials and methods Materials Trypan blue, glutathione (GSH), oxidized glutathione (GSSG), fluoro-2,4-dinitrobenzene, iodoacetic acid, sodium ascorbate, thiobarbituric acid, arachidonic acid and superoxide dismutase were purchased from Sigma (St. Louis, MO). BrCCI,, dithiothreitol ( D T T ) and diethylmaleate (DEM) were obtained from Aldrich (Milwaukee, WI). Collagenase (from Clostridium histoliticum), Hepes and catalase were purchased from Boehringer-Mannheim (Montreal, Quebec). Desferal was a gift from Ciba Geigy Ltd. (Mississauga, Ontario). H.p.1.c. grade solvents were obtained from Caledon (Georgetown, Ontario). Animals Male Sprague-Dawley rats ( 2 W 2 5 0 g) were obtained from Charles River (St. Constant, Quebec) and fed a standard chow diet (Rodent Laboratory Chow #5001, Purina Mills Inc., St. Louis, MO, USA). Isolation and incubation of hepatocytes Hepatocytes were prepared by collagenase perfusion of the liver (Moldeus et al. 1978). Isolated cells were suspended in Krebs-Henseleit buffer, pH 7.4 (1 x lo6cells/ml) containing 1 2 5 mM Hepes and incubated in rotating round bottom flasks in a water bath (37°C) under an atmosphere of 95% 0,/5% CO,. Cell viability (normally 85-95%) was determined by trypan blue exclusion (final concentration 0.16%). Cells were preincubated for 15 min prior to the addition of other chemicals. BrCCl, stock solutions (1 M) were prepared in dimethyl sulphoxide; corresponding levels of dimethyl sulphoxide added to control cells had no effect. Hypoxic cell conditions were created by treating the cells with an atmosphere of 95% NZ/5% CO, for 30 min prior to the addition of the treatment chemicals. After 30min, the amount of oxygen was determined to be approx. 2% using a Clark type oxygen electrode.

GSH and GSSG determination G S H and GSSG levels in the hepatocytes and medium were determined by the h.p.1.c. method of Reed et 01. (1980) after derivatization with iodoacetic acid and fluoro-2,4-dinitrobenzene using a pBondpak NH, column (3.9 mm int. diam. x 30 cm, Waters). Intracellular G S H and GSSG was determined by first trapping extracellular G S H as a mixed disulphide by adding cystine (0.2 mM) to the incubation medium (DiMonte et 01.1987). Then the hepatocyte suspension was centrifuged at 200 x g for one minute to remove the intact cells. After removal of the supernatant, the cells were washed once in cystine-free media and then suspended in cystine-free media. An aliquot of the washed cells and the cystine incubation media were taken for G S H and G S S G analysis. Oxygen uptake Oxygen uptakes were determined using a Clark type oxygen electrode (Yellow Springs Instrument Co., Model 5300) using a thermostated 2ml chamber at 22°C.

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Bromotrichloromethane cytotoxicty

Lipid peroxidation Lipid peroxidation was measured by the thiobarbituric acid test and expressed as the amount of malondialdehyde (MDA) formed using an absorption coefficient of 1.56 x lo5mol cm-' at 535 nm (Buege and Aust 1978).

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Results Bromotrichloromethane caused cytotoxicity to isolated hepatocytes at levels greater than or equal to 2-0mM (figure 1a). A 'blebbing' response preceded the uptake of trypan blue. Carbon tetrachloride at 2.0 m M was not cytotoxic (table 1). In

2

"1

1 1

0 0

2

3

n m of incubation(h) (0)

0

1

Tim of lncubstlon (h) (b) Figure 1. ( a ) Cytotoxicity of bromotrichloromethane to isolated hepatocytes under aerobic conditions. Control cells ( x ) ; BrCCl,: ImM (A);2 m M (0);3 m (H); ~ 4mM ( A ) and 5mM ( 0 ) (b) . Lipid peroxidation in hepatocytes treated with bromotrichloromethane under aerobic conditions. Control cells ( X ) ; BrCCl,: 1 mM ( 0 ) ;2 m M (0): 3mM (H);4mM ( A ) and 5 m M (0).

