- Email: [email protected]
Effect of Inhibitors of Nitric Oxide Synthase on Acetaminophen-Induced Hepatotoxicity in Mice
NITRIC OXIDE: Biology and Chemistry Vol. 6, No. 2, pp. 160 –167 (2002) doi:10.1006/niox.2001.0404, available online at http://www.idealibrary.com on
Effect of Inhibitors of Nitric Oxide Synthase on Acetaminophen-Induced Hepatotoxicity in Mice Jack A. Hinson,* ,1 Thomas J. Bucci,† Lisa K. Irwin,* Sherryll L. Michael,* and Philip R. Mayeux* *Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and †Pathology Associates, Incorporated, National Center for Toxicological Research, Jefferson, Arkansas 72079 Received August 21, 2001; published online October 25, 2001
We recently reported that following a toxic dose of acetaminophen to mice, tyrosine nitration occurs in the protein of cells that become necrotic. Nitration of tyrosine is by peroxynitrite, a species formed from nitric oxide (NO) and superoxide. In this manuscript we studied the effects of the NO synthase inhibitors N-monomethyl-L-arginine (L-NMMA), N-nitro-L-arginine methyl ester (NAME), L-N-(1-iminoethyl)lysine (L-NIL), and aminoguanidine on acetaminophen hepatotoxicity. Acetaminophen (300 mg/kg) increased serum nitrate/nitrite and alanine aminotransferase (ALT) levels, indicating increased NO synthesis and liver necrosis, respectively. None of the NO synthase inhibitors reduced serum ALT levels. In fact, L-NMMA, L-NIL, and aminoguanidine significantly augmented acetaminophen hepatotoxicity at 4 h. A detailed time course indicated that aminoguanidine (15 mg/kg at 0 h and 15 mg/kg at 2 h) significantly increased serum ALT levels over that for acetaminophen alone at 2 and 4 h; however, at 6 and 8 h serum ALT levels in the two groups were identical. At 2 h following acetaminophen plus aminoguanidine NO synthesis was significantly increased; however, at 4, 6, and 8 h NO synthesis was significantly decreased. Aminoguanidine also decreased acetaminophen-induced nitration of tyrosine. Acetaminophen alone did not induce lipid peroxidation, but acetaminophen plus aminoguanidine significantly increased hepatic lipid peroxidation (malondialdehyde levels) at 2, 4, and 6 h. These data are consistent with NO having a critical role in controlling superoxide-mediated lipid peroxidation in acetaminophen hepatotoxicity. Thus, acetaminophen hepatotoxicity may be mediated by either lipid peroxidation or by peroxynitrite. © 2001 Elsevier Science (USA) Key Words: acetaminophen; nitric oxide; peroxynitrite; nitrotyrosine; hepatotoxicity; aminoguanidine.
1 To whom correspondence and reprint requests should be addressed. Fax: (501) 686-8970. E-mail: [email protected].
160
It is becoming increasingly clear that reactive oxygen and nitrogen species play an important role in the development of hepatotoxicity caused by acetaminophen (1– 4). Acetaminophen is a commonly used analgesic/antipyretic that produces necrosis of the centrilobular cells of the liver when taken in overdose. The initial step in toxicity is cytochrome P450 metabolism of acetaminophen to the reactive metabolite N-acetylp-benzoquinone imine (NAPQI). At therapeutic doses this metabolite is efficiently detoxified by glutathione. At large doses conjugation with glutathione leads to depletion of glutathione and NAPQI covalently binds to cysteine residues on proteins as acetaminophenadducts (5). Covalent binding of NAPQI to proteins is an excellent correlate of acetaminophen toxicity. These adducts occur only in the hepatic centrilobular cells that develop necrosis (6, 7). We recently reported that toxic doses of acetaminophen to mice leads to increased NO synthesis and formation of nitrotyrosine-protein adducts. These adducts colocalize with acetaminophenprotein adducts in the centrilobular area (8). Nitrotyrosine-protein adducts are formed by nitration of tyrosine by peroxynitrite, a highly reactive species generated from superoxide and NO (9 –11). It is likely that multiple cell types (Kupffer cells, endothelial cells, and hepatocytes) participate in the synthesis of superoxide and NO in the liver following exposure to toxic doses of acetaminophen (1–3). Administration of gadolinium chloride, dextran sulfate, or dichloromethylene diphosphonate to inactivate Kupffer cells has been shown to decrease acetaminophen toxicity in mice (2, 12–14) in rats and mice, and decreased nitrotyrosine– protein adducts (2). It is known that peroxynitrite is detoxified by GSH (15) and GSH is depleted in livers of acetaminophen-treated mice by conjugation with NAPQI (16, 17). Current therapy for acetaminophen overdose is N-acetylcysteine to increase hepatic GSH biosynthesis. We reasoned that 1089-8603/01 $35.00 © 2001 Elsevier Science (USA) All rights reserved.
iNOS INHIBITION IN ACETAMINOPHEN HEPATOTOXICITY
another approach might be inhibition of peroxynitrite generation by inhibiting NO synthesis. In the present work we studied the effect of inhibitors of nitric oxide synthase (NOS) on the toxicity of acetaminophen in mice. MATERIALS AND METHODS Reagents. Acetaminophen was obtained from Sigma Chemical Company (St. Louis, MO). Universal DAKO LSAB⫹(Labeled Streptavidin-Biotin) Peroxidase kit and DAKO protein block (serum free) were acquired from DAKO Corp. (Carpinteria, CA). Immunopure Peroxidase Suppresser was purchased from Pierce Chemical Company (Rockford, IL). Anti-nitrotyrosine antiserum was raised in rabbits as previously described (18). Animals. Eight-week-old male B6C3F1 male mice having an average weight of 23 g were obtained from Harlan–Sprague–Dawley (Indianapolis, IN). Animals were housed five per cage in clear plastic cages. Mice were fed ad libitum and maintained on a 12-h light/ dark cycle. The mice were acclimatized for 1 week before use. The day before experiment the food was removed from all groups at 5:00 p.m. At 9:00 a.m. the next morning, mice (n ⫽ 5/group) were treated with either 0.4 ml of saline, ip or acetaminophen (300 mg/ kg) at time 0 h. The following nitric oxide inhibitors and doses were used: N-monomethyl-L-arginine (LNMMA) (30 mg/kg), N-nitro-L-arginine methyl ester (L-NAME) (2 mg/kg), L-N-(1-iminoethyl)lysine (L-NIL) (3 mg/kg), and aminoguanidine (15 mg/kg). Those animals receiving NOS inhibitors were administered one dose (ip) at time 0 h. Mice receiving aminoguanidine received a second dose at 2 h. At the indicated time, the mice were anesthetized with CO 2 and blood was taken from the retro-orbital sinus. The blood was allowed to coagulate at room temperature and the samples were then centrifuged. The serum was removed and stored at 4°C prior to analysis. Immediately after bleeding, the mice were sacrificed and the liver was removed. A portion of each liver was weighed and homogenized in a 3:1 v/w of 0.25 M sucrose, 10 mM Hepes, 1 mM EDTA (pH 7.5) buffer. The samples in each of the eight treatment groups were then pooled by equal volumes and protein concentrations were determined using Pierce Coomassie Plus Protein Assay Reagent. Other aliquots of the homogenates were stored at ⫺80°C. Hepatotoxicity and histological assays. Serum alanine aminotransferase (ALT) levels and serum aspartate aminotransferase (AST) levels were used as indi-
161
cators of hepatotoxicity. These assays were performed using a diagnostic kit obtained from Sigma Chemical Company. Liver sections were stained with H&E stain and the relative amount of toxicity determined using the previously published criteria (6). Immunohistochemical analyses were performed as previously described (18). Nitric oxide assay. Nitrate ⫹ Nitrite levels in the serum were determined by precipitation of protein, reduction of nitrate to nitrite by cadmium and quantitation of nitrite using the Griess reagent utilizing a kit obtained from Oxford Biomedical (Oxford, MI). Also, the possibility that nitric oxide may react with lipid hydroperoxy radicals to form an NO adduct was investigated by extracting 0.5 ml of the hepatic homogenate with 3.0 ml of chloroform-methanol (3:1 ratio). This lipid material was washed with an equal volume of water and the chloroform phase evaporated to dryness. The cadmium reduction procedure described above was performed and nitrite was quantified using Griess reagent. The lipid extract was found to test positive with the reagent but acetaminophen treatment of mice did not increase the amount of nitric oxide associated with the hepatic lipid. Liver assays. Lipid peroxidation was assayed by malondialdehyde production in liver homogenate utilizing a thiobarbituric lipid peroxidation assay (19). However, the homogenization of the liver samples for this assay required a special buffer that was pretreated with Chealex 100 to remove iron and contained the antioxidant BHT (0.03%). These treatments decreased spontaneous lipid peroxidation. Statistical analyses. Data are reported as mean ⫾ SE. Comparisons among treatment groups were by analysis of variance followed by the Student– Newman–Keuls post hoc test. RESULTS The model used was one that produces moderately severe hepatotoxicity following administration of acetaminophen at a dose of 300 mg/kg, i.p. (8). The NOS inhibitors were the non-selective inhibitors L-NMMA and L-NAME, and the inducible NOS (iNOS) inhibitors L-NIL and aminoguanidine (20 –23). In the first experiment the effects of L-NAME, L-NMMA, and L-NIL were evaluated. These data are presented in Table I. At 4 h acetaminophen produced a significant increase in serum nitrite/nitrate levels. In the mice receiving acetaminophen plus L-NAME, L-NMMA, or L-NIL, serum
© 2001 Elsevier Science (USA). All rights reserved.
