ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSKS
Vol. 188, No. 1, May, pp. 122-129, 1978
Metabolic Toxicity
Activation
of Bromobenzene
and Hepatotoxicity
in Hepatocytes
Isolated
Diethylmaleate-Treated
HJORDIS JOHAN Department
THOR,
HOGBERG,
PETER
of Forensic Medicine, Received September
J. REED,*
Karolinska
and
Rats’
MOLDgUS,
DONALD
from Phenobarbital-
ROLF HERMANSON, AND
STEN
ORRENIUS
Znstitutet, S-104 01 Stockholm,
15, 1977; revised January
Sweden
16, 1978
Hepatocytes freshly isolated from diethylmaleate-treated rats exhibited a markedly decreased concentration of reduced glutathione (GSH) which increased to the level present in hepatocytes from nontreated rats upon incubation in a complete medium. When bromobenzene was present in the medium, however, this increase in GSH concentration upon incubation was reversed and a further decrease occurred that resulted in GSH depletion and cell death. This was prevented by metyrapone, an inhibitor of the cytochrome P-450linked metabolism of bromobenzene. Bromobenzene metabolism in hepatocytes was accompanied by a fraction of metabolites covalently binding to cellular proteins. The size of this fraction, relative to the amount of total metabolites, was increased in hepatocytes isolated from diethylmaleate-treated rata and in hepatocytes from phenobarbital-treated rats incubated with bromobenzene in the presence of 1,2-epoxy-3,3,3-trichloropropane, an inhibitor of microsomal epoxide hydrase which, however, also acted as a GSH-depleting agent. In addition, the metabolism of bromobenzene by hepatocytes was associated with a marked decrease in various coenzyme levels, including coenzyme A, NAD(H), and NADP(H). Cysteine and cysteamine inhibited the formation of protein-bound metabolites of bromobenzene in microsomes, but did not prevent bromobenzene toxicity in hepatocytes when added at higher concentrations to the incubation medium (containing 0.4 mM cysteine). Metbionine, on the other hand, did not cause a significant effect on bromobenzene metabolism in microsomes and prevented toxicity in bepatocytes, presumably by stimulating GSH synthesis and thereby decreasing the amount of reactive metabolites available for interaction with other cellular nucleophiles. It is concluded that, in contrast to hepatocytes with normal levels of GSH, hepatocytes from diethylmaleate-treated rats were sensitive to bromobenzene toxicity under our incubation conditions. In this system, bromobenzene metabolism led to GSH depletion and was associated with a progressive decrease in coenzyme A and nicotinamide nucleotide levels and a moderate increase in the formation of metabolites covalently bound to protein. Methionine was a potent protective agent which probably acted by enhanced GSH synthesis via the formation of cystathionine.
As discussed in the previous paper (l), bromobenzene-induced hepatotoxicity is probably mediated by the cytochrome P450-dependent formation of the chemically ’ Supported by Grant 03X-2471 from the Swedish Medical Research Council. An American Cancer Society Eleanor Roosevelt-International Cancer Fellowship awarded to D. J. Reed by the International Union Against Cancer is also gratefully acknowledged. ’ Permanent address: Department of Biochemistry and Biophysics, Oregon State University. Corvallis, Oregon 97331.
a Abbreviations used: GSH, glutathione, reduced form; DEM, diethylmaleate; TCPO, 1,2-epoxy-3.3,3trichloropropane; Hepes. N-2-hydroxyethylpiperazinc-iV’-2-ethanesulfonic acid; SKF 525-A, 2-diethylaminoethyl-2,2-diphenylvalerate. 122
0003-9861/78/1881-0122$02,00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
reactive bromobenzene-3,4-epoxide (2) and preceded by GSH3 depletion (2). The production of an electrophilic product during bromobenzene metabolism is indicated not only by GSH depletion, but also by the formation of a fraction of metabolites covalently bound to tissue macromolecules
BROMOBENZENE
TOXICITY
(3). The mechanism(s) by which reactive metabolites such as bromobenzene-3,4epoxide produce cytotoxicity is, however, not known, although it has been speculated that a nucleophilic attack on critical cellular macromolecules may be involved (4). In an attempt to elucidate further the mechanism(s) of action of reactive drug metabolites at the cellular level, we have incubated isolated rat hepatocytes with bromobenzene and monitored metabolite production as well as effects on various cell functions, including plasma membrane integrity. The incubation of hepatocytes with bromobenzene in a complete medium for up to 10 h failed, however, to produce cytotoxicity and it was concluded that resynthesis of GSH during incubation prevented depletion of this thiol and thereby cell death (1). In the present study the rats were pretreated with diethylmaleate to lower the hepatic GSH level (5) prior to the isolation of hepatocytes. Under these conditions subsequent incubation with bromobenzene, even in a complete medium, was associated with a markedly accelerated rate of cell death. Diethylmaleate effects appeared to be limited to depletion of intracellular GSH. Possible mechanisms of bromobenzene-induced cytotoxicity and the protective role of GSH, and GSH resynthesis during incubation, are discussed. MATERIALS
AND
METHODS
Male Sprague-Dawley rats, 200-250 g, were used. Phenobarbital-treated animals were given sodium phenobarbital ip at a daily dose of 80 mg/kg for 3 days. Diethylmaleate (Aldrich), as a 20% solution in corn oil, was administered ip 1 h prior to hepatocyte isolation, at a dose of 0.6 nmol/kg (5). Hepatocyte isolation was performed by collagenase perfusion as previously described (6). The yield of each preparation was 2-4 X 10’ cells, as measured by counting the final cell suspension in a Buerker chamber. Immediately after isolation, the cells excluded both trypan blue and NADH (90-106%) and contained high levels of NADP(H) and ATP. Incubations were performed at 37°C in rotating round-bottomed flasks (7) under a 95% O&j% COz atmosphere, at a cell concentration of 10” cells/ml, in Waymouth medium (MB 752/l, Gibco Biocult Ltd., Scotland) supplemented with horse serum (17.5%), Hepes buffer (25 mM, pH 7.4), heparin (10 Ill/ml), and penicillin (109 IU/ml) (complete medium). The intracellular level of GSH was estimated by
