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TOXICOLOGY
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (M.L.B., T.R.K., H.J.); Liver Research Institute, University of Arizona College of Medicine, Tucson, Arizona (M.L.B., T.R.K., H.J.); and Department of Pathology, University of Texas Health Science Center, Houston, Texas (A.F.)
Received April 2, 2003; accepted June 26, 2003.
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Abstract |
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). The mechanism of toxicity involves the metabolic activation of AAP via the cytochrome P450 system, which results in the formation of an electrophilic reactive metabolite (Jollow et al., 1973), presumably N-acetyl-p-benzoquinone imine (NAPQI) (Dahlin et al., 1984). NAPQI can be conjugated with glutathione (GSH) and excreted. However, after hepatic GSH levels are depleted (Mitchell et al., 1973), NAPQI can covalently bind to cellular proteins (Jollow et al., 1973; Cohen and Khairallah, 1997; Qiu et al., 1998). One of the consequences of protein binding is the development of mitochondrial dysfunction with inhibition of respiration (Meyers et al., 1988; Ramsay et al., 1989), a decrease in hepatic ATP levels (Jaeschke, 1990; Tirmenstein and Nelson, 1990), mitochondrial cytochrome c release (Knight and Jaeschke, 2002), and mitochondrial oxidant stress and peroxynitrite formation (Jaeschke, 1990; Knight et al., 2001). Recent studies showed significantly reduced AAP-induced liver injury after treatment of rats with an inducible nitric oxide synthase (iNOS) inhibitor (Gardner et al., 1998) and in iNOS gene knockout mice (Gardner et al., 2002). However, others did not find protection with similar interventions (Michael et al., 2001; Hinson et al., 2002). On the other hand, we demonstrated recently that scavenging peroxynitrite with GSH strongly attenuated hepatic necrosis during the first 6 h after AAP administration (Knight et al., 2002). The protection was observed in the presence of continued mitochondrial dysfunction and oxidant stress. Despite the controversy about the exact source of nitric oxide, these data suggest that peroxynitrite is a critical mediator of AAP toxicity in mice (Knight et al., 2002). However, a potential long-term benefit of peroxynitrite scavenging has not been demonstrated in this model. Because of the continued mitochondrial oxidant stress in these animals, this is a critical question.
Liver regeneration is a vital process for survival after a toxic insult (Chanda and Mehendale, 1996). Regeneration ensures the replacement of necrotic cells and the full recovery of organ function. Since hepatocytes are mostly in a quiescent state (G0), the regeneration process requires entry into the highly regulated cell cycle (Fausto, 2000). The first step of this process is the priming of hepatocytes by cytokines such as TNF-
and IL-6 (Akerman et al., 1992; Cressman et al., 1996), which makes cells more responsive to growth factors (Fausto et al., 1995). The exposure to growth factors such as hepatocyte growth factor or transforming growth factor- results in the expression of cell cycle proteins (e.g., cyclins and cyclin-dependent kinases) and cell cycle inhibitors (e.g., p21 and p27) (Fausto, 2000). The induction of cyclin D1 is considered a marker of cell cycle progression in hepatocytes (G1 phase) (Albrecht and Hansen, 1999). Once hepatocytes express cyclin D1, they have passed the G1 restriction point and are committed to DNA replication (Fausto, 2000). Interestingly, growth factors also induce inhibitors of the cell cycle (e.g., p21) (Albrecht et al., 1998). The simultaneous expression of activators and repressors of the cell cycle allows a sensitive regulation of liver regeneration (Fausto, 2000).
The aim of our investigation was to evaluate whether a treatment regimen with intravenous GSH that mainly scavenges peroxynitrite has a long-term protective effect against AAP-induced liver injury and enhances survival in this model. Furthermore, we investigated whether the reduced injury and lower mortality is correlated with cell cycle entry (expression of cyclin D1 and p21) and regeneration.
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Materials and Methods |
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Experimental Protocols. At selected times after AAP treatment, the animals were killed by cervical dislocation. Blood, which was drawn from the vena cava with a syringe, was either added to a heparinized tube and centrifuged to obtain plasma or added to a sterile tube and allowed to clot to obtain serum. Plasma was used for 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 glutathione and cell cycle protein expression.
