The Journal of Toxicological Sciences (J. Toxicol. Sci.) 677 Vol.41, No.5, 677-691, 2016 Original Article Metabolism of methiocarb and carbaryl by rat and human livers and plasma, and effect on their PXR, CAR and PPARα activities Chieri Fujino1, Yuki Tamura2, Satoko Tange1, Hiroyuki Nakajima3,4, Seigo Sanoh1, Yoko Watanabe2, Naoto Uramaru2, Hiroyuki Kojima5, Kouichi Yoshinari3,4, Shigeru Ohta1 and Shigeyuki Kitamura2 1Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan 2Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan 3Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-Aoba, Aoba-ku, Sendai 980-8578, Japan 4School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan 5Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819, Japan (Received April 14, 2016; Accepted August 2, 2016) ABSTRACT — The oxidative, reductive, and hydrolytic metabolism of methiocarb and the hydrolyt- ic metabolism of carbaryl by liver microsomes and plasma of rats or humans were examined. The effects of the metabolism of methiocarb and carbaryl on their nuclear receptor activities were also examined. When methiocarb was incubated with rat liver microsomes in the presence of NADPH, methiocarb sul- foxide, and a novel metabolite, methiocarb sulfone were detected. Methiocarb sulfoxide was oxidized to the sulfone by liver microsomes and reduced back to methiocarb by liver cytosol. Thus, the intercon- version between methiocarb and the sulfoxide was found to be a new metabolic pathway for methiocarb by liver microsomes. The product of methiocarb hydrolysis, which is methylthio-3,5-xylenol (MX), was also oxidized to sulfoxide form by rat liver microsomes. The oxidations were catalyzed by human flavin- containing monooxygenase isoform (FMO1). CYP2C19, which is a human cytochrome P450 (CYP) iso- form, catalyzed the sulfoxidations of methiocarb and MX, while CYP1A2 also exhibited oxidase activi- ty toward MX. Methiocarb and carbaryl were not enzymatically hydrolyzed by the liver microsomes, but they were mainly hydrolyzed by plasma and albumin to MX and 1-naphthol, respectively. Both methio- carb and carbaryl exhibited PXR and PPARα agonistic activities; however, methiocarb sulfoxide and sul- fone showed markedly reduced activities. In fact, when methiocarb was incubated with liver microsomes, the receptor activities were decreased. In contrast, MX and 1-naphthol showed nuclear receptor activities equivalent to those of their parent carbamates. Thus, the hydrolysis of methiocarb and carbaryl and the oxidation of methiocarb markedly modified their nuclear receptor activities. Key words: Carbaryl, Hydrolysis, Methiocarb, Oxidative metabolism, PXR, Reductive metabolism INTRODUCTION can be absorbed through the skin and mucous membranes (Bouchard et al., 2008). These pesticides as well as orga- Methiocarb [3,5-dimethyl-4-(methylthio)phenol meth- nophosphate pesticides act as acetylcholinesterase inhib- ylcarbamate] and carbaryl (1-naphthalenol methylcar- itors. The anticholinesterase activity of organophosphate bamate) are esters of carbamic acid and are known as car- pesticides is irreversible while the carbamylation process bamates. They are used as broad-spectrum insecticides. by methiocarb and carbaryl is reversible. Some carbamate Methiocarb and carbaryl are considered as the most effec- pesticides exhibit neurotoxicity via inactivation of acetyl- tive pesticides for a variety of crops, as well as agricul- cholinesterase, which is a key enzyme in nervous trans- tural animals and pets. However, methiocarb and carbaryl mission (Gupta et al., 2007). The neurotoxicity of car- Correspondence: Shigeyuki Kitamura (E-mail: [email protected]) Vol. 41 No. 5 678 C. Fujino et al. bamate pesticides was demonstrated using postnatal rats cell proliferation and transactivation assays using MCF-7 and the potential effect in human infants was discussed human breast cancer cells. Kojima et al. (2004) examined (Vidair, 2004). Furthermore, carbaryl showed immunoto- the estrogenicity of 22 carbamate pesticides and found xic effects and contributed to the development of allergic that methiocarb activates estrogen receptors (ER) α and diseases (Ali Jorsaraei et al., 2014). Methiocarb induc- β. The anti-androgenic activity of methiocarb has also es oxidative stress such as lipid peroxidation and anti- been reported in two other studies (Birkhøj et al., 2004; oxidant defense systems in rats (Ozden et al., 2013). Kojima et al., 2004). Recently, we reported the effect of Some carbamates also influence the reproductive sys- the metabolic transformation of methiocarb and carbaryl tem by inhibiting steroidogenesis in animals and humans. on their estrogenic and