Abstract

Mitochondrial oxidant stress and peroxynitrite formation have been implicated in the pathophysiology of acetaminophen-induced (AAP-induced) liver injury. Therefore, we tested the hypothesis that lipid peroxidation (LPO) might be involved in the injury mechanism. Male C3Heb/FeJ mice fed a diet high in vitamin E (1 g d-α-tocopheryl acetate/kg diet) for 1 week had 6.7-fold higher hepatic tocopherol levels than animals on the control diet (8.2 ± 0.1 nmol/g liver). Treatment of fasted mice with 300 mg/kg AAP caused centrilobular necrosis with high plasma alanine aminotransferase (ALT) activities at 6 h (3280 ± 570 U/l) but no evidence of LPO (hepatic malondialdehyde, 4-hydroxynonenal). Animals on the vitamin E diet had similar injury and LPO as mice on the control diet. To verify a potential effect of the vitamin E diet on drug-induced liver injury, animals were pretreated with a combination of phorone, FeSO4, and allyl alcohol. We observed, 2 h after allyl alcohol, massive LPO and liver cell injury in the livers of animals on the control diet, as indicated by a 32-fold increase in malondialdehyde levels, extensive staining for 4-hydroxynonenal, and ALT activities of 2310 ± 340 U/l. Animals on the vitamin E diet had 40% lower hepatic malondialdehyde levels and 85% lower ALT values. Similar results were obtained when animals were treated for 3 days with α- or γ-tocopherol (0.19 mmol/kg, ip). Both treatments reduced LPO and injury after allyl alcohol but had no effect on AAP hepatotoxicity. Thus, despite the previously shown mitochondrial oxidant stress and peroxynitrite formation, LPO does not appear to be a critical event in AAP-induced hepatotoxicity.

An overdose of the analgesic drug acetaminophen (AAP) can lead to severe liver injury in humans and in experimental animals. Although intensely studied for more than 25 years, the mechanism of this injury is still not entirely clear. It is undisputed that the metabolism of a fraction of the AAP dose by the P450 system is the initial step of the injury process (Jollow et al., 1973). The product of this reaction is a reactive metabolite, presumably N-acetyl-p-benzoquinone imine (NAPQI) (Dahlin et al., 1984), which is detoxified by glutathione (Mitchell et al., 1973). However, if the formation of the reactive metabolite exceeds the capacity of liver glutathione, NAPQI will bind to cellular proteins (Jollow et al., 1973). Over the years, a number of proteins were identified that were modified by NAPQI binding (Cohen et al., 1997). There is increasing evidence to suggest that protein binding is an initiating event of cell injury, which can be amplified through secondary processes (Jaeschke et al., 2003a).

One of these secondary effects of reactive metabolite formation and protein binding is mitochondrial dysfunction (Meyers et al., 1988; Ramsay et al., 1989), which results in ATP depletion and oxidant stress (Jaeschke, 1990; Tirmenstein and Nelson, 1990). Superoxide generated in mitochondria after AAP overdose can dismutate to form molecular oxygen and hydrogen peroxide, which is then reduced to water by glutathione peroxidase using electrons from GSH. The fact that mitochondrial glutathione disulfide (GSSG) levels increase substantially after AAP treatment is strong evidence for the increased hydrogen peroxide formation in mitochondria (Jaeschke, 1990; Knight et al., 2001), although extramitochondrial sources of reactive oxygen cannot be excluded.

Studies with cultured mouse hepatocytes demonstrated that the oxidant stress, measured as increased 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH) fluorescence, precedes cell injury by several hours (Jaeschke et al., 2003b), consistent with an early increase in the GSSG-to-GSH ratio in vivo (Knight et al., 2001). On the other hand, superoxide can react with nitric oxide (NO) to form the potent oxidant peroxynitrite (Squadrito and Pryor, 1998). The rate constant of the reaction between superoxide and NO is several times higher than the rate constant of superoxide dismutation with or without catalysis by superoxide dismutase (Squadrito and Pryor, 1998). Thus, an increased formation of superoxide in the presence of equimolar levels of NO can lead to increased formation of peroxynitrite in addition to hydrogen peroxide generation.