L. G. McGirr et al.

936 Table 1.

Effect of modulators on the cytotoxicity of BrCCI, towards hepatocytes under aerobic conditions. Trypan blue uptake (%) at time Condition

0.5 h

1.0h

2.0 h

3.0h

Control CCI, (2'0mM) BrCCI, (2.0mM) Desferal (100 p ~ ) Desferal (1 m M ) +BHA ( 1 0 0 ~ ~ ) D T T (1 mM) +Ascorbate (1 mM) +DEM ( 3 5 0 ~ ~ )

20f3 20f4 31 k 6 33 & 4 34f3 33f3 35+5 37+6 56&8

23 + 4 22+3 37f8 31 + 3 43f6 37f3 49+6 72f7 87f10

20f5

25+3 24f4 96+4 64+10 46f4 38f4

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+ + +

24f3 69+12 37f6 45f5 31 f 7 53f8 100 100

87f5 100 100

* D T T added at 60min. Results are expressed as the average of three experiments+SD. BHA =butylated hydroxyanisole; D T T = dithiothreitol; DEM = diethylmaleate.

the BrCC1,-treated hepatocytes lipid peroxidation (as determined by malondialdehyde formation) increased without a lag period in a time-dependent manner. Malondialdehyde levels increased with BrCCl, concentration up to 2-0mM BrCCl, (figure 1 b) where they reached a maximum even though the toxicity increased with increasing dosage above 2.0 mM. T o determine the molecular mechanism for the toxicity, different compounds were added to the incubation media to determine their ability to modulate the toxic response. The ferric chelator desferal (100mM or 1mM) and the antioxidant butylated hydroxyanisole (BHA, 1 0 0 ~prevented ~ ) or delayed toxicity over a 3 h time period (see table 1). The thiol reductant dithiothreitol(1 mM) added after 1 h, however, did not prevent or delay cytotoxicity. Depleting GSH with diethylmaleate (3 50 mM) significantly increased the susceptibility of isolated hepatocytes to BrCC1, (table 1). Ascorbic acid (1 mM) markedly increased hepatocyte susceptibility to BrCC1, suggesting that ascorbic acid may be able to reduce BrCCl, to increase the formation of the trichloromethyl radical (table 1). Indeed, the addition of BrCC1, to ascorbic acid solutions resulted in oxygen uptake (table 2) which was catalase resistant (results not shown). Furthermore, oxygen uptake was markedly increased if arachidonic acid Table 2. Effect of ascorbic acid on the reductive activation of BrCCI, and CCI,. Rate of oxygen uptake (nmol O,/min per m)

Reactants BrCCI, (2.0mM) Ascorbate (200jLM)+BrCCI, (2.0mM) Ascorbate (1 mM)+BrCCI, (2.0mM) Ascorbate (200 pM) BrCCI, (2.0m M ) Arachidonate (400 /AM) cc1, (2.0mM) Ascorbate (1 mM) CCI, ( 2 . 0 m ~ ) Ascorbate (5 mM) CCI, (2.0 mM) Ascorbate (1 mM) BrCCI, (2.0mM) Ascorbate (1.0KIM)

+

+

+ + +

0.3fO.l 7.7 0.4 250&3

+

25.0+3 0.3 f0 1 0.7 f 0.2 1.0 k0.3 0.4f 0.1 0.5 f 0.2

Oxygen uptake was determined using a Clark electrode in 2 ml of 0 1 M Tris-HCI buffer, pH 7.4. Results are expressed as an average of three experiments f SD. Reactants were added in the order shown.

Bromotrichloromethane cytotoxicty Table 3.

