Effects of crosstalk between steroid hormones mediated thyroid hormone in zebrafish exposed to 4-tert-octylphenol: Estrogenic and anti-androgenic effects.

Alkylphenols, such as nonylphenol and 4-tert-octylphenol (OP), are byproducts of the biodegradation of alkylphenol ethoxylates and present substantial ecological and health risks in aquatic environments and higher life forms. In this context, our study aimed to explore the effect of OP on reproductive endocrine function in both female and male zebrafish. Over a period of 21 days, the zebrafish were subjected to varying concentrations of OP (0, 0.02, 0.1, and 0.5 µg/L), based on the lowest effective concentration (EC10 = 0.48 µg/L) identified for zebrafish embryos. OP exposure led to a pronounced increase in hepatic vitellogenin (vtg) mRNA expression and 17ß-estradiol biosynthesis in both sexes. Conversely, OP exhibits anti-androgenic properties, significantly diminishes gonadal androgen receptor (ar) mRNA expression, and reduces endogenous androgen (testosterone and 11-ketotestosterone) levels in male zebrafish. Notably, cortisol and thyroid hormone (TH) levels demonstrated concentration-dependent elevations in zebrafish, influencing the regulation of gonadal steroid hormones (GSHs). These findings suggest that prolonged OP exposure may result in sustained reproductive dysfunction in adult zebrafish, which is largely attributable to the intricate reciprocal relationship between hormone levels and the associated gene expression. Our comprehensive biological response analysis of adult zebrafish offers vital insights into the reproductive toxicological effects of OP, thereby enriching future ecological studies on aquatic systems.

Hair Thickness Growth Effect of Adenosine Complex in Male-/Female-Patterned Hair Loss via Inhibition of Androgen Receptor Signaling.

Aging (senescence) is an unavoidable biological process that results in visible manifestations in all cutaneous tissues, including scalp skin and hair follicles. Previously, we evaluated the molecular function of adenosine in promoting alopecia treatment in vitro. To elucidate the differences in the molecular mechanisms between minoxidil (MNX) and adenosine, gene expression changes in dermal papilla cells were examined. The androgen receptor (AR) pathway was identified as a candidate target of adenosine for hair growth, and the anti-androgenic activity of adenosine was examined in vitro. In addition, ex vivo examination of human hair follicle organ cultures revealed that adenosine potently elongated the anagen stage. According to the severity of alopecia, the ratio of the two peaks (terminal hair area/vellus hair area) decreased continuously. We further investigated the adenosine hair growth promoting effect in vivo to examine the hair thickness growth effects of topical 5% MNX and the adenosine complex (0.75% adenosine, 1% penthenol, and 2% niacinamide; APN) in vivo. After 4 months of administration, both the MNX and APN group showed significant increases in hair density (MNX + 5.01% (p < 0.01), APN + 6.20% (p < 0.001)) and thickness (MNX + 5.14% (p < 0.001), APN + 10.32% (p < 0.001)). The inhibition of AR signaling via adenosine could have contributed to hair thickness growth. We suggest that the anti-androgenic effect of adenosine, along with the evaluation of hair thickness distribution, could help us to understand hair physiology and to investigate new approaches for drug development.

Associations of semen quality with pyrethroids in semen and urine samples of men with oligozoospermia: A cross-sectional study in Samsun, Turkey.

Pyrethroids (PYRs) may act as endocrine disrupters and lead to infertility. The aim of the study was to analyze the levels of anti-androgenic PYRs (cypermethrin, deltamethrin, and permethrin) and 3-phenoxy benzoic acid (3-PBA), a general metabolite of PYRs, in both semen and urine samples of men with oligozoospermia. The PYRs and 3-PBA metabolite levels in the semen and urine samples of the men were analyzed through GC-MS. The results indicated that the levels of PYRs in the semen samples of the infertile group were significantly higher than those of the fertile group. It was determined that cypermethrin exposure was associated with changes in sperm count and total sperm motility, while permethrin, deltamethrin, and 3-PBA levels were associated with changes in sperm morphology. It was determined that there was a significant negative correlation between semen deltamethrin levels and sperm morphology and sperm count. In addition, exposure of these patients to deltamethrin (range; 1.53-8.02 µg/l) and having farmer parents were determined to increase the risk of infertility. In conclusion, the findings of this study showed that exposure to environmental PYRs may adversely affect semen quality, especially in terms of sperm morphology, in men with oligozoospermia.

Adult Exposure to Di-N-Butyl Phthalate (DBP) Induces Persistent Effects on Testicular Cell Markers and Testosterone Biosynthesis in Mice.

Studies indicate that phthalates are endocrine disruptors affecting reproductive health. One of the most commonly used phthalates, di-n-butyl phthalate (DBP), has been linked with adverse reproductive health outcomes in men, but the mechanisms behind these effects are still poorly understood. Here, adult male mice were orally exposed to DBP (10 or 100 mg/kg/day) for five weeks, and the testis and adrenal glands were collected one week after the last dose, to examine more persistent effects. Quantification of testosterone, androstenedione, progesterone and corticosterone concentrations by liquid chromatography-mass spectrometry showed that testicular testosterone was significantly decreased in both DBP treatment groups, whereas the other steroids were not significantly altered. Western blot analysis of testis revealed that DBP exposure increased the levels of the steroidogenic enzymes CYP11A1, HSD3ß2, and CYP17A1, the oxidative stress marker nitrotyrosine, and the luteinizing hormone receptor (LHR). The analysis further demonstrated increased levels of the germ cell marker DAZL, the Sertoli cell markers vimentin and SOX9, and the Leydig cell marker SULT1E1. Overall, the present work provides more mechanistic understanding of how adult DBP exposure can induce effects on the male reproductive system by affecting several key cells and proteins important for testosterone biosynthesis and spermatogenesis, and for the first time shows that these effects persist at least one week after the last dose. It also demonstrates impairment of testosterone biosynthesis at a lower dose than previously reported.

Novel toxicity of tris(1,3-dichloro-2-propyl) phosphate in adult male rats.

The widespread use of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) as a flame retardant has led to its release to the environment. Thus, the toxicological effects of TDCIPP on humans and animals are of importance. For better understanding of its potential toxicities, TDCIPP (250, 500, or 650 mg/kg/day) or vehicle control was administrated orally to adult male Wistar-Imamichi rats for 7 days. After the final administration of compounds, organ weights, histopathology, blood biochemistry, and hematology were examined. Hepatic toxicity was observed at doses ≥ 500 mg/kg/day of TDCIPP, and renal toxicity was observed at 650 mg/kg/day. The anti-androgenic activity of TDCIPP was previously confirmed in vitro and in vivo, but weights of epididymis, an androgen-dependent organ, were not affected by TDCIPP treatment in adults. Serum alkaline phosphatase activity was significantly decreased in all TDCIPP-treated rats independent of dose. Hemoglobin concentration, hematocrit, red blood cell count, and reticulocyte count were decreased in all TDCIPP-treated rats, but mean corpuscular volume, total iron-binding capacity, and serum iron were normal, suggesting that renal anemia was caused by TDCIPP. Together with previous reports on effects of anti-androgenic substances on red blood cell indices, anemia caused by TDCIPP could be due to its anti-androgenic activity. These considerations will contribute to further assessment of the toxicity of the compound.

Anti-androgenic, anti-oxidant and anti-apoptotic effects of the aqueous and methanol extracts of Pterorhachis zenkeri (Meliaceae): Evidence from in vivo and in vitro studies.