IN ISOLATED
HEPATOCYTES
123
the method of Saville (8). Measurements were performed on 10” cells, reharvested by gentle centrifugation (8Og) and washed once with Krebs-Henseleit buffer, pH 7.4, containing 2% albumin. One milliliter of 6.5% trichloroacetic acid was added to the reharvested cells and, following centrifugation, a 0.5.ml aliquot of the supernatant was used for analysis. The penetration of NADH into the cells was measured using the lactate dehydrogenase latency test (6, 7). An aliquot of well-mixed hepatocyte suspension was diluted twofold in Krebs-Henseleit buffer, pH 7.4, containing 2% albumin; NADH (0.1 mM final concentration) and pyruvate (0.76 mM final concentration) were then added. The rate of NADH oxidation was recorded at 340 nm, and 100% activity was obtained after lysis of the cells by the addition of Triton X-100 (0.5% final concentration). ATP was assayed in l-ml samples of the hepatocyte incubation mixture after precipitation with 1 N perchloric acid and centrifugation according to Lamprecht and Trautschold (9). Extraction and quantitation of oxidized and reduced nicotinamide nucleotides from hepatocytes (lml samples of the hepatocyte incubation mixture) were performed as described by Klingenberg (10). Coenzyme A (reduced, oxidized, and conjugated) was determined in l-ml aliquots of the hepatocyte incubation mixture according to Michal and Bergmeyer (11). After precipitation with 0.2 ml of 0.1 N perchloric acid and centrifugation, the supernatant was pH adjusted to ~7 with K&O:3 and 0.25 ml was used for analysis. The metabolism of bromobenzene was measured according to Zampaglione et al. (12). [‘4C]Bromobenzene (67 dpm/nmol), dissolved in dimethyl sulfoxide, was added to the incubation mixture to a final concentration of 0.6 mM. In order to keep the bromobenzene concentration constant during incubation, this substance being very volatile, bromobenzene at 40% of the initial concentration had to be added every halfhour. Bromobenzene metabolites were assayed in l-ml aliquotes of the incubation mixture. [‘4C]Bromobenzene covalently bound to protein was determined from the same aliquot using the method of Reid et al. (13). Rat liver microsomes were isolated according to Ernster et al. (14). Incubations with microsomes were performed in sealed flasks in 50 mM Tris-HCl buffer, pH 7.5, containing 10 mM MgCl*, a NADPH-generating system, and 1 mM [‘4C]bromobenzene. Incubations were for up to 15 min and the protein concentration was 1 mg/ml. The metabolism and covalent binding of [‘“Clbromobenzene were measured as described above. Collagenase was obtained from Boehringer/ Mannheim GmbH, Mannheim, Germany, bromobenzene (more than 97% pure) from BDH Chemicals Ltd., England, and [“Clbromobenzene (5.0 Poole, mCi/mmol, 99% purity) from the Radiochemical Center, Amersham, Bucks, England. SKF 525-A was a gift
124
THOR
ET AL.
from Smith, Kline and French Laboratories, Welwyn Garden City, England, as was metyrapone from Ciba-Geigy AG, Basel. All other chemicals were of analytical grade and obtained from local commercial sources. RESULTS
Bromobenzene Toxicity in Hepatocytes from DEM- Treated Rats ATP and NADP(H) levels were similar in hepatocytes freshly isolated from DEMtreated and nontreated rats, and the changes in these levels, and in trypan blue exclusion frequency and NADH penetration, upon incubation in complete medium for up to 5 h were negligible. The intracellular concentration of GSH in hepatocytes from DEM-treated rats was, however, only 20-30% of that in cells from nontreated rats. Upon incubation in complete medium, the concentration of GSH increased at a rate of 5 to 10 nmol/h/lO” cells [cf. Ref. (15)]. Bromobenzene metabolism was somewhat decreased in hepatocytes from rats given a combined treatment of phenobarbital and DEM as compared to those from rats treated with phenobarbital alone. In addition, bromobenzene markedly accelerated cell death after DEM pretreatment. This is illustrated in Fig. 1, which shows that the cells from DEM-treated rats ex-
hOU5
eluded exogenous NADH to approximately 25% of that at zero time after 5 h of incubation with bromobenzene; the corresponding value for cells from non-DEM-treated rats was about 80%. As briefly stated above, the incubation of hepatocytes from DEM-treated rats in complete medium led to a rather rapid and extensive increase in the cellular GSH level. In the presence of bromobenzene, however, there was no increase, but rather a slow, continuous decrease, in GSH concentration, which was associated with enhanced plasma membrane permeability to NADH (Fig. 2). As also shown in Fig. 2, the effects of bromobenzene on both GSH concentration and NADH penetration were prevented when metyrapone was present during incubation. The presence of metyrapone alone had no detectable effect. Production
FIG. 1. Bromobenzene metabolism and its effect on NADH penetration in hepatocytes isolated from phenobarbital and phenobarbitalplus DEM-treated rats. (-) Phenobarbital-treated, (- - -) phenobarbital- plus DEM-treated, (0) metabolites, (0) NADH penetration. One typical experiment of five. Bromobenzene concentration was kept at 0.6 mM.
hOWS
FIG. 2. Effect of bromobenzene metabolism in the presence and absence of metyrapone on GSH level (A) and NADH penetration (B) in hepatocytes isolated from phenobarbitalplus DEM-treated rat. (--) No addition, (---) bromobenzene (0.6 mM), (-.- .-) bromobenzene + metyrapone (0.5 mM). One typical experiment of three.
