Identification and Quantification of in Vivo Metabolites of 9,10- Phenanthrenequinone in Human Urine Associated with Producing Reactive Oxygen Species Miki Asahi, † Mio Kawai, † Takashi Toyama, ‡ Yoshito Kumagai, ‡ Thanyarat Chuesaard, † Ning Tang, † Takayuki Kameda, § Kazuichi Hayakawa, † and Akira Toriba* ,† † Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, 920-1192, Japan ‡ Graduate School of Comprehensive Human Sciences, University of Tukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan § Graduate School of Energy Science, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan * S Supporting Information ABSTRACT: Polycyclic aromatic hydrocarbon quinones (PAHQs) are components in airborne particulate matter (PM) and generate reactive oxygen species (ROS) in a redox cycling process. 9,10-Phenanthrenequinone (9,10-PQ) is a PAHQ found in diesel exhaust particulates and PM. When inhaled, it produces much more ROS than other PAHQs. We hypothesized that urinary metabolites of 9,10-PQ could serve as biomarkers of PAHQ exposure. Here, we describe methods for pretreating urine samples and analyzing 9,10-PQ metabolites by liquid chromatography with tandem mass spectrometry (LC-MS/MS). In urine from rats intra- peritoneally injected with 9,10-PQ, the monoglucuronide of 9,10-dihydroxyphenanthrene (9,10-PQHG) was found to be a major metabolite of 9,10-PQ. 9,10-PQHG was also identified in the urine of a nonoccupationally exposed human by its retention time and MS/MS spectra. Furthermore, the urine contained hardly any free (unmetabolized) 9,10-PQ, but treating it with hydrolytic enzymes released 9,10-PQ from conjugated metabolites such as 9,10-PQHG. The concentrations of 9,10-PQHG in urine samples from nonoccupationally exposed subjects who lived in a suburban area were 2.04-19.08 nmol/mol creatinine. This study is the first to demonstrate the presence of 9,10-PQHG in human urine. Determination of urinary 9,10-PQHG should be useful for determining 9,10-PQ exposure. ■ INTRODUCTION Airborne particulate matter (PM) released from combustion sources such as diesel exhaust is an important pollutant in urban atmospheres. PM is suspected to be a causative factor in several diseases, such as allergies, 1 respiratory diseases, 2,3 and cardiovascular diseases, 4 all of which are considered to be oxidative-stress-related disorders. 5-7 Oxidative stress in an organism arises from excessive generation of reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, and hydroxyl radical or from depletion of antioxidants. 8 The production of ROS can be induced by both endogenous and exogenous factors. 9 Endogenous factors include physiological processes, such as oxidative phosphorylation, P-450 metabo- lism, peroxisomes, and inflammatory cell activation. Exogenous factors include environmental sources such as smoking, diet, and pollution. 10 ROS are mostly scavenged by antioxidative enzymes such as superoxide dismutase (SOD) or antioxidative compounds such as glutathione. However, excessive production of ROS may cause oxidative damage to nucleic acids, proteins, and lipids. 7,8 Oxidative damage is one of the causes of aging, respiratory disease, and cancer. 9,11 Polycyclic aromatic hydrocarbon quinones (PAHQs) have been identified as a PM component and generate ROS in a redox-cycling process. One PAHQ, 9,10-phenanthrenequinone (9,10-PQ), has been identified in diesel exhaust particulates (DEP) 12,13 and PM. 12,14-17 In human A549 cells, 9,10-PQ, one of the ortho-PAHQs, causes massive overproduction of ROS and cytotoxicity compared to other para-PAHQs such as 1,4- PQ. 18 9,10-PQ appears to be a good chemical marker for ROS production associated with PM, and exposure to PAHQs such as 9,10-PQ may cause adverse health effects by the generation of ROS, 19 although their levels in human are unknown. 9,10-PQ is formed by the combustion of fossil fuels, 13,16 by photooxidation of phenanthrene (Phe), 15 and by reactions of Phe with radicals in the atmosphere. 20 9,10-PQ is a major PAHQ in DEP or PM. The concentrations of 9,10-PQ in DEP Received: September 17, 2013 Published: December 10, 2013 Article pubs.acs.org/crt © 2013 American Chemical Society 76 dx.doi.org/10.1021/tx400338t | Chem. Res. Toxicol. 2014, 27, 76-85