Nitrotyrosine is a footprint for peroxynitrite formation (Beckman, 1996). Indeed, nitrotyrosine protein adducts can be detected in vascular endothelial cells and parenchymal cells after AAP overdose before cell injury, i.e., the loss of endothelial cell barrier function (hemorrhage) and ALT release, is observed (Hinson et al., 1998; Knight et al., 2001). Administration of pharmacological doses of glutathione accelerated the recovery of mitochondrial glutathione levels, which effectively scavenged most of the peroxynitrite and protected against cell injury (Knight et al., 2002). These data suggested that peroxynitrite is a critical mediator of AAP hepatotoxicity (Knight et al., 2002). Both hydrogen peroxide (through hydroxyl radical formation by iron-catalyzed Fenton reaction) and peroxynitrite (through hydroxyl radical–like decomposition products) can initiate lipid peroxidation (Radi et al., 1991), which can lead to oncotic necrosis of liver cells. Furthermore, inhibition of peroxynitrite formation by inhibitors of nitric oxide synthase was associated with an increase in lipid peroxidation after AAP treatment (Hinson et al., 2002).

Vitamin E (α-tocopherol) is a lipid-soluble antioxidant that effectively prevents lipid peroxidation (Fariss and Zhang, 2003). Although the level of α-tocopherol in membranes is generally not high enough to be a relevant hydroxyl radical scavenger, it effectively reduces peroxyl radicals (ROO •), one of the less reactive secondary radicals (Fariss and Zhang, 2003). Thus, α-tocopherol prevents the propagation of the radical chain by avoiding the formation of new alkyl radicals. In general, a beneficial effect of α-tocopherol administration is considered evidence for the importance of lipid peroxidation as a mechanism of xenobiotic-induced liver cell injury (Fariss et al., 1985; Jaeschke et al., 1987, 1992; Zhang et al., 2001b). However, the role of lipid peroxidation and potential protection by α-tocopherol remains controversial in AAP hepatotoxicity (Jaeschke et al., 2003a; Smith et al., 1985).

Recently, Fariss and coworkers demonstrated that increasing the levels of α-tocopherol in mitochondria is critical for the protective effect of vitamin E treatment against mitochondria-induced oxidant stress (Fariss and Zhang, 2003; Fariss et al., 2001; Zhang et al., 2001a). In fact, the enrichment of mitochondria with protective α-tocopherol not only prevented lipid peroxidation but also attenuated the production of reactive oxygen species in hepatic mitochondria (Chow et al., 1999; Zhang et al., 2001a). However, γ-tocopherol was shown to react with peroxynitrite (Christen et al., 1997; Hoglen et al., 1997) and reduce protein nitration in vivo (Jiang et al., 2002). Therefore, the objective of this investigation was to test the hypothesis that reactive oxygen and/or peroxynitrite-induced lipid peroxidation may be involved in the mechanism of cell injury after AAP overdose in mice. Our approach was to investigate if animals receiving parental α- or γ-tocopherol or a diet high in d-α-tocopheryl acetate had less lipid peroxidation and suffered less hepatocellular injury than control animals.

MATERIALS AND METHODS

Animals.

Male C3Heb/FeJ mice with an average weight of 18–20 g were purchased from Jackson Laboratory (Bar Harbor, ME). All animals were housed in an environmentally controlled room with 12 h light/12 h dark cycle and allowed free access to food (certified rodent diet no. 8640, Harlan Teklad, Indianapolis, IN) and water. All experimental protocols followed the criteria of University of Arizona and the National Research Council for the care and use of laboratory animals in research. Some animals were fed a diet high in vitamin E (1 g d-α-tocopheryl acetate (TA)/kg diet; Dyets Inc., Bethlehem, PA) compared to a control diet (35 mg TA/kg diet) for 7 days before AAP treatment. In some experiments, animals on the control or high α-tocopherol diet were treated with two doses of the iNOS inhibitor aminoguanidine (15 mg/kg, ip). The first dose was injected at the time of AAP treatment; the second dose was administered 2 h later (Hinson et al., 2002). In other experiments, animals on the control diet received three ip injections of 0.19 mmol/kg α-tocopherol (24.6 mg/ml olive oil), 0.19 mmol/kg γ-tocopherol (23.8 mg/ml), or vehicle at 3, 2, and 1 day(s) before the experiment. Both tocopherol compounds were kind gifts from Henkel Corp. (La Grange, IL). All animals were fasted overnight before the experiments unless stated otherwise. Animals received an ip injection of 300 mg/kg AAP or a combination of 100 mg/kg phorone and 0.35 mmol/kg FeSO4 30 min before the administration of 0.6 mmol/kg allyl alcohol. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless stated otherwise.

