Clofibrate-Induced in Vitro Hepatoprotection against Acetaminophen Is Not Due to Altered Glutathione Homeostasis

Felicity A. Nicholls-Grzemski*,1, Ian C. Calder{dagger}, Brian G. Priestly{ddagger} and Philip C. Burcham*,2

* Department of Clinical and Experimental Pharmacology, The University of Adelaide, Adelaide, South Australia 5005, Australia; {dagger} Environmental Health Branch, South Australian Health Commission, Adelaide, South Australia 5000; and {ddagger} Chemicals and Non-Prescription Medicines Branch, Therapeutic Goods Administration, PO Box 100, Woden, ACT 2606

Received January 10, 2000; accepted March 10, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prior induction of peroxisome proliferation protects mice against the in vivo hepatotoxicity of acetaminophen and various other bioactivation-dependent toxicants. The mechanisms underlying such chemoresistance are poorly understood, although they have been suggested to involve alterations in glutathione homeostasis. To clarify the role of glutathione in this phenomenon, we isolated hepatocytes from mice in which hepatic peroxisome proliferation had been induced with clofibrate. The cells were incubated with a range of acetaminophen concentrations and the extent of cell killing after up to 8 h was assessed by measuring lactate dehydrogenase leakage from the cells. Hepatocytes from clofibrate-pretreated mice were much less susceptible to acetaminophen than cells from vehicle-treated controls. However, the extent of glutathione depletion during exposure to acetaminophen was similar in both cell types, as were rates of excretion of the product of glutathione-mediated detoxication of acetaminophen's quinoneimine metabolite, 3-glutathionyl-acetaminophen. The glutathione-replenishing ability of clofibrate-pretreated cells after a brief exposure to diethyl maleate also resembled that of control cells. More importantly, prior depletion of glutathione by diethyl maleate did not abolish the resistance of clofibrate-pretreated cells to acetaminophen. Taken together, these findings indicate that although glutathione-dependent pathways may contribute to hepatoprotection during peroxisome proliferation, the resistance phenomenon is not due exclusively to this mechanism.

Key Words: peroxisome proliferation; acetaminophen toxicity; hepatoprotection.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisome proliferators are a diverse class of chemicals with a common ability to increase the size and number of hepatocellular peroxisomes (Ashby et al., 1994Go; Bentley et al., 1993Go). In recent years they have attracted considerable attention from molecular biologists interested in the transduction pathways that mediate peroxisome proliferation. This led to the identification of several ligand-activated transcription factors belonging to the steroid hormone receptor superfamily, the so-called peroxisome proliferator-activated receptors (PPARs) (Ashby et al., 1994Go; Mukherjee et al., 1997Go; Schoonjans et al., 1996Go). The various PPARs are thought to mediate most, if not all, of the effects of peroxisome proliferators. In addition to work on their molecular actions, peroxisome proliferators have also received attention from toxicologists, due to their ability to produce hepatocellular carcinomas in rats and mice during chronic exposure (Ashby et al., 1994Go; Elcombe et al., 1985Go; Rao and Reddy, 1987Go; Rao et al., 1988Go; Reddy et al., 1979Go).

A poorly understood property of peroxisome proliferators needing further investigation concerns their ability to diminish the toxicity of various other chemicals. Early work by Salas and associates established that prior treatment with the peroxisome proliferator nafenopin protected rats against hepatic necrosis and steatosis caused by cerium, a rare earth metal (Salas et al., 1976Go; Tuchweber and Salas, 1978Go). Similarly, pretreatment with the plasticiser di-2-ethylhexyl phthalate (DEHP) was shown to protect rats against fatty liver and mortality induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (Tomaszewski et al., 1988Go). In our initial work in this area, we found that prior exposure of mice to clofibrate, silvex, or DEHP dramatically reduced the severity of hepatic necrosis caused by high-dose acetaminophen (N-acetyl aminophenol, AAP) (Nicholls-Grzemski et al., 1991Go; Nicholls-Grzemski et al., 1992Go) and other bioactivation-dependent toxicants, namely carbon tetrachloride and bromobenzene (Nicholls-Grzemski et al., 1993Go; Nicholls-Grzemski et al., 1996Go). These findings were subsequently confirmed by Cohen and associates (Manautou et al., 1994Go; Manautou et al., 1998Go).

