Pathophysiological Role of Poly(ADP-Ribose) Polymerase (PARP) Activation during Acetaminophen-Induced Liver Cell Necrosis in Mice
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《毒物学科学杂志》
Liver Research Institute, University of Arizona, Tucson, Arizona 85737
Departments of Medicine and Pathology, Medical University of Graz, Graz, Austria
Department of Pathology, University of Texas Health Science Center, Houston, Texas 77030
ABSTRACT
DNA fragmentation in hepatocytes occurs early after acetaminophen (AAP) overdose in mice. DNA strandbreaks can induce excessive activation of poly(ADP-ribose) polymerases (PARP), which may lead to oncotic necrosis. Based on controversial findings with chemical PARP inhibitors, the role of PARP-1 activation in AAP hepatotoxicity remains unclear. To investigate PARP-1 activation and evaluate a pathophysiological role of PARP-1, we used both PARP inhibitors (3-aminobenzamide; 5-aminoisoquinolinone) and PARP gene knockout mice (PARP–/–). Treatment of C3Heb/FeJ mice with 300 mg/kg AAP resulted in DNA fragmentation and alanine aminotransferase (ALT) release as early as 3 h, with further increase of these parameters up to 12 h. Few nuclei of hepatocytes stained positive for poly-ADP-ribosylated nuclear proteins (PAR) as indicator for PARP-1 activation at 4.5 h. However, the number of PAR-positive cells and staining intensity increased substantially at 6 and 12 h. Pretreatment with 500 mg/kg 3-aminobenzamide before AAP attenuated hepatic glutathione depletion and completely eliminated DNA fragmentation and liver injury. Delayed treatment several hours after AAP was still partially protective. On the other hand, liver injury was not attenuated in PARP–/– mice compared to wild-type animals. Similarly, the specific PARP-1 inhibitor 5-aminoisoquinolinone (5 mg/kg) was not protective. However, 3-aminobenzamide attenuated liver injury in WT and PARP–/– mice. In summary, PARP-1 activation is a consequence of DNA fragmentation after AAP overdose. However, PARP-1 activation is not a relevant event for AAP-induced oncotic necrosis. The protection of 3-aminobenzamide against AAP-induced liver injury was due to reduced metabolic activation and potentially its antioxidant effect but independent of PARP-1 inhibition.
Key Words: acetaminophen; hepatotoxicity; poly(ADP-ribose) polymerase-1 (PARP-1); DNA fragmentation; 3-aminobenzamide.
INTRODUCTION
Acetaminophen (AAP), a widely used analgesic drug, is safe at therapeutic doses, but an overdose can cause severe liver injury in experimental animals and humans. The toxicity depends on the metabolic activation and formation of a reactive metabolite, presumably N-acetyl-p-benzoquinone imine (NAPQI), which is initially detoxified by reacting with glutathione (GSH) (Nelson, 1990). However, after the cellular GSH content is exhausted, NAPQI covalently binds to cellular proteins (Cohen and Khairallah, 1997; Jollow et al., 1973) including mitochondrial proteins (Qiu et al., 2001; Tirmenstein and Nelson, 1989). This leads to mitochondrial dysfunction, as indicated by inhibition of mitochondrial respiration (Meyers et al., 1988; Ramsay et al., 1989), increased mitochondrial oxidant stress and peroxynitrite formation (Jaeschke, 1990; Knight et al., 2001, 2002), ATP depletion (Jaeschke, 1990; Tirmenstein and Nelson, 1990), Bax translocation to mitochondria (Adams et al., 2001; El-Hassan et al., 2003), and cytochrome c release (Adams et al., 2001; Knight and Jaeschke, 2002). Eventually, the mitochondria form membrane permeability transition pores, and the mitochondrial membrane potential collapses, leading to necrotic cell death (Kon et al., 2004).
In addition to mitochondrial dysfunction, DNA fragmentation has been shown to occur early in the pathophysiology of AAP hepatotoxicity in vivo (Ray et al., 1990, 1993) and in isolated hepatocytes (Shen et al., 1991, 1992). The fact that a general endonuclease inhibitor prevented DNA fragmentation and protected against AAP-induced liver injury (Shen et al., 1992) supported the hypothesis that DNA fragmentation is an important event in the mechanism of cell injury. Although the endonuclease(s) involved in this process have not been conclusively identified, it is unlikely to be the caspase-activated desoxyribonuclease (CAD). There is no relevant caspase-3 activation after AAP overdose (Adams et al., 2001; Gujral et al., 2002; Lawson et al., 1999), and the DNA fragments are different than typically generated during caspase-dependent apoptosis (Jahr et al., 2001). Nevertheless, the DNA damage may lead to activation of poly(ADP-ribose)polymerases (PARP-1) in the nucleus (Szabo and Dawson, 1998). PARP-mediated poly-ADP-ribosylation of nuclear proteins contributes to DNA repair (Szabo and Dawson, 1998). However, excessive activation of PARP depletes cellular NAD+, which subsequently triggers ATP depletion and necrotic cell death (Ha and Snyder, 1999). The potential role of PARP in AAP-induced cell death was previously investigated by using relatively unspecific PARP inhibitors. The results were not consistent. Corcoran and coworkers did not find a protective effect with 3-aminobenzamide (3-AB) in mouse hepatocytes at early time points but observed an aggravation of injury at 12 h after AAP treatment (Shen et al., 1992). In contrast, high doses of 4-aminobenzamide and nicotinamide were shown to prevent AAP hepatotoxicity in vivo (Kroger et al., 1997; Ray et al., 2001). To resolve this controversy and to investigate whether PARP-1 is actually activated and plays a critical role in the early injury phase after AAP overdose, we tested the potential protective effect of 3-aminobenzamide and the novel, more specific PARP inhibitor 5-aminoisoquinolinone (Thiemermann, 2002) in C3Heb/FeJ mice and assessed AAP-induced liver injury in PARP-1 gene knockout mice of the SV129 background strain.