Experimental protocols.

At selected times after AAP or allyl alcohol treatment, the animals were killed by cervical dislocation. Blood was drawn from the vena cava into heparinized syringes and centrifuged. The plasma was used for the determination of alanine aminotransferase (ALT) activities. Immediately after collecting the blood, the livers were excised and rinsed in saline. A small section from each liver was placed in 10% phosphate-buffered formalin to be used in immunohistochemical analysis. A portion of the remaining liver was frozen in liquid nitrogen and stored at −80°C for later analysis of malondialdehyde (MDA) and vitamin E.

Methods.

Plasma ALT activities were determined with the test kit DG 159-UV (Sigma Chemical Co.) and expressed as IU/l. Protein concentrations were assayed using the bicinchoninic acid kit (Pierce, Rockford, IL). Malondialdehyde levels were determined in liver homogenate with a commercial test kit (MDA-586 assay; Oxis International, Inc., Portland, OR).

Vitamin E analysis.

Liver and plasma d-α-tocopherol and d-γ-tocopherol levels were measured by an internal standard (d-δ-tocopherol) method using reversed-phase high-performance liquid chromatography equipped with a McPherson fluorometric detector according to the method of Fariss et al.(1989). Frozen liver was ground to a fine powder and 20–25 mg of liver or 50 μl of plasma was solubilized and extracted with hexane (Fariss et al., 1989, 1993). A standard curve for each tocopherol analog was analyzed with each set of experimental samples. The limit of detection of α-tocopherol and γ-tocopherol by this method is 5 nmol/g tissue and 2 nmol/ml plasma.

Histology and immunohistochemistry.

Formalin-fixed tissue samples were embedded in paraffin and 5 μm sections were cut. Replicate sections were stained with hematoxylin and eosin (H & E) for evaluation of necrosis (Gujral et al., 2002). All sections were obtained from the left lateral lobe. Preliminary studies using several livers showed no difference in necrosis or 4-hydroxynonenal staining between the different lobes of the liver in this model. The percentage necrosis was estimated by evaluating the number of microscopic fields with necrosis compared to the entire cross-section. In general, necrosis was estimated at low power (×100); questionable areas were evaluated at higher magnification (×200 or ×400). The pathologist (A.F.) evaluated all histological sections in a blinded fashion. Formation of nitrotyrosine and 4-hydroxynonenal protein adducts were assessed by immunohistochemistry with the DAKO LSAB Peroxidase Kit (K684; DAKO Corp., Carpinteria, CA) as described previously (Knight et al., 2002). The anti-4-hydroxynonenal antibody was obtained from Calbiochem (San Diego, CA) and the antinitrotyrosine antibody was purchased from Molecular Probes (Eugene, OR).

Statistics.

All results were expressed as mean ± SE. Comparisons between multiple groups were performed with one-way ANOVA or, where appropriate (e.g., Fig. 1), by two-way ANOVA followed by a post hoc Bonferroni test. If the data were not normally distributed, we used the Kruskal-Wallis test (nonparametric ANOVA) followed by Dunn’s Multiple Comparisons test. Statistically, p < 0.05 was considered significant.

RESULTS

Alpha-Tocopherol Diet versus Control Diet

To evaluate the potential protective effect of vitamin E in AAP-induced liver injury, animals were fed a standard rodent diet, which was supplemented with 1 g/kg d-α-tocopheryl acetate. After 1 week on the vitamin E diet, plasma and hepatic α-tocopherol levels increased by 390% and 665% compared to values in animals on the control diet (Fig. 1). Although an overnight fast lowered the α-tocopherol levels in both liver and plasma of animals fed the vitamin E diet, the tocopherol levels were still significantly higher than in fasted animals fed the control diet.

