* Liver Research Institute, University of Arizona, Tucson, Arizona 85737; Departments of Medicine and
Pathology, Medical University of Graz, Graz, Austria; and
Department of Pathology, University of Texas Health Science Center, Houston, Texas 77030
1 To whom correspondence should be addressed at Liver Research Institute, University of Arizona, College of Medicine, 1501 N. Campbell Ave, Room 6309, Tucson, AZ 85724. Fax: (520) 626-5975. E-mail: jaeschke{at}email.arizona.edu.
Received October 5, 2004; accepted December 10, 2004
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ABSTRACT |
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Key Words: acetaminophen; hepatotoxicity; poly(ADP-ribose) polymerase-1 (PARP-1); DNA fragmentation; 3-aminobenzamide.
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INTRODUCTION |
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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.
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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 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.
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RESULTS |
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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Bajt, M. L., Knight, T. R., Farhood, A., and Jaeschke, H. (2003). Scavenging peroxynitrite with glutathione promotes regeneration and enhances survival during acetaminophen-induced liver injury in mice. J. Pharmacol. Exper. Therap. 307, 6773.
Benjamin, R. C., and Gill, D. M. (1980). Poly(ADP-ribose) synthesis in vitro programmed by damaged DNA. A comparison of DNA molecules containing different types of strand breaks. J. Biol. Chem. 255, 1050210508.
Chiu, H., Brittingham, J. A., and Laskin, D. L. (2002). Differential induction of heme oxygenase-1 in macrophages and hepatocytes during acetaminophen-induced hepatotoxicity in the rat: Effects of hemin and biliverdin. Toxicol. Appl. Pharmacol. 181, 106115.[CrossRef][ISI][Medline]
Cohen, S. D., and Khairallah, E. A. (1997). Selective protein arylation and acetaminophen-induced hepatotoxicity. Drug Metab. Rev. 29, 5977.[ISI][Medline]
Czapski, G. A., Cakala, M., Kopczuk, D., and Strosznajder, J. B. (2004). Effect of poly(ADP-ribose) polymerase inhibitors on oxidative stress evoked hydroxyl radical level and macromolecules oxidation in cell free system of rat brain cortex. Neurosci. Lett. 356, 4548.[CrossRef][ISI][Medline]
El-Hassan, H., Anwar, K., Macanas-Pirard, P., Crabtree, M., Chow, S. C., Johnson, V. L., Lee, P. C., Hinton, R. H., Price, S. C., and Kass, G. E. (2003). Involvement of mitochondria in acetaminophen-induced apoptosis and hepatic injury: Roles of cytochrome c, Bax, Bid, and caspases. Toxicol. Appl. Pharmacol. 191, 118129.[CrossRef][ISI][Medline]
Gardner, C. R., Laskin, J. D., Dambach, D. M., Sacco, M., Durham, S. K., Bruno, M. K., Cohen, S. D., Gordon, M. K., Gerecke, D. R., Zhou, P., et al. (2002). Reduced hepatotoxicity of acetaminophen in mice lacking inducible nitric oxide synthase: Potential role of tumor necrosis factor-alpha and interleukin-10. Toxicol. Appl. Pharmacol. 184, 2736.[CrossRef][ISI][Medline]
Gujral, J. S., Knight, T. R., Farhood, A., Bajt, M. L., and Jaeschke, H. (2002). Mode of cell death after acetaminophen overdose in mice: Apoptosis or oncotic necrosis? Toxicol. Sci. 67, 322328.
Ha, H. C., and Snyder, S. H (1999). Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc. Natl. Acad. Sci. USA. 96, 1397813982.
Jaeschke, H. (1990). Glutathione disulfide formation and oxidant stress during acetaminophen-induced hepatotoxicity in mice in vivo: The protective effect of allopurinol. J. Pharmacol. Exp. Ther. 255, 935941.[Abstract]
Jaeschke, H., Knight, T. R., and Bajt, M. L. (2003). The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity. Toxicol. Lett. 144, 279288.[CrossRef][ISI][Medline]
Jahr, S., Hentze, H., Englisch, S., Hardt, D., Fackelmayer, F. O., Hesch, R. D., and Knippers, R. (2001). DNA fragments in the blood plasma of cancer patients: Quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 61, 16591665.
Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gillette, J. R., and Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis. II. Role of covalent binding in vivo. J. Pharmacol. Exp. Ther. 187, 195202.[ISI][Medline]
Khandoga, A., Biberthaler, P., Enders, G., and Krombach, F. (2004). 5-Aminoisoquinolinone, a novel inhibitor of poly(adenosine disphosphate-ribose) polymerase, reduces microvascular liver injury but not mortality rate after hepatic ischemia-reperfusion. Crit. Care Med. 32, 472477.[CrossRef][ISI][Medline]
Knight, T. R., Fariss, M. W., Farhood, A., and Jaeschke, H. (2003). Role of lipid peroxidation as a mechanism of liver injury after acetaminophen overdose in mice. Toxicol. Sci. 76, 229236.
Knight, T. R., Ho, Y. S., Farhood, A., and Jaeschke, H. (2002). Peroxynitrite is a critical mediator of acetaminophen hepatotoxicity in murine livers: Protection by glutathione. J. Pharmacol. Exp. Ther. 303, 468475.
Knight, T. R., and Jaeschke, H. (2002). Acetaminophen-induced inhibition of Fas receptor-mediated liver cell apoptosis: Mitochondrial dysfunction versus glutathione depletion. Toxicol. Appl. Pharmacol. 181, 133141.[CrossRef][ISI][Medline]
Knight, T. R., Kurtz, A., Bajt, M. L., Hinson, J. A., and Jaeschke, H. (2001). Vascular and hepatocellular peroxynitrite formation during acetaminophen-induced liver injury: Role of mitochondrial oxidant stress. Toxicol. Sci. 62, 212220.
Kon, K., Kim, J. S., Jaeschke, H., and Lemasters, J. J. (2004). Mitochondrial permeability transition in acetaminophen-induced necrotic and apoptotic cell death to cultured mouse hepatocytes. Hepatology 40, 11701179.[CrossRef][ISI][Medline]
Kroger, H., Dietrich, A., Ohde, M., Lange, R., Ehrlich, W., and Kurpisz, M. (1997). Protection from acetaminophen-induced liver damage by the synergistic action of low doses of the poly(ADP-ribose) polymerase-inhibitor nicotinamide and the antioxidant N-acetylcysteine or the amino acid L-methionine. Gen. Pharmacol. 28, 257263.[CrossRef][Medline]
Lawson, J. A., Farhood, A., Hopper, R. D., Bajt, M. L., and Jaeschke, H. (2000). The hepatic inflammatory response after acetaminophen overdose: Role of neutrophils. Toxicol. Sci. 54, 509516.
Lawson, J. A., Fisher, M. A., Simmons, C. A., Farhood, A., and Jaeschke, H. (1999). Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice. Toxicol. Appl. Pharmacol. 156, 179186.[CrossRef][ISI][Medline]
Meyers, L. L., Beierschmitt, W. P., Khairallah, E. A., and Cohen, S. D. (1988). Acetaminophen-induced inhibition of mitochondrial respiration in mice. Toxicol. Appl. Pharmacol. 93, 378387.[ISI][Medline]
Nelson, S. D. (1990). Molecular mechanisms of the hepatotoxicity caused by acetaminophen. Semin. Liver Dis. 10, 267278.[ISI][Medline]
Qiu, Y., Benet, L. Z., and Burlingame, A. L. (2001). Identification of hepatic protein targets of the reactive metabolites of the non-hepatotoxic regioisomer of acetaminophen, 3'-hydroxyacetanilide, in the mouse in vivo using two-dimensional gel electrophoresis and mass spectrometry. Adv. Exp. Med. Biol. 500, 663673.[ISI][Medline]
Ramsay, R. R., Rashed, M. S., and Nelson, S. D. (1989). In vitro effects of acetaminophen metabolites and analogs on the respiration of mouse liver mitochondria. Arch. Biochem. Biophys. 273, 449457.[ISI][Medline]
Ray, S. D., Balasubramanian, G., Bagchi, D., and Reddy, C. S. (2001). Ca(2+)-calmodulin antagonist chlorpromazine and poly(ADP-ribose) polymerase modulators 4-aminobenzamide and nicotinamide influence hepatic expression of BCL-XL and P53 and protect against acetaminophen-induced programmed and unprogrammed cell death in mice. Free Radic. Biol. Med. 31, 277291.[CrossRef][ISI][Medline]
Ray, S. D., Kamendulis, L. M., Gurule, M. W., Yorkin, R. D., and Corcoran, G. B. (1993). Ca2+ antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen. FASEB J. 7, 453463.
