In Vitro and In Vivo Studies on Oxygen Free Radical and DNA Adduct Formation in Rat Lung and Liver during Benzo[a]pyrene Metabolism

Abstract

Reactive oxygen species (ROS), possibly produced during the metabolic conversion of benzo(a)pyrene (B[a]P), could be involved in B[a]P-induced genotoxicity and, eventually, carcinogenicity. Therefore, ROS formation by rat lung and liver microsomes was studied in vitro by electron spin resonance (ESR/EPR) spectrometry. B[a]P-mediated generation of ROS was detected in incubations with rat lung, but not with liver microsomes. Inhibition of cytochrome P450 (CYP450) by the non isoform-specific inhibitor SKF-525A resulted in a complete inhibition of B[a]P-dependent ROS formation, whereas ROS formation was not affected by inhibition of prostaglandin H synthase by indomethacin. Subsequently, bulky DNA adduct formation and 8-oxo-dG levels after a single oral dose of B[a]P were examined in vivo in rat lung and liver, in combination with urinary excretion of 8-oxodG. B[a]P exposure resulted in increased urinary 8-oxo-dG levels. On the contrary, 8-oxo-dG levels decreased in liver and lung after B[a]P exposure. Bulky DNA adducts reached higher levels and were more persistent in rat lung than in liver. These results indicate that ROS are generated during the CYP450 dependent metabolism of B[a]P, particularly in the rat lung, but this does not necessarily result in increased levels of oxidative DNA damage in vivo, possibly by induction of DNA repair mechanisms.

• Benzo[a]pyrene
• Reactive oxygen species
• ESR
• DNA damage
• 8-Oxo-dG
• B[a]P, benzo(a)pyrene
• CYP450, cytochrome P450
• dG, 2′-deoxyguanosine
• DMPO, 5,5-dimethyl-1-pyrroline-N-oxide
• DMSO, dimethylsulfoxide
• ESR, electron spin resonance
• HPLC-ECD, high performance liquid chromatography with electrochemical detection
• ROS, reactive oxygen species
• 8-oxodG, 7-hydro-8-oxo-2′-deoxyguanosine
• PAH, polycyclic aromatic hydrocarbon
• POBN, α-(1-oxy-4-pyridyl)-N-tert-butylnitrone