Placental cells also express P

Placental cells also express P450 aromatase (CYP19A1, encoded by CYP19A1). In the placenta, this enzyme requires an external source of androgens, provided by the adrenal gland of the fetus, to form estrogen. The fetal adrenal gland produces dehydroepiandrosterone (DHEA) [9] and, in the placenta, it can be converted into androstenedione by HSD3B1. Aromatase is a cytochrome P450 and is the rate-limiting enzyme in the estrogen biosynthesis. Through interaction with NADPH-cytochrome P450 reductase, aromatase catalyzes three steps of hydroxylation to convert androstenedione to estrone. Estrone is further converted into more potent estrogen, estradiol, by 17β-hydroxysteroid dehydrogenase isoforms (HSD17B1, HSD17B7, and HSD17B12) [10]. Placental estradiol stimulates placental growth and enhances placental blood flow to provide the optimal exchange of gases and nutrients required for the rapidly developing fetus [11]. In the present study, we investigated ziram in inhibition of HSD3B1 and aromatase and explored its potency and mode of action.
Materials and methods
Results
Discussion The association between fungicide exposure and endocrine system has gain awareness in recent years. The identification of environmental endocrine disruptors that interferes with endocrine system such as ziram [3] has led us to know its mechanisms. Ziram has been demonstrated to inhibit 11β-hydroxysteroid dehydrogenase 2 in both human and rats [3], causing the possible link with apparent mineralocorticoid excess due to the incapability of protecting kidney mineralocorticoid receptors by this enzyme [20]. Ziram is also shown to inhibit aldehyde dehydrogenases, the critical enzymes for metabolizing toxic aldehydes and taking part in dopamine metabolite 3,4-dihydroxyphenylacetaldehyde metabolism and the biosynthesis of retinoid HS-173 sale [21] that is critical for regulating germ cell development [22], [23]. In this study, we identified a direct interaction of ziram with human aromatase. Because human aromatase is critical for the biosynthesis of estrogens, which are important for human reproduction, we hypothesized that aromatase inhibition may mediate its endocrine disrupting effects. Although there are several steroid biosynthetic enzymes are present in the placenta, in this study, we tested the effects of ziram on human placental HSD3B1 and aromatase. We found that ziram did not inhibit human placental HSD3B1 activity at ≤100μM while it potently inhibited human aromatase with IC50 value in the nanomolar range. Ziram did not inhibit human placental HSD3B1, which is an NAD+ dehydrogenase. Interestingly, ziram inhibited rat and human 11β-hydroxysteroid dehydrogenase 2 with IC50 values of 35–90μM [24] as well as rat 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase with IC50 values of 1.556 and 1.017μM [3]. However, human aromatase was most sensitive to the inhibition of ziram. Docking study showed that ziram binds to the steroid-binding pocket of human aromatase (Fig. 5). We calculated the free energy for ziram and found that ziram has a free energy of −6.1819kcal. Docking analysis also demonstrates that ziram forms two hydrogen bonds with the hydroxyl group of Arg115 residue and the amide of Met 374 residue of the aromatase (B). According to the literature [15], human aromatase residues comprising the catalytic cleft include Arg 115, Ile 305, Ala 306, Asp 309 and Thr 310, Phe 221, Trp 224, Ile 133, Phe 134, Val 370, Leu 372, Val 373, Met 374, Leu 477, and Ser 478. Met 374 is a critical residue for androstenedione catalysis. Androstenedione was found to bind to the steroid-binding pocket with a hydrogen bond to Met 374 residue of the aromatase. Because ziram forms the hydrogen bond with Met 374 residue of the aromatase, it interferes with the androstenedione binding. We also performed the comparative docking of ziram to other human CYPs, including human CYP11A1 and CYP17A1 and found that ziram was not docked on the heme-binding site and the steroid-binding site (Supplementary Fig. S1), indicating that the inhibition of ziram on CYP19A1 is more specific.