162
HINSON ET AL. TABLE I
Effect of NOS Inhibitors on Acetaminophen-Induced NO Synthesis and Hepatotoxicity Serum Nitrite/Nitrate (M) Experiment 1 Saline Acetaminophen L-NAME Acetaminophen ⫹ L-NAME L-NMMA Acetaminophen ⫹ L-NMMA L-NIL Acetaminophen ⫹ L-NIL Experiment 2 Saline Acetaminophen Aminoguanidine Acetaminophen ⫹ Aminoguanidine
62 ⫾ 8 109 ⫾ 14* 53 ⫾ 6 86 ⫾ 11
Serum ALT (IU/l) 8⫾1 3,255 ⫾ 891* 11 ⫾ 1 3,826 ⫾ 1,090* ,†
36 ⫾ 3* 91 ⫾ 7* ,†
10 ⫾ 2 6,709 ⫾ 1,547* ,** ,†
37 ⫾ 2* 98 ⫾ 11* ,†
10 ⫾ 1 6,040 ⫾ 1,185* ,** ,†
28 ⫾ 5 48 ⫾ 8* 18 ⫾ 2 25 ⫾ 4**
10 ⫾ 3 1,879 ⫾ 934* 8⫾1 4,629 ⫾ 1,117* ,** ,†
Note. The doses were as follows: APAP 300 mg/kg, L-NAME 2 mg/kg, L-NMMA 30 mg/kg, L-NIL 3 mg/kg, and AG 15 mg/kg at 0 and at 2 h. The animals were sacrificed at 4 h. *Different from Saline (P ⬍ 0.05). **Different from acetaminophen alone (P ⬍ 0.05). † Different from inhibitor alone (P ⬍ 0.05).
nitrite/nitrate levels were no different from those in animals receiving acetaminophen alone. L-NMMA and L-NIL did significantly reduce basal levels of serum nitrite/nitrate when compared to saline. Acetaminophen also produced the expected increase in serum ALT, indicating hepatic cytotoxicity. Neither L-NAME, L-NMMA, nor L-NIL was protective against acetaminopheninduced increase in serum ALT. In fact, the combination of L-NMMA or L-NIL with acetaminophen resulted in significantly increased serum ALT levels when compared to acetaminophen alone. This effect of L-NMMA and L-NIL was not due to direct hepatotoxicity because these agents alone did not increase serum ALT levels when compared to saline. In a second set of experiments the effects of aminoguanidine were studied at 4 h. Aminoguanidine has been previously reported to have a half-life of approximately 2 h in the mouse (24). Thus, the mice were administered aminoguanidine at 0 and 2 h and sacrificed at 4 h after administration of acetaminophen (Table I). Acetaminophen produced an increase in serum nitrite/nitrate and ALT levels. Aminoguanidine alone had no effect on serum nitrite/nitrate or ALT levels. However, when administered with acetaminophen, aminoguanidine prevented the increase in serum
nitrite/nitrate levels but augmented the increase in ALT 2.5-fold. To better understand the effects of aminoguanidine a time course study was performed in a third set of experiments where aminoguanidine was administered at a dose of 15 mg/kg at time 0 and at 2 h. Figure 1 shows a time course for the effect of aminoguanidine on acetaminophen-induced hepatotoxicity. ALT levels were significantly higher in the acetaminophen plus aminoguanidine group than the acetaminophen alone group at 2 and 4 h; however, at 6 and 8 h there was no significant difference. Aminoguanidine increased the initial rate of development of the toxicity, but at maximal toxicity (6 – 8 h) there was no difference. Figure 2 shows that at 2 h aminoguanidine did not appear to inhibit the acetaminophen-induced increase in NO synthesis; however, at 4, 6, and 8 h NO synthesis was less in the acetaminophen plus aminoguanidine group than in the acetaminophen alone group. Thus, total NO synthesis was decreased over the 8-h experiment. To determine the effect of aminoguanidine treatment on acetaminophen-induced nitration of tyrosine, liver sections from each time and dose group were stained for nitrotyrosine. Immunohistochemical detection has proven to be an excellent method for detecting nitrotyrosine (25). Figure 3A shows a time course for acetaminophen-induced nitration of liver proteins and Fig. 3B shows the effects of aminoguanidine on acetaminophen-induced nitration of liver proteins. Aminoguanidine appeared to decrease hepatic nitration of tyrosine at each of the times compared to acet-
FIG. 1. Time course for effect of aminoguanidine on acetaminopheninduced hepatotoxicity. Serum alanine aminotransferase levels were determined as a marker hepatotoxicity. Mice (n ⫽ 5) were treated with acetaminophen (300 mg/kg, closed circles), aminoguanidine (15 mg/kg at 0 and at 2 h; closed squares), or acetaminophen plus aminoguanidine (open circles). The 0 time is the saline-treated control mice. *P ⬍ 0.05 compared to acetaminophen alone.
© 2001 Elsevier Science (USA). All rights reserved.
iNOS INHIBITION IN ACETAMINOPHEN HEPATOTOXICITY
FIG. 2. Effect of aminoguanidine on acetaminophen-induced increase in serum levels of nitrate plus nitrite. Serum levels of nitrate plus nitrite were used as a marker of NO synthesis. Mice (n ⫽ 5) were treated with acetaminophen (300 mg/kg, closed circles), aminoguanidine (15 mg/kg at 0 and at 2 h; closed squares), or acetaminophen plus aminoguanidine (open circles). The 0 time is the salinetreated control mice. *P ⬍ 0.05 compared to acetaminophen plus aminoguanidine alone.