Effect of antioxidants on oxygen uptake catalysed by BrCCI, and ascorbic acid. Rate of oxygen uptake (nmol O,/min per m)

Reactants Ascorbate+ BrCCI, Desferal(1M)PM) Desferal (1 mM) +DETAPAC (1 mM) Superoxide dismutase Superoxide dismutase DETAPAC Arachidonic acid (400 ~ L M ) +Arachidonic acid ( 4 0 0 p ~ ) + B H A( 1 0 0 ~ ~ ) +BHA ( 1 0 0 ~ ~ ) D T T (1 mM) +GSH ( 1 0 0 ~ ~ )

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+ + + + + +

937

+

6.9 f 1-0 3.4 & 0.5 1.4f0.3 1.5k0.3 5.6 f 0 2 0.0 19.0f 3.0 9.6 f 2.0 7.1 & 0.5

00 00

Oxygen uptake was determined with a Clark electrode. The reaction mixture contained 2 ml of 0.1 M Tris-HC1 buffer, pH 7.4, BrCCI, (2 mM) and ascorbate ( 0 2 mM). Results expressed as an average of three experiments f S D . BHA =butylated hydroxyanisole; D T T = dithiothreitol; GSH =glutathione.

was present indicating arachidonic acid peroxidation. Oxygen uptake in the absence of arachidonic acid may therefore be attributed to .CCl,. However, the addition of ascorbic acid to BrCCl, (reverse order) did not lead to oxygen uptake, indicating that the effect of ascorbic acid was not the result of direct reduction (table 2). The inhibition of oxygen uptake by iron chelators and/or superoxide dismutase indicates that the reduction was mediated by superoxide radicals (table 3). Since hypoxic conditions have been shown to increase the toxicity of CCl, in rats, the effect of hypoxia on BrCC1,-induced toxicity in hepatocytes were determined. Hypoxia induced by incubating the hepatocytes under a nitrogen atmosphere for the entire 2.5 h BrCC1, exposure period led to a three- to four-fold increase in toxicity (figure 2 a and table 4). Under these conditions control hepatocytes were unaffected but malondialdehyde production was still evident with BrCCl,-treated hepatocytes although the level of malondialdehyde produced was approximately a third less than that produced under oxygen (figure 2 b). However under hypoxic conditions BrCCl, toxicity was still delayed by the iron chelator desferal or the antioxidant butylated hydroxyanisole. Hypoxic conditions did however prevent the enhancement of BrCC1, toxicity by ascorbate. Carbon tetrachloride was less cytotoxic to hepatocytes than BrCCl, under hypoxic conditions (table 4). Associated with the BrCC1,-induced cell death was the relatively slow disappearance of GSH and the appearance of GSSG (figure 3). Since the oxidation of GSH occurred at approximately the same rate as the increased permeability of the cells to trypan blue, an experiment was conducted to determine if GSH oxidation was the result of GSH leaking from the cell and being oxidized in the incubation media. This was accomplished by adding cystine to the incubation media to trap any GSH released into the media as extracellular mixed cysteine-GSH disulphide. With added cystine, it was found that extracellular mixed cysteine-GSH disulphide was formed and only very small amounts of intracellular GSSG were formed (figure 3). This indicates that the GSH leaked from the hepatocyte during cytotoxicity and was autoxidized in the medium. Furthermore GSH depletion and GSH oxidation was markedly inhibited when BrCCl, was added to hepatocytes maintained under hypoxic conditions (results not shown) even though cytotoxicity was markedly enhanced.

L. G. McGirr et al.

938 100

-

80

-

n

60

-

-maa

40

-

20

-

z e

Y

8

c

3

e 8

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$

I-

0 0

2

1

3

nme of incubation(h) (a)

8-

6-

-a

'DO

n

4-

I 2-

0

1

2

3

Time of Incubation(h) (b)

Figure 2. (a)Cytotoxicity of bromotrichloromethaneto isolated hepatocytes under hypoxic conditions. Control cells ( x ); BrCCI,: 0-5mM (0); 1 mM ( 0 )and 2 mM ( A ) . ( b ) Lipid peroxidation in hepatocytes treated with bromotrichloromethane under hypoxic conditions. Control cells ( x ); BrCCI,: 1 mM (0) and 2 m~ (0).