Treatment of fasted animals with 300 mg/kg AAP induced severe liver injury as indicated by the elevated plasma ALT values at 6 h (Fig. 2A). There was no significant difference in plasma ALT activities between animals on the control diet and the vitamin E diet. These findings were confirmed by the histological assessment. Fasted animals on the control diet showed 45 ± 5% necrosis of all hepatocytes. Animals on the vitamin E diet had similar injury (data not shown). As with the fasted animals, fed animals were treated with AAP at 4:00 p.m., where hepatic GSH levels reached the minimum of the diurnal variations (Jaeschke and Wendel, 1985). Again, there was no significant difference in plasma ALT activities between animals on the control diet (4870 ± 1028 U/l, n = 5) and animals on the vitamin E diet (4603 ± 373 U/l, n = 5) 6 h after AAP administration.

As a positive control, animals were treated with allyl alcohol and ferrous iron to induce lipid peroxidation in the liver (Jaeschke et al., 1992). This treatment caused substantial liver injury in animals on the control diet at 2 h after administration of allyl alcohol (Fig. 2A). The same treatment produced significantly less injury in animals on the vitamin E diet (Fig. 2A). Lipid peroxidation as a mechanism of injury was assessed with liver MDA levels and immunohistochemical staining for 4-hydroxynonenal (4-HNE). Based on both parameters, AAP treatment did not induce LPO in fasted animals on the control or the vitamin E diet (Figs. 2B and 3). Similarly, the MDA levels of fed animals on the control diet (5.3 ± 1.2 nmol MDA/g liver, n = 5) were not significantly different from the MDA levels of animals on the vitamin E diet (7.8 ± 1.4, n = 5). In striking contrast, allyl alcohol/iron increased liver MDA levels by more than 30-fold in animals on the control diet (Fig. 2B). However, the same treatment caused significantly less MDA formation in animals on the vitamin E diet (Fig. 2B). Similarly, allyl alcohol/iron dramatically increased staining for 4-HNE in liver tissue sections (Fig. 3). Consistent with the still-elevated MDA levels (Fig. 2B), substantial 4-HNE staining was observed in animals on the vitamin E diet (data not shown).

Parental Administration of α- and γ-Tocopherol

In separate experiments, animals on the control diet received olive oil or 0.19 mmol/kg of α-tocopherol or γ-tocopherol by ip injection for 3 days. Treatment with α-tocopherol selectively enhanced hepatic α-tocopherol levels by 350% (Fig. 4). Treatment with γ-tocopherol increased the hepatic content of γ-tocopherol from below the detection limit (5 nmol/g liver) to 79 ± 20 nmol/g (Fig. 4). When these animals were treated with 300 mg/kg AAP, severe liver injury developed as indicated by the high plasma activities of ALT (Fig. 5A). However, there was no significant difference in injury between animals treated with the vehicle, α-tocopherol, or γ-tocopherol. In contrast, both α-tocopherol and γ-tocopherol treatments attenuated liver injury induced by allyl alcohol/iron (Fig. 5A). Despite similar enhancement of liver tocopherol levels, α-tocopherol was more effective in preventing allyl alcohol/iron–induced liver injury. Consistent with these findings, α-tocopherol was more effective in reducing the elevated hepatic MDA (Fig. 5B). In contrast, neither α-tocopherol nor γ-tocopherol treatment attenuated the centrilobular nitrotyrosine staining after AAP (data not shown).

Lipid Peroxidation after Treatment with the iNOS Inhibitor Aminoguanidine

It was recently hypothesized that hepatic LPO might be responsible for AAP-induced hepatotoxicity after treatment with NOS inhibitors (Hinson et al., 2002). Therefore, animals on the control or vitamin E diet were pretreated with the iNOS inhibitor aminoguanidine at the time of AAP injection and 2 h later. Plasma ALT activities were significantly increased at 6 h after AAP treatment in both controls and aminoguanidine-treated animals on the control or the vitamin E diet (Table 1). Similar to the previous experiment (Fig. 2), there was no increase in liver MDA levels after AAP (Table 1). Pretreatment with aminoguanidine did not enhance hepatic MDA values significantly above control levels (Table 1).