aminophen alone. The apparent decrease in staining observed at 8 h may be a result of loss of nitrotyrosine in the tissue as a result of digestion and reorganization of tissue (6). Figure 4 shows that at 2, 4, and 6 h lipid peroxidation was greater in the acetaminophen plus aminoguanidine group than in the acetaminophen alone group. The data confirm previous reports (26, 27) that acetaminophen alone does not cause lipid peroxidation (hepatic malondialdehyde levels as measured by thiobarbituric acid reactive substances). DISCUSSION GSH depletion and NAPQI binding are well recognized as hallmarks of acetaminophen hepatotoxicity (5). We recently showed that nitrotyrosine adducts also occurred following toxic doses of acetaminophen (8). Thus, it was reasonable to postulate that inhibition of nitric oxide synthesis might decrease hepatotoxicity. Isoform selective and nonselective inhibitors of NOS have been studied as potential therapeutic agents in a number of models of liver and kidney disease (24, 28 – 31) and in humans (32). Unexpectedly, nonselective NOS inhibitors as well as putative selective iNOS inhibitors failed to decreases toxicity. In fact, L-NMMA, L-NIL, and aminoguanidine greatly increased ALT levels when measured at 4 h. However, a detailed time course study indicated that aminoguanidine actually increased the rate of development of the toxicity. Thus, ALT levels were significantly higher at 2 and 4 h, but
163
aminoguanidine did not alter the toxicity at late times (6 – 8 h). Thus, it appears that inhibition of NO synthesis does not decrease the hepatotoxicity of acetaminophen. Consistent with previous reports (26, 27, 33) acetaminophen alone did not cause an increase in lipid peroxidation. However, when acetaminophen was administered with aminoguanidine lipid peroxidation was increased. These findings are consistent with our previous data showing that acetaminophen causes an increase in lipid peroxidation in iNOS knockout mice (34). Taken together these data support the hypothesis that NO serves an important role in inhibiting superoxide-induced lipid peroxidation in acetaminopheninduced hepatotoxicity. The chemical rate constant for reaction of superoxide and nitric oxide is extremely rapid and approaches that of a diffusion-limited rate (35–37). Reaction of nitric oxide with superoxide would form peroxynitrite and this reaction should be a favored reaction. Inhibition of NO synthesis would decrease peroxynitrite formation and could favor superoxide initiated lipid peroxidation (38, 39). Lipid peroxidation is believed to be important mechanistically in various toxicities including the hepatotoxicity produced by carbon tetrachloride and the lung toxicity produced by in paraquat (39, 40). Therefore, we postulate that acetaminophen-induced hepatotoxicity may be mediated by peroxynitrite leading to nitration and oxidation of macromolecules, or by superoxide leading to hydrogen peroxide and lipid peroxidation. Metabolism of acetaminophen to NAPQI leading to depletion of hepatic GSH in the centrilobular cells may be a key factor. GSH is important in detoxification of peroxynitrite (15, 41), hydrogen peroxide, and lipid hydroperoxides (42, 43), and under conditions of GSH depletion these reactions may be inhibited. The zonal nature of the toxicity may be a result of the predominance of CYP2E1 in the centrilobular areas of the liver. CYP2E1 is a critical enzyme involved in metabolism of acetaminophen to NAPQI (7, 44). An alternative mechanism whereby nitric oxide may limit lipid peroxidation is reaction of nitric oxide with lipid radicals to inhibit initiation of lipid peroxidation; however, we could find no evidence for acetaminophen causing an increase in NO associated with the hepatic lipids. The source of superoxide and the mechanism of its presumed increase are not fully understood. Synthesis may be from damaged mitochondria (45), from activated Kupffer cells or other cells (3, 46, 47) from enzymes such as CYP2E1 (48) or from a combination of
© 2001 Elsevier Science (USA). All rights reserved.
164
HINSON ET AL.
FIG. 3. Effect of aminoguanidine on acetaminophen-induced nitration of hepatic tissue. Liver sections from the acetaminophen treated livers (Fig. 1) were processed and stained for nitrated tyrosine residues using anti-nitrotyrosine antiserum. The time course for acetaminophen (APAP)-treated livers is shown in A plus the saline-treated control. The time course for the APAP plus aminoguanidine (AG)-treated is shown in B plus aminoguanidine alone. The data are representative liver sections for each time and treatment group.
© 2001 Elsevier Science (USA). All rights reserved.
iNOS INHIBITION IN ACETAMINOPHEN HEPATOTOXICITY
165
ACKNOWLEDGMENTS This work was supported by a grant (GM58884) from the National Institute for General Medical Sciences, NIH, to J.A.H.
REFERENCES 1. Nakae, D., Yamamoto, K., Yoshiji, H., Kinugasa, T., Maruyama, H., Farber, J. L., and Konishi, Y. (1990). Liposome-encapsulated superoxide dismutase prevents liver necrosis induced by acetaminophen. Am. J. Pathol. 136, 787–795.
FIG. 4. Time course for effect of aminoguanidine on acetaminopheninduced lipid peroxidation. Liver homogenates of the acetaminophen, aminoguanidine, and acetaminophen plus aminoguanidine treated mice were analyzed for malondialdehyde levels. Mice (n ⫽ 5) were treated with acetaminophen (300 mg/kg, closed circles), aminoguanidine (15 mg/kg at 0 and at 2 h; closed squares), or acetaminophen plus aminoguanidine (open circles). The 0 time is the salinetreated control mice. *P ⬍ 0.05 compared to acetaminophen alone.
all of the above. Hepatic NO is produced by constitutively expressed endothelial NOS (eNOS) and by iNOS (49). Whereas eNOS is in endothelial cells, iNOS is in hepatocytes, stellate cells, and Kupffer cells (49). Regardless of the source, acetaminophen causes an increase in NO synthesis. Of the NOS inhibitors tested, only aminoguanidine reduced acetaminophen-stimulated increases in serum nitrite/nitrate concentration despite the ability of L-NMMA and L-NIL to inhibit basal levels in saline-treated animals. Serum nitrate/nitrite levels in saline-treated mice indicate that the status of NO synthesis and perhaps iNOS itself can vary among groups of mice (Table I) and we have observed this variability in newly purchased naı¨ve mice (data not shown). The cause of this variability is not clear. Nonetheless, we postulate that NO can play an important role in determining the mechanism of acetaminophen-induced hepatotoxicity. Acetaminophen appears to stimulate the production of both NO and superoxide. The reaction between NO and superoxide to generate peroxynitrite might serve as a superoxide detoxification reaction. However, acetaminophen metabolism also depletes GSH. This reduces detoxification of peroxynitrite and contributes to toxicity. The data suggest that the reason NOS inhibitors are not only ineffective in reducing acetaminophen hepatotoxicity but actually augment toxicity is because inhibition of NO synthesis shifts the toxicity pathway toward superoxide-derived oxidants.
2. Michael, S. L., Pumford, N. R., Mayeux, P. R., Niesman, M. R., and Hinson, J. A. (1999). Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species. Hepatology 30, 186 –195. 3. Knight, T. R., Kurtz, A., Bajt, M. L., Hinson, J. A., and Jaeschke, H. (2001). Vascular and hepatocellular peroxynitrite formation during acetaminophen toxicity: Role of mitochondrial oxidant stress. Toxicol. Sci. 62, 212–220. 4. Ferret, P. J., Hammoud, R., Tulliez, M., Tran, A., Trebeden, H., Jaffray, P., Malassagne, B., Calmus, Y., Weill, B., and Batteux, F. (2001). Detoxification of reactive oxygen species by a nonpeptidyl mimic of superoxide dismutase cures acetaminopheninduced acute liver failure in the mouse. Hepatology 33, 1173– 1180. 5. Cohen, S. D., Pumford, N. R., Khairallah, E. A., Boekelheide, K., Pohl, L. R., Amouzadeh, H. R., and Hinson, J. A. (1997). Selective protein covalent binding and target organ toxicity. Toxicol. Appl. Pharmacol. 143, 1–12. 6. 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-(cysteinS-yl)-acetaminophen protein adduct in acetaminophen hepatotoxicity. Am. J. Pathol. 138, 359 –371. 7. Hart, S. G., Cartun, R. W., Wyand, D. S., Khairallah, E. A., and Cohen, S. D. (1995). Immunohistochemical localization of acetaminophen in target tissues of the CD-1 mouse: Correspondence of covalent binding with toxicity. Fundam. Appl. Toxicol. 24, 260 –274. 8. Hinson, J. A., Pike, S. L., Pumford, N. R., and Mayeux, P. R. (1998). Nitrotyrosine-protein adducts in hepatic centrilobular areas following toxic doses of acetaminophen in mice. Chem. Res. Toxicol. 11, 604 – 607. 9. Beckman, J. S. (1996). Oxidative damage and tyrosine nitration from peroxynitrite. Chem. Res. Toxicol. 9, 836 – 844. 10. Pryor, W. A., and Squadrito, G. L. (1995). The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268, L699 –L722. 11. Reiter, C. D., Teng, R. J., and Beckman, J. S. (2000). Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J. Biol. Chem. 275, 32460 –32466. 12. Laskin, D. L., and Pendino, K. J. (1995). Macrophages and inflammatory mediators in tissue injury. Annu. Rev. Pharmacol. Toxicol. 35, 655– 677. 13. Goldin, R. D., Ratnayaka, I. D., Breach, C. S., Brown, I. N., and
© 2001 Elsevier Science (USA). All rights reserved.
166
HINSON ET AL.
