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Bisphenol A (BPA) and its analogues are widely used industrial chemicals. Placental 3β-hydroxysteroid dehydrogenases (3β-HSDs) catalyse the conversion of pregnenolone to progesterone. However, the potency of BPA analogues in inhibiting 3β-HSDs activity remains unclear. We investigated the inhibitory effect of 10 BPA analogues on 3β-HSDs activity using an in vitro assay and performed the structure-activity relationship and in silico docking analysis. BPH was the most potent inhibitor of human 3β-HSD1, with an IC 50 value of 0.95 μM. BPFL, BPG, DABPA, BPAP, BPZ, DMBPA, and BPB also inhibited human 3β-HSD1 activity, albeit with lower potency. BPG was the most potent inhibitor of rat 3β-HSD4, with an IC 50 value of 1.14 μM. BPAP, BPFL, BPG, BPH, BPZ, DABPA, and DMBPA are mixed inhibitors of human 3β-HSD1 and they significantly inhibited human JAr cells to secrete progesterone. The LogP values were inversely correlated with the inhibitory effects. Docking analysis showed that most BPA analogues bind to steroid-binding site of both 3β-HSDs. A pharmacophore containing hydrogen bond donor and hydrophobic region was generated for predicting the inhibitory strength of BPA analogues. In conclusion, this study demonstrates that some BPA analogues are potent inhibitors of 3β-HSDs and lipophilicity determines the inhibitory potency., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Benzophenones (BPs) are a class of chemicals found in various personal care and cosmetic products, such as sunscreens and lotions. Their usage is known to cause reproductive and hormonal health risks, but the exact mechanism of action remains unknown. In this study, we investigated the effects of BPs on human and rat placental 3β-hydroxysteroid dehydrogenases (3β-HSDs), which play a crucial role in the biosynthesis of steroid hormones, particularly progesterone. We tested inhibitory effects of 12 BPs, and performed structure-activity relationship (SAR) and in silico docking analysis. The potency of BPs to inhibit human 3β-HSD1 (h3β-HSD1) is BP-1 (IC 50 , 8.37 μM)>BP-2 (9.06 μM)>BP-12 (94.24 μM)>BP-7 (1160 μM) >BP-8 (1257 μM) >BP-6 (1410 μM) > other BPs (ineffective at 100 μM). The potency of BPs on rat r3β-HSD4 is BP-1 (IC 50 , 4.31 μM)>BP-2 (117.3 μM)>BP-6 (669 μM) >BP-3 (820 μM)>other BPs (ineffective at 100 μM). BP-1, BP-2, and BP-12 are mixed h3β-HSD1 inhibitors and BP-1 is a mixed r3β-HSD4 inhibitor. LogP, lowest binding energy, and molecular weight were positively associated with IC 50 for h3β-HSD1, while LogS was negatively associated with IC 50 . The 4-OH substitution in the benzene ring plays a key role in enhancing the effectiveness of inhibiting h3β-HSD1 and r3β-HSD4, possibly through increasing water solubility and decreasing lipophilicity by forming hydrogen bonds. BP-1 and BP-2 inhibited progesterone production in human JAr cells. Docking analysis shows that 2-OH of BP-1 forms hydrogen bonds with catalytic residue Ser125 of h3β-HSD1 and Thr125 of r3β-HSD4. In conclusion, this study demonstrates that BP-1 and BP-2 are moderate inhibitors of h3β-HSD1 and BP-1 is a moderate inhibitor of r3β-HSD4. There is a significant SAR differences for 3β-HSD homologues between BPs and distinct species-dependent inhibition of placental 3β-HSDs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Resveratrol and its analogs are phytochemicals. Human 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) synthesizes steroid hormones for normal pregnancy or promoting cancer metastasis. Whether they inhibit 3β-HSD1 remains unclear. In this study, the inhibitory potency, mode of action, structure-activity relationship, and docking parameters of resveratrol and its analogs on 3β-HSD1 and rat homolog 3β-HSD4 were analyzed. The inhibitory potency of these chemicals on human 3β-HSD1 was 4,4'-dihydroxystilbene (IC 50 , 3.68 μM) > pinostilbene (8.07 μM) > pinosylvin (10.60 μM) > lunularin (26.84 μM) > resveratrol (30.20 μM) > dihydroresveratrol (>100 μM) = oxyresveratrol (>100 μM) > dihydropinosylvin (ineffective at 100 μM). Resveratrol analogs and metabolites are mixed or competitive inhibitors of human 3β-HSD1. Resveratrol and 4,4'-dihydroxystilbene inhibited progesterone secretion by human JAr cells at ≥1 μM. Resveratrol (IC 50 , 32.09 μM) and pinosylvin (34.71 μM) significantly inhibited rat placental 3β-HSD4 activity. Docking analysis shows that resveratrol analogs and metabolites bind the steroid-binding sites of human 3β-HSD1 and rat 3β-HSD4 and interact with the catalytic residues Ser125/Thr125 and Tyr155. The negative correlation of Log P and IC 50 values for human 3β-HSD1 indicates that lipophilicity of chemicals plays a critical role in the inhibitory effect of chemicals. In conclusion, 4,4'-dihydroxystilbene, pinostilbene, and pinosylvin effectively inhibit human 3β-HSD1 depending on their lipophilicity, thereby acting as potential therapeutic agents.