The aim of this study was to evaluate the effects of Pterorhachis zenkeri (Meliaceae) on sex organ growth in immature male rats and, oxidative stress and apoptosis markers in CCL-97 (R2C) Leydig cells. For the in vivo studies, 70 immature male Wistar rats (n = 10/group) were treated for 2 or 4 weeks with: distilled water (10 ml/kg, per os) plus soya oil (1 ml/kg, sc), bicalutamide (10 mg/kg, per os), aqueous or methanol extract of P. zenkeri (10 mg/kg or 62 mg/kg, per os) or testosterone propionate (3 mg/kg, sc). After each treatment period, body and sexual organ weights, plasmatic testosterone, total proteins and total cholesterol levels were measured. In the in vitro test, the effects of the methanol extract of P. zenkeri on cell viability, apoptosis, reactive oxygen species (ROS) production, intracellular calcium release and caspases 3/9 were assessed using CCL-97 Leydig cells. Pterorhachis zenkeri extracts decreased sex organ weights, plasmatic testosterone and protein levels in rats. In the in vitro studies, P. zenkeri inhibited apoptosis, ROS production, calcium release and caspase 3/9 activities. These results suggest that P. zenkeri has anti-androgenic, anti-oxidant and anti-apoptotic activities with methanol extract being the most active and could be an effective alternative for the management of androgen-related diseases.

Combined anti-androgenic effects of mixtures of agricultural pesticides using in vitro and in silico methods.

Humans and wildlife are continuously and simultaneously exposed to various pesticides that have been identified as endocrine disruptors which interfere with regulations of sexual differentiation and fertility. Low-dose effects of combined exposure from mixtures of pesticides have been extensively reported and need to be addressed in the context of human health risk assessment. The objective of the study is to assess the individual and combined anti-androgenic effects of twelve widely used pesticides in MDA-kb2 cells. The order of potency for seven pesticides with moderate anti-androgenic activities was in the order: fenitrothion > dimethomorph > difenoconazole > bromopropylate > prochloraz > imazalil > endosulfan, which was induced by the androgen receptor (AR) antagonism rather than cytotoxicity (with the exception of endosulfan which exhibited the highest cytotoxicity). The other five pesticides exhibited lower anti-androgenic activities. At 10% of AR antagonistic effect, three mixtures comprised of the seven pesticides (Mix-EC10, Mix-EC20, and Mix-EC25) at equi-effect concentrations showed summed concentrations of 6.75E-11, 17.63 and 25.21 µM, respectively. The combined effects were essentially close to the predicted of concentration addition (CA) at realistically low concentrations. In addition, molecular docking simulation indicated that hydrophobic interaction and polar functional groups of the pesticides contributed to the binding energy, which might be responsible for the AR antagonism. Our findings provide a basis for defining similarly acting antagonists in the context of cumulative risk assessment for pesticides in foods.

The anti-androgenic effects of cypermethrin mediated by non-classical testosterone pathway activation of mitogen-activated protein kinase cascade in mouse Sertoli cells.

Previous studies have demonstrated that the anti-androgenic effects of cypermethrin (CYP) are associated with testosterone (T) - related signaling pathway. This study was to investigate the effects of CYP on mouse Sertoli cells (TM4) and clarify whether the mechanisms were mediated by non-classical T signaling pathway activating mitogen-activated protein kinase (MAPK) cascade. The Cell Counting Kit 8 (CCK8) and Real-Time Cell Analysis iCELLigence (RTCA-iCELLigence) system were performed to detect the effects of 10 µM, 20 µM, 40 µM and 80 µM CYP on the viability and proliferation of TM4. The mammalian two hybrid assay, quantitative Real-Time PCR (qRT-PCR) and western blot were conducted to analyze the key genes and proteins involved in T-mediated MAPK signaling pathway. CYP was found to inhibit the viability and proliferation of TM4. Additionally, CYP disturbed the functions of Sertoli cells by inhibiting inhibin B (INH B) expression and facilitating androgen binding protein (ABP) and transferrin (TF) expression. Moreover, CYP suppressed the interaction of AR and Src kinase and inhibited androgen-mediated phosphorylation of Src, epidermal growth factor receptor (EGFR), extracellular-regulated kinase1/2 (ERK1/2) and transcription factor cAMP response element binding protein (CREB). Furthermore, the androgen-induced mRNA and protein expression of CREB-regulated gene early growth response factor (Egr1) decreased after treated with CYP. It is indicated that CYP inhibits the viability and proliferation of Sertoli cells and non-classical T signaling pathway activation of MAPK cascade is involved in anti-androgenic effect of CYP. This study provides a novel insight into the CYP-induced reproductive toxicity.