of Reactive Metabolites
The formation of reactive metabolites during the incubation of hepatocytes with bromobenzene was indicated not only by the decrease in GSH but also by the formation of metabolites strongly bound to protein. As with the total amount of metabolites, this fraction was increased severalfold after phenobarbital pretreatment of the rats and decreased when metyrapone was present during incubation (Table I). Pretreatment with DEM, or the presence of TCPO during incubation, caused a further increase in the amount of protein-
BROMOBENZENE
TOXICITY
IN ISOLATED
bound metabolites, which under these conditions constituted a somewhat larger fraction of the total metabolites. This increase in the amount of protein-bound metabolites after DEM treatment, or in the presence of TCPO, was observable only during the early phase of the incubation, which was presumably due to accelerated cell death and loss of metabolic activity under the influence of these agents. The fact that TCPO was even more efficient than DEM pretreatment in increasing the early formation of protein-bound metabolites of bromobenzene may be ascribed to its dual effect as a GSH-depleting agent and an inhibitor of epoxide hydrase. During the metabolism of bromobenzene TABLE
125
HEPATOCYTES
in hepatocytes isolated from phenobarbitaltreated rats, there was a progressive decrease in the concentration of various coenCoA, NAD(H), and zymes, including NADP(H) (Fig. 3). Both the free and bound forms of CoA and the oxidized and reduced forms of the nicotinamide nucleotides were affected. The decrease was apparent after 2 to 3 h of incubation and pronounced after 5 h, when the concentration of CoA had decreased to 50%, NADP(H) to 55%, and NAD(H) to 35% of the corresponding levels in freshly isolated cells. The effects of bromobenzene metabolism on the coenzyme levels were further enhanced by pretreatment of the rats with DEM (Fig. 3). A high level of ATP in hepatocytes from phenoI
THEFORMATIONOF
PHOTEIN-BOUNDMETABOLITES OFBROMOBENZENEINHEPATOCYTES ANn THEEFFECT OFPRETREATMENTANDINHIBITORS~ Addition (mM) Pretreatment Protein-bound metabolites (nmol/lO” cells)” l-h Incubation
5-h Incubation
None None Metyrapone
(0.5)
0.8 k 0.28 (4) 0.3 (2)
4.4 -t 0.56 (5) 1.1 (2)
None Metyrapone (0.5) TCPO (0.5)
3.4 -t 0.48 (11) 1.2 (2) 7.1 (3)
9.0 f 0.61 (6) 5.1 (2) 9.3 (3)
None Metyrapone
4.6 f 0.38 (6) 0.5 (2)
6.2 f 0.61 (7) 1.2 (2)
Phenobarbital
Phenobarbital and DEM (0.5)
a The final concentration of bromobenzene was 0.6 mM. ’ The values given are means -C SE. The number of experiments
is indicated
within
parentheses.
FIG. 3. Effect of bromobenzene metabolism on NAD(H) (A), NADP(H) (B), and coenzyme A (C) levels in hepatocytes isolated from phenobarbital-treated and phenobarbitaland DEMtreated rats. (M) Phenobarbital-treated, no addition; (M) phenobarbital-treated, 0.6 mM bromobenzene; (W) phenobarbitalplus DEM-treated, 0.6 rnM bromobenzene. In this experiment, the concentrations at zero time were the following: NAD(H), 7.1 nmol/lO” cells, NADP(H), 6.7 nmol/lO“ cells; and CoA, 5.3 nmol/lO” cells (hepatocytes from phenobarhitaltreated rats); and NAD(H), 6.9 nmol/lO” cells, NADP(H), 6.4 nmol/lO” cells; and CoA, 5.0 nmol/lO” cells (hepatocytes from phenobarbitalplus DEM-treated rats).
126
THOR
ET AL.
barbital-treated rats also after 5 h of incubation with bromobenzene indicates that the loss of coenzymes was not due to leakage through a damaged plasma membrane. Protection ity
against
Bromobenzene
Toxic-
As discussed in the previous paper (l), and also indicated by the present results, the level of intracellular GSH correlated well with the degree of bromobenzene toxicity in isolated hepatocytes. Accordingly, the presence of GSH in the microsomal incubates effectively decreased the fraction of protein-bound bromobenzene metabolites (1). Although to a lesser extent, a decrease in the formation of protein-bound metabolites of bromobenzene was also observed when
FIG. 4. Effects of cysteine and cysteamine on overall bromobenzene metabolism (A) and formation of protein-bound metabolites (B) in microsomes from phenobarbital-treated rat. (y) Bromobenzene (1 mM); (---) plus cysteine (5 mM); (-.-) plus cysteamine (1.5 mM); (-. -) phs methionite (3 mM). One typical experiment of three. TABLE
cysteine or cysteamine was added to the microsomal incubate (Fig. 4). Methionine, on the other hand, caused no apparent decrease in the quantity of protein-bound metabolites. Nor was any apparent effect on the formation of protein-bound bromobenzene metabolites in microsomes caused by the addition of serine (3 mM), DNA (0.5 mg/ml), RNA (0.5 mg/ml), or albumin (0.5 mg/ml) to the incubate, whereas the presence of CoA (1 mM) decreased the proportion of protein-bound metabolites. Although both cysteine and cysteamine decreased the size of the fraction of proteinbound metabolites in microsomes, the addition of either of these nucleophiles did not prevent bromobenzene toxicity in hepatocytes from phenobarbitaland DEMtreated rats incubated in a complete medium (Table II). In fact, in our system, cysteamine at a concentration of 1 InM caused an accelerated rate of cell death. Neither did the addition of cysteine (3 mM) to the medium (containing 0.4 mM cysteine) prevent the further decrease in GSH concentration observed in the presence of bromobenzene. In contrast, the addition of methionine (5 mM) to the incubation medium (containing 0.2 mM methionine) markedly decreased bromobenzene toxicity in hepatocytes from phenobarbitaland DEMtreated rats, and the rate and extent of GSH resynthesis were similar to those observed in the absence of bromobenzene and without extra methionine (Fig. 5). In the absence of bromobenzene, the addition of methionine (5 mM) to the medium caused a marked stimulation of both the rate and extent of GSH resynthesis in hepatocytes II
EFFECTS OF CYSTEINE, CYSTEAMINE, AND METHIONINE ON GSH LEVELS AND NADH PENETRATION DURING THE METABOLISM OF BROMOBENZ~NE IN HEPATOCYTES ISOI~ATED FROM PHENOBARBITAL- AND DEM-TREATED RATS” Addition
NADH penetration (% change from zero-time level)
(mM)
l-h Incubation None Bromobenzene Bromobenzene Bromobenzene Bromobenzene
(0.6) + cysteine (3) + cysteamine (1) + methionine (5)
n One typical experiment
of three.