DISCUSSION

The objective of this investigation was to evaluate whether LPO is a relevant component of the mechanism of cell injury after AAP overdose. Using a commercial kit for MDA measurements and immunohistochemical analysis of 4-hydroxynonenal, we could not detect significant increases in LPO. Moreover, enhancing levels of α-tocopherol in the liver by either feeding a diet high in d-α-tocopheryl acetate or by repeated injections of α-tocopherol did not affect AAP-induced liver injury. These results suggest that LPO is not a critical event responsible for AAP hepatotoxicity.

This is somewhat surprising because AAP induces a substantial mitochondrial oxidant stress and peroxynitrite formation (Jaeschke, 1990; Knight et al., 2001). This oxidant stress precedes cell injury by several hours (Jaeschke et al., 2003b) and free radical scavengers attenuate AAP-induced liver injury (Knight et al., 2002; Nakae et al., 1990). These data support the hypothesis that reactive oxygen and reactive nitrogen species are important for the progression of the cell injury after the initial protein binding of NAPQI. The fact that AAP-induced liver injury was not reduced in animals on a high d-α-tocopheryl acetate diet or after repeated injections of α-tocopherol was not due to insufficient loading of liver cells with α-tocopherol. Firstly, both diet and injections effectively increased liver α-tocopherol levels by 5- to 7-fold, consistent with previous observations in rats (Fariss and Zhang, 2003; Fariss et al., 2001). This level of hepatic α-tocopherol enrichment has been shown to protect rat hepatocytes and their mitochondria from toxic oxidative stress (Fariss and Zhang, 2003; Fariss et al., 2001). Secondly, despite higher α-tocopherol levels in fed mice compared to fasted animals, the injury was not reduced. Thirdly, LPO and injury induced by allyl alcohol/iron treatment was significantly attenuated. These data clearly indicate that sufficient α-tocopherol was loaded into liver cells to reduce potential LPO and, if this LPO had been the cause of cell damage, liver injury would have been attenuated.

Based on these findings we can conclude that, despite the significance of a mitochondrial oxidant stress and of peroxynitrite formation after AAP overdose, LPO is not involved in the pathophysiology of AAP-induced hepatotoxicity. Our data are in contrast to a recent study where pretreatment with vitamin E (30 mg/kg) for 30 min before AAP was reported to attenuate AAP-induced liver injury (Sener et al., 2003). However, based on our previous experience (Jaeschke et al., 1992), it is questionable that such a low dose of vitamin E in combination with a short pretreatment time can significantly elevate hepatic vitamin E levels. Thus, the mechanism of this protective effect is unclear.

The differences observed in the effect of fasting on the loss of hepatic and plasma α-tocopherol levels between mice fed a control diet (49 IU vitamin E/kg diet) and a supplemented diet (1000 IU vitamin E/kg diet) are not surprising. A previous report by Machlin and Gabriel (1982) showed that the half-life of α-tocopherol in the liver and plasma of rats fed high levels of vitamin E (approximately 10,000 IU/kg diet) were 0.8 and 1.2 days, respectively. Thus, under overnight fasting conditions (as α-tocopherol is only obtained through the diet), we would expect a substantial depletion of hepatic and plasma α-tocopherol levels as shown in Figures 1A and 1B. The absence of a similar depletion in hepatic and plasma α-tocopherol levels in fasted mice on the control diet suggests the presence of a separate and more stable pool of α-tocopherol in the liver (as plasma α-tocopherol is derived predominately from hepatic α-tocopherol). Still, there was no difference in injury between fasted and fed animals, which were treated at the time of the minimum of the diurnal rhythm of hepatic GSH levels (Jaeschke and Wendel, 1985). These results are similar to those of our previous report in this strain of mice, where we showed that fed animals treated in the afternoon (minimum levels of hepatic GSH: 40–45 nmol/mg protein) with 300 mg/kg AAP suffered severe liver injury (Gujral et al., 2002). In contrast, fed animals treated with AAP in the early morning (maximum of hepatic GSH levels: 60–65 nmol/mg protein) did not show signs of hepatotoxicity up to 24 h. Although the GSH levels of these fed animals were higher than those of fasted animals (20–25 nmol/mg protein), our data suggest that the dose of 300 mg/kg AAP was sufficient to deplete hepatic GSH levels and cause enough protein binding to trigger the secondary injury mechanisms (Gujral et al., 2002).