MATERIALS AND METHODS
Animals. Male C3Heb/FeJ mice with an average weight of 18 to 20 g were purchased from Jackson Laboratory (Bar Harbor, Maine). PARP gene knockout mice (PARP–/–) were kindly provided by Dr. E. F. Wagner (Research Institute of Molecular Pathology, Vienna, Austria). The generation of the PARP–/– mice was described previously (Wang et al., 1995). All animals were housed in an environmentally controlled room with 12 h light/dark cycle and allowed free access to food (certified rodent diet no. 8640, Harlan Teklad, Indianapolis, IN) and water. The experimental protocols followed the criteria of the University of Arizona, the Medical University of Graz, and the National Research Council for the care and use of laboratory animals in research. All animals were fasted overnight before the experiments. Animals
Experimental protocols. At selected times after AAP 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 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.
Methods. Plasma ALT activities were determined with the kinetic test kit 68-B (Biotron Diagnostics, Inc., Hernet, CA) and expressed as IU/liter. Protein concentrations were assayed using the bicinchoninic acid kit (Pierce, Rockford, IL). Total soluble GSH and GSSG were measured in the liver homogenate with a modified method of Tietze as described in detail (Knight et al., 2002). Briefly, the frozen tissue was homogenized at 0°C in 3% sulfosalicylic acid containing 0.1 mM EDTA. After dilution with 0.01 N HCl, the sample was centrifuged, and the supernatant was diluted with 100 mM potassium phosphate buffer (KPP), pH 7.4. The samples were assayed using dithionitrobenzoic acid. All data are expressed in GSH-equivalents. DNA fragmentation was evaluated using the Cell Death Detection ELISA (anti-histone ELISA) (Roche Diagnostics, Indianapolis, IN) as described in detail (Lawson et al., 1999). In this assay, the kinetics (vmax) of product generation is a measure of DNA fragmentation. The vmax values obtained for untreated controls (100%) are compared with those in treated groups. The assay allows the specific quantitation of cytoplasmic histone-associated DNA fragments.
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 between the different lobes of the liver in this model. The percent of necrosis was estimated by evaluating the number of microscopic fields with necrosis compared to the entire cross section. All histological evaluations were done in a blinded fashion by the pathologist (A.F.). Nitrotyrosine staining was assessed by immunohistochemistry with the DAKO LSAB Peroxidase Kit (K684) (DAKO Corp., Carpinteria, CA), which was used according to the manufacturer's instructions (Knight et al., 2002). The anti-nitrotyrosine antibody was obtained from Molecular Probes (Eugene, OR). For PAR-staining, deparaffinized and rehydrated liver sections were incubated in 1x Antigen Retrieval Solution (Dako Cytomation, Carpinteria, CA) for 30 min. at 95°C. Sections were then treated with 10% trichloroacetic acid for 10 min at room temperature. PAR was detected by incubating sections for 2 h with an anti-pADPr, IgY antibody (1:50, Tulip Biolabs, West Point, PA) followed by 1 h incubation with a biotinylated goat anti-chicken antibody (1:100, Vector, Burlingame, CA). Antibody binding was visualized using the Vectastain Elite ABC (peroxidase) Standard Kit using AEC (Dako) as a substrate.
Statistics. All results were expressed as mean ± SE. Comparisons between multiple groups were performed with one-way ANOVA followed by Bonferroni t-test. If the data were not normally distributed, we used the Kruskal-Wallis Test (nonparametric ANOVA) followed by Dunn's Multiple Comparisons Test; p < 0.05 was considered significant.
RESULTS
DNA Fragmentation and PARP Activation
Treatment of C3Heb/FeJ mice with a hepatotoxic dose of acetaminophen (300 mg/kg) (Bajt et al., 2003; Knight et al., 2002; Lawson et al., 2000) resulted in a time-dependent activation of PARP-1, as indicated by the immunohistochemical detection of poly-ADP-ribosylation (PAR) in hepatocellular nuclei (Figs. 1 and 2). PAR staining was not detectable in any cells up to 3 h after AAP (Figs. 1B and 2A). At 4.5 h, a few PAR-positive could be observed. The number of PAR-positive cells increased substantially at 6 and even more at 12 h, affecting many cells in the centrilobular area (Figs. 1 and 2A). The staining intensity was low at the early time points and increased considerably between 6 and 12 h (Figs. 1C and 1D). Mainly, damaged cells with morphological evidence of nuclear fragmentation showed positive staining for PAR (Figs. 1C and 1D). Using an anti-histone ELISA to sensitively monitor AAP-induced DNA damage (Lawson et al., 1999), progressive DNA fragmentation was observed beginning at 3 h after AAP overdose (Fig. 2B). DNA fragmentation and cell injury (indicated by increased plasma ALT activities) showed a close temporary correlation (Figs. 2B and 2C). PAR staining was delayed compared to DNA damage (Fig. 2A).