The mechanism(s) underlying the hepatoprotection afforded by peroxisome proliferators have yet to be clarified. In the case of AAP, its toxicity involves a CYP450-generated reactive quinoneimine (NAPQI) that forms upon saturation of conjugative Phase II metabolism (Dahlin et al., 1984Go). The toxic metabolite is rapidly conjugated with glutathione, although under overdose situations glutathione stores are depleted, and NAPQI forms covalent adducts with a range of cellular proteins (Hinson et al., 1994Go; Jollow et al., 1973Go; Mitchell et al., 1973aGo; Mitchell et al., 1973bGo).

Given the crucial role of glutathione during AAP toxicity, treatments that replete cellular stores of the tripeptide are effective antidotes against AAP (Oak and Choi, 1998Go; Uhlig and Wendel, 1990Go). Consequently, one explanation of the hepatoprotection afforded by peroxisome proliferators could be that the livers of pretreated animals more readily detoxicate NAPQI via glutathione-dependent pathways, perhaps due to an enhanced ability to maintain glutathione homeostasis during exposure to toxic electrophiles. In support of such a mechanism, Manautou and coworkers reported that the biliary excretion of 3-glutathionyl-AAP was enhanced about 2-fold in clofibrate-pretreated mice, and was accompanied by diminished adduction of hepatic proteins (Manautou et al., 1996aGo).

To further investigate the mechanisms underlying the hepatoprotection against AAP afforded by peroxisome proliferation, and to clarify the role of altered glutathione homeostasis in the resistance phenomenon, an in vitro model of isolated hepatocytes was used in the present study. Although hepatocytes from peroxisome-proliferated rodents have been used to investigate the toxicity of substances as diverse as hydrogen peroxide (Garberg et al., 1992Go) and chlorinated acetic acid analogues (Bruschi and Bull, 1993Go), their use in the investigation of AAP toxicity has not been reported. Because they permit close correlation of cell death with other biochemical changes, isolated liver cells are particularly useful during study of AAP toxicity (Adamson and Harman, 1989Go; Dawson et al., 1984Go; Mitchell et al., 1985Go; Shen et al., 1992Go). In addition, as sulfhydryl-altering agents can be used to modulate the capacity of hepatocytes to metabolize AAP via glutathione-dependent pathways, the role of this tripeptide in clofibrate-induced hepatoprotection can be examined (Dawson et al., 1984Go; Harman and Self, 1986Go; Massey and Racz, 1981Go; Mitchell et al., 1985Go). Furthermore, the use of isolated hepatocytes removes in vivo pharmacokinetic factors that influence the amount of xenobiotic delivered to the liver cell in vivo, thus removing a factor that can complicate interpretation of in vivo findings. Collectively, our present findings indicate that hepatocytes from clofibrate-pretreated mice resist cell killing by AAP, and that such hepatoprotection does not primarily involve altered glutathione homeostasis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Clofibrate was obtained from Serva Feinbiochemica, Heidelberg, Germany. Diethylmaleate was from Merck Biochemicals, Munich, Germany. Collagenase and acetaminophen came from the Sigma Chemical Co., St. Louis, MO, USA; powdered RPMI-1640 media was purchased from ICN Pharmaceutical Inc., Cleveland, OH, USA. All other chemicals used were of the highest quality commercially available and came from standard suppliers.

Animal treatment.
To achieve peroxisome proliferation, adult male Swiss mice (35–40 g) received daily doses of 500 mg/kg clofibrate (i.p) over a 10-day period. Control mice were treated in the same fashion with an equivalent volume of olive oil vehicle. During these treatments the mice were supplied ad libitum with standard rodent chow and water. They were housed in the University of Adelaide Medical School Animal Services Facility on a 12-h light/dark cycle.

Hepatocyte isolation and incubation.
Hepatocytes were prepared by collagenase digestion of the liver using a 2-step method described by Adamson and Harman (1989). Cell viability was assessed by the Trypan blue exclusion method and was routinely 80% or higher. Following 2 or 3 washing steps with Krebs-Henseleit solution, the cells were resuspended in RPMI-1640 media containing HEPES (10 mM), penicillin (100 Units/ml) and streptomycin (100 µg/ml). The cell suspensions were added to rat tail tendon collagen-coated dishes and then maintained in 5% CO2 at 37°C in a Thermoline 60A humidified incubator. After a 2-h period to allow attachment of viable cells, the plates were washed several times with Hanks-buffered saline (HBS). The medium was replaced with fresh RPMI-1640 containing various concentrations of acetaminophen (AAP, 10–3000 µM). At 1, 2, 4, and 8 h after commencing exposure to AAP, 60 µl aliquots of culture medium were taken for the determination of lactate dehydrogenase (LDH) activity. The latter was determined by measuring NADH formation during the enzymatic oxidation of lactate to pyruvate. Briefly, 50 µl of extracellular media was added to 0.5 ml of reagent containing 50 mM lactic acid and 7 mM NAD+ in 0.25 M tris-HCl buffer, pH 8.9. The change in absorbance at 340 nm was recorded over 1 min using a Hitachi U-2000 spectrophotometer. LDH activity at each time point was expressed as percentage of the total LDH activity on the plate at the completion of the experiment (to determine the latter, cells were lysed by adding 240 µl of 2% Triton-X100 to each plate).