Recent findings suggest that γ-tocopherol can react with peroxynitrite (Christen et al., 1997; Hoglen et al., 1997) and even reduce protein nitration in vivo (Jiang et al., 2002). However, despite increased levels of γ-tocopherol in the liver after repeated injections of this antioxidant, allyl alcohol–induced LPO and injury were attenuated but AAP-induced liver injury was not reduced. Since there is evidence for the importance of peroxynitrite formation in the pathophysiology of AAP hepatotoxicity (Gardner et al., 2002; Knight et al., 2002), these data suggest that peroxynitrite levels were unaffected by high γ-tocopherol content in vivo. The most likely reason for this observation is the fact that the lipid-soluble γ-tocopherol is membrane-bound, where it protected against LPO. However, peroxynitrite is a water-soluble compound, which most likely reacts more readily with water-soluble antioxidants (e.g., GSH) or proteins. Consistent with this conclusion, we did not find a reduction in nitrotyrosine staining with γ-tocopherol but a very effective attenuation of nitrotyrosine formation with late treatment of GSH (Knight et al., 2002). Thus, our data do not support the hypothesis that γ-tocopherol is an effective scavenger for peroxynitrite in the liver in vivo.

We used the combination of allyl alcohol/ferrous iron to induce massive LPO and liver injury. The metabolism of allyl alcohol by alcohol dehydrogenase causes the formation of acrolein, a highly reactive unsaturated aldehyde that depletes hepatic glutathione levels, and an excessive amount of NADH, which can trigger a reductive mobilization of iron (Jaeschke et al., 1987, 1992). Consistent with previous findings (Jaeschke et al., 1987, 1992; Mathews et al., 1994; Wendel et al., 1982), only massive LPO, as indicated by a more than 30-fold increase of liver MDA levels, induced significant liver injury within 1–2 h. The increased levels of α-tocopherol attenuated LPO only by about 50% but almost completely prevented liver injury. These findings support previous conclusions that moderate LPO with a 1- to-2-fold increase of LPO parameters over baseline, as encountered during most pathophysiological situations with oxidant stress in vivo, is quantitatively not sufficient to cause cell injury (Jaeschke, 1995; Mathews et al., 1994). Therefore, recent suggestions that, under conditions of iNOS inhibition, the mechanism of AAP hepatotoxicity may switch from a peroxynitrite-induced injury mechanism to LPO (Hinson et al., 2002) could not be supported by our findings. Firstly, the reported minor increase in plasma levels of MDA in animals treated with the iNOS inhibitor aminoguanidine compared to AAP alone (Hinson et al., 2002) was not detected in the liver (Table 1). Secondly, loading the liver with α-tocopherol had no effect on the injury. Thus, even under conditions of iNOS inhibition, LPO is not a relevant mechanism of AAP-induced liver injury.

In summary, we demonstrated that an AAP overdose caused serious liver injury but no LPO in control animals or after treatment with the iNOS inhibitor aminoguanidine. Consequently, enhancing the hepatic content of α- or γ-tocopherol by feeding a diet high in d-α-tocopheryl acetate or direct administration for several days did not reduce AAP-induced liver injury. On the other hand, increasing hepatic levels of α- or γ-tocopherol significantly attenuated allyl alcohol/iron–induced LPO and liver injury. These results suggest that hepatocytes were enriched with sufficient tocopherol to inhibit potential LPO. We conclude that, despite the previously shown mitochondrial oxidant stress and peroxynitrite formation after AAP overdose, LPO is not a critical event in the mechanism of injury and, therefore, lipid-soluble antioxidants are ineffective in reducing AAP-induced liver injury. Thus, water-soluble radical scavengers, for example, GSH (Knight et al., 2002), may be more promising as therapeutic agents in preventing the progression of AAP-induced liver injury.