2 5 7 25 5
5-h Incubation
GSH (nmol/lOfi l-h Incubation
3 73 75 97
12 7 6 6
11
9
cells)
5-h Incubation 33 5 3 3 27
BROMOBENZENE
TOXICITY
FIG. 5. Effect of methionine on GSH level (A) and NADH penetration (B) in hepatocytes isolated from phenobarbitaland DEM-treated rats and incubated in the presence or absence of bromobenzene. (-) No addition; (---) bromobenzene (0.6 mM); (-.-) me(-- --) bromobenzene plus methiothionine (5 mM); nine. One typical experiment of three.
from phenobarbital[cf. Ref. (15)].
and DEM-treated
rats
DISCUSSION
The results of several in uiuo studies have shown that hepatotoxicity produced by bromobenzene, acetaminophen, and a number of other agents correlates well with prior GSH depletion (2). Further, lowering the hepatic GSH level by pretreatment with other GSH-depleting compounds, e.g., DEM (5), has been found to potentiate the effects of these hepatotoxins. Conversely, a decreased toxicity of these agents has been observed under conditions that facilitate GSH biosynthesis (16). The results of the present study further underline the critical role of GSH in preventing bromobenzene toxicity in hepatocytes. In fact, bromobenzene-induced toxicity was only observed in hepatocytes with a lowered level of GSH obtained by pretreatment of the animals with DEM. In hepatocytes from non-DEM-treated rats incubated with bromobenzene in complete medium, the concentration of GSH never fell below 30% (ca. 10 nmol/106 cells) of that in freshly isolated cells (l), a level which appeared critical in our experiments. Cysteine and methionine, two amino acid precursors of GSH, and cysteamine have been extensively used to protect against liver damage caused by reactive drug metabolites, particularly metabolites of acetaminophen (17, 18). Cysteamine has been
IN ISOLATED
HEPATOCYTES
127
found to be the most efficient of these agents and is also widely used therapeutically in acetaminophen overdosage (17). Of the two amino acid precursors of GSH, methionine has been shown to be more effective (17). The addition to the incubation medium of cysteine or cysteamine, at concentrations that caused a significant decrease in the formation of protein-bound bromobenzene metabolites with microsomes, did not protect against bromobenzene toxicity in the hepatocytes (cf. Table II). Neither did the addition of extra cysteine (3 mM) to the medium stimulate GSH synthesis in hepatocytes from DEM-treated rats, indicating that the original concentration of this amino acid in the medium (0.4 mM) was not rate-limiting for GSH resynthesis in hepatocytes incubated with bromobenzene. However, extracellular cysteine may be a less effective GSH precursor than extracellular methionine (15). Upon the incubation of hepatocytes from DEM-treated rats in complete medium in the absence of bromobenzene, there was a rapid resynthesis of GSH, which was further stimulated by an increased concentration of methionine in the medium (cf. Fig. 5). Also, with bromobenzene present, the rate of increase in GSH concentration at this higher level of methionine (5 mM) was substantial and comparable to the rate observed in the absence of bromobenzene at a methionine concentration of 0.2 mM. As might be expected, the stimulatory effect of the higher concentration of methionine on GSH biosynthesis in hepatocytes from DEM-treated rats was associated with a marked decrease in the cytotoxic effect of bromobenzene (cf. Fig. 5). These data support the conclusion that, with isolated hepatocytes, methioninederived from protein turnover and the incubation medium-may provide a substantial part of the sulfur for GSH biosynthesis during extensive GSH utilization (15). They are also in accordance with the previous observations that methionine was equally effective as cysteine in the formation of mercapturic acid derivatives with bromobenzene in dogs (19, 20) and that methionine prevented the depletion of hepatic
128
THOR
GSH by ethionine in rats (21). Taken together, these findings indicate that a facilitated resynthesis of GSH may well explain the protective effect of methionine on hepatotoxicity produced by bromobenzene and certain other agents. Although it appears likely that bromobenzene toxicity is mediated by a chemically reactive metabolite, the mechanism(s) by which this metabolite produces toxicity is as yet unknown. The observations that liver necrosis caused by hepatoxins like bromobenzene and acetaminophen is preceded by GSH depletion and associated with an increase in the amount of metabolites covalently bound to liver protein (2) suggest that cytotoxicity is the result of a change in the balance between the formation of the reactive metabolite and its subsequent inactivation by GSH conjugation. They do not imply, however, that covalent labeling of tissue protein as such is the toxic mechanism; in our study, there was, for example, no substantial increase in the overall formation of protein-bound metabolites when hepatocytes were incubated with bromobenzene under conditions associated with accelerated cell death, i.e., when the hepatocytes were isolated from rats pretreated with both phenobarbital and DEM. The target may well be any one of a number of cellular components, including specific proteins, other macromolecules, and even smaller molecules such as the coenzymes investigated in this study [cf. Ref. (ZS)]. Since both CoA and the nicotinamide nucleotides have previously been found to undergo alkylation under similar conditions [cf. Ref. (23)], it seems quite possible that the decreased concentrations we have observed during bromobenzene metabolism in hepatocytes may have been due to nucleophilic attack by the reactive metabolite and perhaps also may have been contributory to the cytotoxic effect. The limited effect of TCPO on the formation of protein-bound metabolites of bromobenzene in microsomes suggests that the electrophilic metabolite formed, mainly bromobenzene-3,4-epoxide (2), may be a poor substrate for epoxide hydrase and thus probably a very reactive species. This is further indicated by the reactivity of this species with GSH, also in the absence of
ET AL.
