In vitro metabolism of di(2-ethylhexyl) phthalate (DEHP) by various tissues and cytochrome P450s of human and rat Kyoungju Choi ⇑ , Hyun Joo, Jerry L. Campbell Jr., Rebecca A. Clewell, Melvin E. Andersen, Harvey J. Clewell III The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709, United States article info Article history: Received 19 October 2011 Accepted 2 December 2011 Available online 13 December 2011 Keywords: DEHP In vitro metabolism Human tissues Rat tissues Cytochrome P450 LC–MS/MS abstract In vitro metabolism of DEHP by subcellular fractions of human brain, intestine, kidney, liver, lung, skin, testis, rat liver and recombinant CYP isoforms of human and rat was investigated using LC–MS/MS. DEHP was rapidly hydrolyzed to mono(2-ethylhexyl) phthalate (MEHP) in 12 microsomal/cytosolic fractions of selected 7 human organs and rat liver but not in microsomal fractions of human brain and human female skin. MEHP was metabolized to CYP-mediated oxidative and dealkylated metabolites in human and rat liver and at a lower rate in human intestine. Measurable amounts of mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH MEHP), mono(2-ethyl-5-oxohexyl) phthalate (5-Oxo MEHP), mono(2-ethyl-5-carboxy- pentyl) phthalate (5-carboxy MEPP), mono(2-carboxymethyl-hexyl) phthalate (2-carboxy MMHP) and phthalic acid (PA) were formed by human liver fractions. Human CYP2C9 / 1, CYP2C19 and rat CYP2C6 were the major CYP isoforms producing 5-OH MEHP and 5-Oxo MEHP metabolites; however, only human CYP2C9 / 1 and 2C9 / 2 produced 5-carboxy MEPP from MEHP. Additionally, human CYP3A4 and rat CYP3A2 were the primary enzymes for PA production via heteroatom dealkylation of MEHP. Percent total normal- ized rates (%TNR) by CYP2C9 / 1 in human liver microsomes (HLM) were 94%, 98% and 100%, respectively, for 5-OH MEHP, 5-Oxo MEHP, 5-carboxy MEPP, and 76% for PA production by CYP3A4. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer in polyvinyl chloride (PVC) products, in cosmetics and in various consumer products, including building materials, automobiles, and clothing and toys (CDC, 2009). It is the only approved plasticizer by Food and Drug Administration (FDA) for use in medical devices (ATSDR, 2002 and FDA, 2001). Worldwide produc- tion of phthalates is 2.7 million metric tons annually (Bauer and Herrmann, 1997) and DEHP concentration may reach up to 40% by total weight of PVC plastics (CDC, 2009). Since phthalates are not covalently bound to the polymer, they leach out and have become widely distributed in the environment through manufac- turing, processing and disposal (CDC, 2009). The exposure routes of human to phthalates are inhalation, ingestion and intravenous (medical) exposure. Dermal exposure may occur; however, skin absorption of DEHP is limited (Wester et al., 1998). Ingestion is considered as the major exposure route, followed by an inhalation of indoor air (CDC, 2009). Selected phthalate monoesters, such as mono(2-ethylhexyl) phthalate (MEHP) and mono-n-butyl phthalate (MBP), respectively, that are produced in vivo after exposures to DEHP and di-n-butyl phthalate (DBP), are reproductive and developmen- tal toxicants (Crinnion, 2010). In vitro studies with rat and mouse testis and Leydig cell lines showed that MEHP decreased testoster- one production and increased gonocytes apoptosis (Clewell et al., 2010; Chauvigné et al., 2009; Lehraiki et al., 2009). In contrast, in vitro studies of MEHP with human fetal testis showed no inhib- itory effect on testosterone production but reduced germ cell num- bers (Lambrot et al., 2009). Epidemiological studies in human populations have suggested an association between phthalate exposure and adverse health outcomes. Hauser et al. (2005, 2007) found that the sperm DNA damage was significantly correlated to urinary levels of MEHP, 5-OH MEHP and 5-Oxo MEHP in male partners of infertile couples. The prenatal exposure to phthalates such as diethyl phthalate, DBP, benzyl butyl phthalate, DEHP and di-isobutyl phthalate was significantly associated with reduced anogenital distance (AGD) in human male infants (Swan et al., 2005). However, the strongest association in the study was with monoethyl phthalate (MEP) which has not shown endocrine disruption even at high doses in rodents. Hydrolysis of DEHP, oxidative metabolism of MEHP and organ- specific metabolites of DEHP have been examined in laboratory animals (Albro et al., 1982; Driven et al., 1993; Ito et al., 2005; Peck and Albro, 1982; Rowland, 1974). After ingestion, DEHP is rapidly hydrolyzed to MEHP and 2-ethylhexanol (2-EH) in the blood, liver and gut, which are the substrates for x- and x-1 oxidation. The 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.12.002 ⇑ Corresponding author. Address: The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709, United States. Tel.: +1 919 558 1369; fax: +1 919 558 1300. E-mail address: kchoi@thehamner.org (K. Choi). Toxicology in Vitro 26 (2012) 315–322 Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit