Abstract
Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [14C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [14C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment.
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Abbreviations
- ABT:
-
1-Aminobenzotriazole
- HRN™:
-
Hepatic cytochrome P450 reductase null mice
- WT:
-
Wild-type C57BL6 J mice
- P450:
-
Cytochrome P450
- NADPH:
-
Reduced nicotinamide adenine dinucleotide phosphate
- UDPGA:
-
Uridine 5′-diphospho-glucuronic acid
- ALT:
-
Alanine aminotransferase
- AST:
-
Aspartate aminotransferase
- GLDH:
-
Glutamate dehydrogenase
- ALP:
-
Alkaline phosphatase
- NAFLD:
-
Non-alcoholic fatty liver disease
References
Alkhouri N, Dixon LJ, Feldstein AE (2009) Lipotoxicity in nonalcoholic fatty liver disease: not all lipids are created equal. Expert Rev Gastroenterol Hepatol 3:445–451
Boelsterli UA (2003) Diclofenac-induced liver injury: a paradigm of idiosyncratic drug toxicity. Toxicol Appl Pharmacol 192:307–322
Boelsterli UA, Ramirez-Alcantara V (2011) NSAID acyl glucuronides and enteropathy. Curr Drug Metab 12:245–252
Boelsterli UA, Redinbo MR, Saitta KS (2013) Multiple NSAID-induced hits injure the small intestine: underlying mechanisms and novel strategies. Toxicol Sci 131:654–667
Claria J, Titos E (2004) Kupffer cell. Gastroenterol Hepatol 27:264–273
Day SH, Mao A, White R, Schulz-Utermoehl T, Miller R, Beconi MG (2005) A semi-automated method for measuring the potential for protein covalent binding in drug discovery. J Pharmacol Toxicol Methods 52:278–285
Dunk AA, Walt RP, Jenkins WJ, Sherlock SS (1982) Diclofenac hepatitis. Br Med J (Clin Res Ed) 284:1605–1606
Elcombe CR, Odum J, Foster JR, Stone S, Hasmall S, Soames AR, Kimber I, Ashby J (2002) Prediction of rodent nongenotoxic carcinogenesis: evaluation of biochemical and tissue changes in rodents following exposure to nine nongenotoxic NTP carcinogens. Environ Health Perspect 110:363–375
Ferre N, Claria J (2006) New insights into the regulation of liver inflammation and oxidative stress. Mini Rev Med Chem 6:1321–1330
Gan TJ (2010) Diclofenac: an update on its mechanism of action and safety profile. Curr Med Res Opin 26:1715–1731
Gutthann SP, Garcia Rodriguez LA, Raiford DS (1997) Individual nonsteroidal antiinflammatory drugs and other risk factors for upper gastrointestinal bleeding and perforation. Epidemiology 8:18–24
Henderson CJ, Otto DM, Carrie D, Magnuson MA, McLaren AW, Rosewell I, Wolf CR (2003) Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase. J Biol Chem 278:13480–13486
Hickey EJ, Raje RR, Reid VE, Gross SM, Ray SD (2001) Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death. Free Radic Biol Med 31:139–152
Kalgutkar AS, Soglia JR (2005) Minimising the potential for metabolic activation in drug discovery. Expert Opin Drug Metab Toxicol 1(1):91–142 Review
Lorbek G, Lewinska M, Rozman D (2012) Cytochrome P450s in the synthesis of cholesterol and bile acids-from mouse models to human diseases. FEBS J 279:1516–1533
Miyamoto G, Zahid N, Uetrecht JP (1997) Oxidation of diclofenac to reactive intermediates by neutrophils, myeloperoxidase, and hypochlorous acid. Chem Res Toxicol 10(4):414–419
Mizuno H, Sakamoto C, Matsuda K, Wada K, Uchida T, Noguchi H, Akamatsu T, Kasuga M (1997) Induction of cyclooxygenase 2 in gastric mucosal lesions and its inhibition by the specific antagonist delays healing in mice. Gastroenterology 112:387–397
Mutch DM, Klocke B, Morrison P, Murray CA, Henderson CJ, Seifert M, Williamson G (2007) The disruption of hepatic cytochrome p450 reductase alters mouse lipid metabolism. J Proteome Res 6:3976–3984