glutathione-S-transferase. On the other hand, there seems to exist a certain specificity in regard to its interaction with various macromolecules; it bound to protein, but not to a measurable extent to lipid or nucleic acids. Whether these preliminary observations reveal properties of importance for the cytotoxic effect remains, however, to be established. REFERENCES 1. THOR, H., MOLDIXIS, P., KRISTOFF,HSON, A., HoaBERG, J., REED, D. J., AND ORRENI~JS, S. (1978) Arch. Biochem. Biophys. 188, 122. 2. JOI.I
OF BIOCHEMISTRY
AND BIOPHYSKS
Vol. 188, No. 1, May, pp. 122-129, 1978
Metabolic Toxicity
Activation
of Bromobenzene
and Hepatotoxicity
in Hepatocytes
Isolated
Diethylmaleate-Treated
HJORDIS JOHAN Department
THOR,
HOGBERG,
PETER
of Forensic Medicine, Received September
J. REED,*
Karolinska
and
Rats’
MOLDgUS,
DONALD
from Phenobarbital-
ROLF HERMANSON, AND
STEN
ORRENIUS
Znstitutet, S-104 01 Stockholm,
15, 1977; revised January
Sweden
16, 1978
Hepatocytes freshly isolated from diethylmaleate-treated rats exhibited a markedly decreased concentration of reduced glutathione (GSH) which increased to the level present in hepatocytes from nontreated rats upon incubation in a complete medium. When bromobenzene was present in the medium, however, this increase in GSH concentration upon incubation was reversed and a further decrease occurred that resulted in GSH depletion and cell death. This was prevented by metyrapone, an inhibitor of the cytochrome P-450linked metabolism of bromobenzene. Bromobenzene metabolism in hepatocytes was accompanied by a fraction of metabolites covalently binding to cellular proteins. The size of this fraction, relative to the amount of total metabolites, was increased in hepatocytes isolated from diethylmaleate-treated rata and in hepatocytes from phenobarbital-treated rats incubated with bromobenzene in the presence of 1,2-epoxy-3,3,3-trichloropropane, an inhibitor of microsomal epoxide hydrase which, however, also acted as a GSH-depleting agent. In addition, the metabolism of bromobenzene by hepatocytes was associated with a marked decrease in various coenzyme levels, including coenzyme A, NAD(H), and NADP(H). Cysteine and cysteamine inhibited the formation of protein-bound metabolites of bromobenzene in microsomes, but did not prevent bromobenzene toxicity in hepatocytes when added at higher concentrations to the incubation medium (containing 0.4 mM cysteine). Metbionine, on the other hand, did not cause a significant effect on bromobenzene metabolism in microsomes and prevented toxicity in bepatocytes, presumably by stimulating GSH synthesis and thereby decreasing the amount of reactive metabolites available for interaction with other cellular nucleophiles. It is concluded that, in contrast to hepatocytes with normal levels of GSH, hepatocytes from diethylmaleate-treated rats were sensitive to bromobenzene toxicity under our incubation conditions. In this system, bromobenzene metabolism led to GSH depletion and was associated with a progressive decrease in coenzyme A and nicotinamide nucleotide levels and a moderate increase in the formation of metabolites covalently bound to protein. Methionine was a potent protective agent which probably acted by enhanced GSH synthesis via the formation of cystathionine.
As discussed in the previous paper (l), bromobenzene-induced hepatotoxicity is probably mediated by the cytochrome P450-dependent formation of the chemically ’ Supported by Grant 03X-2471 from the Swedish Medical Research Council. An American Cancer Society Eleanor Roosevelt-International Cancer Fellowship awarded to D. J. Reed by the International Union Against Cancer is also gratefully acknowledged. ’ Permanent address: Department of Biochemistry and Biophysics, Oregon State University. Corvallis, Oregon 97331.
a Abbreviations used: GSH, glutathione, reduced form; DEM, diethylmaleate; TCPO, 1,2-epoxy-3.3,3trichloropropane; Hepes. N-2-hydroxyethylpiperazinc-iV’-2-ethanesulfonic acid; SKF 525-A, 2-diethylaminoethyl-2,2-diphenylvalerate. 122
0003-9861/78/1881-0122$02,00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
reactive bromobenzene-3,4-epoxide (2) and preceded by GSH3 depletion (2). The production of an electrophilic product during bromobenzene metabolism is indicated not only by GSH depletion, but also by the formation of a fraction of metabolites covalently bound to tissue macromolecules
BROMOBENZENE
TOXICITY
(3). The mechanism(s) by which reactive metabolites such as bromobenzene-3,4epoxide produce cytotoxicity is, however, not known, although it has been speculated that a nucleophilic attack on critical cellular macromolecules may be involved (4). In an attempt to elucidate further the mechanism(s) of action of reactive drug metabolites at the cellular level, we have incubated isolated rat hepatocytes with bromobenzene and monitored metabolite production as well as effects on various cell functions, including plasma membrane integrity. The incubation of hepatocytes with bromobenzene in a complete medium for up to 10 h failed, however, to produce cytotoxicity and it was concluded that resynthesis of GSH during incubation prevented depletion of this thiol and thereby cell death (1). In the present study the rats were pretreated with diethylmaleate to lower the hepatic GSH level (5) prior to the isolation of hepatocytes. Under these conditions subsequent incubation with bromobenzene, even in a complete medium, was associated with a markedly accelerated rate of cell death. Diethylmaleate effects appeared to be limited to depletion of intracellular GSH. Possible mechanisms of bromobenzene-induced cytotoxicity and the protective role of GSH, and GSH resynthesis during incubation, are discussed. MATERIALS
AND
METHODS