Glutathione repletion experiments.
Diethyl maleate (DEM), a carbonyl compound that rapidly depletes cytosolic glutathione (Maellaro et al., 1994Go; Mitchell et al., 1985Go) was used to compare the glutathione-replenishing abilities of control versus clofibrate-pretreated cells. Briefly, hepatocyte monolayers were exposed to 500 µM DEM for 30 min to achieve glutathione depletion. The media containing DEM was then removed and the cells were washed with HBS before the medium was replaced with DEM-free RPMI-1640 media. After 1, 2, and 4 h, the culture media was removed, and the plates were washed three times with HBS before 1 ml of trichloroacetic acid (6.5%) was added to precipitate cellular proteins. The cellular matter was then scraped from the plates and transferred to Eppendorf tubes. After centrifugation, the acidic supernatant was analyzed for glutathione using a procedure based on the method of Saville (1958). The remaining pellets were dissolved in 0.5 ml of 0.5 M sodium hydroxide, and incubated at 40°C for 1 h; protein was then determined using the Pierce BCA kit with bovine serum albumin used as a standard. Glutathione levels were expressed as nmol/mg protein.

Determination of acetaminophen metabolites.
Glutathionyl-AAP was measured in hepatocyte culture media using an HPLC method described by Madhu and Klassen (1991). Briefly, 200-µl aliquots of culture media obtained from hepatocytes after 8-h incubations with AAP (10–3000 µM) were transferred to Eppendorf tubes that contained 200 µl cold methanol. The samples were then resolved on a phenyl µBondpak column using an isocratic pump connected to a Waters 490 UV detector set at 254 nm. The column was eluted at 1 ml/min with a mobile phase comprising water/methanol/acetic acid (17.75:3.00:0.27). The retention times were 7.1 min for AAP and 15.9 min for 3-glutathionyl-AAP. Authentic 3-glutathionyl-AAP and acetaminophen O-glucuronide, which were used as standards during the HPLC assay, were a kind gift of Dr R. S. Andrews (Winthrop Laboratories, Newcastle, England).

Statistical analysis.
All data obtained was analyzed by one-way ANOVA. When differences (p < 0.05) were observed between control and clofibrate-pretreated groups, Bonferroni's post hoc test was used to analyze data at specific time points.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Validation of Hepatocyte Model
A hepatocyte model in which peroxisome numbers were increased was needed for our investigation of the effect of peroxisome proliferation on acetaminophen (AAP) toxicity in vitro. A number of strategies can be used to obtain such cells, with one straightforward approach simply involving addition of peroxisome proliferators to the culture media soon after hepatocyte isolation (Gray et al., 1983Go; Kocarek and Feller 1989Go; Tomaszewski et al., 1990Go). However, because it takes several days to achieve peroxisome proliferation under these conditions, this approach was judged unsuitable for our work in light of the knowledge that CYP450 levels decline steadily after hepatocyte isolation, hampering study of the toxicity of bioactivation-dependent compounds such as AAP.

Although the strategy we chose was more time consuming, involving a 10-day treatment with clofibrate prior to hepatocyte isolation, it nonetheless provided high yields of viable, bioactivation-competent cells. To confirm that the pretreatment regimen produced consistent peroxisome proliferation, liver weights were determined after a 10-day exposure of mice to clofibrate or vehicle. Clofibrate induced significant hepatomegaly (average liver weights increased 30% over controls) as well as a 4.5-fold elevation in the hepatic activity of palmitoyl CoA oxidase, a widely used peroxisomal marker (n = 4, p < 0.05). A slight (16%) increase in the glutathione content was also evident in the livers of clofibrate-pretreated mice relative to controls (p < 0.05) after this 10-day pretreatment regimen. Collagenase digestion of these livers provided high yields of hepatocytes that maintained their viability during our experiments, typically exhibiting less than 5% LDH leakage after 8-h incubation in RPMI-1640 culture media.