Bisphenols (BPs) as endocrine-disrupting compounds have drawn attention to their health hazards. Whether a BP interferes with glucocorticoid metabolism remains unclear. 11β-Hydroxysteroid dehydrogenase 2 (11β-HSD2) is a key glucocorticoid-metabolizing enzyme that controls fetal glucocorticoid levels across the placental barrier and mineralocorticoid receptor specificity in the kidney. In this study, 11 BPs were tested to inhibit human placental and rat renal 11β-HSD2 and were analyzed for inhibitory potency, mode action, and docking parameters. BPs had inhibitory potency against human 11β-HSD2: BPFL>BPAP>BPZ>BPB>BPC>BPAF>BPA>TDP and the IC 10 values were 0.21, 0.55, 1.04, 2.04, 2.43, 2.57, 14.43, and 22.18 μM, respectively. All BPs are mixed inhibitors except BPAP, which is a competitive inhibitor for human 11β-HSD2. Some BPs also inhibited rat renal 11β-HSD2, with BPB (IC 50 , 27.74 ± 0.95) > BPZ (42.14 ± 0.59) > BPAF (54.87 ± 1.73) > BPA (77.32 ± 1.20) > other BPs (about 100 μM). Docking analysis showed that all BPs bound to the steroid-binding site, interacting with the catalytic residue Tyr232 of both enzymes and the most potent human 11β-HSD2 inhibitor BPFL acts possibly due to its large fluorene ring that has hydrophobic interaction with residues Glu172 and Val270 and π-stacking interaction with catalytic residue Tyr232. The increase in the size of substituted alkanes and halogenated groups in the methane moiety of the bridge of BPs increases its inhibitory potency. Regressions of the lowest binding energy with inhibition constant indicated that there was an inverse regression. These results indicated that BPs significantly inhibited human and rat 11β-HSD2 activity and that there were species-dependent differences., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Human 3β-hydroxysteroid dehydrogenase type I (HSD3B1) and rat type IV (HSD3B4) in placentas catalyze the conversion of pregnenolone to progesterone, which plays a key role in maintaining pregnancy. Many phenolic compounds potentially inhibit HSD3B in placentas as endocrine disruptors. In this study, the effects of 16 phenolic compounds on the activity of human HSD3B1 and rat HSD3B4 were determined and the structure-activity relationship was compared. HSD3B1 in human placental microsomes and HSD3B4 in rat placental microsomes were used to measure their activities and pregnenolone and NAD + were used as substrates. Of the 16 phenolic compounds, 4-nonylphenol, pentabromophenol, and 2-bromophenol resulted in residual human HSD3B1 activity lower than 50 % and 4-nonylphenol and pentabromophenol resulted in residual rat HSD3B4 activity lower than 50 %. 4-Nonylphenol, pentabromophenol, and 2-bromophenol were mixed inhibitors of human HSD3B1, with Ki values of 2.31, 3.58 and 4.86 µM, respectively, while 4-nonylphenol and pentabromophenol were noncompetitive inhibitors of rat HSD3B4 with Ki values of 20.86 and 141.8 µM. Molecular docking showed that 4-nonylphenol, pentabromophenol, and 2-bromophenol docked to the active sites of human HSD3B1 and rat HSD3B4, and the shift of residue S125 in human HSD3B1 to T125 in rat HSD3B4 could explain the species-dependent difference in their inhibitory potency and mode of action. This study demonstrates that 4-nonylphenol, pentabromophenol, and 2-bromophenol are mixed inhibitors of human placental HSD3B1, while 4-nonylphenol and pentabromophenol are noncompetitive inhibitors of rat HSD3B4, possibly blocking the placental steroidogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Tebuconazole (TEB) is one of the widely used broad-spectrum triazole fungicides. Its accumulation in mammals leads to various endocrine disruptions. However, it is unclear whether the exposure of TEB during pregnancy affects the growth and development of fetus and placenta. Here, TEB was exposed to pregnant Sprague-Dawley female rats from gestational days 12-21 of 0, 25, 50 or 100 mg/kg for 10 days. TEB reduced placental estradiol levels. TEB disrupted the structure and function of the placenta, leading to hypertrophy, fibrin exudation, edema, calcification, arterial fibroblast proliferation, and trophoblastic