Male Sprague-Dawley rats, 200-250 g, were used. Phenobarbital-treated animals were given sodium phenobarbital ip at a daily dose of 80 mg/kg for 3 days. Diethylmaleate (Aldrich), as a 20% solution in corn oil, was administered ip 1 h prior to hepatocyte isolation, at a dose of 0.6 nmol/kg (5). Hepatocyte isolation was performed by collagenase perfusion as previously described (6). The yield of each preparation was 2-4 X 10’ cells, as measured by counting the final cell suspension in a Buerker chamber. Immediately after isolation, the cells excluded both trypan blue and NADH (90-106%) and contained high levels of NADP(H) and ATP. Incubations were performed at 37°C in rotating round-bottomed flasks (7) under a 95% O&j% COz atmosphere, at a cell concentration of 10” cells/ml, in Waymouth medium (MB 752/l, Gibco Biocult Ltd., Scotland) supplemented with horse serum (17.5%), Hepes buffer (25 mM, pH 7.4), heparin (10 Ill/ml), and penicillin (109 IU/ml) (complete medium). The intracellular level of GSH was estimated by
IN ISOLATED
HEPATOCYTES
123
the method of Saville (8). Measurements were performed on 10” cells, reharvested by gentle centrifugation (8Og) and washed once with Krebs-Henseleit buffer, pH 7.4, containing 2% albumin. One milliliter of 6.5% trichloroacetic acid was added to the reharvested cells and, following centrifugation, a 0.5.ml aliquot of the supernatant was used for analysis. The penetration of NADH into the cells was measured using the lactate dehydrogenase latency test (6, 7). An aliquot of well-mixed hepatocyte suspension was diluted twofold in Krebs-Henseleit buffer, pH 7.4, containing 2% albumin; NADH (0.1 mM final concentration) and pyruvate (0.76 mM final concentration) were then added. The rate of NADH oxidation was recorded at 340 nm, and 100% activity was obtained after lysis of the cells by the addition of Triton X-100 (0.5% final concentration). ATP was assayed in l-ml samples of the hepatocyte incubation mixture after precipitation with 1 N perchloric acid and centrifugation according to Lamprecht and Trautschold (9). Extraction and quantitation of oxidized and reduced nicotinamide nucleotides from hepatocytes (lml samples of the hepatocyte incubation mixture) were performed as described by Klingenberg (10). Coenzyme A (reduced, oxidized, and conjugated) was determined in l-ml aliquots of the hepatocyte incubation mixture according to Michal and Bergmeyer (11). After precipitation with 0.2 ml of 0.1 N perchloric acid and centrifugation, the supernatant was pH adjusted to ~7 with K&O:3 and 0.25 ml was used for analysis. The metabolism of bromobenzene was measured according to Zampaglione et al. (12). [‘4C]Bromobenzene (67 dpm/nmol), dissolved in dimethyl sulfoxide, was added to the incubation mixture to a final concentration of 0.6 mM. In order to keep the bromobenzene concentration constant during incubation, this substance being very volatile, bromobenzene at 40% of the initial concentration had to be added every halfhour. Bromobenzene metabolites were assayed in l-ml aliquotes of the incubation mixture. [‘4C]Bromobenzene covalently bound to protein was determined from the same aliquot using the method of Reid et al. (13). Rat liver microsomes were isolated according to Ernster et al. (14). Incubations with microsomes were performed in sealed flasks in 50 mM Tris-HCl buffer, pH 7.5, containing 10 mM MgCl*, a NADPH-generating system, and 1 mM [‘4C]bromobenzene. Incubations were for up to 15 min and the protein concentration was 1 mg/ml. The metabolism and covalent binding of [‘“Clbromobenzene were measured as described above. Collagenase was obtained from Boehringer/ Mannheim GmbH, Mannheim, Germany, bromobenzene (more than 97% pure) from BDH Chemicals Ltd., England, and [“Clbromobenzene (5.0 Poole, mCi/mmol, 99% purity) from the Radiochemical Center, Amersham, Bucks, England. SKF 525-A was a gift
124
THOR
ET AL.
from Smith, Kline and French Laboratories, Welwyn Garden City, England, as was metyrapone from Ciba-Geigy AG, Basel. All other chemicals were of analytical grade and obtained from local commercial sources. RESULTS
Bromobenzene Toxicity in Hepatocytes from DEM- Treated Rats ATP and NADP(H) levels were similar in hepatocytes freshly isolated from DEMtreated and nontreated rats, and the changes in these levels, and in trypan blue exclusion frequency and NADH penetration, upon incubation in complete medium for up to 5 h were negligible. The intracellular concentration of GSH in hepatocytes from DEM-treated rats was, however, only 20-30% of that in cells from nontreated rats. Upon incubation in complete medium, the concentration of GSH increased at a rate of 5 to 10 nmol/h/lO” cells [cf. Ref. (15)]. Bromobenzene metabolism was somewhat decreased in hepatocytes from rats given a combined treatment of phenobarbital and DEM as compared to those from rats treated with phenobarbital alone. In addition, bromobenzene markedly accelerated cell death after DEM pretreatment. This is illustrated in Fig. 1, which shows that the cells from DEM-treated rats ex-
hOU5
eluded exogenous NADH to approximately 25% of that at zero time after 5 h of incubation with bromobenzene; the corresponding value for cells from non-DEM-treated rats was about 80%. As briefly stated above, the incubation of hepatocytes from DEM-treated rats in complete medium led to a rather rapid and extensive increase in the cellular GSH level. In the presence of bromobenzene, however, there was no increase, but rather a slow, continuous decrease, in GSH concentration, which was associated with enhanced plasma membrane permeability to NADH (Fig. 2). As also shown in Fig. 2, the effects of bromobenzene on both GSH concentration and NADH penetration were prevented when metyrapone was present during incubation. The presence of metyrapone alone had no detectable effect. Production
FIG. 1. Bromobenzene metabolism and its effect on NADH penetration in hepatocytes isolated from phenobarbital and phenobarbitalplus DEM-treated rats. (-) Phenobarbital-treated, (- - -) phenobarbital- plus DEM-treated, (0) metabolites, (0) NADH penetration. One typical experiment of five. Bromobenzene concentration was kept at 0.6 mM.
hOWS
FIG. 2. Effect of bromobenzene metabolism in the presence and absence of metyrapone on GSH level (A) and NADH penetration (B) in hepatocytes isolated from phenobarbitalplus DEM-treated rat. (--) No addition, (---) bromobenzene (0.6 mM), (-.- .-) bromobenzene + metyrapone (0.5 mM). One typical experiment of three.