Effect of the PARP Inhibitor 3-Aminobenzamide (3-AB) on AAP Hepatotoxicity
Since conflicting reports regarding the potential efficacy of AB against AAP-induced liver injury have been published (Ray et al., 2001; Shen et al., 1992), we treated mice with 500 mg/kg 3-AB either 0.5 h before AAP or 1.5 or 2.5 h after AAP administration. Pretreatment with 3-AB resulted in a complete protection against AAP hepatotoxicity at 6 h (Figs. 3A and 3B). Since hepatic GSH depletion is significantly reduced in 3-AB-pretreated animals during the first 20 min after AAP injection (Fig. 4), the drug protected most likely because it inhibited the metabolic activation of AAP. However, treatment with 3-AB was still significantly protective when administered at 1.5 or 2.5 h after AAP (Fig. 3). 3-AB pretreatment completely prevented AAP-induced DNA fragmentation (Fig. 5A) and the appearance of any PAR-positive cells in the tissue (Fig. 5B). On the other hand, post-AAP treatment with 3-AB significantly attenuated DNA fragmentation (Fig. 5A) and reduced the number of PAR-positive cells by >50% (Fig. 5B). In addition, the staining intensity of PAR-positive cells was strongly attenuated (Fig. 1E). Furthermore, nitrotyrosine staining, which reflects reactive nitrogen formation (Gardner et al., 2002; Knight et al., 2001, 2002), was completely prevented with 3-AB pretreatment and was significantly attenuated with posttreatment (data not shown). The data suggest that other mechanisms in addition to PARP-1 inhibition may be responsible for the protective effects of 3-AB against AAP hepatotoxicity.
Acetaminophen Hepatotoxicity in PARP Gene Knockout Mice
To further assess a potential role of PARP in the mechanism of AAP-induced cell death, we compared AAP hepatotoxicity in SV129 wild-type (WT) and PARP–/– mice 6 h after a dose of 300 mg/kg AAP. Using immunohistochemical detection of PAR, we could confirm AAP-induced PARP activation in wild-type (72 ± 16 PAR-positive cells/20 HPF) but not in PARP–/– animals (0 PAR-positive cells/20 HPF). However, plasma ALT activities and histological assessment of necrosis did not reveal any significant protection in PARP–/– mice compared to wild-type animals (Figs. 6A and 6B). In contrast, plasma ALT levels and the area of necrosis were moderately increased in PARP–/– mice. On the other hand, treatment with 3-AB (at 1.5 h after AAP) significantly protected against AAP-induced liver injury in wild-type animals (Fig. 6). Interestingly, 3-AB even attenuated liver injury in the absence of PARP gene expression (Fig. 6), strongly suggesting that the effect of 3-AB appears to be mainly independent of PARP activation. To evaluate if the results would be different at a lower dose of AAP, we treated WT and PARP–/– mice with 225 mg/kg AAP. There was no significant difference in injury, as indicated by a similar increase in plasma ALT activities at 6 h after AAP (WT: 5890 ± 545 U/l; PARP–/–: 4620 ± 1050 U/l; p > 0.05).
Effect of the specific PARP Inhibitor 5-Aminoisoquinolinone (5-AIQ)
Recently, more specific, water-soluble PARP inhibitors such as 5-AIQ became available (Thiemermann, 2002). Treatment with 5 mg/kg AIQ 1.5 h after AAP had no significant effect on AAP-induced increase in plasma ALT activities and the overall area of necrosis (Table 1). Although the number of PAR-positive cells was not significantly reduced (Table 1), the staining intensity of parallel stained tissues of AIQ-treated animals was reduced (Fig. 1F), suggesting that AIQ at least partially inhibited PARP in these experiments.
DISCUSSION
The main objectives of this investigation were to (a) document the time course of PARP-1 activation after AAP overdose and to (b) evaluate a potential role of PARP in AAP-induced cell death. PARP-1 is activated by DNA damage, in particular double-strand breaks (Benjamin and Gill, 1980). PARP-1 activity leads to ADP-ribosylation of nuclear proteins as initiation of DNA repair and in ADP-ribosylation of endonucleases to prevent further DNA damage (Yakovlev et al., 2000). Whereas moderate PARP-1 activation is a critical step for DNA repair, excessive PARP-1 activation leads to depletion of cellular NAD+ and ATP content and can cause oncotic necrosis (Ha and Snyder, 1999; Szabo and Dawson, 1998). Corcoran and coworkers clearly demonstrated that DNA fragmentation was a critical early event in the pathophysiology of AAP-induced cell death in vivo (Ray et al., 1990, 1993) and in cultured hepatocytes (Shen et al., 1991, 1992). Using an anti-histone ELISA and the TUNEL assay, we could confirm these findings (Gujral et al., 2002; Lawson et al., 1999). In addition, we now provide direct evidence for AAP-induced PARP-1 activation by immunohistochemical detection of ADP-ribosylation of nuclear proteins. Consistent with the hypothesis that DNA damage activates PARP-1, PAR staining was only observed in centrilobular cells showing early signs of oncosis. In addition, the increase in the number of PAR-positive cells and the staining intensity followed the increase in DNA fragmentation. In fact, the highest PAR staining intensity was observed at 12 h after AAP (i.e., well after most centrilobular hepatocytes lost viability as judged by ALT release and histology). Moreover, if cell injury is either prevented or the extent of necrosis is attenuated, PARP-1 activation is either eliminated or attenuated, respectively. Thus, PARP-1 activation during AAP hepatotoxicity occurs mainly in damaged cells, which are in the process of undergoing oncotic necrosis.