Analysis of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA) as a new potential biomarker of exposure to vinclozolin in urine.

Vinclozolin (V) is a fungicide with anti-androgenic properties whose metabolism is not fully understood, and data on urinary elimination of either V or its metabolites are limited. Therefore the kinetics of urinary elimination of V and its metabolites, after an oral dose in adult male rats were investigated. A single oral dose of V (100 mg/kg) suspended in corn oil was administered to male adult Wistar rats, and urine was collected at different times after dosing. V and its metabolites were extracted from urine, then enzymatically hydrolyzed using ß-glucuronidase/sulfatase of H. pomatia, and analyzed by HPLC/DAD. Urinary pharmacokinetic parameters were calculated using the analyte concentrations adjusted by creatinine levels. V and its metabolites 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA, formerly denoted as M5), 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), 3,5-dichloroaniline (M3), and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) were efficiently detected. The mean urine concentrations of V and M1 metabolite were fitted to a two-compartmental model for pharmacokinetic analysis. DTMBA approximately represented 88% of the total excreted metabolites, it was easily detected up to 168 h after dosing and its half-lives were 21.5 and 74.1 h, respectively. M1 was the second most abundant metabolite and was detected up to 144 h after being void. V and M3 were detected before 48 h, and M2 exhibited the lowest levels during the first 8 h after dosing. DTMBA, the most abundant V metabolite is quickly eliminated by urine, it is chemically stable, specific and could represent a useful alternative to be used as a biomarker of exposure to V.

Parallel assessment of the effects of bisphenol A and several of its analogs on the adult human testis.

STUDY QUESTION: Are bisphenol A (BPA) and BPA analogs (BPA-A) safe for male human reproductive function? SUMMARY ANSWER: The endocrine function of human testes explants [assessed by measuring testosterone and insulin-like factor 3 (INSL3)] was impacted by exposure of the human adult testis explants to BPA/BPA-A. WHAT IS KNOWN ALREADY: The few epidemiologic studies performed suggest that bisphenols have potential endocrine disruptive properties, but they did not identify clear and direct patterns of endocrine disruption. STUDY DESIGN, SIZE, DURATION: Adult human testis explants in culture were exposed to BPA and the analogs bisphenol F (BPF), bisphenol S (BPS), bisphenol E (BPE), bisphenol B (BPB) and bisphenol A diglycidyl ether (BADGE) at 10-9-10-5 M for 24 or 48 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human adult testes were obtained from prostate cancer patients who had no hormone therapy, or from multiorgan donors. After ex vivo exposure to the investigated bisphenols, the measured outcomes were related to histopathology (gross morphology and germ cell viability determined by anti-caspase three immunohistochemistry), and the levels of testosterone, INSL3 and inhibin B were measured using immunoassays. The levels of mRNA encoding key enzymes of bisphenol biotransformation were investigated by quantitative PCR: UGT2B15 UDP (glucuronosyltransferase two family, polypeptide B15), GUSB (glucuronidase beta), SULT1A1 and 3 (sulfotransferase family 1 A member 1 and 3) and STS (steroid sulfatase). MAIN RESULTS AND THE ROLE OF CHANCE: A significant dose-dependent inhibition was found between testosterone levels measured in the culture medium and concentrations of BPA (P = 0.00778 at 24 h and P = 0.0291 at 48 h), BPE (P = 0.039) and BPF (P = 0.00663). The observed BPA and BPA-A-induced inhibition of testosterone production varied according to duration of exposure and BPA/BPA-A concentrations. BPA (10-9 M; P < 0.05), BPB (10-9 M; P < 0.05), BPS (10-9 and 10-8 M; P < 0.05) and BADGE (10-5 M; P < 0.05) increased Leydig cell INSL3 production. By contrast, BPE dose dependently inhibited INSL3 (P = 0.0372). Conversely, Sertoli cell function (inhibin B) and germ cell viability were not significantly affected by either bisphenols. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Environmental compounds cannot be deliberately administered to men, justifying the use of an ex vivo approach. A relatively low number of testes samples were available for analysis (n = 3, except for testosterone secretion with n = 5). The active concentrations of BPA and BPA-A used in the study were higher than those found in human biological fluids. WIDER IMPLICATIONS OF THE FINDINGS: Under our experimental conditions, direct exposure to BPA or BPA-A can result in endocrine disturbance in the adult human testis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Inserm (Institut National de la Santé et de la Recherche Médicale), EHESP-School of Public Health, University of Rennes1, by grants from the Agence Nationale de la Recherche (ANR; grant#ANR-13-CESA-0012-03 NEWPLAST) and Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail (ANSES; grant#EST-2010/2/046 (BPATESTIS)). All authors declare they have no current or potential competing financial interests.