Discussion Incubation of isolated hepatocytes with BrCCI, (2 mM) under aerobic conditions led to cell death as determined by trypan blue uptake. However, the same concentration of CCl, was not cytotoxic. Other investigators found CCI, to be cytotoxic to hepatocytes at this concentration if the hepatocytes were prepared from phenobarbital- or diethyl maleate-pretreated rats (Smith et al. 1983). T h e metabolic activation of halomethanes is believed to involve a reductive dehalogenation catalysed by reduced cytochrome P-450 which results in the homolytic cleavage of the halomethane to free radicals (Sipes et al. 1977). In the case of CCI, and BrCCI,,

Bromotrichloromethae cytotoxicty Table 4.

939

Effect of modulators on the cytotoxicity of BrCCI, towards hepatocytes under hypoxic conditions. Trypan blue uptake (%) at time Condition

05h

Control CCI, (20mM) BrCCI, (2.0mM) Desferal (100p ~ ) Desferal (1 mM) +BHA ( 1 0 0 ~ ~ ) +Ascorbate (1 mM)

18kl 21f3 61 f 18 51f12 29+4 29f2 59k9

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+ +

1.0 h

1.5 h

2.0 h

21fl 25+3 90k7 80+9 66f6 39+4 95+3

17+3 34f4 100 89f9 76+6 38+3

18+5 43+4 100 100 87+5 77+5

100

100

Results are expressed as an average of three experimentsf SD. BHA =butylated hydroxyanisole.

0

1

2

Tlme of lncubatlon (h) Figure 3. Glutathione depletion and formation of oxidized glutathione on treatment of hepatocytes with 2 mM bromotrichloromethane. Total GSH (A); Total GSSG corrected for extracellular GSSG at time 0 (A)and Extracellular GSSG ( x ) formed in the presence of cystine (0.2 mM) added to the incubation medium. In the absence of BrCC13total GSH in control hepatocyteswas depleted 10%.

the trichloromethyl radical (CCl,) is formed. T h e bond dissociation energies of BrCCl, (49 kcal/mol) is less than CCI, (68 kcal/mol) (Walling 1957) and indicates that BrCCI, would be more readily reduced than CCl,. Bromotrichloromethane was also more effective than CC1, at inducing lipid peroxidation in vivo or in tissue slices (Sagai and Tappel 1979, Gavino et al. 1984). Lipid peroxidation (as determined by malonaldehyde) occurred immediately on addition of BrCCl, to hepatocytes under aerobic conditions and continued until cytotoxicity ensued. Lipid peroxidation however, was maximal at 2mM BrCC1, while cell death continued to increase at concentrations up to 5 mM. This may indicate that BrCC1, activation mechanisms are saturated at levels above 2 mM and/or that toxicity at concentrations above 2 mM could also involve solvent effects (Recknagel et al. 1989). One other note of caution is that the CCl, and BrCCl, probably undergo evaporation in our model system particularly because the procedure involves continuous gassing