of Reactive Metabolites
The formation of reactive metabolites during the incubation of hepatocytes with bromobenzene was indicated not only by the decrease in GSH but also by the formation of metabolites strongly bound to protein. As with the total amount of metabolites, this fraction was increased severalfold after phenobarbital pretreatment of the rats and decreased when metyrapone was present during incubation (Table I). Pretreatment with DEM, or the presence of TCPO during incubation, caused a further increase in the amount of protein-
BROMOBENZENE
TOXICITY
IN ISOLATED
bound metabolites, which under these conditions constituted a somewhat larger fraction of the total metabolites. This increase in the amount of protein-bound metabolites after DEM treatment, or in the presence of TCPO, was observable only during the early phase of the incubation, which was presumably due to accelerated cell death and loss of metabolic activity under the influence of these agents. The fact that TCPO was even more efficient than DEM pretreatment in increasing the early formation of protein-bound metabolites of bromobenzene may be ascribed to its dual effect as a GSH-depleting agent and an inhibitor of epoxide hydrase. During the metabolism of bromobenzene TABLE
125
HEPATOCYTES
in hepatocytes isolated from phenobarbitaltreated rats, there was a progressive decrease in the concentration of various coenCoA, NAD(H), and zymes, including NADP(H) (Fig. 3). Both the free and bound forms of CoA and the oxidized and reduced forms of the nicotinamide nucleotides were affected. The decrease was apparent after 2 to 3 h of incubation and pronounced after 5 h, when the concentration of CoA had decreased to 50%, NADP(H) to 55%, and NAD(H) to 35% of the corresponding levels in freshly isolated cells. The effects of bromobenzene metabolism on the coenzyme levels were further enhanced by pretreatment of the rats with DEM (Fig. 3). A high level of ATP in hepatocytes from phenoI
THEFORMATIONOF
PHOTEIN-BOUNDMETABOLITES OFBROMOBENZENEINHEPATOCYTES ANn THEEFFECT OFPRETREATMENTANDINHIBITORS~ Addition (mM) Pretreatment Protein-bound metabolites (nmol/lO” cells)” l-h Incubation
5-h Incubation
None None Metyrapone
(0.5)
0.8 k 0.28 (4) 0.3 (2)
4.4 -t 0.56 (5) 1.1 (2)
None Metyrapone (0.5) TCPO (0.5)
3.4 -t 0.48 (11) 1.2 (2) 7.1 (3)
9.0 f 0.61 (6) 5.1 (2) 9.3 (3)
None Metyrapone
4.6 f 0.38 (6) 0.5 (2)
6.2 f 0.61 (7) 1.2 (2)
Phenobarbital
Phenobarbital and DEM (0.5)
a The final concentration of bromobenzene was 0.6 mM. ’ The values given are means -C SE. The number of experiments
is indicated
within
parentheses.
FIG. 3. Effect of bromobenzene metabolism on NAD(H) (A), NADP(H) (B), and coenzyme A (C) levels in hepatocytes isolated from phenobarbital-treated and phenobarbitaland DEMtreated rats. (M) Phenobarbital-treated, no addition; (M) phenobarbital-treated, 0.6 mM bromobenzene; (W) phenobarbitalplus DEM-treated, 0.6 rnM bromobenzene. In this experiment, the concentrations at zero time were the following: NAD(H), 7.1 nmol/lO” cells, NADP(H), 6.7 nmol/lO“ cells; and CoA, 5.3 nmol/lO” cells (hepatocytes from phenobarhitaltreated rats); and NAD(H), 6.9 nmol/lO” cells, NADP(H), 6.4 nmol/lO” cells; and CoA, 5.0 nmol/lO” cells (hepatocytes from phenobarbitalplus DEM-treated rats).
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THOR
ET AL.
barbital-treated rats also after 5 h of incubation with bromobenzene indicates that the loss of coenzymes was not due to leakage through a damaged plasma membrane. Protection ity
against
Bromobenzene
Toxic-
As discussed in the previous paper (l), and also indicated by the present results, the level of intracellular GSH correlated well with the degree of bromobenzene toxicity in isolated hepatocytes. Accordingly, the presence of GSH in the microsomal incubates effectively decreased the fraction of protein-bound bromobenzene metabolites (1). Although to a lesser extent, a decrease in the formation of protein-bound metabolites of bromobenzene was also observed when
FIG. 4. Effects of cysteine and cysteamine on overall bromobenzene metabolism (A) and formation of protein-bound metabolites (B) in microsomes from phenobarbital-treated rat. (y) Bromobenzene (1 mM); (---) plus cysteine (5 mM); (-.-) plus cysteamine (1.5 mM); (-. -) phs methionite (3 mM). One typical experiment of three. TABLE
cysteine or cysteamine was added to the microsomal incubate (Fig. 4). Methionine, on the other hand, caused no apparent decrease in the quantity of protein-bound metabolites. Nor was any apparent effect on the formation of protein-bound bromobenzene metabolites in microsomes caused by the addition of serine (3 mM), DNA (0.5 mg/ml), RNA (0.5 mg/ml), or albumin (0.5 mg/ml) to the incubate, whereas the presence of CoA (1 mM) decreased the proportion of protein-bound metabolites. Although both cysteine and cysteamine decreased the size of the fraction of proteinbound metabolites in microsomes, the addition of either of these nucleophiles did not prevent bromobenzene toxicity in hepatocytes from phenobarbitaland DEMtreated rats incubated in a complete medium (Table II). In fact, in our system, cysteamine at a concentration of 1 InM caused an accelerated rate of cell death. Neither did the addition of cysteine (3 mM) to the medium (containing 0.4 mM cysteine) prevent the further decrease in GSH concentration observed in the presence of bromobenzene. In contrast, the addition of methionine (5 mM) to the incubation medium (containing 0.2 mM methionine) markedly decreased bromobenzene toxicity in hepatocytes from phenobarbitaland DEMtreated rats, and the rate and extent of GSH resynthesis were similar to those observed in the absence of bromobenzene and without extra methionine (Fig. 5). In the absence of bromobenzene, the addition of methionine (5 mM) to the medium caused a marked stimulation of both the rate and extent of GSH resynthesis in hepatocytes II
EFFECTS OF CYSTEINE, CYSTEAMINE, AND METHIONINE ON GSH LEVELS AND NADH PENETRATION DURING THE METABOLISM OF BROMOBENZ~NE IN HEPATOCYTES ISOI~ATED FROM PHENOBARBITAL- AND DEM-TREATED RATS” Addition
NADH penetration (% change from zero-time level)
(mM)
l-h Incubation None Bromobenzene Bromobenzene Bromobenzene Bromobenzene
(0.6) + cysteine (3) + cysteamine (1) + methionine (5)
n One typical experiment
of three.
