Acetaminophen-Induced Hepatotoxicity in Mice Lacking Inducible Nitric Oxide Synthase Activity

doi:10.1006/niox.2001.0385

Nitric Oxide

Volume 5, Issue 5, October 2001, Pages 432-441

https://doi.org/10.1006/niox.2001.0385 Get rights and content

Abstract

We recently reported that nitrotyrosine and acetaminophen (APAP)-cysteine protein adducts colocalize in the hepatic centrilobular cells following a toxic dose of APAP to mice. Whereas APAP-adducts are formed by reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine, nitrotyrosine residues are formed by reaction of tyrosine with peroxynitrite. Peroxynitrite is formed from nitric oxide (NO) and superoxide. This manuscript examines APAP (300 mg/kg) hepatotoxicity in mice lacking inducible nitric oxide synthase activity (NOS2 null or knockout mice; C57BL/6-Nos2^tm1Lau) and in the wildtype mice. In a time course the ALT levels in the exposed NOS2 null mice were approximately 50% of the wildtype mice; however, histological examination of liver sections indicated similar levels of centrilobular hepatic necrosis in both wild-type and NOS2 null mice. Serum nitrate plus nitrite levels (NO synthesis) were identical in saline-treated NOS2 null and wild-type mice (53 ± 2 μM). APAP increased NO synthesis in wild-type mice only. The increases paralleled the increases in ALT levels with peak levels of serum nitrate plus nitrite at 6 h (168 ± 27 μM). In wild-type mice hepatic tyrosine nitration was greatly increased relative to saline treated controls. Tyrosine nitration increased in NOS2 null mice also, but the increase was much less. APAP increased hepatic malonaldehyde levels (lipid peroxidation) in NOS2 null mice only. The results suggest the presence of multiple pathways to APAP-mediated hepatic necrosis, one via nitrotyrosine, as in the wild-type mice, and another that is not dependent upon inducible nitric oxide synthase activity, but which may involve increased superoxide.

