Research Evidence That Humans Metabolize Benzene via Two Pathways Stephen M. Rappaport,1 Sungkyoon Kim,2,* Qing Lan,3 Roel Vermeulen,4 Suramya Waidyanatha,2 Luoping Zhang,1 Guilan Li,5 Songnian Yin,5 Richard B. Hayes,3 Nathaniel Rothman,3 and Martyn T. Smith1 1School of Public Health, University of California at Berkeley, Berkeley, California, USA; 2School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA; 3National Cancer Institute, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, Maryland, USA; 4Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands; 5Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China Background : Recent evidence has shown that humans metabolize benzene more efficiently at environmental air concentrations than at concentrations > 1 ppm. This led us to speculate that an unidentified metabolic pathway was mainly responsible for benzene metabolism at ambient levels. o Bjectiv : We statistically tested whether human metabolism of benzene is better fitted by a kinetic model having two pathways rather than one. words Methods : We fit Michaelis-Menten-like models to levels of urinary benzene metabolites and the corresponding air concentrations for 263 nonsmoking Chinese females. Estimated benzene con- centrations ranged from less than 0.001 ppm to 299 ppm, with 10th and 90th percentile values of 0.002 ppm and 8.97 ppm, respectively. r sulte : Using values of Akaike’s information criterion obtained under the two models, we found strong statistical evidence favoring two metabolic pathways, with respective affinities (benzene air concentrations analogous to Km values) of 301 ppm for the low-affinity pathway (probably domi- nated by cytochrome P450 enzyme 2E1) and 0.594 ppm for the high-affinity pathway (unknown). The exposure-specific metabolite level predicted by our two-pathway model at nonsaturating con- centrations was 184 µM/ppm of benzene, a value close to an independent estimate of 194 µM/ppm for a typical nonsmoking Chinese female. Our results indicate that a nonsmoking woman would metabolize about three times more benzene from the ambient environment under the two-pathway model (184 µM/ppm) than under the one-pathway model (68.6 µM/ppm). In fact, 73% of the ambient benzene dose would be metabolized via the unidentified high-affinity pathway. c onclusi : Because regulatory risk assessments have assumed nonsaturating metabolism of ben- zene in persons exposed to air concentrations well above 10 ppm, our findings suggest that the true leukemia risks could be substantially greater than currently thought at ambient levels of exposure— about 3-fold higher among nonsmoking females in the general population. k ey : benzene, biomonitoring, cancer risk, cytochrome P450, metabolism. Environ Health Perspect 117:946–952 (2009). doi:10.1289/ehp.0800510 available via http://dx.doi.org/ [Online 19 February 2009] Benzene is an important industrial chem- ical that is also present in gasoline, engine exhausts, wood smoke, and tobacco smoke [International Agency for Research on Cancer (IARC) 1989]. In fact, benzene is truly ubiq- uitous in the environment, with air concen- trations ranging from parts per billion in rural and urban settings to parts per million in some workplaces (IARC 1989; Wallace 1996). This is worrisome because benzene causes leukemia and probably other lympho- hematopoietic cancers in humans (Hayes et al. 1997; Infante et al. 1977; Rinsky et al. 1987), and there is evidence that benzene is hema- totoxic at levels < 1 ppm (3.2 mg/m3) (Lan et al. 2004), the current permissible expo- sure limit in the United States (Occupational Safety and Health Administration 1987). Benzene also produces malignant tumors at multiple sites in rodents (Huff et al. 1989; Maltoni et al. 1989). The toxicology of few chemicals has been pursued as vigorously as that of benzene. A PubMed search of the keywords “benzene” and “toxicity” returned more than 2,700 publications. Reports of benzene’s propen- sity to damage human blood-forming tissues 946 emerged as early as 1897 (Santesson 1897), and the first evidence of its leukemoge- nicity was published in 1928 (Delore and Borgomano 1928). Although benzene must be metabolized to exert toxicity, the metabolism is complex, and particular roles played by the various metabolites have not been fully elucidated (Ross 1996; Smith 1996; Snyder 2002). As shown in Figure 1, benzene is oxidized by cytochrome P450 (CYP) enzymes to benzene oxide, which exists in equilibrium with its tau- tomer oxepin. Spontaneous rearrangement of benzene oxide produces phenol that is either excreted or oxidized by CYP enzymes to hyd- roquinone, which is excreted or oxidized to 1,4-benzoquinone. Other major metabolites include catechol (which can be oxidized to 1,2-benzoquinone), after hydrolysis of ben- zene oxide and aromatization of benzene dihydrodiol, and E,E-muconic acid (hereafter “muconic acid”), after oxidation of oxepin and ring opening. Reaction between benzene oxide and glutathione, possibly mediated by glutathione-S-transferases, can produce the minor metabolite S-phenylmercapturic acid (SPMA). For humans exposed to benzene at air concentrations between 0.1 and 10 ppm, phenol represents 70–85% of urinary ben- zene metabolites, whereas hydroquinone, muconic acid, and catechol each represent 5–10%, and SPMA represents less than 1% (Kim et al. 2006b). Benzene oxide, the benzoquinones, muconaldehydes, and benzene diol epoxides (formed from CYP oxidation of benzene dihy- drodiol) are electrophiles that readily react with peptides and proteins (Bechtold et al. 1992; Henderson et al. 2005; McDonald et al. 1993; Waidyanatha et al. 2005) and can thereby interfere with cellular function (Smith 1996). Reactions between 1,4-benzoquinone and critical nucleophilic loci in topoisomerase II, an important enzyme involved in DNA replication and maintenance, are men- tioned as likely key events in the cascade of effects related to benzene’s ability to damage chromosomes (Chen and Eastmond 1995; Lindsey et al. 2004). Address correspondence to S.M. Rappaport, School of Public Health, University of California, Berkeley, CA 94720-7356 USA. Telephone: (510) 642-4355. Fax: (510) 642-0427. E-mail: srappaport@berkeley.edu *Current address: School of Public Health, Seoul National University, Seoul, Korea. We are indebted to M. Dosemici, who compiled some of the benzene exposure data used in this investi- gation; to M. Shin, who helped assemble a portion of the database; and to P. Egeghy, L. Nylander-French, K. Gwin, and I. Hertz-Picciotto, who contributed to a previous study from which we used data to estimate benzene uptake at sub-parts-per-million exposure lev- els. We also appreciate the helpful comments of M. Andersen, who reviewed a draft of the manuscript, and of S. Selvin, who provided helpful discussion regarding the statistical analyses. This research was supported by the National Institute for Environmental Health Sciences through grants P42ES05948 and P30ES10126 to S.M.R. and RO1ES06721 and P42ES04705 to M.T.S. and by funds from the intramural research program of the National Institutes of Health, National Cancer Institute. S.M.R. has received consulting and expert tes- timony fees from law firms representing plain- tiffs’ cases involving exposure to benzene, and he has received research support from the American Petroleum Institute and the American Chemistry Council. G.L. has received funds from the American Petroleum Institute for consulting on benzene-re- lated health research. M.T.S. has received consulting and expert testimony fees from law firms represent- ing both plaintiffs and defendants in cases involving exposure to benzene. The other authors declare they have no competing financial interests. Received 19 December 2008; accepted 18 February 2009. ovlume 117 | number 6 | June 2009 • Environmental Health Perspectives