Park B-J, Lee Y-J, Lee H-L (2014) Chronic liver inflammation: clinical implications beyond alcoholic liver disease. World J Gastroenterol 20:2168–2175
Perez-Estrada LA, Malato S, Gernjak W, Aguera A, Thurman EM, Ferrer I, Fernandez-Alba AR (2005) Photo-fenton degradation of diclofenac: identification of main intermediates and degradation pathway. Environ Sci Technol 39(21):8300–8306
Pickup K, Gavin A, Jones HB, Karlsson E, Page C, Ratcliffe K, Sarda S, Schulz-Utermoehl T, Wilson I (2012) The hepatic reductase null mouse as a model for exploring hepatic conjugation of xenobiotics: application to the metabolism of diclofenac. Xenobiotica 42:195–205
Planaguma A, Claria J, Miquel R, Lopez-Parra M, Titos E, Masferrer JL, Rodes V, Arroyo J (2005) The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPARgamma activation. Faseb J 19:1120–1122
Purcell P, Henry D, Melville G (1991) Diclofenac hepatitis. Gut 32:1381–1385
Ramirez-Alcantara V, LoGuidice A, Boelsterli UA (2009) Protection from diclofenac-induced small intestinal injury by the JNK inhibitor SP00125 in a mouse model of NSAID-associated enterophathy. Am J Physiol Gastrointest Liver Physiol 297:G990–G998
Salama A, Kroll H, Wittmann G, Mueller-Eckhardt C (1996) Diclofenac-induced immune haemolytic anaemia: simultaneous occurrence of red blood cell autoantibodies and drug-dependent antibodies. Br J Haematol 95:640–644
Sarda S, Page C, Pickup K, Schulz-Utermoehl T, Wilson I (2012) Diclofenac metabolism in the mouse: novel in vivo metabolites identified by high performance liquid chromatography coupled to linear ion trap mass spectrometry. Xenobiotica 42:179–194
Seitz S, Boelsterli UA (1998) Diclofenac acyl glucuronide, a major biliary metabolite, is directly involved in small intestinal injury in rats. Gastroenterology 115:1476–1482
Shipkova M, Armstrong VW, Oellerich M, Wieland E (2003) Acyl glucuronide drug metabolites: toxicological and analytical implications. Ther Drug Monit 25(1):1–16
Stiborova M, Arlt VM, Henderson CJ, Wolf CR, Kotrbova V, Moserova M, Hudecek J, Phillips DH, Frei E (2008) Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: studies with the hepatic NADPH:cytochrome P450 reductase null mouse. Toxicol Appl Pharmacol 226:318–327
Usui T, Mise M, Hashizume T, Yabuki M, Komuro S (2009) Evaluation of the potential for drug-induced liver injury based on in vitro covalent binding to human liver proteins. Drug Metab Dispos 37:2383–2392
Wang XJ, Chamberlain M, Vassieva O, Henderson CJ, Wolf CR (2005) Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice. Biochem J 388:857–867
Wheeler MD (2003) Endotoxin and Kupffer cell activation in alcoholic liver disease. Alcohol Res Health 27:300–306
Zhu Y, Zhang QY (2012) Role of intestinal cytochrome P450 enzymes in diclofenac-induced toxicity in the small intestine. J Pharmacol Exp Ther 343:362–370
Acknowledgments
We thank Jonathan Greenall, Elizabeth Johnson and Peter Cotton for their excellent technical assistance, James Noakes for directing the in vivo study and Marie South for statistical advice.
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Akingbasote, J.A., Foster, A.J., Wilson, I. et al. Hepatic effects of repeated oral administration of diclofenac to hepatic cytochrome P450 reductase null (HRN™) and wild-type mice. Arch Toxicol 90, 853–862 (2016). https://doi.org/10.1007/s00204-015-1505-x
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DOI: https://doi.org/10.1007/s00204-015-1505-x