Methods. Plasma ALT activities were determined with the test kit DG 159-UV (Sigma-Aldrich) and expressed as IU/liter. Serum levels of interleukin-6 were determined with a murine IL-6 enzyme-linked immunosorbent assay kit (R & D Systems). Total soluble GSH and GSSG were measured in the liver homogenate with a modified method of Tietze as described in detail previously (Jaeschke and Mitchell, 1990). Briefly, the frozen tissue was homogenized on ice in 3% sulfosalicylic acid containing 0.1 mM EDTA. An aliquot of the homogenate was added to 10 mM N-ethylmaleimide (NEM) in potassium phosphate buffer (KPP), and another aliquot was added to 0.01 N HCl. The NEM-KPP sample was centrifuged, and the supernatant was passed through a C18 cartridge to remove free NEM and NEM-GSH adducts (Sep-Pak; Waters, Milford, MA). The HCl sample was centrifuged, and the supernatant was diluted with KPP. All samples were assayed using dithionitrobenzoic acid. All data are expressed in GSH equivalents.
Histology and Immunohistochemistry. Formalin-fixed tissue samples were embedded in paraffin, and 5-µm sections were cut. Replicate sections were stained with H&E for evaluation of oncotic necrosis (Gujral et al., 2001, 2002). Necrotic cells were identified using the following morphological criteria: increased eosinophilia, cell swelling, cell lysis, karyorhexis, and karyolysis. The percentage of necrosis was estimated by evaluating the number of microscopic fields with necrosis compared with the entire cross-section. In general, necrosis was estimated at low power (x100); questionable areas were evaluated at higher magnification (x200 or x400). The pathologist evaluated all histological sections in a blinded fashion. Sections were also stained for the proliferating cell nuclear antigen (PCNA) using a rabbit polyclonal anti-PCNA antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and standard immunohistochemical procedures as described in detail previously (Chosay et al., 1998).
Western Blot Analyses. Expression of the cell cycle proteins cyclin D1 and p21 as well as the PCNA were analyzed by Western blotting as described in detail previously (Bajt et al., 2000). Briefly, freshly excised liver was homogenized in 25 mM HEPES buffer, pH 7.5, containing 5 mM EDTA, 2 mM dithiothreitol, 1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, and 1 µg/ml pepstatin, leupeptin, and aprotinin. Homogenates were centrifuged at 14,000g for 20 min at 4°C. Protein concentrations in the cytosolic extracts were determined using the bicinchoninic acid kit (Pierce Biotechnology, Rockford, IL). Cytosolic extracts (50 µg per lane) were resolved by 4 to 20% SDS-polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to polyvinylidine difluoride membranes (Immobilin-P; Millipore Corporation, Bedford, MA) that were then blocked with 5% milk in Tris-buffered saline (20 mM Tris and 0.15 M NaCl, pH 7.4), containing 0.1% Tween 20, and 0.1% bovine serum albumin overnight at 4°C. After washing with Tris-buffered saline, membranes were then incubated with a mouse monoclonal anti-PCNA antibody, a mouse monoclonal anti-cyclin D1 antibody, or a goat polyclonal anti-p21 antibody (all from Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. The membranes were then washed again and incubated with the secondary antibody horseradish peroxidase-coupled anti-mouse or anti-goat IgG (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences Inc., Piscataway, NJ) according to the manufacturer's instructions.
Statistics. All results were expressed as mean ± S.E. Comparisons between multiple groups were performed with one-way analysis of variance followed by Bonferroni t test. If the data were not normally distributed, we used the Kruskal-Wallis test (nonparametric analysis of variance) followed by Dunn's Multiple Comparisons test. P < 0.05 was considered significant.
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). In agreement with these previous data, we observed significant increases in plasma ALT levels and histological evidence of centrilobular necrosis at 6 h after AAP treatment alone (Fig. 1, A and B). Although the plasma ALT activities did not further increase at later time points (Fig. 1A), the area of necrosis expanded to 80% of all hepatocytes at 12 to 24 h after AAP treatment (Fig. 1B). All animals survived up to 12 h. However, only 53% (8 of 15) of the AAP-treated mice were alive at 24 h and only 1 of 15 animals (6%) survived for 48 h. In striking contrast, animals treated with GSH showed significantly less liver injury at all time points up to 48 h, as indicated by the significantly lower plasma ALT values and the more than 60% reduction in the area of necrosis (Fig. 1). In addition, none of the animals (0 of 15) died during the 48-h experiment. In a separate long-term experiment, all animals treated with AAP + GSH survived for 7 days (5 of 5 animals) with ALT levels not significantly different from untreated animals (100 ± 28 U/liter).