Wickramasinghe, S. N. (1996). Role of macrophages in acetaminophen (paracetamol)-induced hepatotoxicity. J. Pathol. 179, 432– 435. 14. Blazka, M. E., Germolec, D. R., Simeonova, P., Bruccoleri, A., Pennypacker, K. R., and Luster, M. I. (1995). Acetaminopheninduced hepatotoxicity is associated with early changes in NF-kB and NF-IL6 DNA binding activity. J. Inflamm. 47, 138 – 150. 15. Sies, H., Sharov, V. S., Klotz, L. O., and Briviba, K. (1997). Glutathione peroxidase protects against peroxynitrite-mediated oxidations. A new function for selenoproteins as peroxynitrite reductase. J. Biol. Chem. 272, 27812–27817. 16. Hinson, J. A., Mays, J. B., and Cameron, A. M. (1983). Acetaminophen-induced hepatic glycogen depletion and hyperglycemia in mice. Biochem. Pharmacol. 32, 1979 –1988. 17. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211–217. 18. Hinson, J. A., Michael, S. L., Ault, S. G., and Pumford, N. R. (2000). Western blot analysis for nitrotyrosine protein adducts in livers of saline-treated and acetaminophen-treated mice [In Process Citation]. Toxicol. Sci. 53, 467– 473. 19. Buege, J. A., and Aust, S. D. (1978). Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. 20. Rees, D. D., Palmer, R. M., Schulz, R., Hodson, H. F., and Moncada, S. (1990). Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br. J. Pharmacol. 101, 746 –752. 21. Bryk, R., and Wolff, D. J. (1998). Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1iminoethyl)lysine. Biochemistry 37, 4844 – 4852. 22. Moore, W. M., Webber, R. K., Jerome, G. M., Tjoeng, F. S., Misko, T. P., and Currie, M. G. (1994). L-N6-(1-iminoethyl)lysine: A selective inhibitor of inducible nitric oxide synthase. J. Med. Chem. 37, 3886 –3888. 23. Faraci, W. S., Nagel, A. A., Verdries, K. A., Vincent, L. A., Xu, H., Nichols, L. E., Labasi, J. M., Salter, E. D., and Pettipher, E. R. (1996). 2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo. Br. J. Pharmacol. 119, 1101–1108. 24. Bowman, M. A., Simell, O. G., Peck, A. B., Cornelius, J., Luchetta, R., Look, Z., Maclaren, N. K., and Atkinson, M. A. (1996). Pharmacokinetics of aminoguanidine administration and effects on the diabetes frequency in nonobese diabetic mice. J. Pharmacol. Exp. Ther. 279, 790 –794. 25. Viera, L., Ye, Y. Z., Estevez, A. G., and Beckman, J. S. (1999). Immunohistochemical methods to detect nitrotyrosine. Methods Enzymol. 301, 373–381.
induced hepatotoxicity: Comparison with carbon tetrachloride. Toxicol. Lett. 66, 7–12. 28. Ou, J., Carlos, T. M., Watkins, S. C., Saavedra, J. E., Keefer, L. K., Kim, Y. M., Harbrecht, B. G., and Billiar, T. R. (1997). Differential effects of nonselective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis, ICAM-1 expression, and neutrophil accumulation during endotoxemia. Nitric Oxide 1, 404 – 416. 29. Uhlenius, N., Tikkanen, I., Tikkanen, T., Miettinen, A., Tornroth, T., and Fyhrquist, F. (1996). Chronic inhibition of nitric oxide synthase in Heymann nephritis. Nephron 74, 144 –149. 30. Walker, L. M., Walker, P. D., Imam, S. Z., Ali, S. F., and Mayeux, P. R. (2000). Evidence for peroxynitrite formation in renal ischemia-reperfusion injury: Studies with the inducible nitric oxide synthase inhibitor L-N(6)-(1-Iminoethyl)lysine. J. Pharmacol. Exp. Ther. 295, 417– 422. 31. Hock, C. E., Yin, K., Yue, G., and Wong, P. Y. (1997). Effects of inhibition of nitric oxide synthase by aminoguanidine in acute endotoxemia. Am. J. Physiol. 272, H843–H850. 32. Petros, A., Lamb, G., Leone, A., Moncada, S., Bennett, D., and Vallance, P. (1994). Effects of a nitric oxide synthase inhibitor in humans with septic shock. Cardiovasc. Res. 28, 34 –39. 33. Mitchell, J. R., Corcoran, G. B., Smith, C. V., Hughes, H., and Lauterburg, B. H. (1981). Alkylation and peroxidation injury from chemically reactive metabolites. Adv. Exp. Med. Biol. 136, 199 –223. 34. Michael, S. L., Mayeux, P. R., Bucci, T. J., Warbritton, A. R., Irwin, L. K., Pumford, N. R., and Hinson, J. A. (2001). Acetaminophen-induced hepatotoxicity in mice lacking inducible nitric oxide synthase activity. Nitric Oxide 5, 432– 441. 35. Huie, R. E., and Padmaja, S. (1993). The reaction of no with superoxide. Free Rad. Res. Commun. 18, 195–199. 36. Goldstein, S., and Czapski, G. (1995). The reaction of NO • with O2 •⫺ and HO2 •: A pulse radiolysis study. Free Rad. Biol. Med. 19, 505–510. 37. Thomson, L., Trujillo, M., Telleri, R., and Radi, R. (1995). Kinetics of cytochrome c2⫹ oxidation by peroxynitrite: Implications for superoxide measurements in nitric oxide-producing biological systems. Arch. Biochem. Biophys. 319, 491– 497. 38. Pederson, T. C., and Aust, S. D. (1975). The mechanism of liver microsomal lipid peroxidation. Biochim. Biophys. Acta 385, 232– 241. 39. Bus, J. S., Aust, S. D., and Gibson, J. E. (1976). Paraquat toxicity: Proposed mechanism of action involving lipid peroxidation. Environ. Health Perspect. 16, 139 –146. 40. Recknagel, R. O., Glende, E. A., Dolak, J. A., and Waller, R. L. (1989). Mechanisms of carbon tetrachloride toxicity. Pharmacol. Ther. 43, 139 –154. 41. Briviba, K., Kissner, R., Koppenol, W. H., and Sies, H. (1998). Kinetic study of the reaction of glutathione peroxidase with peroxynitrite. Chem. Res. Toxicol. 11, 1398 –1401.
26. Younes, M., Cornelius, S., and Siegers, C. P. (1986). Ferrous ion supported in vivo lipid peroxidation induced by paracetamol—Its relation to hepatotoxicity. Res. Commun. Chem. Pathol. Pharmacol. 51, 89 –99.
42. Brigelius-Flohe, R. (1999). Tissue-specific functions of individual glutathione peroxidases. Free Rad. Biol. Med. 27, 951–965.
27. Kamiyama, T., Sato, C., Liu, J., Tajiri, K., Miyakawa, H., and Marumo, F. (1993). Role of lipid peroxidation in acetaminophen-
43. Arthur, J. R. (2000). The glutathione peroxidases. Cell Mol. Life Sci. 57, 1825–1835.
© 2001 Elsevier Science (USA). All rights reserved.
iNOS INHIBITION IN ACETAMINOPHEN HEPATOTOXICITY
167
44. Anundi, I., Lahteenmaki, T., Rundgren, M., Moldeus, P., and Lindros, K. O. (1993). Zonation of acetaminophen metabolism and cytochrome P450 2E1-mediated toxicity studied in isolated periportal and perivenous hepatocytes. Biochem. Pharmacol. 45, 1251–1259. 45. Jaeschke, H., and Mitchell, J. R. (1989). Mitochondria and xanthine oxidase both generate reactive oxygen species in isolated perfused rat liver after hypoxic injury. Biochem. Biophys. Res. Commun. 160, 140 –147.
aminophen hepatotoxicity. Toxicol. Appl. Pharmacol. 133, 43– 52.
48. Koop, D. R. (1992). Oxidative and reductive metabolism by cytochrome P450 2E1. FASEB J. 6, 724 –730.
46. Blazka, M. E., Wilmer, J. L., Holladay, S. D., Wilson, R. E., and Luster, M. I. (1995). Role of proinflammatory cytokines in acet-
49. Muriel, P. (2000). Regulation of nitric oxide synthesis in the liver. J. Appl. Toxicol. 20, 189 –195.
47. Laskin, D. L., Gardner, C. R., Price, V. F., and Jollow, D. J. (1995). Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology 21, 1045– 1050.