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940

L. G. McGirr et al.

at 37°C. As would be expected this evaporation was considerable when chloroform (b.p. 60°C) was used (Ekstrom and Hogberg 1980). Bromotrichloromethane (b.p. 105OC) and particularly CCl, (b.p. 76°C) may therefore be more toxic in a closed system than the results shown here. Incubation of isolated hepatocytes under a nitrogen atmosphere increased susceptibility to BrCCl, several fold even though lower levels of lipid peroxidation ensued. Carbon tetrachloride was also more cytotoxic under hypoxic conditions but was less cytotoxic than BrCCl, under hyperoxic conditions. Enhanced toxicity of CCl, and BrCCl, under hypoxic conditions has been demonstrated in several systems (Shen et al. 1982, LaCagnin et al. 1988). Although CC1,-induced lipid peroxidation in isolated hepatocytes has been shown to be 10-fold higher at 7 mmHg of oxygen, cytotoxicity was highest at 1mmHg (No11 et al. 1987). Oxygen therefore not only inhibits the reductive activation of CCl, by reduced cytochrome P-450 but is also required for lipid peroxidation. The much high susceptibility of hypoxic hepatocytes to BrCCl,, even though lipid peroxidation is more extensive under aerobic conditions, may indicate that a more specific and cytotoxic lipid peroxidation is induced in hypoxic hepatocytes. Previous investigators using isolated or cultured hepatocytes to study the molecular cytotoxic mechanisms for the hepatotoxin CCl, have shown that the antioxidants promethazine and propyl gallate prevented lipid peroxidation completely and protected against hepatocyte glucose 6-phosphatase inactivation (Poli et al. 1981, DeGroot and Haas 1981). An early cytotoxic effect (5 min) observed in CC1,-treated hepatocytes is an increase in cytosolic CaZ+ resulting from inhibition of the endoplasmic reticulum and mitochondria1 calcium pumps (Poli et al. 1988, Long and Moore 1986 a, b). The inhibition of microsomal Ca2+ sequestration has been attributed to lipid peroxidation more than to covalent binding (Waller et al. 1983). However, antioxidants did not prevent hepatocyte cytochrome P-450 destruction indicating that oxygen-independent covalent binding of CCl, to cytochrome P-450 may be responsible for the destruction (Frank et al. 1982). Evidence that lipid peroxidation also plays an important role in the process leading to cell death is that the antioxidant BHA was able to delay or protect the cells against BrCC1,-induced cytotoxicity under hyperoxic or hypoxic conditions. Butylated hydroxyanisole does not interact with CCl, or CC1,OO. thus, its effect appears to be specifically related to lipid peroxidation in this system. The connection between cell death and lipid peroxidation may be related to the specific location of the lipid peroxidation. With CCl, and BrCCI,, lipid peroxidation occurs primarily within the endoplasmic reticulum as a result of the reactivity of the radicals produced by reduced cytochrome P-450 or NADPH-cytochrome P-450 reductase. Lipid peroxidation eventually involves the plasma membrane (Ungemach 1985). Other compounds producing lipid peroxidation may induce more generalized and less cytotoxic lipid peroxidation. Previous investigators have shown that the antioxidant vitamin E prevented both CC1,-induced lipid peroxidation and cytotoxicity in isolated hepatocytes (Smith et al. 1983). However, the antioxidants disulphiram and diethyldithiocarbamate were recently shown to prevent lipid peroxidation without affecting hepatocyte injury (Dogterom et al. 1988) indicating that these antioxidants become cytotoxic during CCl, metabolism. Further evidence that lipid peroxidation plays an important role in BrCC1,induced cell death is the protective effect of the iron chelator, desferal. Desferroxamine has also previously been shown to protect against CC1,-induced hepatotoxic-

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Bromotrichloromethane cytotoxicty