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APAP overdose induced acute liver injury is the second most common cause that often requires liver transplantation worldwide, for which N-acetyl cysteine is the only synthetic drug clinically approved as an antidote. So, it was felt that there is a need for the novel therapeutic approach for the treatment of liver diseases with less adverse effects. This review provides detailed analysis of the different plant extracts; phytochemicals and herbal formulations for the amelioration of APAP-induced liver injury.
The data was collected using different online resources including PubMed, ScienceDirect, Google Scholar, Springer, and Web of Science using keywords given below.
Over the past decades various reports have revealed that plant-based approaches may be a better treatment choice for the APAP-induced hepatotoxicity in pre-clinical experimental conditions. Moreover, herbal compounds provide several advantages over the synthetic drugs with fewer side effects, easy availability and less cost for the treatment of life-threatening diseases.
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The hepatotoxicity induced by APAP is caused by the excessive production of N-acetyl-para-benzoquinone imine (NAPQI), which, when reacting with hepatic proteins proved to cause irreversible lesions. Associated with this process, an intense inflammatory process is also evidenced, characterized by the increased cell influx and production/release of inflammatory mediators. Trans anethole, an aromatic compounds has been showed anti-inflammatory efficacy by inhibit the cellular recruitment and synthesis/releases of many proinflammatory mediators such as prostaglandin (PGE₂), cytokines (TNF, IL-1) and nitrico oxide (NO). The aim of this study is to investigate the effect of trans anethole on some inflammatory parameters that are involved in hepatotoxicity induced by high doses of acetaminophen. Our results demonstrate that treatment with AN at doses 125 and 250 mg/kg once a day for seven days prevented the changes caused by the APAP overdose, showing less intensity in the histological changes (necrosis, size of hepatocyte area and inflammatory infiltration), and corroborating the findings of serum activities of transaminases and phosphatases and the activity of the enzyme myeloperoxidase. In addition, the treatment prevented the up-regulation of proinflammatory mediators such as NO, TNF, IL-1α, MIP-1α and MCP-1 and induced the up-regulation of anti-inflammatory cytokines (IL-4 and IL-10). Thus, our results demonstrate a possible protective effect of trans anethole on the hepatotoxicity induced by APAP.
Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes
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The hepatotoxicity of acetaminophen (APAP) occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO) and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1) inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP), a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein), reactive oxygen formation (superoxide), loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction.
The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes
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3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N′-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1 mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5–3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5–3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5–3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.
S-nitrosothiol signaling regulates liver development and improves outcome following toxic liver injury
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Citation Excerpt :
Studies have shown that nitric oxide synthase 2 (Nos2, inducible isoform) is induced by APAP, and that nitrosative stress, as measured by nitrotyrosine, is increased in the centrilobular region of the liver (Hinson et al., 1998; Knight et al., 2001; Saito et al., 2010). Furthermore, mice deficient in either Nos1 (neuronal isoform) or Nos2 are protected from APAP, suggesting that the Nos isoforms may exacerbate liver injury (Agarwal et al., 2012; Michael et al., 2001). However, there is also evidence that NO signaling can be beneficial in the setting of ischemia-reperfusion injury (Cottart et al., 1999; Elrod et al., 2008) or during liver regeneration following partial hepatectomy (Kurokawa et al., 2012; Mei and Thevananther, 2011; Rai et al., 1998).
Toxic liver injury is a leading cause of liver failure and death because of the organ’s inability to regenerate amidst massive cell death, and few therapeutic options exist. The mechanisms coordinating damage protection and repair are poorly understood. Here, we show that S-nitrosothiols regulate liver growth during development and after injury in vivo; in zebrafish, nitric-oxide (NO) enhanced liver formation independently of cGMP-mediated vasoactive effects. After acetaminophen (APAP) exposure, inhibition of the enzymatic regulator S-nitrosoglutathione reductase (GSNOR) minimized toxic liver damage, increased cell proliferation, and improved survival through sustained activation of the cytoprotective Nrf2 pathway. Preclinical studies of APAP injury in GSNOR-deficient mice confirmed conservation of hepatoprotective properties of S-nitrosothiol signaling across vertebrates; a GSNOR-specific inhibitor improved liver histology and acted with the approved therapy N-acetylcysteine to expand the therapeutic time window and improve outcome. These studies demonstrate that GSNOR inhibitors will be beneficial therapeutic candidates for treating liver injury.
Role of connexin 32 in acetaminophen toxicity in a knockout mice model
2014, Experimental and Toxicologic Pathology
Citation Excerpt :
Mice given saline alone (20 mL/kg) in the same way served as the corresponding controls (saline). The dose levels of APAP were selected to achieve mild to moderate hepatocellular injury, but not excessive toxicity (Michael et al., 2001; James et al., 2003b). One and 24 h after dosing, blood was collected via the inferior vena cava under ether anesthesia; all mice were euthanized by exsanguination.
Gap junctional intercellular communication (GJIC), by which glutathione (GSH) and inorganic ions are transmitted to neighboring cells, is recognized as being largely involved in toxic processes of chemicals. We examined acetaminophen (APAP)-induced hepatotoxicity clinicopathologically using male wild-type mice and mice lacking the gene for connexin32, a major gap junction protein in the liver [knockout (Cx32KO) mice]. When APAP was intraperitoneally administered at doses of 100, 200, or 300 mg/kg, hepatic centrilobular necrosis with elevated plasma aminotransferase activities was observed in wild-type mice receiving 300 mg/kg, and in Cx32KO mice given 100 mg/kg or more. At 200 mg/kg or more, hepatic GSH and GSSG contents decreased significantly and the effect was more severe in wild-type mice than in Cx32KO mice. On the other hand, markedly decreased GSH staining was observed in the hepatic centrilobular zones of Cx32KO mice compared to that of wild-type mice. These results demonstrate that Cx32KO mice are more susceptible to APAP hepatotoxicity than wild-type mice, and indicate that the distribution of GSH of the centrilobular zones in the hepatic lobules, rather than GSH and GSSG contents in the liver, is important in APAP hepatotoxicity. In conclusion, Cx32 protects against APAP-induced hepatic centrilobular necrosis in mice, which may be through the GSH transmission to neighboring hepatocytes by GJIC.

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Regular ArticleAcetaminophen-Induced Hepatotoxicity in Mice Lacking Inducible Nitric Oxide Synthase Activity

Abstract

Toxicol. Appl. Pharmacol.

Fund. Appl. Toxicol.

Hepatology

Methods Enzymol.

Biochem. Pharmacol.

Toxicol. Lett.

Chem. Biol. Interact.

J. Biol. Chem.

Toxicol. Appl. Pharmacol

Pharmacol. Ther.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Immunohistochemical localization and quantification of the 3-(cystein-S-yl)-acetaminophen protein adduct in acetaminophen hepatotoxicity

Am. J. Pathol.

Kupffer cells: Their activation and role in animal models of liver injury and human liver disease

Semin. Liver Dis.

Macrophages and inflammatory mediators in tissue injury

Annu. Rev. Pharmacol. Toxicol.

Role of macrophages in acetaminophen (paracetamol)-induced hepatotoxicity

J. Pathol.

Acetaminophen-induced hepatotoxicity is associated with early changes in NF-kB and NF-IL6 DNA binding activity

J. Inflamm.

Regular Article
Acetaminophen-Induced Hepatotoxicity in Mice Lacking Inducible Nitric Oxide Synthase Activity