© 2001 Elsevier Science (USA). All rights reserved.
Effect of Inhibitors of Nitric Oxide Synthase on Acetaminophen-Induced Hepatotoxicity in Mice Jack A. Hinson,* ,1 Thomas J. Bucci,† Lisa K. Irwin,* Sherryll L. Michael,* and Philip R. Mayeux* *Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and †Pathology Associates, Incorporated, National Center for Toxicological Research, Jefferson, Arkansas 72079 Received August 21, 2001; published online October 25, 2001
We recently reported that following a toxic dose of acetaminophen to mice, tyrosine nitration occurs in the protein of cells that become necrotic. Nitration of tyrosine is by peroxynitrite, a species formed from nitric oxide (NO) and superoxide. In this manuscript we studied the effects of the NO synthase inhibitors N-monomethyl-L-arginine (L-NMMA), N-nitro-L-arginine methyl ester (NAME), L-N-(1-iminoethyl)lysine (L-NIL), and aminoguanidine on acetaminophen hepatotoxicity. Acetaminophen (300 mg/kg) increased serum nitrate/nitrite and alanine aminotransferase (ALT) levels, indicating increased NO synthesis and liver necrosis, respectively. None of the NO synthase inhibitors reduced serum ALT levels. In fact, L-NMMA, L-NIL, and aminoguanidine significantly augmented acetaminophen hepatotoxicity at 4 h. A detailed time course indicated that aminoguanidine (15 mg/kg at 0 h and 15 mg/kg at 2 h) significantly increased serum ALT levels over that for acetaminophen alone at 2 and 4 h; however, at 6 and 8 h serum ALT levels in the two groups were identical. At 2 h following acetaminophen plus aminoguanidine NO synthesis was significantly increased; however, at 4, 6, and 8 h NO synthesis was significantly decreased. Aminoguanidine also decreased acetaminophen-induced nitration of tyrosine. Acetaminophen alone did not induce lipid peroxidation, but acetaminophen plus aminoguanidine significantly increased hepatic lipid peroxidation (malondialdehyde levels) at 2, 4, and 6 h. These data are consistent with NO having a critical role in controlling superoxide-mediated lipid peroxidation in acetaminophen hepatotoxicity. Thus, acetaminophen hepatotoxicity may be mediated by either lipid peroxidation or by peroxynitrite. © 2001 Elsevier Science (USA) Key Words: acetaminophen; nitric oxide; peroxynitrite; nitrotyrosine; hepatotoxicity; aminoguanidine.
1 To whom correspondence and reprint requests should be addressed. Fax: (501) 686-8970. E-mail: [email protected].
160
It is becoming increasingly clear that reactive oxygen and nitrogen species play an important role in the development of hepatotoxicity caused by acetaminophen (1– 4). Acetaminophen is a commonly used analgesic/antipyretic that produces necrosis of the centrilobular cells of the liver when taken in overdose. The initial step in toxicity is cytochrome P450 metabolism of acetaminophen to the reactive metabolite N-acetylp-benzoquinone imine (NAPQI). At therapeutic doses this metabolite is efficiently detoxified by glutathione. At large doses conjugation with glutathione leads to depletion of glutathione and NAPQI covalently binds to cysteine residues on proteins as acetaminophenadducts (5). Covalent binding of NAPQI to proteins is an excellent correlate of acetaminophen toxicity. These adducts occur only in the hepatic centrilobular cells that develop necrosis (6, 7). We recently reported that toxic doses of acetaminophen to mice leads to increased NO synthesis and formation of nitrotyrosine-protein adducts. These adducts colocalize with acetaminophenprotein adducts in the centrilobular area (8). Nitrotyrosine-protein adducts are formed by nitration of tyrosine by peroxynitrite, a highly reactive species generated from superoxide and NO (9 –11). It is likely that multiple cell types (Kupffer cells, endothelial cells, and hepatocytes) participate in the synthesis of superoxide and NO in the liver following exposure to toxic doses of acetaminophen (1–3). Administration of gadolinium chloride, dextran sulfate, or dichloromethylene diphosphonate to inactivate Kupffer cells has been shown to decrease acetaminophen toxicity in mice (2, 12–14) in rats and mice, and decreased nitrotyrosine– protein adducts (2). It is known that peroxynitrite is detoxified by GSH (15) and GSH is depleted in livers of acetaminophen-treated mice by conjugation with NAPQI (16, 17). Current therapy for acetaminophen overdose is N-acetylcysteine to increase hepatic GSH biosynthesis. We reasoned that 1089-8603/01 $35.00 © 2001 Elsevier Science (USA) All rights reserved.
iNOS INHIBITION IN ACETAMINOPHEN HEPATOTOXICITY
another approach might be inhibition of peroxynitrite generation by inhibiting NO synthesis. In the present work we studied the effect of inhibitors of nitric oxide synthase (NOS) on the toxicity of acetaminophen in mice. MATERIALS AND METHODS Reagents. Acetaminophen was obtained from Sigma Chemical Company (St. Louis, MO). Universal DAKO LSAB⫹(Labeled Streptavidin-Biotin) Peroxidase kit and DAKO protein block (serum free) were acquired from DAKO Corp. (Carpinteria, CA). Immunopure Peroxidase Suppresser was purchased from Pierce Chemical Company (Rockford, IL). Anti-nitrotyrosine antiserum was raised in rabbits as previously described (18). Animals. Eight-week-old male B6C3F1 male mice having an average weight of 23 g were obtained from Harlan–Sprague–Dawley (Indianapolis, IN). Animals were housed five per cage in clear plastic cages. Mice were fed ad libitum and maintained on a 12-h light/ dark cycle. The mice were acclimatized for 1 week before use. The day before experiment the food was removed from all groups at 5:00 p.m. At 9:00 a.m. the next morning, mice (n ⫽ 5/group) were treated with either 0.4 ml of saline, ip or acetaminophen (300 mg/ kg) at time 0 h. The following nitric oxide inhibitors and doses were used: N-monomethyl-L-arginine (LNMMA) (30 mg/kg), N-nitro-L-arginine methyl ester (L-NAME) (2 mg/kg), L-N-(1-iminoethyl)lysine (L-NIL) (3 mg/kg), and aminoguanidine (15 mg/kg). Those animals receiving NOS inhibitors were administered one dose (ip) at time 0 h. Mice receiving aminoguanidine received a second dose at 2 h. At the indicated time, the mice were anesthetized with CO 2 and blood was taken from the retro-orbital sinus. The blood was allowed to coagulate at room temperature and the samples were then centrifuged. The serum was removed and stored at 4°C prior to analysis. Immediately after bleeding, the mice were sacrificed and the liver was removed. A portion of each liver was weighed and homogenized in a 3:1 v/w of 0.25 M sucrose, 10 mM Hepes, 1 mM EDTA (pH 7.5) buffer. The samples in each of the eight treatment groups were then pooled by equal volumes and protein concentrations were determined using Pierce Coomassie Plus Protein Assay Reagent. Other aliquots of the homogenates were stored at ⫺80°C. Hepatotoxicity and histological assays. Serum alanine aminotransferase (ALT) levels and serum aspartate aminotransferase (AST) levels were used as indi-
161
cators of hepatotoxicity. These assays were performed using a diagnostic kit obtained from Sigma Chemical Company. Liver sections were stained with H&E stain and the relative amount of toxicity determined using the previously published criteria (6). Immunohistochemical analyses were performed as previously described (18). Nitric oxide assay. Nitrate ⫹ Nitrite levels in the serum were determined by precipitation of protein, reduction of nitrate to nitrite by cadmium and quantitation of nitrite using the Griess reagent utilizing a kit obtained from Oxford Biomedical (Oxford, MI). Also, the possibility that nitric oxide may react with lipid hydroperoxy radicals to form an NO adduct was investigated by extracting 0.5 ml of the hepatic homogenate with 3.0 ml of chloroform-methanol (3:1 ratio). This lipid material was washed with an equal volume of water and the chloroform phase evaporated to dryness. The cadmium reduction procedure described above was performed and nitrite was quantified using Griess reagent. The lipid extract was found to test positive with the reagent but acetaminophen treatment of mice did not increase the amount of nitric oxide associated with the hepatic lipid. Liver assays. Lipid peroxidation was assayed by malondialdehyde production in liver homogenate utilizing a thiobarbituric lipid peroxidation assay (19). However, the homogenization of the liver samples for this assay required a special buffer that was pretreated with Chealex 100 to remove iron and contained the antioxidant BHT (0.03%). These treatments decreased spontaneous lipid peroxidation. Statistical analyses. Data are reported as mean ⫾ SE. Comparisons among treatment groups were by analysis of variance followed by the Student– Newman–Keuls post hoc test. RESULTS The model used was one that produces moderately severe hepatotoxicity following administration of acetaminophen at a dose of 300 mg/kg, i.p. (8). The NOS inhibitors were the non-selective inhibitors L-NMMA and L-NAME, and the inducible NOS (iNOS) inhibitors L-NIL and aminoguanidine (20 –23). In the first experiment the effects of L-NAME, L-NMMA, and L-NIL were evaluated. These data are presented in Table I. At 4 h acetaminophen produced a significant increase in serum nitrite/nitrate levels. In the mice receiving acetaminophen plus L-NAME, L-NMMA, or L-NIL, serum
© 2001 Elsevier Science (USA). All rights reserved.