infarction. RNA-seq analysis showed that TEB mainly down-regulated the expression of iron transport genes and up-regulated the expression of genes for immune/inflammatory responses. Further qPCR showed that TEB down-regulated Tfrc, Hamp, Eif2ak2 and up-regulated the expression of Cd34, Cd36, Jag1, Pln, Cyp1a1, Esrra, and Aqp1 at 50 and 100 mg/kg. Western blot and semi-quantitative immunohistochemical staining also demonstrated that TEB lowered the levels of TFRC and EIF2AK2 and increased the levels of CD34, CD36, JAG1, CYP1A1, and ESRRA at 50 and 100 mg/kg. In conclusion, TEB severely damages the structure and function of the placenta, leading to hypertrophy of the placenta, low birth weight and feminization of the male fetus possibly via several pathways including iron transport and TNF signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Zearalenone is an estrogenic mycotoxin produced by a variety of Fusarium fungi. There is evidence that exposure to zearalenone can cause intrauterine growth restriction, but little is known about the mechanism in the rat placenta caused by zearalenone. From gestational day 14-21, female Sprague Dawley rats (60 days old) were gavaged with zearalenone (0, 2.5, 5, 10, and 20 mg/kg/day body weight). Zearalenone dose-dependently reduced serum LH and FSH levels of dams at ≥ 5 mg/kg. RNA-seq and qPCR showed that zearalenone significantly down-regulated Slc38a1 expression at 2.5 mg/kg, Echs1 and Pc at 10 mg/kg, as well as Slc1a5, Cd36, Ldlr, Hadhb, and Cyp17a1 expression at a dose of 20 mg/kg, while it up-regulated the expression of Notch signal (Dvl1 and Jag 1). After zearalenone treatment, their proteins showed a similar trend. Zearalenone reduced the phosphorylation of AKT1, ERK1/2, and mTOR at 5 mg/kg or higher and 4EBP1 at 5 mg/kg. Zearalenone also increased BECLIN1, LC3B, and p62 levels and elevated BAX/BCL2 and CASP3/PROCASP3 ratios. In conclusion, zearalenone disrupts placental function such as reduction of nutrient transport and lipid metabolism possibly via AKT1/ERK1/2/mTOR-mediated autophagy and apoptosis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Human placental 3β-hydroxysteroid dehydrogenase/steroid Δ5, 4-isomerase 1 (HSD3B1), a high-affinity type I enzyme, uses pregnenolone to make progesterone, which is critical for maintenance of pregnancy. HSD3B1 is located in the mitochondrion and the smooth endoplasmic reticulum of placental cells and is encoded by HSD3B1 gene. HSD3B1 contains GATA and TEF-5 regulatory elements. Many endocrine disruptors, including phthalates, methoxychlor and its metabolite, organotins, and gossypol directly inhibit placental HSD3B1 thus blocking progesterone production. In this review, we discuss the placental HSD3B1, its gene regulation, biochemistry, subcellular location, and inhibitors from the environment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human placental CYP19A1 catalyzes the estrogen synthesis from androgens. The enzyme is encoded by CYP19A1 gene located in chromosome 15q21. This enzyme is a monooxygenase in the smooth endoplasmic reticulum. The various promoters of the CYP19A1 gene determine its expression in different tissues and the distal promoter I.1 controls its expression in the placenta and retinoids can regulate the expression. Many food components and environmental chemicals inhibit CYP19A1 activity via different modes of action. These chemicals include gossypol, flavones, flavanones, chalconoids, resveratrol, and tobacco alkaloids derived from foods as well as phthalates, insecticides, fungicides, and biocides in the contaminated foods. The inhibition of placental CYP19A1 could impair pregnancy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