Peroxisome Proliferation Protects Hepatocytes against AAP Toxicity
Our earlier work indicated that clofibrate-pretreated mice were resistant to the in vivo hepatotoxicity of high-dose AAP (Nicholls-Grzemski et al., 1992Go). Importantly, the data shown at the 4-h time point in Figure 1Go indicates that the protection against AAP is retained in hepatocytes isolated from clofibrate-pretreated mice. In control cells from vehicle-treated mice, AAP produced a pronounced concentration-dependent loss of viability, with 3 mM AAP producing 90% LDH leakage after 4 h (Fig. 1Go). In contrast, the same concentration of AAP produced less than 40% LDH leakage in hepatocytes from clofibrate pretreated mice. The protection persisted until the end of the experiments (i.e., 8 h), as 1 mM and higher concentrations of AAP produced 100% cell death in control cells by this time, but just 50% lethality in clofibrate-pretreated cells (data not shown). Collectively, these findings indicate that clofibrate-induced hepatoprotection is conserved during the hepatocyte isolation process, and the protective effect is associated with the parenchymal cell fraction. Consequently, it seems to involve a biochemical change within the hepatocyte and is not simply due to in vivo factors (e.g., changes in AAP disposition) that may accompany clofibrate-induced hepatomegaly.



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FIG. 1. LDH leakage from isolated mouse hepatocytes after 4-h incubation in the presence of various concentrations of AAP (10–3000 µM). Cells were isolated from either vehicle- (open circles) or clofibrate-pretreated (filled circles) mice. Each data point represents the mean ± SEM of 15 independent observations. Asterisks (*) indicate a difference in LDH leakage between control and clofibrate-pretreated cells at a given concentration of AAP (* p < 0.05; *** p < 0.001, Bonferroni post hoc test).

 
Clofibrate-Induced Hepatoprotection Is Not Due to Altered Glutathione Homeostasis
AAP-induced liver toxicity involves conversion of the drug to an electrophilic metabolite, NAPQI (Dahlin et al., 1984Go). Although NAPQI is normally detoxicated via conjugation with reduced glutathione, during AAP intoxication cellular glutathione reserves are exhausted, allowing adduction of proteins (Corcoran et al., 1985Go; Massey and Racz, 1981Go). One explanation for the hepatoprotection afforded by clofibrate could thus be that the cells are less prone to glutathione depletion during exposure to AAP. To investigate this possibility, we measured cellular glutathione levels in hepatocytes from control or clofibrate-pretreated mice after 1- and 4-h exposure to a range of AAP concentrations (Fig. 2Go). At the commencement of the experiment, no differences were seen in the glutathione content of cells from control (40.2 ± 7 nmol/mg; n = 8) compared to clofibrate-pretreated mice (38.7 ± 7 nmol/mg; n = 8). Still more importantly, no significant differences in glutathione levels were evident in clofibrate-pretreated cells relative to controls after either 1- or 4-h exposures to a range of AAP concentrations (Fig. 2Go). Thus, although they were less susceptible to AAP-induced cell killing (Fig. 1Go), the clofibrate-pretreated cells were just as prone to glutathione depletion during AAP intoxication as the control cells.



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FIG. 2. Levels of glutathione in hepatocytes after 1-h (circles) and 4-h (squares) exposure to a range of AAP concentrations (10–3000 µM). Cells were isolated from either vehicle-treated controls (open symbols) or clofibrate-pretreated mice (filled symbols). Values are expressed as a percentage of the glutathione content of the respective cells prior to exposure to AAP. The data points on the left side of the curve represent cells incubated in the absence of AAP (i.e., designated 0 mM AAP). Each data point represents the mean ± SEM of 4 independent observations.