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We previously showed that GSH treatment at 1.5 h after AAP did not prevent the mitochondrial oxidant stress 6 h after AAP administration (Knight et al., 2002). Compared with control animals, mice treated with AAP had substantially reduced hepatic GSH levels and 4.5-fold higher GSSG concentrations at 24 h after AAP (Fig. 2, A and B). This resulted in an increase of the hepatic GSSG-to-GSH ratio from 0.4 ± 0.1 in controls to 6.5 ± 1.2 in AAP-treated mice (P < 0.05). Animals receiving GSH had liver glutathione content similar to untreated controls (Fig. 2A) but showed significantly elevated levels of GSSG (Fig. 2B). The GSSG-to-GSH ratio (1.7 ± 0.1) was significantly reduced compared with AAP alone.
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To investigate how the time of treatment with GSH affected the protective effect, animals were treated with GSH at the same time as AAP or at 1.5, 2.25, or 3 h after AAP. Based on plasma ALT activities and histological evaluation of necrosis, the best protection was observed with GSH treatment at the same time as AAP (Fig. 3, A and B). A significantly reduced injury at 24 h was also found when the animals were treated 1.5 h after AAP. However, neither treatment at 2.25 nor at 3 h had a statistically significant effect on plasma ALT activities (Fig. 3, A and B). Although not significant, the area of necrosis still showed a trend to reduced injury (Fig. 3B). In addition, none of the GSH-treated animals died. This was in contrast to the 50% mortality in animals that received only AAP.
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To investigate whether the improved liver injury and survival of GSH-treated animals correlated with evidence of cell cycle activation and regeneration, cyclin D1 and PCNA levels were analyzed by Western blotting. Cyclin D1 was not detectable in livers of controls or animals treated only with AAP at 6 to 24 h (Fig. 4A). In contrast, cyclin D1 expression was clearly observed in GSH-treated animals at 12, 24, and 48 h after AAP administration (Fig. 4B). All controls and AAP-treated animals had low hepatic protein levels of PCNA (Fig. 5A). In contrast, hepatic PCNA levels progressively increased in GSH-treated animals from 12 to 48 h after AAP (Fig. 5B). Since it is known that cell cycle inhibitors are also activated during regeneration (Albrecht et al., 1998), we evaluated the expression of the cell cycle inhibitor p21. This protein was not expressed in controls and was not up-regulated after AAP treatment (Fig. 6A). However, in livers of GSH-treated animals, p21 expression was faintly detectable at 6 and 12 h and strongly expressed at 24 and 48 h after AAP (Fig. 6B). To assess when GSH has to be administered to induce cell cycle activation, animals were treated with GSH at various times after AAP injection. Analysis for cyclin D1 expression showed that GSH treatment at the time of AAP administration or at 1.5 and 2.25 h after AAP caused cyclin D1 expression at 24 h (Fig. 7). GSH administration at 3 h had no effect.
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Although the increased expression of cyclin D1, p21, and PCNA clearly indicate that liver cells in animals treated with GSH entered the cell cycle, the Western blot analysis did not reveal which cells are affected within the liver lobule. To investigate this issue, liver sections were immunostained for PCNA antigen. In agreement with the Western blot analyses, control livers did not stain for PCNA (Fig. 8A). Treatment with AAP alone did not increase PCNA immunoreactivity at any time point from 6 to 24 h (Fig. 8B and data not shown). In contrast, substantial PCNA staining was observed in hepatocytes surrounding the necrotic areas after treatment with AAP in combination with GSH (1.5 h) for 24 h (Fig. 8C).
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It is well established that liver regeneration is a multistep process. Priming of hepatocytes by cytokines such as TNF- and IL-6 is the initial event (Fausto, 2000). To investigate a potential involvement of IL-6 in the regeneration process of AAP/GSH-treated animals, IL-6 formation was measured. Serum concentrations of IL-6 progressively increased after AAP administration, reaching peak levels of almost 400% above baseline at 24 h (Fig. 9). In contrast, serum IL-6 values in GSH-treated animals did not significantly increase during the entire 48-h observation period (Fig. 9). Since pretreatment with IL-6 can enhance regeneration and protect against ischemia-reperfusion injury (Selzner et al., 1999), we treated the animals with similar doses of murine recombinant IL-6 (450 µg/kg) 24 h before AAP. However, IL-6 treatment had no effect on AAP-induced severe centrilobular necrosis (data not shown).