941

ity in mice (Younes and Siegers 1985). The source of iron involved in the BrCCl, toxic mechanism is unknown but the haem protein cytochrome P-450 undergoes rapid inactivation as a result of the covalent binding by CCl, (Poli et al. 1981). Pretreatment of the hepatocytes with diethyl maleate to decrease intracellular GSH levels markedly increased hepatocyte sensitivity to BrCCl,. A similar enhancement was reported for CCl, (Poli et al. 1981). Although GSH has been shown to interact with CCl,OO- (Slater et al. 1985) and forms a radical adduct with CCl, (Connor et al. 1990), no significant changes in GSH levels in hepatocytes occurred. GSH leaked from the cell after membrane damage and was oxidized to GSSG in the media. This could also explain the slow decrease in G S H observed in CC1,-treated hepatocytes (Dogterom et al. 1988). Depletion of GSH as a result of conjugation with secondary lipid peroxidation products like 4-hydroxyalkenals (Alin et al. 1985) or with phosgene (Reiter and Burk 1988) was therefore not apparent. Liver GSH levels in rats were also unchanged by BrCC1, administration (Docks and Krishna 1976). The increased sensitivity to BrCCl, of hepatocytes with lower GSH levels could indicate that GSH oxidation and futile redox cycling occurs. Others have shown that GSH prevents BrCCl, binding to microsomes (Sipes et al. 1977). Glutathione oxidation would not be detectable if the rate of G S H oxidation (e.g. by radicals or during lipid peroxidation) was slower than the rate of GSSG reduction. Ascorbate also increased the BrCCl, cytotoxicity towards hepatocytes indicating that ascorbate increases the formation of cytotoxic CCl, or CCl,OO-. The addition of BrCCl, to a solution of ascorbic acid resulted in oxygen uptake which was catalaseinsensitive indicating that the oxygen uptake resulted from the reaction of CCl, with 0,to give CC1,OO.. In addition, this system resulted in additional oxygen uptake in the presence of arachidonic acid. The formation of CCl, was not the result of the direct reduction of BrCCl, by ascorbate since the addition of ascorbate to BrCCl, (reverse order) did not result in extensive oxygen uptake. Also, this system was sensitive to superoxide dismutase and the iron chelators, desferal and DETAPAC indicating the involvement of iron in the formation of superoxide. Superoxide radicals have been shown to reduce both CCl, and BrCCl, to the trichloromethyl radical (Janzen et al. 1985, Roberts and Sawyer 1981). Whether this occurs extracellularly or intracellularly is not clear. This type of activation mechanism would probably not play much of a role in BrCCl, hepatocyte toxicity because GSH prevents this activation mechanism. In conclusion, although cytotoxicity does not always correlate with the extent of lipid peroxidation, peroxidation of essential lipids is one of the pathways for bromotrichloromethane-induced cytotoxicity in isolated hepatocytes and GSH plays a protective role.

Acknowledgements This work was supported by the Medical Research Council and the Natural Sciences and Engineering Research Council of Canada. References ALIN, P., DANIELSON, U. H., and MANNERVIK, B., 1985, 4-Hydroxyalk-2-enals are substrates for glutathione transferase. Federation of European Biological Societies Letters, 179, 267-270. BECKER, E., MESSNER, B., and BERNDT,J., 1987, Two mechanisms of CCl, induced fatty liver. Free Radical Research Communications, 3. 299-308.

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BUEGE, J. A,, and AUST,S. D., 1978,Microsomal lipid peroxidation. In Methods in Enzymology, Vol. 52, edited by S. Fleischer and L. Packer, (New York: Academic Press), pp. 302-310. BURDINO, E., GRAVELA, F., UGAZIO, G., VANNINI, V., and CALLIGARO, A., 1973, Initiation of free radical reactions and hepatoxocity in rats poisoned with carbon tetrachloride or bromotrichloromethane. Agents and Actions, 4, 244-253. CAWTHORNE, M. A., BUNYAN, J., SENNITT, M. W., and GREEN,J., 1970,Vitamin E and hepatotoxic agents. Vitamin E, synthetic antioxidants and carbon tetrachloride toxicity in the rat. British Journal of Nutrition, 24, 357-384. CONNOR, H. D., LACAGNIN, L. B., KNECHT, K. T., THURMAN, R. G., and MASON, R. P., 1990,Reaction of glutathione with a free radical metabolite of carbon tetrachloride. Molecular Pharmacology, 37,

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Bromotrichloromethane cytotoxicty

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Molecular mechanisms for bromotrichloromethane cytotoxicity in isolated rat hepatocytes.

1. Bromotrichloromethane added to isolated rat hepatocytes resulted in increased cell death as determined by trypan blue uptake. Toxicity increased in...
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