Protein assay for heme oxygenase-1 (HO-1) induced by chemicals in HepG2 cells.
Levels of heme oxygenase-1 (HO-1), a stress response protein, were measured to examine oxidative stress induced by several chemicals in HepG2 cells with and without S9mix using an ELISA. CdCl(2), heme, and diclofenac sodium salt (diclofenac) were used as inducers of HO-1. Acetaminophen (AAP) and cyclophosphamide (CP) were used as oxidative stress inducers. Stannic mesoporphyrin (SnMP) was used as an inhibitor of HO activity. Cytotoxicity was determined, and HO-1 levels were measured in HepG2 cells exposed to chemicals other than CP with non-metabolic activation without S9mix, and to diclofenac, AAP and CP with metabolic activation with S9mix. HO-1 levels were increased by CdCl(2) (7.5 microM), heme (10, 100 microM), and stannic mesoporphyrin (SnMP) (10 microM), but were not changed by AAP, and were decreased by diclofenac. HO-1 levels were increased by diclofenac (300 microM), and CP (36 microM), but were unaffected by AAP because of low sensitivity in HepG2 cells. The induction of HO-1 expression was first observed in cultured HepG2 cells treated with CP under conditions involving metabolic activation. These results showed the measurement of HO-1 protein levels in this system is useful when assessing oxidative stress as a tool for detecting drug toxicity.
Miyamoto
Yohei
Y
Ohshida
Keiyu
K
Sasago
Kaori
K
D000082:Acetaminophen / Q000633:toxicity; D019256:Cadmium Chloride / Q000633:toxicity*; D002478:Cells, Cultured; D003520:Cyclophosphamide / Q000633:toxicity; D004008:Diclofenac / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006418:Heme / Q000633:toxicity*; D051547:Heme Oxygenase-1 / Q000032:analysis* / Q000096:biosynthesis*; D056945:Hep G2 Cells; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects*
D004008:Diclofenac; D000082:Acetaminophen; D006418:Heme; D003520:Cyclophosphamide; D051547:Heme Oxygenase-1; D019256:Cadmium Chloride