Investigations of putative reproductive toxicity of low-dose exposures to vinclozolin in Wistar rats.

The current investigation examines whether the fungicide vinclozolin, which has an anti-androgenic mode of action, is capable of disrupting endocrine homeostasis at very low doses. The data generated clarify whether a non-monotonic dose-response relationship exists to enhance the current debate about the regulation of endocrine disruptors. Moreover, it is part of a series of investigations assessing the dose-response relationship of single and combined administration of anti-androgenic substances. A pre-postnatal in vivo study design was chosen which was compliant with regulatory testing protocols. The test design was improved by additional endpoints addressing hormone levels, morphology and histopathological examinations. Doses were chosen to represent an effect level (20 mg/kg bw/d), the current NOAEL (4 mg/kg bw/d), and a dose close to the "ADI" (0.005 mg/kg bw/d) for the detection of a possible non-monotonic dose-response curve. Anti-androgenic changes were observable at the effect level but not at lower exposures. Nipple/areola counts appeared to be the most sensitive measure of effect, followed by male sex organ weights at sexual maturation, and finally gross and histopathological findings. The results indicate the absence of evidence for effects at low or very low dose levels. A non-monotonic dose-response relationship was not evident.

Investigations on the dose-response relationship of combined exposure to low doses of three anti-androgens in Wistar rats.

The current investigation examines whether combined exposure to three anti-androgens (flutamide, prochloraz, vinclozolin) result in interference with endocrine homeostasis when applied at very low dose levels, and whether the results of combined exposure are more pronounced than to the individual compounds. A pre-post-natal in vivo study design was chosen with more parameters than regulatory testing protocols require (additional endpoints addressing hormone levels, morphology and histopathological examinations). Dose levels were chosen to represent the lowest observed adverse effect level (LOAEL), the no observed adverse effect level (NOAEL), and the acceptable daily intake for each individual substance. Anti-androgenic changes were observable at the effect level (LOAEL) but not at lower exposures. Nipple/areola counts appeared to be a sensitive measure of effect, in addition to male sex organ weights at sexual maturation, and finally gross findings. The results indicate the absence of evidence for effects at low or very low dose levels. No (adverse) effects were seen at the NOAEL dose. A non-monotonic dose-response relationship was not evident. Combined exposure at LOAEL level resulted in enhanced responses for anogenital index, number of areolas/nipples, delayed preputial separation and reduced ventral prostate weight in comparison to the individual compounds.