3β-Hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase 1 (3β-HSD1) plays a critical role in the biosynthesis of progesterone from pregnenolone in the human placenta to maintain normal pregnancy. Whether they inhibit placental 3β-HSD1 and mode of inhibition remains unclear. In this study, we screened 21 pesticides and fungicides in five classes to inhibit human 3β-HSD1 and compared them to rat homolog 3β-HSD4. 3β-HSD activity was measured by catalyzing pregnenolone to progesterone in the presence of NAD+. Of the 21 chemicals, azoles (difenoconazole), thiocarbamates (thiram and ferbam) and organochlorine (hexachlorophene) significantly inhibited human 3β-HSD1 with half maximal inhibitory concentration (IC 50) values of 2.77, 0.24, 0.68, and 17.96 μM, respectively. We also found that difenoconazole, ferbam and hexachlorophene are mixed/competitive inhibitors of 3β-HSD1 while thiram is a mixed/noncompetitive inhibitor. Docking analysis showed that difenoconazole and hexachlorophene bound steroid-binding site. Difenoconazole and hexachlorophene except thiram and ferbam also significantly inhibited rat 3β-HSD4 activity with IC 50 of 1.12 and 2.28 µM, respectively. Thiram and ferbam significantly inhibited human 3β-HSD1 possibly by interfering with cysteine residues, while they had no effects on rat 3β-HSD4. In conclusion, some pesticides potently inhibit placental 3β-HSD, leading to the reduction of progesterone formation. [Display omitted] • Difenoconazole, thiram, and ferbam are potent human 3β-HSD1 inhibitors. • Hexachlorophene is a moderate human 3β-HSD1 inhibitor. • The species-dependent difference for inhibition of placental 3β-HSD. • Thiram and ferbam inhibit human 3β-HSD1 by targeting cysteine residues. • Thiram and ferbam do not inhibit rat 3β-HSD4. [ABSTRACT FROM AUTHOR]

Triadimefon is a fungicide that is broadly used to treat fungal diseases of plants. It causes developmental toxicity in the animal model. Whether triadimefon disrupts the placental function and the underlying mechanism remains unclear. Thirty-six female pregnant Sprague-Dawley rats were randomly assigned into four groups and were orally administered via gavage of triadimefon (0, 25, 50, and 100 mg/kg/day) for 10 days from gestational day (GD) 12–21. Triadimefon disrupted the structure of the placenta, leading to hypertrophy, abnormal hemodynamics, including fibrin exudation, edema, hemorrhage, infarction, and inflammation. RNA-seq analysis showed that triadimefon down-regulated the expression of developmental and metabolic genes, while up-regulating the immune/inflammatory genes. The qPCR showed that triadimefon markedly down-regulated the expression of Cpt1c , Scd2 , Ldlr , Dvl1 , Flt4 , and Vwf and their proteins, while up-regulating the expression of Cyp1a1 , Star , Ccl5, and Cx3cr1 and their proteins at 25–100 mg/kg. Western blot showed that triadimefon reduced the level of STAT3 at doses of 50 and 100 mg/kg and the phosphorylation of AMPK at 100 mg/kg. In conclusion, triadimefon severely damages the structure and function of the placenta, leading to placental hypertrophy, local blood circulation disorders, and inflammation and this may be associated with its down-regulation of genes related to metabolism and nutrient transport and the up-regulation of inflammatory genes via STAT3 and AMPK signals. [Display omitted] • Triadimefon reduces placental efficiency after gestational exposure. • Triadimefon increases placental thickness. • Triadimefon down-regulates the expression of nutrient transporter genes. • Triadimefon up-regulates the expression of inflammatory genes. • Triadimefon reduces AMPK phosphorylation and STAT3. [ABSTRACT FROM AUTHOR]

Many insecticides and fungicides are endocrine-disrupting compounds, which possibly interfere with the placental endocrine system. In the placenta, 3β-hydroxysteroid dehydrogenase/Δ5,4-isomerase type 1 (HSD3B1) is the major steroidogenic enzyme, which makes progesterone from pregnenolone to support the placental stability. In this study, we screened 12 classes of insecticides and fungicides to inhibit placental HSD3B1 activity and compared them to the rat homolog type 4 (HSD3B4) isoform. Human HSD3B1 activity and rat HSD3B4 activity were measured in the presence of 200 nM pregnenolone and 0.2 mM NAD+ and 100 μM of test chemical. Triclosan, triflumizole, dichlone, and oxine at 100 μM significantly inhibited human HSD3B1 activity with the residual activity being less than 50% of the control. Further study showed that the half-maximal inhibitory concentration (IC 50) values of