The functional significance of PARP-1 was studied in PARP-1 gene knockout mice and with chemical inhibitors. Previous studies reported contradictory findings with PARP inhibitors. Corcoran's group found no protection against AAP-induced cell damage with 3 mM 3-AB in cultured murine hepatocytes at early time points but observed an aggravation of cell death at 12 h after AAP treatment (Shen et al., 1992). Consistent with these findings, we found a moderate increase in liver injury in PARP–/– mice compared to wild-type animals after 300 mg/kg AAP. These data suggest that PARP activation could be beneficial under certain conditions by allowing less severely injured cells to recover. In contrast to these observations, others reported a protective effect of high doses of nicotinamide and of 4-aminobenzamide in vivo (Kroger et al., 1997; Ray et al., 2001). Treatment with 500 mg/kg 3-AB also was effective in our studies. The extent of protection ranged from complete prevention of AAP-induced liver injury, DNA fragmentation, and PARP activation after pretreatment with 3-AB to a partial efficacy when 3-AB was administered at 1.5 or even 2.5 h after AAP. However, pretreatment with 3-AB significantly reduced the depletion of hepatic GSH levels during the first 20 min after AAP administration. The depletion of hepatic GSH content is caused by the formation of the reactive metabolite NAPQI (Nelson, 1990). In support of this hypothesis, we showed previously that the exponential loss of hepatic GSH during the first 30 min after intraperitoneal injection of AAP correlates with the biliary excretion of the GSH-AAP conjugate (Jaeschke, 1990). Since the early phase of GSH loss reflects the formation of the reactive metabolite NAPQI, we conclude that pretreatment with 3-AB inhibited reactive metabolite formation and, therefore, most likely prevented the initiation of toxicity. On the other hand, 3-AB also protected when given at 1.5 h or 2.5 h after AAP. Protein binding of NAPQI peaks at 1 h after an intraperitoneal injection of 300 mg/kg AAP (Roberts et al., 1991), and treatment with N-acetylcysteine at 1 or 3 h after AAP did not attenuate protein binding (Salminen et al., 1998). Furthermore, treatment with GSH at 1.5 h or later did not affect AAP-induced mitochondrial dysfunction and oxidant stress (Knight et al., 2002). Thus, it is unlikely that 3-AB significantly affected AAP metabolism at this later stage. On the other hand, delayed treatment with 3-AB protected even PARP–/– mice, which suggests that the hepatoprotective effect of 3-AB does not depend on PARP-1 inhibition. 3-AB also attenuated DNA fragmentation and reduced nitrotyrosine staining, which is an indicator for peroxynitrite generation (Knight et al., 2002). These findings support the hypothesis that 3-AB acted upstream of PARP-1 activation. Since a mitochondria-derived oxidant stress and peroxynitrite formation is critical in AAP hepatotoxicity (reviewed in Jaeschke et al., 2003), one possible mechanism of action could be that 3-AB worked as an antioxidant (Czapski et al., 2004). Although the antioxidant efficiency of 3-AB in brain homogenate is considerably lower compared to -tocopherol (Czapski et al., 2004), this comparison may be of limited relevance. Neither the enrichment of hepatocytes with -tocopherol nor with -tocopherol protected against AAP-induced hepatotoxicity in vivo (Knight et al., 2003). In contrast, an increase in water-soluble antioxidants in the liver (e.g., glutathione or biliverdin) scavenged peroxynitrite and effectively attenuated AAP-mediated liver injury (Bajt et al., 2003; Chiu et al., 2002; Knight et al., 2002). The similarities in the protection against AAP-induced hepatotoxicity between 3-AB and other water-soluble antioxidants support the hypothesis that 3-AB can act as peroxynitrite scavenger in vivo. Further investigations are necessary to support this hypothesis.
The conclusion that PARP-1 activation may not be relevant for AAP-induced cell death was further supported by the observation that the specific PARP inhibitor 5-AIQ (Thiemermann, 1999) did not protect against AAP-induced liver injury. The staining intensity in 5-AIQ-treated livers was moderately reduced compared to AAP alone, suggesting that 5-AIQ partially inhibited PARP activity. However, our data indicate that, even with a potent and specific inhibitor, it is difficult to completely eliminate PARP activity after the severe AAP-induced DNA damage. Nevertheless, the same dose of 5-AIQ as we used in our study proved to be effective against ischemia-reperfusion injury in the liver (Khandoga et al., 2004). Thus, neither the results with PARP–/– mice nor the use of a specific PARP inhibitor supported the hypothesis that PARP-1 activation is critical for AAP-induced cell death.
In summary, our data demonstrated a progressive activation of PARP-1 in response to DNA damage in hepatocytes of AAP-treated animals. However, neither PARP–/– mice nor animals treated with the specific PARP-1 inhibitor 5-AIQ were protected against AAP-induced liver injury. This suggests that PARP-1 activation does not contribute to AAP-induced cell death under the experimental conditions used in this study. On the other hand, treatment with 3-AB prevented AAP hepatotoxicity by mechanisms independent of PARP-1 inhibition such as inhibiting metabolic activation of AAP and potentially by an antioxidant effect. Thus, tissue protection observed after treatment with an unspecific PARP-1 inhibitor such as 3-AB should be interpreted with caution.