At 10 years of chlormadinone use in Latin America: a review.

Chlormadinone acetate (CMA) is a progesterone derivative (17α-acetoxy-6-chloro-4,6-pregnadiene-3,20-dione), first synthesized in 1961. It was used as progestin-based hormone replacement therapy; since 1999 it was first used for oral contraception combined with ethinyl estradiol (EE). CMA exerts a potent progestagenic effect, about one third higher than that observed with endogenous progesterone. CMA is also an anti-estrogen, showing no androgenic effects (at birth control dose). Unlike progesterone, it has a mild glucosteroidal effect with no anti-mineralocorticoid effect at all. These biological actions have allowed CMA to have a role for therapeutic use in dysmenorrhea, hyperandrogenism, and as a contraceptive agent. In addition, CMA has exhibited beneficial neuroendocrine effects on women's mood. CMA-EE combination has shown excellent contraceptive efficacy, high tolerability, and compliance due to its risk-benefit profile, having additional benefits on skin and hair, such as reduction of seborrhea and acne. Metabolic tolerance of CMA has been demonstrated in several clinical studies. Currently, CMA is formulated to be taken as oral caplets in a 21 caplets package containing 0.03 mg/EE and 2 mg CMA per pill with/without seven placebo additional pills. Another presentation has 24 caplets containing 0.02 mg/EE and 2 mg CMA plus four placebo pills.

Metabolism of UV-filter benzophenone-3 by rat and human liver microsomes and its effect on endocrine-disrupting activity.

Benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) is widely used as sunscreen for protection of human skin and hair from damage by ultraviolet (UV) radiation. In this study, we examined the metabolism of BP-3 by rat and human liver microsomes, and the estrogenic and anti-androgenic activities of the metabolites. When BP-3 was incubated with rat liver microsomes in the presence of NADPH, 2,4,5-trihydroxybenzophenone (2,4,5-triOH BP) and 3-hydroxylated BP-3 (3-OH BP-3) were newly identified as metabolites, together with previously detected metabolites 5-hydroxylated BP-3 (5-OH BP-3), a 4-desmethylated metabolite (2,4-diOH BP) and 2,3,4-trihydroxybenzophenone (2,3,4-triOH BP). In studies with recombinant rat cytochrome P450, 3-OH BP-3 and 2,4,5-triOH BP were mainly formed by CYP1A1. BP-3 was also metabolized by human liver microsomes and CYP isoforms. In estrogen reporter (ER) assays using estrogen-responsive CHO cells, 2,4-diOH BP exhibited stronger estrogenic activity, 2,3,4-triOH BP exhibited similar activity, and 5-OH BP-3, 2,4,5-triOH BP and 3-OH BP-3 showed lower activity as compared to BP-3. Structural requirements for activity were investigated in a series of 14 BP-3 derivatives. When BP-3 was incubated with liver microsomes from untreated rats or phenobarbital-, 3-methylcholanthrene-, or acetone-treated rats in the presence of NADPH, estrogenic activity was increased. However, liver microsomes from dexamethasone-treated rats showed decreased estrogenic activity due to formation of inactive 5-OH BP-3 and reduced formation of active 2,4-diOH BP. Anti-androgenic activity of BP-3 was decreased after incubation with liver microsomes.

Investigations of putative reproductive toxicity of low-dose exposures to flutamide in Wistar rats.

