Glutathione transferases EC also referred to as glutathione

Glutathione transferases (EC 2.5.1.18; also referred to as glutathione S-transferases, GSTs) are ubiquitous and promiscuous enzymes that catalyze various kinds of reactions involving a wide variety of substrates that are toxic and chemically reactive (Angelucci et al., 2005). The primary function of GSTs, particularly in higher organisms, is the detoxification of both endobiotics and xenobiotics through their conjugation to reduced glutathione (GSH) for the maintenance of normal redox homeostasis (Armstrong, 1991). GSTP1-1, one of the cytosolic GSTs, in addition to its primary function of detoxification regulates cell survival and apoptosis by interacting with c-Jun N-terminal kinase-1 (JNK1), maintaining it in an inactive form, thereby protecting the JNJ-10198409 against hydrogen peroxide-induced cell death (Adler et al., 1999). A recent study revealed that GSTP1-1 overexpression interferes with prostate cancer cell motility and viability by interacting with MYC and shutting down the MEK–ERK1/2 pathway (Wang et al., 2017). Typically, cytosolic GSTs are homodimeric proteins consisting of two 22–30 kDa-sized subunits (Board and Menon, 2013; Turk et al., 2015). Each of the GST subunits has its own active site which is made up of a GSH-binding site (G-site) and an electrophilic substrate-binding site (H-site) (Prade et al., 1997). Deltamethrin (DEL) is a common name for the synthetic pyrethroid insecticide S-cyano-3-phenoxybenzyl-cis-(1R,3R)-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate. It is also known by the trade names Decis®, Decasyn®, Butox®, K-Othrine®, Kordon®, and Sadethrin®. Pyrethroids are the only class of insecticides recommended by both the Centre for Disease Control and Prevention (CDC) and the World Health Organization (WHO) to treat protective nets against mosquitoes (Pennetier et al., 2008). Mosquito nets impregnated in DEL have been used successfully all over the world to control malaria (Joshi et al., 2003). Molecular targets of pyrethroids in mammals are much the same as those in insects and consist of voltage-gated ion channels for Na+, Cl–, and Ca2+, GABA-gated chloride channels, nicotinic receptors for acetylcholine, and intercellular gap junctions. The modulation of these channels/receptors by pyrethroid insecticides induces axonal hyperexcitability, leading to prolonged depolarization and burst firing (Gupta and Crissman, 2013). Intriguingly, there exists an increased risk of pyrethroid neurotoxicity in developing mammals, which results, at least in part, from the differential expression pattern of voltage-gated sodium channel isoforms in neonates versus adults (Wakeling et al., 2012). It may be worth mentioning in this context that authorities on teratology recommend caution, stating that occupational, environmental and medicinal exposures to pyrethroid products should be minimized during pregnancy (Barlow et al., 2015). Although in general DEL and other pyrethroids are considered safe for human use (Rehman et al., 2014), experimental data concerning the effects of DEL on human enzymatic systems are quite lacking. The aim of this study, therefore, is to elucidate the interaction of human placental GSTP1-1 (hpGSTP1-1) with DEL.
Materials and methods
Results and discussion First, the purity of commercially purchased human placental GSTP1-1 (hpGSTP1-1) was determined using native and SDS–PAGE (Laemmli, 1970). The enzyme yielded a single band both on native and SDS–polyacrylamide gels, with a subunit Mr of 21,380 Da (not shown). The study of the inhibitory action of DEL on hpGSTP1-1 was carried out at fixed [CDNB] and [GSH] (1 mM each). The presence of low amounts of xylene (up to 0.03%, v/v) and ethanol (up to 7%, v/v) in the reaction mixture did not alter enzyme activity. From the data obtained, SOS response was evident that hpGSTP1-1 was subject to inhibition by DEL in a concentration-dependent manner, with an IC50 value of 6.1 μM (Fig. 1). Considering the linearity of the resulting Hill plot, it is plausible to assume either that the entire surface of the dimeric enzyme bears one unique inhibitor-binding site or that there are two equivalent inhibitor-binding sites distributed at the two monomers, each altering the optimal arrangement of the nearby substrate-binding site individually yet equally so as to hamper catalysis. Recently, the interaction of hpGSTP1-1 with several antidepressants has been elucidated by our team (Dalmizrak et al., 2016, 2011). The antidepressants studied involved the tricyclic compounds clomipramine and amitriptyline and also the selective serotonin reuptake inhibitor fluoxetine. At fixed [CDNB] and variable [GSH] and at fixed [GSH] and variable [CDNB], the corresponding Ki values were 5.62 ± 4.37 mM and 8.09 ± 1.27 mM, respectively for fluoxetine (Dalmizrak et al., 2016). For amitriptyline, the Ki was 1.75 ± 0.37 mM at fixed [CDNB] and variable [GSH], and 3.90 ± 2.26 mM for clomipramine under the same condition. But when the substrate varied was CDNB, the Ki value for amitriptyline was 4.90 ± 0.68 mM and that for clomipramine was 3.37 ± 0.39 mM (Dalmizrak et al., 2011). In the current study, the Ki values for DEL at fixed [CDNB]–varied [GSH], and at fixed [GSH]–varied [CDNB] were found to be 5.61 ± 0.32 μM and 7.96 ± 0.97 μM, respectively. The exhibition by DEL of Ki values in the low-micro molar range suggests that this pyrethroid pesticide is a more potent inhibitor of hpGSTP1-1 than the entire set of antidepressants studied.