2,3,7,8-Tetrachlorodibenzo-p-dioxin induced cytochrome P450s alter the formation of reactive oxygen species in liver cells.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was classified by the International Agency for Research on Cancer as a carcinogen in humans. It acts through an aryl hydrocarbon receptor-mediated mechanism, inducing the transcription of numerous genes, including various cytochrome P450s (CYPs - CYP1A1, 1A2, 1B1). Induction of CYPs may lead to genotoxicity by generating reactive oxygen species (ROS) which can damage DNA directly and/or via the generation of reactive metabolites. We determined ROS formation with the 2',7'-dihydrodichlorofluorescein diacetate fluorescence assay after incubation of HepG2 hepatoma cells or primary rat hepatocytes with TCDD. The amount of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA was measured using HPLC-MS/MS, the amount of CYP1A1 protein by Western blotting. The catalytic activity of CYP1A enzymes was determined as 7-ethoxyresorufin-O-deethylase (EROD) activity. Incubation of cells with TCDD for 48 h caused increased levels of ROS in primary rat hepatocytes as well as increased levels of 8-oxo-dG in DNA compared to untreated cells. In the HepG2 cell line no significant effects were observed for both ROS formation and 8-oxo-dG levels. Both effects were in good agreement with the extent of induction of CYP1A1 protein and EROD activity, suggesting that CYP1 induction is a major source of ROS formation in TCDD-treated hepatocytes.
Knerr
Stefanie
S
Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Germany.
Schaefer
Johanna
J
Both
Saskia
S
Mally
Angela
A
Dekant
Wolfgang
W
Schrenk
Dieter
D
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis; D004790:Enzyme Induction / Q000187:drug effects; D005452:Fluoresceins / Q000032:analysis; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008113:Liver Neoplasms; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*
C110400:2',7'-dichlorodihydrofluorescein diacetate; D005452:Fluoresceins; D000072317:Polychlorinated Dibenzodioxins; D017382:Reactive Oxygen Species; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1; D003849:Deoxyguanosine