Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls

Acetaminophen(APAP)is a widely used analgesic and antipyretic drug,which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses.The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology.As a result,this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions.However,the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic.However,many studies using this model are flawed,which jeopardizes the scientific and clinical relevance.The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained.The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism,mitochondrial dysfunction,necrotic cell death,autophagy and the sterile inflammatory response.In addition,the most frequently made mistakes when using this model are discussed.Thus,considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

Protective effects of 5-methoxypsoralen against acetaminophen-induced hepatotoxicity in mice

AIM:To investigate the hepatic protective effects of 5-methoxypsoralen(5-MOP) and to learn if 5-MOP causes hepatotoxicity at protective doses.METHODS:C57BL/6J mice were administrated orally with 5-MOP at doses of 12.5,25 and 50 mg/kg body weight respectively every morning for 4 d before given acetaminophen(APAP) subcutaneously at a dose of 500 mg/kg.The 5-MOP alone group was treated with 5-MOP orally at a dose of 50 mg/kg body weight for 4 d without APAP.Twenty-four hours after APAP administration,blood samples of mice were analyzed for serum enzyme alanine transaminase(ALT),aspartate transaminase(AST),lactate dehydrogenase(LDH) levels,and malondialdehyde(MDA),reduced glutathione(GSH) and oxidized glutathione(GSSG) of liver tissues were measured and histopathologic changes of the liver were observed.RESULTS:Compared with the vehicle control group,the serum levels(IU/L) of ALT,AST and LDH were all increased significantly in APAP group(8355 ± 3940 vs 30 ± 21,P < 0.05;6482 ± 4018 vs 146 ± 58,P <0.05;24627 ± 10975 vs 1504 ± 410,P < 0.05).Compared with APAP group,the serum ALT levels(IU/L)(1674 ± 1810 vs 8355 ± 3940,P < 0.05;54 ± 39 vs 8355 ± 3940,P < 0.05;19 ± 9 vs 8355 ± 3940,P < 0.05),AST levels(IU/L)(729 ± 685 vs 6482 ± 4108,P < 0.05;187 ± 149 vs 6482 ± 4108,P < 0.05;141 ± 12 vs 6482 ± 4108,P < 0.05) and LDH levels(IU/L)(7220 ± 6317 vs 24 627 ± 10 975,P < 0.05;1618 ± 719 vs 24 627 ± 10 975,P < 0.05;1394 ± 469 vs 24 627 ± 10 975,P < 0.05) were all decreased drastically in the three-dosage 5-MOP pretreatment groups.Pretreatment of 5-MOP could attenuate histopathologic changes induced by APAP,including hepatocellular necrosis and infiltration of inflammatory cells,and the effect was dose-dependent.MDA levels(nmol/mg) were decreased by 5-MOP in a dose-dependent manner(0.98 ± 0.45 vs 2.15 ± 1.07,P > 0.05;0.59 ± 0.07 vs 2.15 ± 1.07,P < 0.05;0.47 ± 0.06 vs 2.15 ± 1.07,P < 0.05).The pretreatment of 5-MOP could also increase the GSH/GSSG ratio(3.834 ± 0.340 vs 3.306 ± 0.282,P > 0.05;5.330 ± 0.421 vs 3.306 ± 0.282,P < 0.05;6.180 ± 0.212 vs 3.306 ± 0.282,P < 0.05).In the group treated with 5-MOP but without APAP,the serum enzyme levels,the liver histopathologic manifestation,and the values of MDA and GSH/GSSG ratio were all normal.CONCLUSION:5-MOP can effectively protect C57BL/6J mice from APAP-induced hepatotoxicity and possesses an antioxidative activity,and does not cause liver injury at the protective doses.

Mitochondrial damage and biogenesis in acetaminophen-induced liver injury

Liver injury and acute liver failure caused by acetaminophen(APAP)overdose is the clinically most important drug toxicity in Western countries.Mechanistic investigations have revealed a central role of mitochondria in the pathophysiology.Excess formation of the reactive metabolite N-acetyl-p-benzoquinone imine(NAPQI)after an overdose leads to hepatic glutathione depletion,mitochondrial protein adducts formation and an initial oxidant stress,which triggers the activation of mitogen activated protein(MAP)kinase cascade ultimately leading to c-jun N-terminal kinase(JNK)phosphorylation.Phospho-JNK translocates to the mitochondria and amplifies the oxidative and nitrosative stress eventually causing the mitochondrial membrane permeability transition pore opening and cessation of adenosine triphosphate(ATP)synthesis.In addition,mitochondrial matrix swelling ruptures the outer membrane and releases endonucleases,which cause nuclear deoxyribonucleic acid(DNA)fragmentation.Together,the nuclear DNA damage and the extensive mitochondrial dysfunction result in necrotic cell death.However,the procell death signaling events are counteracted by adaptive responses such as autophagy and mitochondrial biogenesis.The improved mechanistic insight into the pathophysiology leads to better understanding of the mechanisms of action of the existing antidote N-acetylcysteine and justifies the clinical testing of novel therapeutics such as 4-methylpyrazole and calmangafodipir.