ACKNOWLEDGMENTS
This work was supported in part by National Institutes of Health grant AA 12916 (H.J.), by grant P-15502 from the Austrian Science Foundation (M.T.), and by a GEN-AU project grant from the Austrian Ministry of Science (M.T.).
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Departments of Medicine and Pathology, Medical University of Graz, Graz, Austria
Department of Pathology, University of Texas Health Science Center, Houston, Texas 77030
ABSTRACT
DNA fragmentation in hepatocytes occurs early after acetaminophen (AAP) overdose in mice. DNA strandbreaks can induce excessive activation of poly(ADP-ribose) polymerases (PARP), which may lead to oncotic necrosis. Based on controversial findings with chemical PARP inhibitors, the role of PARP-1 activation in AAP hepatotoxicity remains unclear. To investigate PARP-1 activation and evaluate a pathophysiological role of PARP-1, we used both PARP inhibitors (3-aminobenzamide; 5-aminoisoquinolinone) and PARP gene knockout mice (PARP–/–). Treatment of C3Heb/FeJ mice with 300 mg/kg AAP resulted in DNA fragmentation and alanine aminotransferase (ALT) release as early as 3 h, with further increase of these parameters up to 12 h. Few nuclei of hepatocytes stained positive for poly-ADP-ribosylated nuclear proteins (PAR) as indicator for PARP-1 activation at 4.5 h. However, the number of PAR-positive cells and staining intensity increased substantially at 6 and 12 h. Pretreatment with 500 mg/kg 3-aminobenzamide before AAP attenuated hepatic glutathione depletion and completely eliminated DNA fragmentation and liver injury. Delayed treatment several hours after AAP was still partially protective. On the other hand, liver injury was not attenuated in PARP–/– mice compared to wild-type animals. Similarly, the specific PARP-1 inhibitor 5-aminoisoquinolinone (5 mg/kg) was not protective. However, 3-aminobenzamide attenuated liver injury in WT and PARP–/– mice. In summary, PARP-1 activation is a consequence of DNA fragmentation after AAP overdose. However, PARP-1 activation is not a relevant event for AAP-induced oncotic necrosis. The protection of 3-aminobenzamide against AAP-induced liver injury was due to reduced metabolic activation and potentially its antioxidant effect but independent of PARP-1 inhibition.
Key Words: acetaminophen; hepatotoxicity; poly(ADP-ribose) polymerase-1 (PARP-1); DNA fragmentation; 3-aminobenzamide.
INTRODUCTION
Acetaminophen (AAP), a widely used analgesic drug, is safe at therapeutic doses, but an overdose can cause severe liver injury in experimental animals and humans. The toxicity depends on the metabolic activation and formation of a reactive metabolite, presumably N-acetyl-p-benzoquinone imine (NAPQI), which is initially detoxified by reacting with glutathione (GSH) (Nelson, 1990). However, after the cellular GSH content is exhausted, NAPQI covalently binds to cellular proteins (Cohen and Khairallah, 1997; Jollow et al., 1973) including mitochondrial proteins (Qiu et al., 2001; Tirmenstein and Nelson, 1989). This leads to mitochondrial dysfunction, as indicated by inhibition of mitochondrial respiration (Meyers et al., 1988; Ramsay et al., 1989), increased mitochondrial oxidant stress and peroxynitrite formation (Jaeschke, 1990; Knight et al., 2001, 2002), ATP depletion (Jaeschke, 1990; Tirmenstein and Nelson, 1990), Bax translocation to mitochondria (Adams et al., 2001; El-Hassan et al., 2003), and cytochrome c release (Adams et al., 2001; Knight and Jaeschke, 2002). Eventually, the mitochondria form membrane permeability transition pores, and the mitochondrial membrane potential collapses, leading to necrotic cell death (Kon et al., 2004).
In addition to mitochondrial dysfunction, DNA fragmentation has been shown to occur early in the pathophysiology of AAP hepatotoxicity in vivo (Ray et al., 1990, 1993) and in isolated hepatocytes (Shen et al., 1991, 1992). The fact that a general endonuclease inhibitor prevented DNA fragmentation and protected against AAP-induced liver injury (Shen et al., 1992) supported the hypothesis that DNA fragmentation is an important event in the mechanism of cell injury. Although the endonuclease(s) involved in this process have not been conclusively identified, it is unlikely to be the caspase-activated desoxyribonuclease (CAD). There is no relevant caspase-3 activation after AAP overdose (Adams et al., 2001; Gujral et al., 2002; Lawson et al., 1999), and the DNA fragments are different than typically generated during caspase-dependent apoptosis (Jahr et al., 2001). Nevertheless, the DNA damage may lead to activation of poly(ADP-ribose)polymerases (PARP-1) in the nucleus (Szabo and Dawson, 1998). PARP-mediated poly-ADP-ribosylation of nuclear proteins contributes to DNA repair (Szabo and Dawson, 1998). However, excessive activation of PARP depletes cellular NAD+, which subsequently triggers ATP depletion and necrotic cell death (Ha and Snyder, 1999). The potential role of PARP in AAP-induced cell death was previously investigated by using relatively unspecific PARP inhibitors. The results were not consistent. Corcoran and coworkers did not find a protective effect with 3-aminobenzamide (3-AB) in mouse hepatocytes at early time points but observed an aggravation of injury at 12 h after AAP treatment (Shen et al., 1992). In contrast, high doses of 4-aminobenzamide and nicotinamide were shown to prevent AAP hepatotoxicity in vivo (Kroger et al., 1997; Ray et al., 2001). To resolve this controversy and to investigate whether PARP-1 is actually activated and plays a critical role in the early injury phase after AAP overdose, we tested the potential protective effect of 3-aminobenzamide and the novel, more specific PARP inhibitor 5-aminoisoquinolinone (Thiemermann, 2002) in C3Heb/FeJ mice and assessed AAP-induced liver injury in PARP-1 gene knockout mice of the SV129 background strain.
