In Vitro and In Vivo Studies on Oxygen Free Radical and DNA Adduct Formation in Rat Lung and Liver during Benzo[a]pyrene Metabolism JACOB J. BRIEDE ´ , ROGERW.L. GODSCHALK, MARIJN T.G. EMANS, THEO M.C.M. DE KOK, EBIENUS VAN AGEN, JAN M.S. VAN MAANEN*, FREDERIK-JAN VAN SCHOOTEN and JOS C.S. KLEINJANS † Department of Health Risk Analysis and Toxicology, Maastricht University, PO Box 616, 6200 MD Maastricht, the Netherlands Accepted by Dr A. Collins (Received 24 September 2003; In revised form 7 May 2004) 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 gene- ration 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 indo- methacin. 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 Abbreviations: B[a]P, benzo(a)pyrene; CYP450, cytochrome P450; dG, 2 0 -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 0 -deoxyguanosine; PAH, polycyclic aromatic hydrocarbon; POBN, a-(1-oxy-4-pyridyl)-N-tert-butyl- nitrone INTRODUCTION Benzo[a]pyrene (B[a]P) is a well studied polycyclic aromatic hydrocarbon (PAH), which is known to be metabolized by cytochrome P450 (CYP450) to certain reactive diol-epoxides that can interact with DNA. [1] However, other metabolic routes have been described, which might be of biological relevance. For instance, B[a]P may be metabolized by the peroxidative activity of CYP450 or other enzymes (e.g. prostaglandin H synthase) via a one-electron oxidation pathway to a B[a]P-radical cation [2] that may covalently bind to DNA. [3] Additionally, B[a]P radical-cations are thought to be precursors for the labile phenol, 6-OH-B[a]P. [4] Auto-oxidation of this derivative may ultimately result in the formation of B[a]P quinones. B[a]P quinones are almost invariably found after metabolism of B[a]P, sometimes account- ing for more than 50% of the total metabolic yield. [5] The B[a]P quinones formed are 6,12-, 1,6- and 3,6- B[a]P dione, which can undergo redox-cycling to their corresponding B[a]P diols and so may produce superoxide [6] (ROS) (Fig. 1) that can be converted into the more reactive hydroxyl radicals via the Haber–Weiss reaction. In particular, hydroxyl free ISSN 1071-5762 print/ISSN 1029-2470 online q 2004 Taylor & Francis Ltd DOI: 10.1080/10715760400000976 *Jan van Maanen died unexpectedly on November 5th, 2002. This article is to honor his memory. † Corresponding author. Tel.: þ31-433881096/97. Fax: þ31-433884146. E-mail: j.kleinjans@grat.unimaas.nl Free Radical Research, Volume 38 Number 9 (September 2004), pp. 995–1002 Free Radic Res Downloaded from informahealthcare.com by University of Maastricht on 10/15/12 For personal use only.