Immunohistochemical Localization of Acetaminophen in Target Tissues of the CD-1 Mouse: Correspondence of Covalent Binding with Toxicity

doi:10.1006/faat.1995.1029

Fundamental and Applied Toxicology

Volume 24, Issue 2, February 1995, Pages 260-274

Fundamental and Appl...

https://doi.org/10.1006/faat.1995.1029 Get rights and content

Abstract

Immunohistochemical Localization of Acetaminophen in Target Tissues of the CD-1 Mouse: Correspondence of Covalent Binding with Toxicity. Emeigh Hart, S. G., Cartun, R. W., Wyand, D. S., Khairallah, E. A., and Cohen, S. D. (1995). Fundam. Appl. Toxicol. 24, 260-274.

Administration of hepatotoxic doses of acetaminophen (APAP) to mice results in necrosis, not only of liver cells but of renal proximal tubules and bronchiolar and olfactory epithelium. In the liver, covalent binding is localized to the centrilobular hepatocytes which later undergo necrosis. This study was undertaken to compare the cellular distribution of bound APAP in all four major target tissues with that of cytochrome P4502E1 (a P450 isoenzyme commonly associated with APAP bioactivation), with emphasis on the cell types which later undergo necrosis. Tissues were collected from mice at selected times after APAP administration (600 mg/kg, po) and fixed by microwave irradiation for immunohistochemistry, or in formalin for histopathological study. Immunohistochemical localization of bound APAP was performed on 5 μm paraffin sections using an affinity-purified anti-APAP antibody. Similar tissues from naive mice were used for immunohistochemical localization of cytochrome P4502E1 (using a polyclonal sheep anti-P4502E1 antibody). Positive staining with both the anti-APAP and the anti-P4502E1 antibodies was similar in distribution, being present in the cell types which become damaged by APAP in all four target tissues. These results demonstrate that covalent binding and subsequent necrosis are localized in common with cytochrome P4502E1, suggesting that, as in the liver, toxicity in extrahepatic targets is also related to the ability of these tissues to activate APAP in situ.

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Acetaminophen (APAP) overdose causes liver injury and acute liver failure, as well as acute kidney injury, which is not prevented by the clinical antidote N-acetyl-L-cysteine (NAC). The absence of therapeutics targeting APAP-induced nephrotoxicity is due to gaps in understanding the mechanisms of renal injury. APAP metabolism through Cyp2E1 drives cell death in both the liver and kidney. We demonstrate that Cyp2E1 is localized to the proximal tubular cells in mouse and human kidneys. Virtually all the Cyp2E1 in kidney cells is in the endoplasmic reticulum (ER), not in mitochondria. By contrast, hepatic Cyp2E1 is in both the ER and mitochondria of hepatocytes. Consistent with this subcellular localization, a dose of 600 mg/kg APAP in fasted C57BL/6J mice induced the formation of APAP protein adducts predominantly in mitochondria of hepatocytes, but the ER of the proximal tubular cells of the kidney. We found that reactive metabolite formation triggered ER stress-mediated activation of caspase-12 and apoptotic cell death in the kidney. While co-treatment with 4-methylpyrazole (4MP; fomepizole) or the caspase inhibitor Ac-DEVD-CHO prevented APAP-induced cleavage of procaspase-12 and apoptosis in the kidney, treatment with NAC had no effect. These mechanisms are clinically relevant because 4MP but not NAC also significantly attenuated APAP-induced apoptotic cell death in primary human kidney cells. We conclude that reactive metabolite formation by Cyp2E1 in the ER results in sustained ER stress that causes activation of procaspase-12, triggering apoptosis of proximal tubular cells, and that 4MP but not NAC may be an effective antidote against APAP-induced kidney injury.
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Citation Excerpt :
Previous data showed that human proximal tubular cells express cytochrome P450 enzymes (Cummings et al., 2000). In addition, APAP undergoes a P450-mediated metabolism, which results in NAPQI formation, GSH depletion and protein binding in the kidney (Emeigh Hart et al., 1991; Hart et al., 1994, 1995). Immunohistochemistry studies have also demonstrated positive staining with both anti-APAP and anti-CYP2E1 antibodies in renal proximal tubules (Hart et al., 1995).
Acetaminophen (APAP) hepatotoxicity is the most common cause of acute liver failure in the United States, and while a significant percentage of APAP overdose patients develop kidney injury, molecular mechanisms involved in APAP-induced nephrotoxicity are relatively unknown. We have shown that 4-methylpyrazole (4MP, Fomepizole) protects against APAP-induced liver injury by inhibiting reactive metabolite formation through Cyp2E1, and analysis of data from APAP overdose patients indicated that kidney dysfunction strongly correlated with severe liver injury. Since Cyp2E1 is also expressed in the kidney, this study explored protection by 4MP against APAP-induced nephrotoxicity. Male C57BL/6 J mice were treated with either 300 or 600 mg/kg APAP with or without 4MP for 2, 6 or 24 h, followed by measurement of APAP metabolism and tissue injury. Interestingly, levels of APAP and its non-oxidative metabolites were significantly higher in kidneys when compared to the liver. APAP-protein adducts were present in both tissues within 2 h, but were absent in kidney mitochondria, unlike in the liver. While GSH depletion was seen in both tissues, activation of c-jun N-terminal kinase and its translocation to the mitochondria, which is a critical feature of APAP-induced liver injury, was not detected in the kidney. Treatment with 4MP attenuated APAP oxidative metabolite generation, GSH depletion as well as kidney injury indicating its potential use in protection against APAP-induced nephrotoxicity. In conclusion, since reactive metabolite formation seems to be common in both liver and kidney, 4MP mediated inhibition of Cyp2E1 protects against APAP-induced nephrotoxicity. However, downstream mechanisms of APAP-induced nephrotoxicity seem distinct from the liver.
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Regular ArticleImmunohistochemical Localization of Acetaminophen in Target Tissues of the CD-1 Mouse: Correspondence of Covalent Binding with Toxicity

Abstract

Regular Article
Immunohistochemical Localization of Acetaminophen in Target Tissues of the CD-1 Mouse: Correspondence of Covalent Binding with Toxicity