MATERIALS AND METHODS
Animals. Male C3Heb/FeJ mice with an average weight of 18 to 20 g were purchased from Jackson Laboratory (Bar Harbor, Maine). PARP gene knockout mice (PARP–/–) were kindly provided by Dr. E. F. Wagner (Research Institute of Molecular Pathology, Vienna, Austria). The generation of the PARP–/– mice was described previously (Wang et al., 1995). All animals were housed in an environmentally controlled room with 12 h light/dark cycle and allowed free access to food (certified rodent diet no. 8640, Harlan Teklad, Indianapolis, IN) and water. The experimental protocols followed the criteria of the University of Arizona, the Medical University of Graz, and the National Research Council for the care and use of laboratory animals in research. All animals were fasted overnight before the experiments. Animals
Experimental protocols. At selected times after AAP 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 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.
Methods. Plasma ALT activities were determined with the kinetic test kit 68-B (Biotron Diagnostics, Inc., Hernet, CA) and expressed as IU/liter. Protein concentrations were assayed using the bicinchoninic acid kit (Pierce, Rockford, IL). Total soluble GSH and GSSG were measured in the liver homogenate with a modified method of Tietze as described in detail (Knight et al., 2002). Briefly, the frozen tissue was homogenized at 0°C in 3% sulfosalicylic acid containing 0.1 mM EDTA. After dilution with 0.01 N HCl, the sample was centrifuged, and the supernatant was diluted with 100 mM potassium phosphate buffer (KPP), pH 7.4. The samples were assayed using dithionitrobenzoic acid. All data are expressed in GSH-equivalents. DNA fragmentation was evaluated using the Cell Death Detection ELISA (anti-histone ELISA) (Roche Diagnostics, Indianapolis, IN) as described in detail (Lawson et al., 1999). In this assay, the kinetics (vmax) of product generation is a measure of DNA fragmentation. The vmax values obtained for untreated controls (100%) are compared with those in treated groups. The assay allows the specific quantitation of cytoplasmic histone-associated DNA fragments.
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 between the different lobes of the liver in this model. The percent of necrosis was estimated by evaluating the number of microscopic fields with necrosis compared to the entire cross section. All histological evaluations were done in a blinded fashion by the pathologist (A.F.). Nitrotyrosine staining was assessed by immunohistochemistry with the DAKO LSAB Peroxidase Kit (K684) (DAKO Corp., Carpinteria, CA), which was used according to the manufacturer's instructions (Knight et al., 2002). The anti-nitrotyrosine antibody was obtained from Molecular Probes (Eugene, OR). For PAR-staining, deparaffinized and rehydrated liver sections were incubated in 1x Antigen Retrieval Solution (Dako Cytomation, Carpinteria, CA) for 30 min. at 95°C. Sections were then treated with 10% trichloroacetic acid for 10 min at room temperature. PAR was detected by incubating sections for 2 h with an anti-pADPr, IgY antibody (1:50, Tulip Biolabs, West Point, PA) followed by 1 h incubation with a biotinylated goat anti-chicken antibody (1:100, Vector, Burlingame, CA). Antibody binding was visualized using the Vectastain Elite ABC (peroxidase) Standard Kit using AEC (Dako) as a substrate.
Statistics. All results were expressed as mean ± SE. Comparisons between multiple groups were performed with one-way ANOVA followed by Bonferroni t-test. If the data were not normally distributed, we used the Kruskal-Wallis Test (nonparametric ANOVA) followed by Dunn's Multiple Comparisons Test; p < 0.05 was considered significant.
RESULTS
DNA Fragmentation and PARP Activation
Treatment of C3Heb/FeJ mice with a hepatotoxic dose of acetaminophen (300 mg/kg) (Bajt et al., 2003; Knight et al., 2002; Lawson et al., 2000) resulted in a time-dependent activation of PARP-1, as indicated by the immunohistochemical detection of poly-ADP-ribosylation (PAR) in hepatocellular nuclei (Figs. 1 and 2). PAR staining was not detectable in any cells up to 3 h after AAP (Figs. 1B and 2A). At 4.5 h, a few PAR-positive could be observed. The number of PAR-positive cells increased substantially at 6 and even more at 12 h, affecting many cells in the centrilobular area (Figs. 1 and 2A). The staining intensity was low at the early time points and increased considerably between 6 and 12 h (Figs. 1C and 1D). Mainly, damaged cells with morphological evidence of nuclear fragmentation showed positive staining for PAR (Figs. 1C and 1D). Using an anti-histone ELISA to sensitively monitor AAP-induced DNA damage (Lawson et al., 1999), progressive DNA fragmentation was observed beginning at 3 h after AAP overdose (Fig. 2B). DNA fragmentation and cell injury (indicated by increased plasma ALT activities) showed a close temporary correlation (Figs. 2B and 2C). PAR staining was delayed compared to DNA damage (Fig. 2A).