The current investigation examines whether the model anti-androgenic substance flutamide is capable of disrupting endocrine homeostasis at very low doses. The data generated clarify whether a non-monotonic dose-response relationship exists to enhance the current debate about the regulation of endocrine disruptors. Moreover, it is part of a series of investigations assessing the dose-response relationship of single and combined administration of anti-androgenic substances. A pre-postnatal in vivo study design was chosen, which was compliant with regulatory testing protocols. The test design was improved by additional endpoints addressing hormone levels, morphology, and histopathological examinations. Doses were chosen to represent a clear effect level (2.5 mg/kg bw/d), a low endocrine effect level (LOAEL, 0.25 mg/kg bw/d), a NOAEL for endocrine effects (0.025 mg/kg bw/d), a further dose at 0.0025 mg/kg bw/d flutamide, as well as an "ADI" (0.00025 mg/kg bw/d or 100-fold below the NOAEL) for the detection of a possible non-monotonic dose-response curve. Anti-androgenic changes were observable at LOAEL and the clear effect dose level but not at lower exposures. Nipple retention appeared to be the most sensitive measure of anti-androgenic effects, followed by age at sexual maturation, anogenital distance/anogenital index and male sex organ weights, as well as gross and histopathological findings. The results of all five doses indicate the absence of evidence for effects at very low dose levels. A non-monotonic dose-response relationship was not evident for the anti-androgenic drug flutamide.

Reduced testosterone production in TM3 Leydig cells treated with Aspalathus linearis (Rooibos) or Camellia sinensis (tea).

Flavonoids are major compounds of Aspalathus linearis and Camellia sinensis. They are classified as endocrine disruptors and some have been shown to inhibit testosterone production. TM3 Leydig cell cultures were treated with 250-5000 µg mL(-1) A. linearis (unfermented or fermented rooibos) or Camellia sinensis (green or black tea) for 24 h in the absence or presence of 6 mIU/200 µl human chorionic gonadotropin (hCG). Under nonstimulated conditions, all teas tend to decrease testosterone production (3.9-31.8%). However, under hCG-stimulation, a significant reduction in testosterone production was observed at all concentrations by both rooibos and tea (16.3-37.9%). MTT assay and phase contrast microscopy, revealed that at 250-1000 µg ml(-1) , both plants maintained the viability, proliferation and morphology of the cells, while 5000 µg ml(-1) was cytotoxic to the cells (P < 0.05). In conclusion, the results here demonstrate the anti-androgenic property of A. linearis and C. sinensis.

Antioxidative, anti-androgenic, and inhibitory activities of ethanolic extract of Annona muricata leaf on sex hormones-induced benign prostate hyperplasia through in vivo and in silico studies.

Benign prostate hyperplasia (BPH) remains one of the major age-related urological problems in the world. Annona muricata (soursop) leaf has been reported to exhibit antiproliferative, antioxidant, and anti-inflammatory activities, among others, in the literature. Here, we aimed to unravel the antioxidative, antiandrogenic, and inhibitory activities of the ethanol extract of Annona muricata leaf (EEAML) on sex hormones-induced benign prostate hyperplasia (BPH) through in vivo and in silico studies. Thirty-six male rats were segmented into six groups of six animals each, the control group received water, and the BPH group and the remaining four groups were parentally infused with testosterone (T) and oestradiol (E2) (0.08 and 0.04 mg/Kgbwt) once daily for 28 days to induce BPH. After that, the control and BPH groups received water and normal saline, while the remaining four groups received finasteride (FIN) (0.1 mg/kgbwt) and EEAML (200, 400, and 800 mg/kgbwt) for another 28 days before sacrifice, and serum was collected for biochemical analysis. Additionally, the active ingredients of EEAML were identified using a Gas Chromatography Flame Ionisation Detector (GC-FID) followed by molecular docking (MD) against the human androgen receptor (hAR) target, and ADMET analysis of selected EEAML compounds was carried out. EEAML (200, 400, and 800 mg/kgbwt) restored the T and E2-induced depletion of reduced glutathione level, superoxide dismutase and catalase activities, and elevation of malondialdehyde, prostate-specific antigen, testosterone, and dihydrotestosterone levels in the serum of BPH rats. GC-FID analysis of EEAML showed the presence of 21 compounds from which 15 compounds were subjected to MD revealing that flavone, followed by ribalinidine, flavonone, anthocyanin, and naringenin displayed desirable binding affinities against the hAR target. ADMET analysis of these top-five EEAML compounds revealed that they were excellent oral bioavailable drug candidates with predicted minimal toxicities. In conclusion, EEAML exhibited antioxidative, antiandrogenic, and inhibitory activities owing to its phytoconstituents, which in turn could serve as drug templates for much better efficacy.