anti-androgenic activities (Tange Carbaryl inhibits progesterone biosynthesis by prima- et al., 2016). In addition to endocrine-disruption by the ry human granulosa-lutein cells (Cheng et al., 2006). transcriptional activation of hormonal receptors, the dis- In mice, there was a significant decrease in the number ruption of hormone levels via nuclear receptors that are of estrous cycles, duration of the phases of each cycle, associated with the regulation of hormone levels should and number of healthy follicles (Baligar and Kaliwal, also be considered. The nuclear receptors include pregnane 2002). Carbamates are considered safe because they are X receptor (PXR), constitutive androstane receptor (CAR), easily metabolized and degraded in the environment. and peroxisome proliferator-activated receptor α (PPARα). However, carbamates have been reported to accumulate PXR plays a critical role in the transcriptional regu- in fish and invertebrates (Soler et al., 2007). With regard lation of CYP3A. Many compounds including xenobiot- to toxicity to wildlife, carbaryl has shown cardiac effects ics such as clinical drugs and pesticides have been shown in zebrafish embryos (Lin et al., 2007). Exposure of car- to be PXR agonists. CAR is a nuclear receptor close- baryl to catfish resulted in the suppression of serum lev- ly related with PXR. The activation of CAR results in an el of thyroxin (Sinha et al., 1991). Sun et al. (2008) have increased expression of CYP2B and CYP2C genes. The reported of the inhibitory effects of carbaryl, 1-naphthol, CAR ligand-binding pocket is smaller and less flexible and 2-naphthol on the receptor-mediated transcription of than that of PXR (Kretschmer and Baldwin, 2005; Timsit thyroid hormones. Carbaryl also induces oxidative stress and Negishi, 2007). Some compounds have both PXR and in snails (Leomanni et al., 2015). CAR activities whereas others are either PXR agonists or Carbamate pesticides are metabolized via hydroly- CAR agonists. PPARα functions as the receptor for the sis and/or oxidation in animals and humans. Carbamates induction of CYP4A gene, which is involved in fatty acid are hydrolyzed in plasma. The oxidation of sulfur-con- metabolism (Reddy and Hashimoto, 2001). PPARα ago- taining carbamates such as ethiofencarb, methiocarb, and nists have been reported to induce the gene expression of aldicarb to sulfoxide derivatives is a well-known pathway 17β-hydroxysteroid dehydrogenase type IV, which cata- (Montesissa et al., 1994; Pelekis and Krishnan, 1997; lyzes the conversion of estradiol to its inactive metabo- Risher et al., 1987), reportedly catalyzed by cyto- lite estrone (Fan et al., 1998). Furthermore, these nucle- chrome P450 (CYP) and flavin-containing monooxygen- ar receptors regulate the expression of phase II enzymes, ase (FMO) (Buronfosse et al., 1995; Furnes and Schlenk, which act as regulators of hormonal metabolism. Some 2005; Schlenk et al., 2002; Usmani et al., 2004; Hajjar carbamates have been reported to activate both PXR and and Hodgson, 1980). However, the metabolic fate of the CAR. As for the activation of PXR and CAR of methio- sulfoxide formed has not been extensively investigated. carb and carbaryl, both positive and negative results have In this study, the metabolism of methiocarb to the sulfox- been reported (Kojima et al., 2010; Abass et al., 2012). ide, the oxidation of methiocarb sulfoxide to the sulfone, There are no reports currently available on the effects of and the reduction of the sulfoxide to methiocarb by liver carbamates on PPARα. preparations were examined in rats and humans. Regard- The effects of metabolic modification of chemicals ing the endocrine-disrupting activities of the carbamates, toward ER and androgen receptor (AR) activities have which occur by the transcriptional activation of hormonal been investigated. Some chemicals such as methoxychlor, receptors, some positive results with respect to hormonal trans-stilbene, benzo[a]pyrene, 2-nitrofluorene, and sty- receptor-disruption have been reported. Klotz et al. (1997) rene oligomers are negative in in vitro estrogen screening reported that aldicarb, bendiocarb, and carbaryl weakly tests but exhibit estrogenic activity after they are metab- activated estrogen- or progesterone-responsive reporter olized by the microsomal CYP system (Kitamura et al., genes in breast MCF-7 and endometrial (Ishikawa) can- 2008). The anti-androgenic activity of fenthion, which is cer cells. Andersen et al. (2002) also reported that methi- an organophosphate pesticide, was markedly decreased ocarb exhibits estrogenic and anti-androgenic activities in after its oxidative metabolism by liver microsomes Vol. 41 No.