 
To further investigate the role of altered glutathione homeostasis in clofibrate-induced hepatoprotection, we compared the ability of the cells to replenish glutathione levels after a brief exposure to the glutathione-depleting reagent DEM. As Figure 3Go indicates, exposure of both control and clofibrate-pretreated cells to 0.5 mM DEM for 30 min resulted in pronounced (60–70%) glutathione depletion. The cells were then washed and incubated in DEM-free media for up to 4 h, and glutathione levels were determined after 1, 2, and 4 h (Fig. 3Go). In both types of cells, glutathione levels were suppressed for 2 h and then slowly recovered, doubling their 2-h levels by the end of the experiment at 4 h. Sampling at later time points was not possible, as DEM, although having no effect on clofibrate-pretreated cells, was toxic to the control cells (data not shown). At each time point examined, there were no differences between the glutathione content of controls compared to cells from clofibrate-pretreated mice, indicating an enhanced ability to replenish cell glutathione following acute depletion was an unlikely explanation of the hepatoprotection produced by clofibrate.



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FIG. 3. Glutathione repletion in mouse hepatocytes following acute depletion by diethyl maleate (DEM). After a 30-min exposure to 0.5 mM DEM, cells were washed with HBS and fresh media was added (0 h). Data was obtained in cells isolated from either vehicle-treated (open circles) or clofibrate-pretreated mice (filled circles). Values are expressed as a percentage of the glutathione content of the respective cells prior to exposure to DEM. Each data point represents the mean ± SEM of four independent observations.

 
Prior Glutathione Depletion Does Not Abolish Hepatoprotection
To more definitively address the role of glutathione in cytoprotection, the effect of depleting cell glutathione prior to exposure to AAP was examined. We reasoned that if the protective effect was primarily glutathione dependent, prior depletion would restore the sensitivity of clofibrate-pretreated cells to AAP toxicity to that of control cells. DEM was again used in these experiments, as it potentiates AAP toxicity in hepatocytes (Hayes et al., 1986Go; Mitchell et al., 1985Go). The pretreatment regimen (0.5 mM DEM, 30 min) was shown earlier to deplete cell glutathione stores by 60–70% (Fig. 3Go).

The effect of DEM-pretreatment on AAP toxicity (assessed via measurements of LDH leakage) in hepatocytes from control and clofibrate-pretreated mice is shown in Figure 4Go. In keeping with observations in other laboratories (Hayes et al., 1986Go; Mitchell et al., 1985Go), DEM strongly enhanced AAP lethality after 4 h in the control cells (Fig. 4Go), although the DEM treatment alone caused a modest (~15%) increase in LDH leakage in cells that were not exposed to AAP. However, the most striking finding of these experiments was that prior treatment with DEM did not enhance AAP toxicity in clofibrate-pretreated cells (Fig. 4Go), and the extent of toxicity produced by AAP in these cells was virtually identical to that seen in clofibrate-pretreated cells that had not undergone DEM pretreatment. Incidentally, we also confirmed these observations during a small-scale in vivo experiment. Vehicle- and clofibrate-pretreated mice (n = 4) were administered 0.9g/ kg DEM 30 min prior to a nonhepatotoxic dose of AAP (200 mg/kg). Whereas all vehicle-treated mice either died or exhibited extensive liver damage, none of the clofibrate-treated mice showed any adverse effects (change in serum enzymes or histopathology) to AAP (Fig. 5Go), despite reduction of hepatic glutathione to negligible levels (less than 5% of control values). Collectively, these results strongly suggest that although glutathione is very important in preventing AAP toxicity under normal conditions, some other factor must explain the protective effect that accompanies pretreatment of mice with clofibrate.



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FIG. 4. The effect of pretreatment with diethyl maleate (DEM) on the extent of LDH leakage from isolated mouse hepatocytes after incubation with a range of AAP concentrations (10–1000 µM). Cells were isolated from either vehicle-treated (open symbols) or clofibrate-pretreated (filled symbols) mice. Data from cells not pretreated with DEM are shown as circles; data from cells pretreated with DEM (0.5 mM, 30 min) are represented as squares. The data points on the left side of the curve represent cells incubated in the absence of AAP. Each data point represents the mean ± SEM of four independent observations. Crosshatches (#) indicate that a difference in LDH leakage was evident between vehicle-pretreated control cells and DEM-pretreated control cells at a given concentration of AAP (# p < 0.05, ## p < 0.01). DEM pretreatment had no effect on LDH leakage from clofibrate-pretreated cells at any AAP concentration.

 


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FIG. 5. Effect of pretreatment with diethyl maleate (DEM) on plasma sorbitol dehydrogenase (SDH) activities in the plasma of vehicle- or clofibrate-pretreated mice 24 h after administration of 100 or 200 mg/kg acetaminophen (AAP). Vehicle and clofibrate pretreated mice received DEM (0.9 g/kg, i.p.) 30 min prior to administration of 2 nonhepatotoxic doses of AAP. Blood samples were collected 24 h later and hepatotoxicity was determined by plasma sorbitol dehydrogenase (SDH) activity. Data indicate mean ± SEM; n = 1–4. Note that 25% and 75% mortality was observed in vehicle-pretreated mice that received 100 mg/kg and 200 mg/kg AAP, respectively.

