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

Authors: Jacob J. Briedé; Roger W.L. Godschalk; Marijn T.G. Emans; Theo M.C.M. de Kok; Ebienus van Agen; Jan M.S. van Maanen; Frederik-Jan van Schooten; Jos C.S. Kleinjans

Source: Free Radical Research, Volume 38, Number 9, September, 2004 , pp. 995-1002(8)

Publisher: Informa Healthcare

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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.

Keywords: Benzo[a]pyrene; Reactive oxygen species; ESR; DNA damage; 8-Oxo-dG; B[a]P, benzo(a)pyrene; CYP450, cytochrome P450; dG, 2 prime -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 prime -deoxyguanosine; PAH, polycyclic aromatic hydrocarbon; POBN, alpha -(1-oxy-4-pyridyl)-N-tert-butylnitrone