triclosan, triflumizole, dichlone, and oxine were 85.53 ± 9.14, 73.75 ± 3.42, 2.54 ± 0.40, and 102.93 ± 6.10 μM, respectively. In the presence of pregnenolone, triclosan, triflumizole, and dichlone were mixed inhibitors of HSD3B1, while oxine was a noncompetitive inhibitor. In the presence of NAD+, triclosan exhibited competitive inhibition while triflumizole possessed uncompetitive inhibition. Docking analysis showed that triclosan bound NAD+-binding site, while triflumizole, dichlone, and oxine mostly bound steroid-binding site. When the effect of these insecticides on rat placental HSD3B4 activity was screened in the presence of 200 nM pregnenolone, atrazine, triclosan, triflumizole, oxine, cyprodinil, and diphenyltin at 100 μM significantly inhibited rat HSD3B4 activity, with IC 50 values of triclosan, triflumizole, oxine, and cyprodinil were 82.99 ± 6.48, 35.45 ± 2.73, 105.59 ± 12.04, and 43.37 ± 3.00 μM, respectively. The mode action analysis showed that t riflumizole and cyprodinil were almost competitive inhibitors, while triclosan and oxine were almost noncompetitive inhibitors of rat HSD3B4. Docking analysis showed that triclosan and oxine bound cofactor NAD+ binding residues more than steroid-binding residues of rat HSD3B4 while triflumizole and cyprodinil bound most pregnenolone-interactive residues. In conclusion, some insecticides such as triclosan, triflumizole, and oxine can effectively inhibit both human and rat placental HSD3B activity and they have unique mode action due to the structure difference. [Display omitted] • Triclosan, triflumizole, and dichlone are mixed inhibitors of human HSD3B1. • Triclosan, triflumizole, cyprodinil are mixed inhibitors of rat HSD3B4. • Oxine is a non-competitive inhibitor of human HSD3B1 and rat HSD3B4. • Primary structure difference in catalytic residue S125T may explain the species-dependent difference of these chemicals. [ABSTRACT FROM AUTHOR]

Placenta contains 3β-hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase (HSD3B), which catalyzes pregnenolone to progesterone for maintaining pregnancy. Perfluoroalkyl carbonic acids (PFC) are subclass of perfluoroalkyl substances containing 4–14 carbons (C4-C14) in the carbon backbone and are potential endocrine disruptors. Whether PFC inhibit HSD3B and structure-activity relationship (SAR) remains unclear. Herein, we screened 11 PFC for inhibiting human type I HSD3B (HSD3B1) and rat type IV HSD3B (HSD3B4) activities and determined SAR and mode of inhibition. HSD3B was measured by converting pregnenolone to progesterone assisted by NAD+ in placental microsomes. Of the 11 PFC, C9-C14 significantly inhibited human HSD3B1 activity at 100 μM. Half-maximal inhibitory concentration (IC 50) values of C9-C14 compounds were 363.56 ± 12.14, 12.78 ± 0.69, 6.54 ± 0.65, 20.88 ± 0.41, 118.35 ± 0.16, and 149.26 ± 21.67 μM, respectively. We determined Ki values and mode of inhibition of three most potent PFC (C10-C12), and found that they were mixed inhibitors against pregnenolone, with Ki values of 5.57 ± 4.37, 2.04 ± 2.26, and 9.93 ± 7.71, respectively. Docking analysis showed that they bound steroid-binding site. Effects of PFC on rat placental HSD3B4 were performed. Of the 11 PFC, C10-C12 significantly inhibited rat HSD3B4 activity at 100 μM. IC 50 values of C10-C12 compounds were 45.85 ± 1.49, 36.08 ± 1.50, and 88.74 ± 1.99 µM, respectively. Ki values and inhibition modes of the three most potent PFC (C10-C12) were studied. It was found that they were mixed inhibitors against pregnenolone, with Ki values of 48.16 ± 20.44, 36.28 ± 53.07, and 91.79 ± 21.75 μM, respectively. Docking analysis showed that they bound steroid-binding site of rat HSD3B4. In conclusion, PFC showed significant SAR differences. The potency of inhibiting HSD3B activity increased from C9 to C11, and then declined. Human HSD3B1 was more sensitive to the inhibition of rat HSD3B4. [Display omitted] • Some perfluoroalkyl carbonic acids are inhibitors of human HSD3B1 and rat HSD3B4. • PFC inhibit human and rat HSD3B depending on the carbon chain length. • The potency of PFC is increased from C9 to C11 and then is declined. [ABSTRACT FROM AUTHOR]