 
This conclusion was further strengthened by measurements of 3-glutathionyl-AAP excretion into the culture media of AAP-treated control and clofibrate-pretreated cells both with and without DEM-pretreatment (Fig. 6Go). The glutathione conjugate was measured by UV-HPLC as described in the Materials and Methods section. In the control cells that had not undergone DEM pretreatment, a concentration-dependent increase in excretion of 3-glutathionyl-AAP was evident after a 4-h incubation, although its production appeared to plateau at 0.3 mM AAP and higher concentrations (Fig. 6Go). The latter effect is presumably due to cell death, which exceeded 60% at the highest AAP concentrations (Fig. 1Go). As CYP450-mediated AAP bioactivation requires NADPH, a cofactor produced only by viable hepatocytes, cell death could be expected to diminish NAPQI formation. In clofibrate-pretreated cells that were not pretreated with DEM, 3-glutathionyl-AAP formation was the same as in controls at all AAP concentrations, although the plateau at 0.3 and 1.0 mM was not evident, presumably because more of the cells remained viable (Fig 1Go). In keeping with its ability to deplete cellular glutathione, pretreatment with DEM strongly attenuated 3-glutathionyl-AAP formation in hepatocytes from control and clofibrate-pretreated mice, an effect that was statistically significant at 0.1 mM AAP and greater concentrations (Fig. 6Go). However, there was no difference between the extent of metabolite formation between control- and clofibrate-pretreated cells at any concentration of AAP (Fig. 6Go). Furthermore, at nontoxic concentrations of AAP, no significant differences were evident in the formation of the AAP-glucuronide conjugate between control and clofibrate-pretreated cells (data not shown). Taken together, these findings indicate that an increased ability to metabolize AAP or its reactive metabolite via Phase II pathways does not explain the hepatoprotection afforded by clofibrate.



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FIG. 6. Formation of 3-glutathionyl-AAP in mouse hepatocytes after 4-h exposure to a range of AAP concentrations (10–1000 µM) in control (circles) or diethyl maleate (DEM)-pretreated cells (squares). Cells were isolated from either vehicle-treated (open symbols) or clofibrate-pretreated mice (filled symbols). Each data point represents the mean ± SEM of four independent observations. Crosshatches (#) indicate a difference due to DEM treatment on metabolite excretion in cells from vehicle-treated cells at a given AAP concentration; asterisks (*) indicate a difference in metabolite excretion due to DEM treatment in clofibrate-pretreated cells (* or #, p < 0.05; ** or ##, p < 0.01, Bonferroni post hoc test for differences related to DEM depletion within treatment groups).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results from this study have extended previous observations of a protective effect against acetaminophen (AAP) toxicity during clofibrate-induced peroxisome proliferation in mice (Nicholls-Grzemski et al., 1991Go; Nicholls-Grzemski et al., 1992Go; Manautou et al., 1994Go; Manautou et al., 1996aGo). Our finding that the protection persists upon hepatocyte isolation suggests that it is not simply due to dispositional or metabolic zonation factors that are associated with the in vivo situation, but rather involves a biochemical alteration in the cells. Identifying the exact biochemical and molecular change(s) responsible for hepatoprotection is not a trivial task, as peroxisome proliferators increase the abundance of over 100 different proteins in mouse liver (Anderson et al., 1996Go). Any number of these may be involved in the hepatoprotective response.

Although not identifying the mechanisms underlying hepatoprotection, our present findings help focus future investigation by ruling out a role for altered glutathione homeostasis as the primary cytoprotective mechanism. Several findings led to this conclusion, namely; 1) levels of glutathione were depleted to the same extent during poisoning with AAP in both control and clofibrate-pretreated cells; 2) rates of glutathione replenishment following short-term exposure to diethyl maleate (DEM) were similar in both cell types, and 3) excretion of the initial product of reactions between glutathione and NAPQI, 3-glutathionyl-AAP, were similar in both cell types. Typically, the levels of 3-glutathionyl-AAP in culture media are very sensitive indicators of the glutathione-conjugating capacity of isolated hepatocytes, changing in response to pretreatments that modify the cellular thiol status (Harman and Self, 1986Go; Massey and Racz, 1981Go; Moldeus et al., 1982Go).