162
HINSON ET AL. TABLE I
Effect of NOS Inhibitors on Acetaminophen-Induced NO Synthesis and Hepatotoxicity Serum Nitrite/Nitrate (M) Experiment 1 Saline Acetaminophen L-NAME Acetaminophen ⫹ L-NAME L-NMMA Acetaminophen ⫹ L-NMMA L-NIL Acetaminophen ⫹ L-NIL Experiment 2 Saline Acetaminophen Aminoguanidine Acetaminophen ⫹ Aminoguanidine
62 ⫾ 8 109 ⫾ 14* 53 ⫾ 6 86 ⫾ 11
Serum ALT (IU/l) 8⫾1 3,255 ⫾ 891* 11 ⫾ 1 3,826 ⫾ 1,090* ,†
36 ⫾ 3* 91 ⫾ 7* ,†
10 ⫾ 2 6,709 ⫾ 1,547* ,** ,†
37 ⫾ 2* 98 ⫾ 11* ,†
10 ⫾ 1 6,040 ⫾ 1,185* ,** ,†
28 ⫾ 5 48 ⫾ 8* 18 ⫾ 2 25 ⫾ 4**
10 ⫾ 3 1,879 ⫾ 934* 8⫾1 4,629 ⫾ 1,117* ,** ,†
Note. The doses were as follows: APAP 300 mg/kg, L-NAME 2 mg/kg, L-NMMA 30 mg/kg, L-NIL 3 mg/kg, and AG 15 mg/kg at 0 and at 2 h. The animals were sacrificed at 4 h. *Different from Saline (P ⬍ 0.05). **Different from acetaminophen alone (P ⬍ 0.05). † Different from inhibitor alone (P ⬍ 0.05).
nitrite/nitrate levels were no different from those in animals receiving acetaminophen alone. L-NMMA and L-NIL did significantly reduce basal levels of serum nitrite/nitrate when compared to saline. Acetaminophen also produced the expected increase in serum ALT, indicating hepatic cytotoxicity. Neither L-NAME, L-NMMA, nor L-NIL was protective against acetaminopheninduced increase in serum ALT. In fact, the combination of L-NMMA or L-NIL with acetaminophen resulted in significantly increased serum ALT levels when compared to acetaminophen alone. This effect of L-NMMA and L-NIL was not due to direct hepatotoxicity because these agents alone did not increase serum ALT levels when compared to saline. In a second set of experiments the effects of aminoguanidine were studied at 4 h. Aminoguanidine has been previously reported to have a half-life of approximately 2 h in the mouse (24). Thus, the mice were administered aminoguanidine at 0 and 2 h and sacrificed at 4 h after administration of acetaminophen (Table I). Acetaminophen produced an increase in serum nitrite/nitrate and ALT levels. Aminoguanidine alone had no effect on serum nitrite/nitrate or ALT levels. However, when administered with acetaminophen, aminoguanidine prevented the increase in serum
nitrite/nitrate levels but augmented the increase in ALT 2.5-fold. To better understand the effects of aminoguanidine a time course study was performed in a third set of experiments where aminoguanidine was administered at a dose of 15 mg/kg at time 0 and at 2 h. Figure 1 shows a time course for the effect of aminoguanidine on acetaminophen-induced hepatotoxicity. ALT levels were significantly higher in the acetaminophen plus aminoguanidine group than the acetaminophen alone group at 2 and 4 h; however, at 6 and 8 h there was no significant difference. Aminoguanidine increased the initial rate of development of the toxicity, but at maximal toxicity (6 – 8 h) there was no difference. Figure 2 shows that at 2 h aminoguanidine did not appear to inhibit the acetaminophen-induced increase in NO synthesis; however, at 4, 6, and 8 h NO synthesis was less in the acetaminophen plus aminoguanidine group than in the acetaminophen alone group. Thus, total NO synthesis was decreased over the 8-h experiment. To determine the effect of aminoguanidine treatment on acetaminophen-induced nitration of tyrosine, liver sections from each time and dose group were stained for nitrotyrosine. Immunohistochemical detection has proven to be an excellent method for detecting nitrotyrosine (25). Figure 3A shows a time course for acetaminophen-induced nitration of liver proteins and Fig. 3B shows the effects of aminoguanidine on acetaminophen-induced nitration of liver proteins. Aminoguanidine appeared to decrease hepatic nitration of tyrosine at each of the times compared to acet-
FIG. 1. Time course for effect of aminoguanidine on acetaminopheninduced hepatotoxicity. Serum alanine aminotransferase levels were determined as a marker hepatotoxicity. Mice (n ⫽ 5) were treated with acetaminophen (300 mg/kg, closed circles), aminoguanidine (15 mg/kg at 0 and at 2 h; closed squares), or acetaminophen plus aminoguanidine (open circles). The 0 time is the saline-treated control mice. *P ⬍ 0.05 compared to acetaminophen alone.
© 2001 Elsevier Science (USA). All rights reserved.
iNOS INHIBITION IN ACETAMINOPHEN HEPATOTOXICITY
FIG. 2. Effect of aminoguanidine on acetaminophen-induced increase in serum levels of nitrate plus nitrite. Serum levels of nitrate plus nitrite were used as a marker of NO synthesis. Mice (n ⫽ 5) were treated with acetaminophen (300 mg/kg, closed circles), aminoguanidine (15 mg/kg at 0 and at 2 h; closed squares), or acetaminophen plus aminoguanidine (open circles). The 0 time is the salinetreated control mice. *P ⬍ 0.05 compared to acetaminophen plus aminoguanidine alone.