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Discussion |
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). In contrast, GSH treatment at the same time as AAP administration had similar effects but eliminated the mitochondrial oxidant stress. We concluded from these results that injection of GSH at 1.5 h or later minimally affected the detoxification of the reactive metabolite NAPQI but mainly acted as a scavenger of peroxynitrite (Knight et al., 2002). This conclusion is supported by the observation that a dose of 400 mg AAP/kg, injected i.p., caused maximal protein adduct formation during the first hour with no relevant further increase during the second hour (Roberts et al., 1991). This suggests that under conditions similar to ours, most of the reactive metabolite formation took place during the first hour after AAP administration. However, results by Corcoran et al. (1985a) indicate that after oral doses of 1000 mg AAP/kg, maximal covalent binding of the reactive metabolite is observed at 4 to 6 h and the biliary glutathione conjugate excretion is still strong at 4 h (Corcoran et al., 1985b). Although this suggests that reactive metabolite formation may occur later than 1 to 2 h after AAP, part of the reason for these results is the delayed absorption of an oral dose compared with i.p. injection. In addition, the very high dose of AAP used in these studies may have prolonged the metabolism. However, after i.p. administration of AAP, hepatic glutathione levels are exhausted in less than 30 min (Jaeschke, 1990; Knight et al., 2001) and covalent binding is maximal between 1 to 2 h (Roberts et al., 1991). Intravenously administered GSH is rapidly degraded in the kidney (half-life of plasma glutathione in fasted mice, 5 min) (Wendel and Jaeschke, 1982), the amino acids are reabsorbed in the kidney, redistributed, and taken up into hepatocytes, and GSH is resynthesized. Because of the multiple steps involved, it takes at least 30 to 60 min to measure a relevant increase and up to 2 h to fully restore hepatic glutathione levels in starved control animals (Wendel and Jaeschke, 1982). Because of the delay in treatment and the time needed for de novo GSH synthesis in the liver, it is most likely that the main impact of GSH administered at 1.5 h after AAP or later was not the detoxification of NAPQI but the scavenging of peroxynitrite and potentially other reactive oxygen species. It is well documented that GSH and other sulfhydryl-containing compounds, such as N-acetylcysteine, are potent scavengers of peroxynitrite, hydrogen peroxide, and hypochlorous acid in vitro (Liu et al., 1994; Knight et al., 2001, 2002). However, under in vivo conditions, both intravenously administered GSH or N-acetylcysteine will stimulate hepatic GSH synthesis by supplying the essential amino acid cysteine.
The administration of GSH after AAP treatment restored cytosolic and mitochondrial GSH levels to values slightly higher than those of untreated fasted animals at 6 h (Knight et al., 2002). In contrast, animals treated with AAP had still significantly lower levels in both the cytosol and in particular the mitochondria (Knight et al., 2002). This indicated that GSH administration accelerated the recovery of the critical GSH pools at the time when substantial peroxynitrite was formed in hepatocytes (Knight et al., 2001). Having less liver injury, AAP/GSH-treated animals ate similar to controls when food was offered again at 9 h after AAP administration. Consequently, these animals had supraphysiological levels of hepatic GSH at 24 h. These data are consistent with previous fasting/refeeding experiments (Wendel and Jaeschke, 1988). In contrast, the hepatic GSH levels in AAP-treated animals only recovered to levels of starved animals, suggesting that animals with severe liver injury did not eat, which may have contributed to the progressive demise of these animals. Despite the lower GSH levels, GSSG concentrations and the GSSG-to-GSH ratio remained very high in AAP-treated animals. This indicates that there is a continuing oxidant stress in these hepatocytes. We showed previously that the majority of GSSG is located in mitochondria at 6 (Knight et al., 2001) as well as 24 h (Jaeschke, 1990). Although the GSH-treated animals had elevated GSSG levels, the GSSG-to-GSH ratio was only modestly increased. These data suggest a reduced mitochondrial oxidant stress in AAP/GSH-treated animals at 24 h compared with AAP alone. Thus, the increased oxidant stress in both groups of animals at 6 h got worse in AAP-treated animals but improved in the AAP/GSH-treated animals.