Effect of the PARP Inhibitor 3-Aminobenzamide (3-AB) on AAP Hepatotoxicity
Since conflicting reports regarding the potential efficacy of AB against AAP-induced liver injury have been published (Ray et al., 2001; Shen et al., 1992), we treated mice with 500 mg/kg 3-AB either 0.5 h before AAP or 1.5 or 2.5 h after AAP administration. Pretreatment with 3-AB resulted in a complete protection against AAP hepatotoxicity at 6 h (Figs. 3A and 3B). Since hepatic GSH depletion is significantly reduced in 3-AB-pretreated animals during the first 20 min after AAP injection (Fig. 4), the drug protected most likely because it inhibited the metabolic activation of AAP. However, treatment with 3-AB was still significantly protective when administered at 1.5 or 2.5 h after AAP (Fig. 3). 3-AB pretreatment completely prevented AAP-induced DNA fragmentation (Fig. 5A) and the appearance of any PAR-positive cells in the tissue (Fig. 5B). On the other hand, post-AAP treatment with 3-AB significantly attenuated DNA fragmentation (Fig. 5A) and reduced the number of PAR-positive cells by >50% (Fig. 5B). In addition, the staining intensity of PAR-positive cells was strongly attenuated (Fig. 1E). Furthermore, nitrotyrosine staining, which reflects reactive nitrogen formation (Gardner et al., 2002; Knight et al., 2001, 2002), was completely prevented with 3-AB pretreatment and was significantly attenuated with posttreatment (data not shown). The data suggest that other mechanisms in addition to PARP-1 inhibition may be responsible for the protective effects of 3-AB against AAP hepatotoxicity.
Acetaminophen Hepatotoxicity in PARP Gene Knockout Mice
To further assess a potential role of PARP in the mechanism of AAP-induced cell death, we compared AAP hepatotoxicity in SV129 wild-type (WT) and PARP–/– mice 6 h after a dose of 300 mg/kg AAP. Using immunohistochemical detection of PAR, we could confirm AAP-induced PARP activation in wild-type (72 ± 16 PAR-positive cells/20 HPF) but not in PARP–/– animals (0 PAR-positive cells/20 HPF). However, plasma ALT activities and histological assessment of necrosis did not reveal any significant protection in PARP–/– mice compared to wild-type animals (Figs. 6A and 6B). In contrast, plasma ALT levels and the area of necrosis were moderately increased in PARP–/– mice. On the other hand, treatment with 3-AB (at 1.5 h after AAP) significantly protected against AAP-induced liver injury in wild-type animals (Fig. 6). Interestingly, 3-AB even attenuated liver injury in the absence of PARP gene expression (Fig. 6), strongly suggesting that the effect of 3-AB appears to be mainly independent of PARP activation. To evaluate if the results would be different at a lower dose of AAP, we treated WT and PARP–/– mice with 225 mg/kg AAP. There was no significant difference in injury, as indicated by a similar increase in plasma ALT activities at 6 h after AAP (WT: 5890 ± 545 U/l; PARP–/–: 4620 ± 1050 U/l; p > 0.05).
Effect of the specific PARP Inhibitor 5-Aminoisoquinolinone (5-AIQ)
Recently, more specific, water-soluble PARP inhibitors such as 5-AIQ became available (Thiemermann, 2002). Treatment with 5 mg/kg AIQ 1.5 h after AAP had no significant effect on AAP-induced increase in plasma ALT activities and the overall area of necrosis (Table 1). Although the number of PAR-positive cells was not significantly reduced (Table 1), the staining intensity of parallel stained tissues of AIQ-treated animals was reduced (Fig. 1F), suggesting that AIQ at least partially inhibited PARP in these experiments.
DISCUSSION
The main objectives of this investigation were to (a) document the time course of PARP-1 activation after AAP overdose and to (b) evaluate a potential role of PARP in AAP-induced cell death. PARP-1 is activated by DNA damage, in particular double-strand breaks (Benjamin and Gill, 1980). PARP-1 activity leads to ADP-ribosylation of nuclear proteins as initiation of DNA repair and in ADP-ribosylation of endonucleases to prevent further DNA damage (Yakovlev et al., 2000). Whereas moderate PARP-1 activation is a critical step for DNA repair, excessive PARP-1 activation leads to depletion of cellular NAD+ and ATP content and can cause oncotic necrosis (Ha and Snyder, 1999; Szabo and Dawson, 1998). Corcoran and coworkers clearly demonstrated that DNA fragmentation was a critical early event in the pathophysiology of AAP-induced cell death in vivo (Ray et al., 1990, 1993) and in cultured hepatocytes (Shen et al., 1991, 1992). Using an anti-histone ELISA and the TUNEL assay, we could confirm these findings (Gujral et al., 2002; Lawson et al., 1999). In addition, we now provide direct evidence for AAP-induced PARP-1 activation by immunohistochemical detection of ADP-ribosylation of nuclear proteins. Consistent with the hypothesis that DNA damage activates PARP-1, PAR staining was only observed in centrilobular cells showing early signs of oncosis. In addition, the increase in the number of PAR-positive cells and the staining intensity followed the increase in DNA fragmentation. In fact, the highest PAR staining intensity was observed at 12 h after AAP (i.e., well after most centrilobular hepatocytes lost viability as judged by ALT release and histology). Moreover, if cell injury is either prevented or the extent of necrosis is attenuated, PARP-1 activation is either eliminated or attenuated, respectively. Thus, PARP-1 activation during AAP hepatotoxicity occurs mainly in damaged cells, which are in the process of undergoing oncotic necrosis.