aminophen alone. The apparent decrease in staining observed at 8 h may be a result of loss of nitrotyrosine in the tissue as a result of digestion and reorganization of tissue (6). Figure 4 shows that at 2, 4, and 6 h lipid peroxidation was greater in the acetaminophen plus aminoguanidine group than in the acetaminophen alone group. The data confirm previous reports (26, 27) that acetaminophen alone does not cause lipid peroxidation (hepatic malondialdehyde levels as measured by thiobarbituric acid reactive substances). DISCUSSION GSH depletion and NAPQI binding are well recognized as hallmarks of acetaminophen hepatotoxicity (5). We recently showed that nitrotyrosine adducts also occurred following toxic doses of acetaminophen (8). Thus, it was reasonable to postulate that inhibition of nitric oxide synthesis might decrease hepatotoxicity. Isoform selective and nonselective inhibitors of NOS have been studied as potential therapeutic agents in a number of models of liver and kidney disease (24, 28 – 31) and in humans (32). Unexpectedly, nonselective NOS inhibitors as well as putative selective iNOS inhibitors failed to decreases toxicity. In fact, L-NMMA, L-NIL, and aminoguanidine greatly increased ALT levels when measured at 4 h. However, a detailed time course study indicated that aminoguanidine actually increased the rate of development of the toxicity. Thus, ALT levels were significantly higher at 2 and 4 h, but
163
aminoguanidine did not alter the toxicity at late times (6 – 8 h). Thus, it appears that inhibition of NO synthesis does not decrease the hepatotoxicity of acetaminophen. Consistent with previous reports (26, 27, 33) acetaminophen alone did not cause an increase in lipid peroxidation. However, when acetaminophen was administered with aminoguanidine lipid peroxidation was increased. These findings are consistent with our previous data showing that acetaminophen causes an increase in lipid peroxidation in iNOS knockout mice (34). Taken together these data support the hypothesis that NO serves an important role in inhibiting superoxide-induced lipid peroxidation in acetaminopheninduced hepatotoxicity. The chemical rate constant for reaction of superoxide and nitric oxide is extremely rapid and approaches that of a diffusion-limited rate (35–37). Reaction of nitric oxide with superoxide would form peroxynitrite and this reaction should be a favored reaction. Inhibition of NO synthesis would decrease peroxynitrite formation and could favor superoxide initiated lipid peroxidation (38, 39). Lipid peroxidation is believed to be important mechanistically in various toxicities including the hepatotoxicity produced by carbon tetrachloride and the lung toxicity produced by in paraquat (39, 40). Therefore, we postulate that acetaminophen-induced hepatotoxicity may be mediated by peroxynitrite leading to nitration and oxidation of macromolecules, or by superoxide leading to hydrogen peroxide and lipid peroxidation. Metabolism of acetaminophen to NAPQI leading to depletion of hepatic GSH in the centrilobular cells may be a key factor. GSH is important in detoxification of peroxynitrite (15, 41), hydrogen peroxide, and lipid hydroperoxides (42, 43), and under conditions of GSH depletion these reactions may be inhibited. The zonal nature of the toxicity may be a result of the predominance of CYP2E1 in the centrilobular areas of the liver. CYP2E1 is a critical enzyme involved in metabolism of acetaminophen to NAPQI (7, 44). An alternative mechanism whereby nitric oxide may limit lipid peroxidation is reaction of nitric oxide with lipid radicals to inhibit initiation of lipid peroxidation; however, we could find no evidence for acetaminophen causing an increase in NO associated with the hepatic lipids. The source of superoxide and the mechanism of its presumed increase are not fully understood. Synthesis may be from damaged mitochondria (45), from activated Kupffer cells or other cells (3, 46, 47) from enzymes such as CYP2E1 (48) or from a combination of
© 2001 Elsevier Science (USA). All rights reserved.
164
HINSON ET AL.
FIG. 3. Effect of aminoguanidine on acetaminophen-induced nitration of hepatic tissue. Liver sections from the acetaminophen treated livers (Fig. 1) were processed and stained for nitrated tyrosine residues using anti-nitrotyrosine antiserum. The time course for acetaminophen (APAP)-treated livers is shown in A plus the saline-treated control. The time course for the APAP plus aminoguanidine (AG)-treated is shown in B plus aminoguanidine alone. The data are representative liver sections for each time and treatment group.
© 2001 Elsevier Science (USA). All rights reserved.
iNOS INHIBITION IN ACETAMINOPHEN HEPATOTOXICITY
165
ACKNOWLEDGMENTS This work was supported by a grant (GM58884) from the National Institute for General Medical Sciences, NIH, to J.A.H.
REFERENCES 1. Nakae, D., Yamamoto, K., Yoshiji, H., Kinugasa, T., Maruyama, H., Farber, J. L., and Konishi, Y. (1990). Liposome-encapsulated superoxide dismutase prevents liver necrosis induced by acetaminophen. Am. J. Pathol. 136, 787–795.
FIG. 4. Time course for effect of aminoguanidine on acetaminopheninduced lipid peroxidation. Liver homogenates of the acetaminophen, aminoguanidine, and acetaminophen plus aminoguanidine treated mice were analyzed for malondialdehyde levels. Mice (n ⫽ 5) were treated with acetaminophen (300 mg/kg, closed circles), aminoguanidine (15 mg/kg at 0 and at 2 h; closed squares), or acetaminophen plus aminoguanidine (open circles). The 0 time is the salinetreated control mice. *P ⬍ 0.05 compared to acetaminophen alone.
all of the above. Hepatic NO is produced by constitutively expressed endothelial NOS (eNOS) and by iNOS (49). Whereas eNOS is in endothelial cells, iNOS is in hepatocytes, stellate cells, and Kupffer cells (49). Regardless of the source, acetaminophen causes an increase in NO synthesis. Of the NOS inhibitors tested, only aminoguanidine reduced acetaminophen-stimulated increases in serum nitrite/nitrate concentration despite the ability of L-NMMA and L-NIL to inhibit basal levels in saline-treated animals. Serum nitrate/nitrite levels in saline-treated mice indicate that the status of NO synthesis and perhaps iNOS itself can vary among groups of mice (Table I) and we have observed this variability in newly purchased naı¨ve mice (data not shown). The cause of this variability is not clear. Nonetheless, we postulate that NO can play an important role in determining the mechanism of acetaminophen-induced hepatotoxicity. Acetaminophen appears to stimulate the production of both NO and superoxide. The reaction between NO and superoxide to generate peroxynitrite might serve as a superoxide detoxification reaction. However, acetaminophen metabolism also depletes GSH. This reduces detoxification of peroxynitrite and contributes to toxicity. The data suggest that the reason NOS inhibitors are not only ineffective in reducing acetaminophen hepatotoxicity but actually augment toxicity is because inhibition of NO synthesis shifts the toxicity pathway toward superoxide-derived oxidants.
2. Michael, S. L., Pumford, N. R., Mayeux, P. R., Niesman, M. R., and Hinson, J. A. (1999). Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species. Hepatology 30, 186 –195. 3. Knight, T. R., Kurtz, A., Bajt, M. L., Hinson, J. A., and Jaeschke, H. (2001). Vascular and hepatocellular peroxynitrite formation during acetaminophen toxicity: Role of mitochondrial oxidant stress. Toxicol. Sci. 62, 212–220. 4. Ferret, P. J., Hammoud, R., Tulliez, M., Tran, A., Trebeden, H., Jaffray, P., Malassagne, B., Calmus, Y., Weill, B., and Batteux, F. (2001). Detoxification of reactive oxygen species by a nonpeptidyl mimic of superoxide dismutase cures acetaminopheninduced acute liver failure in the mouse. Hepatology 33, 1173– 1180. 5. Cohen, S. D., Pumford, N. R., Khairallah, E. A., Boekelheide, K., Pohl, L. R., Amouzadeh, H. R., and Hinson, J. A. (1997). Selective protein covalent binding and target organ toxicity. Toxicol. Appl. Pharmacol. 143, 1–12. 6. 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-(cysteinS-yl)-acetaminophen protein adduct in acetaminophen hepatotoxicity. Am. J. Pathol. 138, 359 –371. 7. Hart, S. G., Cartun, R. W., Wyand, D. S., Khairallah, E. A., and Cohen, S. D. (1995). Immunohistochemical localization of acetaminophen in target tissues of the CD-1 mouse: Correspondence of covalent binding with toxicity. Fundam. Appl. Toxicol. 24, 260 –274. 8. Hinson, J. A., Pike, S. L., Pumford, N. R., and Mayeux, P. R. (1998). Nitrotyrosine-protein adducts in hepatic centrilobular areas following toxic doses of acetaminophen in mice. Chem. Res. Toxicol. 11, 604 – 607. 9. Beckman, J. S. (1996). Oxidative damage and tyrosine nitration from peroxynitrite. Chem. Res. Toxicol. 9, 836 – 844. 10. Pryor, W. A., and Squadrito, G. L. (1995). The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268, L699 –L722. 11. Reiter, C. D., Teng, R. J., and Beckman, J. S. (2000). Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J. Biol. Chem. 275, 32460 –32466. 12. Laskin, D. L., and Pendino, K. J. (1995). Macrophages and inflammatory mediators in tissue injury. Annu. Rev. Pharmacol. Toxicol. 35, 655– 677. 13. Goldin, R. D., Ratnayaka, I. D., Breach, C. S., Brown, I. N., and
© 2001 Elsevier Science (USA). All rights reserved.
166
HINSON ET AL.