In addition to the reduced oxidant stress and tissue necrosis, all animals treated with GSH showed clear evidence of cell cycle activation and tissue regeneration. In order for hepatocytes to divide (mitosis), they have to leave the resting state (G0) and go through the cell cycle with DNA synthesis (S phase) and mitosis (M phase) (Fausto, 2000). To go through the sequence of these steps, the cell has to pass a number of checkpoints. Expression of cyclin D1 is a reliable marker that the cell has overcome a restriction point in G1 and is irreversibly committed to DNA synthesis (G1/S transition) (Albrecht and Hansen, 1999). Furthermore, PCNA is synthesized in the G1 and S phases, resulting in prominent nuclear staining for this antigen in proliferating cells (Bravo and Macdonald-Bravo, 1987). In addition to promoters of the cell cycle, cell cycle inhibitors are also expressed in the regenerating liver (Albrecht et al., 1998). All three proteins (cyclin D1, p21, and PCNA) were expressed in livers of AAP/GSH-treated animals as early as 12 h after AAP administration. In contrast, none of these proteins increased in animals treated only with AAP. Interestingly, cell cycle protein expression at 24 h did not correlate with the severity of the injury at 24 h (Fig. 4). In contrast, cell cycle protein expression was observed in livers where peroxynitrite formation was prevented (GSH treatment at t = 0) or peroxynitrite was effectively scavenged (GSH treatment at t = 1.5 or 2.25 h) (Knight et al., 2002). Thus, we can conclude that GSH treatment, predominantly by scavenging peroxynitrite, attenuated the progression of AAP-induced liver injury and prevented acute liver failure, thereby allowing cell cycle activation and regeneration. This is critical for a full functional recovery of the injured liver and long-term survival.
Hepatocyte regeneration is a multistep process requiring cytokines and growth factors for its initiation (Fausto et al., 1995). Cytokines prime hepatocytes for the activating effect of growth hormones (Fausto, 2000). Neutralizing TNF- or eliminating the synthesis of IL-6 prevented a regenerative response after partial hepatectomy (Akerman et al., 1992; Cressman et al., 1996). In addition, pretreatment with recombinant IL-6 promoted liver regeneration in IL-6 gene-deficient mice (Cressman et al., 1996) and attenuated liver injury during ischemia reperfusion (Selzner et al., 1999). Interestingly, IL-6 formation was only detectable in AAP-treated animals, which did not show signs of regeneration during the first 24 h. In contrast, GSH treatment eliminated IL-6 formation in AAP-treated mice. Moreover, pretreatment with a high dose of murine IL-6, similar to previous successful experiments (Cressman et al., 1996; Selzner et al., 1999), had no effect on AAP-induced liver injury. In contrast to these results, Chanda et al. (1995) showed that forcing hepatocytes into cell division with a low dose of thioacetamide protected rat livers from subsequent AAP-induced hepatotoxicity. This demonstrates that an early regeneration response can reduce toxic liver injury. On the other hand, treatment with growth factors that stimulate mitosis did not prevent acetaminophen-induced liver failure in dogs (Francavilla et al., 1993). These data suggest that regeneration cannot rescue severely injured cells. Our data agree with these results. The severely damaged hepatocytes after AAP were not able to enter the cell cycle and mount a regenerative response despite the formation of IL-6. However, blunting the progression of the injury by GSH treatment preserved the capacity of the liver to regenerate and therefore to recover from the insult. Interestingly, in contrast to the regenerative response after partial hepatotectomy (Cressman et al., 1996), regeneration after AAP-induced liver injury appears to be independent of IL-6. One possible explanation for these findings could be that the TNF- generated in AAP-treated animals may stimulate hepatic regeneration by directly, i.e., independent of IL-6, inducing the expression of transforming growth factor-, which is a potent mitogen for hepatocytes (Gallucci et al., 2000).
In summary, our data demonstrated that treatment with GSH after a hepatotoxic dose of AAP attenuated the intracellular oxidant stress and liver cell necrosis and consequently prevented death from liver failure. In contrast to animals that received AAP alone, all livers of GSH-treated animals showed clear evidence for cell cycle activation and regeneration. We conclude that accelerated restoration of cellular GSH levels at the time of peroxynitrite formation can attenuate the progression of AAP-induced liver injury and allows liver regeneration and ultimately, survival.