Our conclusion that altered glutathione homeostasis plays a minor role in clofibrate-induced hepatoprotection differs in some respects to work by Cohen and associates, who attributed the in vivo protective effect, at least in the early stages of AAP intoxication, to accelerated plasma clearance of AAP secondary to enhanced biliary excretion of 3-glutathionyl-AAP (Manautou et al., 1996aGo). However, they also reported that 4 h after a toxic dose of AAP there was no significant decrease in hepatic glutathione in clofibrate-pretreated mice (Manautou et al., 1994Go). This finding differed from our earlier in vivo observations, as AAP-induced hepatic glutathione depletion was similar in control and clofibrate-pretreated mice, at least when measured 3 h after the administration of doses of 300 mg/kg AAP and above (Nicholls-Grzemski et al., 1991Go; Nicholls-Grzemski et al., 1992Go). It is difficult to account for the different role the two groups have assigned to glutathione in the hepatoprotective effect of PxP. However, it may be significant that one study by the Cohen group, in which they examined the effect of a single dose of clofibrate on AAP hepatotoxicity, did suggest a glutathione-independent mechanism of hepatoprotection (Manautou et al., 1996bGo). The fact that they did not observe a significant role for this pathway in the repeated clofibrate dosing experiments may reflect differences in experimental procedure between their group and ours. In addition to the difference in the strain of mice used by our groups, Manautou and associates routinely treat mice with a higher dose of AAP (800 mg/kg) than we used in our earlier in vivo studies. Furthermore, Manautou et al. routinely administer AAP to mice after an 18-h fast, which is known to alter hepatic glutathione homeostasis (Wendel and Jaeschke, 1983Go). In addition, Manautou et al. administer AAP in a vehicle containing 50% polyethylene glycol. Because the latter can inhibit the CYP450-catalyzed metabolism of AAP (Snawder et al., 1993Go; Thomsen et al., 1995Go), this factor may further contribute to the different conclusions drawn by our respective groups concerning the role of glutathione in clofibrate-induced hepatoprotection. A further possibility, not addressed in the present study, could be that clofibrate pretreatment alters glutathione homeostasis in a minor yet toxicologically significant subcellular pool. One possibility that could be addressed in future studies is that clofibrate alters the glutathione content of mitochondria, a pool that is resistant to DEM but is important to hepatocyte viability during in vitro oxidative stress (Meredith and Reed, 1982Go).

An alternative explanation for the protective effect could be that clofibrate pretreatment diminishes the expression and/or activity of CYP2E1 and CYP1A2, the main isoforms that bioactivate AAP to NAPQI in rodents (Patten et al., 1993Go). However this seems unlikely, as the levels of 3-glutathionyl-AAP excreted into the culture medium of cells from clofibrate-pretreated mice were essentially the same as in control cells (Fig. 6Go). In addition to reflecting the glutathione status of cells, levels of 3-glutathionyl-AAP excreted from hepatocytes are sensitive to changes in CYP450 activity, increasing in response to CYP450 induction, and decreasing on treatment with CYP450 inhibitors (Harman and Fischer, 1983Go; Moldeus, 1978Go). Our finding that 3-glutathionyl-AAP excretion from clofibrate-pretreated hepatocytes was not elevated suggests the bioactivation capacity of these cells was unaltered, ruling out such a change as the major hepatoprotective mechanism.

A more likely explanation for the hepatoprotection could be that clofibrate pretreatment disrupts the biochemical events involved in the progression of cell death during AAP toxicity. However, due to the broad range of proposals concerning the fundamental biochemical lesion involved in AAP toxicity, establishing which of these is altered by clofibrate pretreatment is a complex task. Although not complete, the list of mechanisms advanced to account for AAP toxicity includes enzyme dysfunction due to arylation of critical cell proteins (Bartelone et al., 1989Go); disruption of calcium homeostasis and activation of calcium-dependent degradative pathways (Moore et al., 1985Go; Shen et al., 1992Go); overproduction of oxygen radicals (Adamson and Harman, 1993Go); enhanced lipid peroxidation (Wendel and Feuerstein, 1981Go); inhibition of cell regeneration and repair (Boulares et al., 1999Go); production of cytotoxic mediators by activated Kupffer and immune cells (Laskin et al., 1995Go); mitochondrial dysfunction secondary to respiratory chain damage (Burcham and Harman, 1991Go); and induction of apoptosis (Ray et al., 1996Go). As many of these toxic processes are intertwined, establishing which are attenuated during peroxisome proliferation is a challenging task.