Wickramasinghe, S. N. (1996). Role of macrophages in acetaminophen (paracetamol)-induced hepatotoxicity. J. Pathol. 179, 432– 435. 14. Blazka, M. E., Germolec, D. R., Simeonova, P., Bruccoleri, A., Pennypacker, K. R., and Luster, M. I. (1995). Acetaminopheninduced hepatotoxicity is associated with early changes in NF-kB and NF-IL6 DNA binding activity. J. Inflamm. 47, 138 – 150. 15. Sies, H., Sharov, V. S., Klotz, L. O., and Briviba, K. (1997). Glutathione peroxidase protects against peroxynitrite-mediated oxidations. A new function for selenoproteins as peroxynitrite reductase. J. Biol. Chem. 272, 27812–27817. 16. Hinson, J. A., Mays, J. B., and Cameron, A. M. (1983). Acetaminophen-induced hepatic glycogen depletion and hyperglycemia in mice. Biochem. Pharmacol. 32, 1979 –1988. 17. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211–217. 18. Hinson, J. A., Michael, S. L., Ault, S. G., and Pumford, N. R. (2000). Western blot analysis for nitrotyrosine protein adducts in livers of saline-treated and acetaminophen-treated mice [In Process Citation]. Toxicol. Sci. 53, 467– 473. 19. Buege, J. A., and Aust, S. D. (1978). Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. 20. Rees, D. D., Palmer, R. M., Schulz, R., Hodson, H. F., and Moncada, S. (1990). Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br. J. Pharmacol. 101, 746 –752. 21. Bryk, R., and Wolff, D. J. (1998). Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1iminoethyl)lysine. Biochemistry 37, 4844 – 4852. 22. Moore, W. M., Webber, R. K., Jerome, G. M., Tjoeng, F. S., Misko, T. P., and Currie, M. G. (1994). L-N6-(1-iminoethyl)lysine: A selective inhibitor of inducible nitric oxide synthase. J. Med. Chem. 37, 3886 –3888. 23. Faraci, W. S., Nagel, A. A., Verdries, K. A., Vincent, L. A., Xu, H., Nichols, L. E., Labasi, J. M., Salter, E. D., and Pettipher, E. R. (1996). 2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo. Br. J. Pharmacol. 119, 1101–1108. 24. Bowman, M. A., Simell, O. G., Peck, A. B., Cornelius, J., Luchetta, R., Look, Z., Maclaren, N. K., and Atkinson, M. A. (1996). Pharmacokinetics of aminoguanidine administration and effects on the diabetes frequency in nonobese diabetic mice. J. Pharmacol. Exp. Ther. 279, 790 –794. 25. Viera, L., Ye, Y. Z., Estevez, A. G., and Beckman, J. S. (1999). Immunohistochemical methods to detect nitrotyrosine. Methods Enzymol. 301, 373–381.
induced hepatotoxicity: Comparison with carbon tetrachloride. Toxicol. Lett. 66, 7–12. 28. Ou, J., Carlos, T. M., Watkins, S. C., Saavedra, J. E., Keefer, L. K., Kim, Y. M., Harbrecht, B. G., and Billiar, T. R. (1997). Differential effects of nonselective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis, ICAM-1 expression, and neutrophil accumulation during endotoxemia. Nitric Oxide 1, 404 – 416. 29. Uhlenius, N., Tikkanen, I., Tikkanen, T., Miettinen, A., Tornroth, T., and Fyhrquist, F. (1996). Chronic inhibition of nitric oxide synthase in Heymann nephritis. Nephron 74, 144 –149. 30. Walker, L. M., Walker, P. D., Imam, S. Z., Ali, S. F., and Mayeux, P. R. (2000). Evidence for peroxynitrite formation in renal ischemia-reperfusion injury: Studies with the inducible nitric oxide synthase inhibitor L-N(6)-(1-Iminoethyl)lysine. J. Pharmacol. Exp. Ther. 295, 417– 422. 31. Hock, C. E., Yin, K., Yue, G., and Wong, P. Y. (1997). Effects of inhibition of nitric oxide synthase by aminoguanidine in acute endotoxemia. Am. J. Physiol. 272, H843–H850. 32. Petros, A., Lamb, G., Leone, A., Moncada, S., Bennett, D., and Vallance, P. (1994). Effects of a nitric oxide synthase inhibitor in humans with septic shock. Cardiovasc. Res. 28, 34 –39. 33. Mitchell, J. R., Corcoran, G. B., Smith, C. V., Hughes, H., and Lauterburg, B. H. (1981). Alkylation and peroxidation injury from chemically reactive metabolites. Adv. Exp. Med. Biol. 136, 199 –223. 34. Michael, S. L., Mayeux, P. R., Bucci, T. J., Warbritton, A. R., Irwin, L. K., Pumford, N. R., and Hinson, J. A. (2001). Acetaminophen-induced hepatotoxicity in mice lacking inducible nitric oxide synthase activity. Nitric Oxide 5, 432– 441. 35. Huie, R. E., and Padmaja, S. (1993). The reaction of no with superoxide. Free Rad. Res. Commun. 18, 195–199. 36. Goldstein, S., and Czapski, G. (1995). The reaction of NO • with O2 •⫺ and HO2 •: A pulse radiolysis study. Free Rad. Biol. Med. 19, 505–510. 37. Thomson, L., Trujillo, M., Telleri, R., and Radi, R. (1995). Kinetics of cytochrome c2⫹ oxidation by peroxynitrite: Implications for superoxide measurements in nitric oxide-producing biological systems. Arch. Biochem. Biophys. 319, 491– 497. 38. Pederson, T. C., and Aust, S. D. (1975). The mechanism of liver microsomal lipid peroxidation. Biochim. Biophys. Acta 385, 232– 241. 39. Bus, J. S., Aust, S. D., and Gibson, J. E. (1976). Paraquat toxicity: Proposed mechanism of action involving lipid peroxidation. Environ. Health Perspect. 16, 139 –146. 40. Recknagel, R. O., Glende, E. A., Dolak, J. A., and Waller, R. L. (1989). Mechanisms of carbon tetrachloride toxicity. Pharmacol. Ther. 43, 139 –154. 41. Briviba, K., Kissner, R., Koppenol, W. H., and Sies, H. (1998). Kinetic study of the reaction of glutathione peroxidase with peroxynitrite. Chem. Res. Toxicol. 11, 1398 –1401.
26. Younes, M., Cornelius, S., and Siegers, C. P. (1986). Ferrous ion supported in vivo lipid peroxidation induced by paracetamol—Its relation to hepatotoxicity. Res. Commun. Chem. Pathol. Pharmacol. 51, 89 –99.
42. Brigelius-Flohe, R. (1999). Tissue-specific functions of individual glutathione peroxidases. Free Rad. Biol. Med. 27, 951–965.
27. Kamiyama, T., Sato, C., Liu, J., Tajiri, K., Miyakawa, H., and Marumo, F. (1993). Role of lipid peroxidation in acetaminophen-
43. Arthur, J. R. (2000). The glutathione peroxidases. Cell Mol. Life Sci. 57, 1825–1835.
© 2001 Elsevier Science (USA). All rights reserved.
iNOS INHIBITION IN ACETAMINOPHEN HEPATOTOXICITY
167
44. Anundi, I., Lahteenmaki, T., Rundgren, M., Moldeus, P., and Lindros, K. O. (1993). Zonation of acetaminophen metabolism and cytochrome P450 2E1-mediated toxicity studied in isolated periportal and perivenous hepatocytes. Biochem. Pharmacol. 45, 1251–1259. 45. Jaeschke, H., and Mitchell, J. R. (1989). Mitochondria and xanthine oxidase both generate reactive oxygen species in isolated perfused rat liver after hypoxic injury. Biochem. Biophys. Res. Commun. 160, 140 –147.
aminophen hepatotoxicity. Toxicol. Appl. Pharmacol. 133, 43– 52.
48. Koop, D. R. (1992). Oxidative and reductive metabolism by cytochrome P450 2E1. FASEB J. 6, 724 –730.
46. Blazka, M. E., Wilmer, J. L., Holladay, S. D., Wilson, R. E., and Luster, M. I. (1995). Role of proinflammatory cytokines in acet-
49. Muriel, P. (2000). Regulation of nitric oxide synthesis in the liver. J. Appl. Toxicol. 20, 189 –195.
47. Laskin, D. L., Gardner, C. R., Price, V. F., and Jollow, D. J. (1995). Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology 21, 1045– 1050.
© 2001 Elsevier Science (USA). All rights reserved.