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Footnotes |
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ABBREVIATIONS: AAP, acetaminophen; ALT, alanine aminotransferase; GSH, reduced glutathione; GSSG, glutathione disulfide; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; KPP, potassium phosphate buffer; NAPQI, N-acetyl-p-benzoquinone imine; NEM, N-ethylmaleimide; PCNA, proliferating cell nuclear antigen; TNF, tumor necrosis factor.
Address correspondence to: Dr. Mary Lynn Bajt, Liver Research Institute, University of Arizona, College of Medicine, 1501 N. Campbell Avenue, Room 6309, Tucson, AZ 85724. E-mail: mlb3{at}email.arizona.edu
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  L. M. Aleksunes, S. N. Campion, M. J. Goedken, and J. E. Manautou Acquired Resistance to Acetaminophen Hepatotoxicity is Associated with Induction of Multidrug Resistance-Associated Protein 4 (Mrp4) in Proliferating Hepatocytes Toxicol. Sci., August 1, 2008; 104(2): 261 - 273. [Abstract] [Full Text] [PDF]
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M. L. Bajt, H.-M. Yan, A. Farhood, and H. Jaeschke Plasminogen Activator Inhibitor-1 Limits Liver Injury and Facilitates Regeneration after Acetaminophen Overdose Toxicol. Sci., August 1, 2008; 104(2): 419 - 427. [Abstract] [Full Text] [PDF]
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 M. L. Bajt, A. Farhood, J. J. Lemasters, and H. Jaeschke Mitochondrial Bax Translocation Accelerates DNA Fragmentation and Cell Necrosis in a Murine Model of Acetaminophen Hepatotoxicity J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 8 - 14. [Abstract] [Full Text] [PDF]
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 P. Pacher, J. S. Beckman, and L. Liaudet Nitric Oxide and Peroxynitrite in Health and Disease Physiol Rev, January 1, 2007; 87(1): 315 - 424. [Abstract] [Full Text] [PDF]
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 J.-H. Zhu, X. Zhang, J. P. McClung, and X. G. Lei Impact of Cu,Zn-Superoxide Dismutase and Se-Dependent Glutathione Peroxidase-1 Knockouts on Acetaminophen-Induced Cell Death and Related Signaling in Murine Liver Experimental Biology and Medicine, December 1, 2006; 231(11): 1726 - 1732. [Abstract] [Full Text] [PDF]
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 D. Botta, S. Shi, C. C. White, M. J. Dabrowski, C. L. Keener, S. L. Srinouanprachanh, F. M. Farin, C. B. Ware, W. C. Ladiges, R. H. Pierce, et al. Acetaminophen-induced Liver Injury Is Attenuated in Male Glutamate-cysteine Ligase Transgenic Mice J. Biol. Chem., September 29, 2006; 281(39): 28865 - 28875. [Abstract] [Full Text] [PDF]
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H. Jaeschke and M. L. Bajt Intracellular Signaling Mechanisms of Acetaminophen-Induced Liver Cell Death Toxicol. Sci., January 1, 2006; 89(1): 31 - 41. [Abstract] [Full Text] [PDF]
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C. Cover, A. Mansouri, T. R. Knight, M. L. Bajt, J. J. Lemasters, D. Pessayre, and H. Jaeschke Peroxynitrite-Induced Mitochondrial and Endonuclease-Mediated Nuclear DNA Damage in Acetaminophen Hepatotoxicity J. Pharmacol. Exp. Ther., November 1, 2005; 315(2): 879 - 887. [Abstract] [Full Text] [PDF]
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C. Cover, P. Fickert, T. R. Knight, A. Fuchsbichler, A. Farhood, M. Trauner, and H. Jaeschke Pathophysiological Role of Poly(ADP-Ribose) Polymerase (PARP) Activation during Acetaminophen-Induced Liver Cell Necrosis in Mice Toxicol. Sci., March 1, 2005; 84(1): 201 - 208. [Abstract] [Full Text] [PDF]
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M. L. Bajt, T. R. Knight, J. J. Lemasters, and H. Jaeschke Acetaminophen-Induced Oxidant Stress and Cell Injury in Cultured Mouse Hepatocytes: Protection by N-Acetyl Cysteine Toxicol. Sci., August 1, 2004; 80(2): 343 - 349. [Abstract] [Full Text] [PDF]
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, P < 0.05 (compared with controls, t = 0); #, P < 0.05 (compared with AAP).