Although the toxic metabolite NAPQI appears to trigger the deterioration of a wide range of cell constituents, a consensus seems to be emerging concerning a central role for oxidative cell damage in AAP hepatotoxicity. Such an involvement was suggested by early observations that the oxygen radical metabolizing enzymes superoxide dismutase and catalase can protect hepatocytes against AAP (Kyle et al., 1987Go) and that inhibition of the peroxide reducing enzyme glutathione peroxidase with gold thioglucose increased susceptibility to AAP (Adamson and Harman, 1989Go). In addition, an increase in chemiluminence indicative of an elevation of reactive oxygen species has been observed following AAP exposure in vitro (Lores Arnaiz et al., 1995Go; Minamide et al., 1998Go). Finally, a number of antioxidants (including {alpha}-tocopherol, butylated hydroxyanisole, desferrioximine) protect against AAP toxicity in both in vivo and in vitro models (Fairhurst et al., 1982Go; Harman, 1985Go; Ito et al., 1994Go; Lake et al., 1981Go; Rosenbaum et al., 1984Go). Such protection can occur without altering either glutathione depletion or protein adduction (Gerson et al., 1985Go; Sakaida et al., 1995Go).

More recently, although their earlier efforts suggested oxidative protein damage was not involved in AAP hepatotoxicity (Gibson et al., 1996Go), Hinson and coworkers identified a dramatic increase in the levels of heme-adducted proteins in AAP-intoxicated mouse livers (Michael et al., 1999Go). The latter involve covalent attachment of heme prosthetic groups to proteins and form during oxidative protein damage (Vuletich and Osawa, 1998Go). The generation of these adducts was accompanied by an increase in nitrotyrosine adducts, apparently formed by peroxynitrite, a byproduct of the oxidative burst of activated macrophages that infiltrate the AAP-intoxicated mouse liver (Michael et al., 1999Go).

Although such findings suggest that oxidative damage occurs during AAP hepatotoxicity due to recruitment of immune cells, evidence for an intrinsic oxidative stress within hepatocytes has also recently strengthened. Previously, a mechanistic basis for an enhanced oxidative stress in AAP toxicity was weak, due in part to the inability of several groups to detect redox cycling by AAP's toxic metabolite NAPQI (Dahlin et al., 1984Go; Powis et al., 1984Go). However, new insights into the mechanisms underlying oxidative stress during AAP toxicity were recently provided by Burlingame and associates, who employed definitive 2-D gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry to identify NAPQI-adducted proteins in the livers of AAP-intoxicated mice (Qiu et al., 1998Go). Although a range of new protein targets were identified, comparative studies of the protein damage produced by AAP and its nontoxic 3`-regioisomer indicated mitochondrial glutathione peroxidase was a particularly pronounced target for NAPQI. This finding concurs with a prior demonstration of an early loss of glutathione peroxidase activity in mouse liver following AAP intoxication (Tirmenstein and Nelson, 1990Go). In addition, an increase in mitochondrial superoxide anion as well as hydrogen peroxide formation occurs soon after AAP intoxication (Lores Arnaiz et al., 1995Go). Collectively, these findings appear to suggest that a diminished ability of hepatocytes to detoxicate oxidants via glutathione peroxidase may underlie the toxicity of AAP.

In light of this emerging consensus concerning a role for oxidative events in AAP hepatotoxicity, one explanation for the hepatoprotection afforded by clofibrate and other PxPs could be that the ability of the hepatocytes to withstand oxidative stress is enhanced. This would confer resistance to damage by other prooxidant chemicals, thus explaining why protection has been seen following pretreatment with peroxisome proliferators against carbon tetrachloride, bromobenzene, and chloroform toxicities (Manautou et al., 1998Go; Nicholls-Grzemski et al., 1993Go). Although the exact mechanism is not known, the resistance to oxidative cell killing is evidently not simply due to an increase in glutathione availability, and apparently involves changes to other cellular constituents.


    NOTES
 
1 Present address: Department of Pharmaceutical Sciences, Washington State University, Pullman, WA 99164–6534. Back

2 To whom correspondence should be addressed. Fax: 61-8-8224-0685. E-mail: philip.burcham{at}adelaide.edu.au. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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