The functional significance of PARP-1 was studied in PARP-1 gene knockout mice and with chemical inhibitors. Previous studies reported contradictory findings with PARP inhibitors. Corcoran's group found no protection against AAP-induced cell damage with 3 mM 3-AB in cultured murine hepatocytes at early time points but observed an aggravation of cell death at 12 h after AAP treatment (Shen et al., 1992). Consistent with these findings, we found a moderate increase in liver injury in PARP–/– mice compared to wild-type animals after 300 mg/kg AAP. These data suggest that PARP activation could be beneficial under certain conditions by allowing less severely injured cells to recover. In contrast to these observations, others reported a protective effect of high doses of nicotinamide and of 4-aminobenzamide in vivo (Kroger et al., 1997; Ray et al., 2001). Treatment with 500 mg/kg 3-AB also was effective in our studies. The extent of protection ranged from complete prevention of AAP-induced liver injury, DNA fragmentation, and PARP activation after pretreatment with 3-AB to a partial efficacy when 3-AB was administered at 1.5 or even 2.5 h after AAP. However, pretreatment with 3-AB significantly reduced the depletion of hepatic GSH levels during the first 20 min after AAP administration. The depletion of hepatic GSH content is caused by the formation of the reactive metabolite NAPQI (Nelson, 1990). In support of this hypothesis, we showed previously that the exponential loss of hepatic GSH during the first 30 min after intraperitoneal injection of AAP correlates with the biliary excretion of the GSH-AAP conjugate (Jaeschke, 1990). Since the early phase of GSH loss reflects the formation of the reactive metabolite NAPQI, we conclude that pretreatment with 3-AB inhibited reactive metabolite formation and, therefore, most likely prevented the initiation of toxicity. On the other hand, 3-AB also protected when given at 1.5 h or 2.5 h after AAP. Protein binding of NAPQI peaks at 1 h after an intraperitoneal injection of 300 mg/kg AAP (Roberts et al., 1991), and treatment with N-acetylcysteine at 1 or 3 h after AAP did not attenuate protein binding (Salminen et al., 1998). Furthermore, treatment with GSH at 1.5 h or later did not affect AAP-induced mitochondrial dysfunction and oxidant stress (Knight et al., 2002). Thus, it is unlikely that 3-AB significantly affected AAP metabolism at this later stage. On the other hand, delayed treatment with 3-AB protected even PARP–/– mice, which suggests that the hepatoprotective effect of 3-AB does not depend on PARP-1 inhibition. 3-AB also attenuated DNA fragmentation and reduced nitrotyrosine staining, which is an indicator for peroxynitrite generation (Knight et al., 2002). These findings support the hypothesis that 3-AB acted upstream of PARP-1 activation. Since a mitochondria-derived oxidant stress and peroxynitrite formation is critical in AAP hepatotoxicity (reviewed in Jaeschke et al., 2003), one possible mechanism of action could be that 3-AB worked as an antioxidant (Czapski et al., 2004). Although the antioxidant efficiency of 3-AB in brain homogenate is considerably lower compared to -tocopherol (Czapski et al., 2004), this comparison may be of limited relevance. Neither the enrichment of hepatocytes with -tocopherol nor with -tocopherol protected against AAP-induced hepatotoxicity in vivo (Knight et al., 2003). In contrast, an increase in water-soluble antioxidants in the liver (e.g., glutathione or biliverdin) scavenged peroxynitrite and effectively attenuated AAP-mediated liver injury (Bajt et al., 2003; Chiu et al., 2002; Knight et al., 2002). The similarities in the protection against AAP-induced hepatotoxicity between 3-AB and other water-soluble antioxidants support the hypothesis that 3-AB can act as peroxynitrite scavenger in vivo. Further investigations are necessary to support this hypothesis.
The conclusion that PARP-1 activation may not be relevant for AAP-induced cell death was further supported by the observation that the specific PARP inhibitor 5-AIQ (Thiemermann, 1999) did not protect against AAP-induced liver injury. The staining intensity in 5-AIQ-treated livers was moderately reduced compared to AAP alone, suggesting that 5-AIQ partially inhibited PARP activity. However, our data indicate that, even with a potent and specific inhibitor, it is difficult to completely eliminate PARP activity after the severe AAP-induced DNA damage. Nevertheless, the same dose of 5-AIQ as we used in our study proved to be effective against ischemia-reperfusion injury in the liver (Khandoga et al., 2004). Thus, neither the results with PARP–/– mice nor the use of a specific PARP inhibitor supported the hypothesis that PARP-1 activation is critical for AAP-induced cell death.
In summary, our data demonstrated a progressive activation of PARP-1 in response to DNA damage in hepatocytes of AAP-treated animals. However, neither PARP–/– mice nor animals treated with the specific PARP-1 inhibitor 5-AIQ were protected against AAP-induced liver injury. This suggests that PARP-1 activation does not contribute to AAP-induced cell death under the experimental conditions used in this study. On the other hand, treatment with 3-AB prevented AAP hepatotoxicity by mechanisms independent of PARP-1 inhibition such as inhibiting metabolic activation of AAP and potentially by an antioxidant effect. Thus, tissue protection observed after treatment with an unspecific PARP-1 inhibitor such as 3-AB should be interpreted with caution.
ACKNOWLEDGMENTS
This work was supported in part by National Institutes of Health grant AA 12916 (H.J.), by grant P-15502 from the Austrian Science Foundation (M.T.), and by a GEN-AU project grant from the Austrian Ministry of Science (M.T.).
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