TABLE 1

Effect of Aminoguanidine on Acetaminophen-Induced Lipid Peroxidation and Liver Injury

Treatment ALT (U/l) MDA (nmol/mg protein) 
Note. Animals were fed a control diet (CD) or a diet high in d-α-tocopheryl acetate (VitED) for 1 week. Then all animals received 300 mg/kg acetaminophen (AAP) and were sacrificed 6 h later. Some of the animals were treated with 15 mg/kg of the iNOS inhibitor aminoguanidine (AG) at the time of AAP injection and 2 h later. Plasma ALT activities and liver malondialdehyde (MDA) levels were measured as indicators of liver injury and lipid peroxidation, respectively. Data represent means ± SE of n = 5 animals per group. 
*p < 0.05 (compared to controls). 
Controls 25 ± 8 10.7 ± 2.1 
AAP (CD) 2960 ± 616* 16.1 ± 1.7 
AAP (VitED) 3195 ± 319* 12.7 ± 0.5 
AAP (CD) + AG 3879 ± 607* 15.7 ± 1.5 
AAP (VitED) + AG 3136 ± 693* 14.4 ± 1.6 
Treatment ALT (U/l) MDA (nmol/mg protein) 
Note. Animals were fed a control diet (CD) or a diet high in d-α-tocopheryl acetate (VitED) for 1 week. Then all animals received 300 mg/kg acetaminophen (AAP) and were sacrificed 6 h later. Some of the animals were treated with 15 mg/kg of the iNOS inhibitor aminoguanidine (AG) at the time of AAP injection and 2 h later. Plasma ALT activities and liver malondialdehyde (MDA) levels were measured as indicators of liver injury and lipid peroxidation, respectively. Data represent means ± SE of n = 5 animals per group. 
*p < 0.05 (compared to controls). 
Controls 25 ± 8 10.7 ± 2.1 
AAP (CD) 2960 ± 616* 16.1 ± 1.7 
AAP (VitED) 3195 ± 319* 12.7 ± 0.5 
AAP (CD) + AG 3879 ± 607* 15.7 ± 1.5 
AAP (VitED) + AG 3136 ± 693* 14.4 ± 1.6 
FIG. 1.

(A) Liver and (B) plasma α-tocopherol levels were measured in untreated mice, which were fed a rodent diet with standard levels (35 mg/kg diet, CD) or high levels (1 g/kg, α-Toc) of d-α-tocopheryl acetate for 7 days. Fed animals and animals fasted overnight are compared. Data represent means ± SE of n = 4 animals per group. *p < 0.05 (compared to CD). #p < 0.05 (fed vs. fasted).

FIG. 1.

(A) Liver and (B) plasma α-tocopherol levels were measured in untreated mice, which were fed a rodent diet with standard levels (35 mg/kg diet, CD) or high levels (1 g/kg, α-Toc) of d-α-tocopheryl acetate for 7 days. Fed animals and animals fasted overnight are compared. Data represent means ± SE of n = 4 animals per group. *p < 0.05 (compared to CD). #p < 0.05 (fed vs. fasted).

FIG. 2.

(A) Plasma ALT activities and (B) hepatic malondialdehyde (MDA) content were determined in control C3Heb/FeJ mice (Co), 6 h after ip injection of acetaminophen (300 mg AAP/kg) or 2 h after treatment with a combination of phorone/FeSO4/allyl alcohol (AA). All animals were starved overnight. The animals were either fed a rodent diet with standard levels (35 mg/kg diet, CD) or high levels (1 g/kg, α-Toc) of d-α-tocopheryl acetate for 7 days. Data represent means ± SE of n = 5 animals per group. *p < 0.05 (compared to controls, Co). #p < 0.05 (CD vs. α-Toc).

FIG. 2.

