Evaluation of three-dimensional cultured HepG2 cells in a nano culture plate system: an in vitro human model of acetaminophen hepatotoxicity.
Overdoses of acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) cause severe liver injury, yet there is no common or high throughput in vitro human APAP model. This study examined the characteristics and usefulness of HepG2 cells grown in a nano culture plate (NCP) system, a three-dimensional culture method, as an in vitro human model for APAP-induced hepatotoxicity. The NCP-cultured HepG2 cells showed higher expression of mRNA and protein levels of cytochrome P450 2E1, which metabolizes APAP to a toxic metabolite, APAP-cysteine adduct formation, and higher sensitivity against APAP-induced cell injury compared with conventionally cultured cells. We demonstrated that treatment of APAP in NCP-cultured HepG2 cells shows key mechanistic features of APAP-induced hepatotoxicity, such as decreases in intracellular glutathione and mitochondrial membrane potential, activation of JNK, and cellular injury; and pharmacological agents, such as Cyclosporine A (a mitochondrial permeability transition inhibitor) and SP600125 (a JNK inhibitor), prevented cell injury induced by APAP exposure. In addition, the antidote of APAP-induced hepatotoxicity, N-acetylcysteine, could attenuate cellular injury induced by APAP in NCP-cultured HepG2 cells. We suggest that cellular injury induced by APAP treatment using an NCP-HepG2 system is a useful human model to study mechanisms and screen drug candidates of APAP-induced hepatotoxicity.
Department of Clinical Chemistry and Informatics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Japan.
Kohei
K
Aritomi
Yoichi
Y
Ishitsuka
Yoshiro
Y
Tomishima
Daisuke
D
Shimizu
Nazuki
N
Abe
Tsuyoshi
T
Shuto
Mitsuru
M
Irikura
Hirofumi
H
Kai
Tetsumi
T
Irie
D000082:Acetaminophen / Q000037:antagonists & inhibitors / Q000633:toxicity*; D000111:Acetylcysteine / Q000494:pharmacology; D000873:Anthracenes / Q000494:pharmacology; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism / Q000502:physiology; D004353:Drug Evaluation, Preclinical; D005978:Glutathione / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D036103:Nanotechnology / Q000379:methods*; D018675:Toxicity Tests / Q000379:methods*
D000873:Anthracenes; C432165:pyrazolanthrone; D000082:Acetaminophen; D016572:Cyclosporine; D019392:Cytochrome P-450 CYP2E1; D048670:MAP Kinase Kinase 4; D005978:Glutathione; D000111:Acetylcysteine