2 5 7 25 5
5-h Incubation
GSH (nmol/lOfi l-h Incubation
3 73 75 97
12 7 6 6
11
9
cells)
5-h Incubation 33 5 3 3 27
BROMOBENZENE
TOXICITY
FIG. 5. Effect of methionine on GSH level (A) and NADH penetration (B) in hepatocytes isolated from phenobarbitaland DEM-treated rats and incubated in the presence or absence of bromobenzene. (-) No addition; (---) bromobenzene (0.6 mM); (-.-) me(-- --) bromobenzene plus methiothionine (5 mM); nine. One typical experiment of three.
from phenobarbital[cf. Ref. (15)].
and DEM-treated
rats
DISCUSSION
The results of several in uiuo studies have shown that hepatotoxicity produced by bromobenzene, acetaminophen, and a number of other agents correlates well with prior GSH depletion (2). Further, lowering the hepatic GSH level by pretreatment with other GSH-depleting compounds, e.g., DEM (5), has been found to potentiate the effects of these hepatotoxins. Conversely, a decreased toxicity of these agents has been observed under conditions that facilitate GSH biosynthesis (16). The results of the present study further underline the critical role of GSH in preventing bromobenzene toxicity in hepatocytes. In fact, bromobenzene-induced toxicity was only observed in hepatocytes with a lowered level of GSH obtained by pretreatment of the animals with DEM. In hepatocytes from non-DEM-treated rats incubated with bromobenzene in complete medium, the concentration of GSH never fell below 30% (ca. 10 nmol/106 cells) of that in freshly isolated cells (l), a level which appeared critical in our experiments. Cysteine and methionine, two amino acid precursors of GSH, and cysteamine have been extensively used to protect against liver damage caused by reactive drug metabolites, particularly metabolites of acetaminophen (17, 18). Cysteamine has been
IN ISOLATED
HEPATOCYTES
127
found to be the most efficient of these agents and is also widely used therapeutically in acetaminophen overdosage (17). Of the two amino acid precursors of GSH, methionine has been shown to be more effective (17). The addition to the incubation medium of cysteine or cysteamine, at concentrations that caused a significant decrease in the formation of protein-bound bromobenzene metabolites with microsomes, did not protect against bromobenzene toxicity in the hepatocytes (cf. Table II). Neither did the addition of extra cysteine (3 mM) to the medium stimulate GSH synthesis in hepatocytes from DEM-treated rats, indicating that the original concentration of this amino acid in the medium (0.4 mM) was not rate-limiting for GSH resynthesis in hepatocytes incubated with bromobenzene. However, extracellular cysteine may be a less effective GSH precursor than extracellular methionine (15). Upon the incubation of hepatocytes from DEM-treated rats in complete medium in the absence of bromobenzene, there was a rapid resynthesis of GSH, which was further stimulated by an increased concentration of methionine in the medium (cf. Fig. 5). Also, with bromobenzene present, the rate of increase in GSH concentration at this higher level of methionine (5 mM) was substantial and comparable to the rate observed in the absence of bromobenzene at a methionine concentration of 0.2 mM. As might be expected, the stimulatory effect of the higher concentration of methionine on GSH biosynthesis in hepatocytes from DEM-treated rats was associated with a marked decrease in the cytotoxic effect of bromobenzene (cf. Fig. 5). These data support the conclusion that, with isolated hepatocytes, methioninederived from protein turnover and the incubation medium-may provide a substantial part of the sulfur for GSH biosynthesis during extensive GSH utilization (15). They are also in accordance with the previous observations that methionine was equally effective as cysteine in the formation of mercapturic acid derivatives with bromobenzene in dogs (19, 20) and that methionine prevented the depletion of hepatic
128
THOR
GSH by ethionine in rats (21). Taken together, these findings indicate that a facilitated resynthesis of GSH may well explain the protective effect of methionine on hepatotoxicity produced by bromobenzene and certain other agents. Although it appears likely that bromobenzene toxicity is mediated by a chemically reactive metabolite, the mechanism(s) by which this metabolite produces toxicity is as yet unknown. The observations that liver necrosis caused by hepatoxins like bromobenzene and acetaminophen is preceded by GSH depletion and associated with an increase in the amount of metabolites covalently bound to liver protein (2) suggest that cytotoxicity is the result of a change in the balance between the formation of the reactive metabolite and its subsequent inactivation by GSH conjugation. They do not imply, however, that covalent labeling of tissue protein as such is the toxic mechanism; in our study, there was, for example, no substantial increase in the overall formation of protein-bound metabolites when hepatocytes were incubated with bromobenzene under conditions associated with accelerated cell death, i.e., when the hepatocytes were isolated from rats pretreated with both phenobarbital and DEM. The target may well be any one of a number of cellular components, including specific proteins, other macromolecules, and even smaller molecules such as the coenzymes investigated in this study [cf. Ref. (ZS)]. Since both CoA and the nicotinamide nucleotides have previously been found to undergo alkylation under similar conditions [cf. Ref. (23)], it seems quite possible that the decreased concentrations we have observed during bromobenzene metabolism in hepatocytes may have been due to nucleophilic attack by the reactive metabolite and perhaps also may have been contributory to the cytotoxic effect. The limited effect of TCPO on the formation of protein-bound metabolites of bromobenzene in microsomes suggests that the electrophilic metabolite formed, mainly bromobenzene-3,4-epoxide (2), may be a poor substrate for epoxide hydrase and thus probably a very reactive species. This is further indicated by the reactivity of this species with GSH, also in the absence of
ET AL.
glutathione-S-transferase. On the other hand, there seems to exist a certain specificity in regard to its interaction with various macromolecules; it bound to protein, but not to a measurable extent to lipid or nucleic acids. Whether these preliminary observations reveal properties of importance for the cytotoxic effect remains, however, to be established. REFERENCES 1. THOR, H., MOLDIXIS, P., KRISTOFF,HSON, A., HoaBERG, J., REED, D. J., AND ORRENI~JS, S. (1978) Arch. Biochem. Biophys. 188, 122. 2. JOI.I