(A) Plasma ALT activities and (B) hepatic malondialdehyde (MDA) content were determined in control C3Heb/FeJ mice (Co), 6 h after ip injection of acetaminophen (300 mg AAP/kg) or 2 h after treatment with a combination of phorone/FeSO4/allyl alcohol (AA). All animals were starved overnight. The animals were either fed a rodent diet with standard levels (35 mg/kg diet, CD) or high levels (1 g/kg, α-Toc) of d-α-tocopheryl acetate for 7 days. Data represent means ± SE of n = 5 animals per group. *p < 0.05 (compared to controls, Co). #p < 0.05 (CD vs. α-Toc).

FIG. 3.

Immunohistochemical staining of liver sections for 4-hydroxynonenal (4-HNE) as an index of lipid peroxidation. (A) There was no positive staining for 4-HNE in livers of control animals. (B) In contrast, massive, predominantly periportal staining was observed after treatment for 2 h with phorone/FeSO4/allyl alcohol. (C and D). However, no relevant staining was observed 6 h after acetaminophen. pv, portal vein; cv, central vein; (A–C) ×200; (D) ×400.

FIG. 3.

Immunohistochemical staining of liver sections for 4-hydroxynonenal (4-HNE) as an index of lipid peroxidation. (A) There was no positive staining for 4-HNE in livers of control animals. (B) In contrast, massive, predominantly periportal staining was observed after treatment for 2 h with phorone/FeSO4/allyl alcohol. (C and D). However, no relevant staining was observed 6 h after acetaminophen. pv, portal vein; cv, central vein; (A–C) ×200; (D) ×400.

FIG. 4.

Liver α- and γ-tocopherol levels were measured in untreated mice, which either received vehicle (olive oil), 0.19 mmol/kg α-tocopherol, or 0.19 mmol/kg γ-tocopherol by ip injection daily for 3 days. The animals were starved overnight and sacrificed 18 h after the last treatment. Data represent means ± SE of n = 3–4 animals per group. *p < 0.05 (compared to controls).

FIG. 4.

Liver α- and γ-tocopherol levels were measured in untreated mice, which either received vehicle (olive oil), 0.19 mmol/kg α-tocopherol, or 0.19 mmol/kg γ-tocopherol by ip injection daily for 3 days. The animals were starved overnight and sacrificed 18 h after the last treatment. Data represent means ± SE of n = 3–4 animals per group. *p < 0.05 (compared to controls).

FIG. 5.

(A) Plasma ALT activities and (B) hepatic malondialdehyde (MDA) content were determined in control C3Heb/FeJ mice (Co), 6 h after ip injection of acetaminophen (300 mg AAP/kg) or 2 h after treatment with a combination of phorone/FeSO4/allyl alcohol (AA). Animals received vehicle (olive oil), 0.19 mmol/kg α-tocopherol, or 0.19 mmol/kg γ-tocopherol by ip injection daily for 3 days before treatment with AAP or AA. Data represent means ± SE of n = 4 animals per group. *p < 0.05 (compared to Co). #p < 0.05 (compared to AAP or AA alone).

FIG. 5.

(A) Plasma ALT activities and (B) hepatic malondialdehyde (MDA) content were determined in control C3Heb/FeJ mice (Co), 6 h after ip injection of acetaminophen (300 mg AAP/kg) or 2 h after treatment with a combination of phorone/FeSO4/allyl alcohol (AA). Animals received vehicle (olive oil), 0.19 mmol/kg α-tocopherol, or 0.19 mmol/kg γ-tocopherol by ip injection daily for 3 days before treatment with AAP or AA. Data represent means ± SE of n = 4 animals per group. *p < 0.05 (compared to Co). #p < 0.05 (compared to AAP or AA alone).

1
To whom correspondence should be addressed at the University of Arizona, College of Medicine, Liver Research Institute, 1501 North Campbell Avenue, Room 6309, Tucson, AZ 85724. Fax: (520) 626-5975. E-mail: jaeschke@email.arizona.edu.

This investigation was supported in part by National Institutes of Health Grant AA12916 (to H.J.) and the Gasper and Irene Lazzara Foundation (M.W.F.).

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