Ray, S. D., Sorge, C. L., Raucy, J. L., and Corcoran, G. B. (1990). Early loss of large genomic DNA in vivo with accumulation of Ca2+ in the nucleus during acetaminophen-induced liver injury. Toxicol. Appl. Pharmacol. 106, 346351.[CrossRef][ISI][Medline]
Roberts, D. W., Bucci, T. J., Benson, R. W., Warbritton, A. R., McRae, T. A., Pumford, N. R., and Hinson, J. A. (1991). Immunohistochemical localization and quantification of the 3-(cystein-S-yl)-acetaminophen protein adduct in acetaminophen hepatotoxicity. Am. J. Pathol. 138, 359371.[Abstract]
Salminen, W. F., Jr, Voellmy, R., and Roberts, S. M. (1998). Effect of N-acetylcysteine on heat shock protein induction by acetaminophen in mouse liver. J. Pharmacol. Exp. Ther. 286, 519524.
Shen, W., Kamendulis, L. M., Ray, S. D., and Corcoran, G. B. (1991). Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: Correlation of nuclear Ca2+ accumulation and early DNA fragmentation with cell death. Toxicol. Appl. Pharmacol. 111, 242254.[ISI][Medline]
Shen,W., Kamendulis, L. M., Ray, S. D., and Corcoran, G. B. (1992). Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: Effects of Ca(2+)-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death. Toxicol. Appl. Pharmacol. 112, 3240.[CrossRef][ISI][Medline]
Szabo, C., and Dawson, V. L. (1998). Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion. Trends Pharmacol. Sci. 19, 287298.[CrossRef][ISI][Medline]
Thiemermann, C. (2002). Development of novel, water-soluble inhibitors of poly (adenosine 5'-diphosphate ribose) synthetase activity for use in shock and ischemia-reperfusion injury. Crit. Care Med. 30, 11631165.[CrossRef][ISI][Medline]
Tirmenstein, M. A., and Nelson, S. D. (1989). Subcellular binding and effects on calcium homeostasis produced by acetaminophen and a nonhepatotoxic regioisomer, 3'-hydroxyacetanilide, in mouse liver. J. Biol. Chem. 264, 98149819.
Tirmenstein, M. A., and Nelson, S. D. (1990). Acetaminophen-induced oxidation of protein thiols. Contribution of impaired thiol-metabolizing enzymes and the breakdown of adenine nucleotides. J. Biol. Chem. 265, 30593065.
Wang, Z. Q., Auer, B., Stingl, L., Berghammer, H., Haidacher, D., Schweiger, M., and Wagner, E. F. (1995). Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease. Genes Dev. 9, 509520.[Abstract]
Yakovlev, A. G., Wang, G., Stoica, B. A., Boulares, H. A., Spoonde, A. Y., Yoshihara, K., and Smulson, M. E. (2000). A role of the Ca2+ /Mg2+-dependent endonuclease in apoptosis and its inhibition by Poly(ADP-ribose) polymerase. J. Biol. Chem. 275, 2130221308.