Methodology for developing data-rich Key Event Relationships for Adverse Outcome Pathways exemplified by linking decreased androgen receptor activity with decreased anogenital distance.

The Adverse Outcome Pathway (AOP) framework has gained widespread acceptance in toxicological disciplines as a tool for aiding chemical hazard assessment. Despite increased activity in AOP development, progress towards a high volume of fully endorsed AOPs has been slow, partly due to the challenging task of constructing complete AOPs according to the AOP Developer's Handbook. To facilitate greater uptake of new knowledge units onto the open-source AOP-wiki platform, a pragmatic approach was recently proposed. This approach involves considering Key Event Relationships (KERs) for individual development through systematic approaches, as they represent essential units of knowledge from which causality can be inferred; from low complexity test data to adverse outcomes in intact organisms. However, more broadly adopted harmonized methodologies for KER development would be desirable. Using the AOP Developer's Handbook as a guide, a KER linking 'decreased androgen receptor (AR) activity' with 'reduced anogenital distance (AGD)' was developed to demonstrate a methodology applicable for future developments of KERs requiring systematic literature retrieval approaches.

Tricresyl phosphate inhibits fertilization in Japanese medaka (Oryzias latipes): Emphasizing metabolic toxicity.

As tricresyl phosphate (TCrP) is commonly found in global water sources, its potential reproductive toxicity to fish is of increasing concern. Japanese medaka larvae were exposed to TCrP at 657.9, 1,511, and 4042 ng/L for 100 days. We identified significant fertilization inhibition (6.9%-12.8%) in all exposure groups. Intersex was significantly induced at 4042 ng/L, with an incidence of 22.0%. TCrP exposure also caused dilation of the efferent duct in the testes with maximum duct widths of 83.3, 93.2, and 149.7 µm in the 657.9, 1,511, and 4042 ng/L exposure groups, respectively. These widths were all significantly larger than that observed in the control group (37.7 µm) and likely contributed substantially to fertilization inhibition. The TCrP metabolites 4-OH-MDTP and 3-OH-MDTP, were detected at high concentrations in the liver and elicited 5.8-fold and 5.3-fold greater androgen receptor antagonistic activity than that elicited by TCrP (39.8 µM), which may explain the intersex observed in low exposure groups. 4-OH-MDTP and 3-OH-MDTP elicited anti-estrogenic activities by blocking the estrogen receptor, and the concentrations at which its responses were equal to the IC20 of tamoxifen were 16.1 µM and 18.9 µM, respectively, as detected using the yeast two-hybrid assay. Such anti-estrogenic activities were likely the main driver of dilation of the efferent duct. Observed adverse outcomes after exposure to TCrP all occurred under environmentally relevant concentrations, suggesting considerable ecological risk to wild fish.

Toxicity and endocrine-disrupting potential of PM2.5: Association with particulate polycyclic aromatic hydrocarbons, phthalate esters, and heavy metals.

The adverse effects of fine atmospheric particulate matter with aerodynamic diameters of ≤2.5 µm (PM2.5) are closely associated with particulate chemicals. In this study, PM2.5 samples were collected from highway and industry sites in Hangzhou, China, during the autumn and winter, and their cytotoxicity and pulmonary toxicity and endocrine-disrupting potential (EDP) were evaluated in vitro and in vivo; the particulate polycyclic aromatic hydrocarbons (PAHs), phthalate esters (PAEs), and heavy metals were then characterized. The toxicological results suggested that the PM2.5 from highway site induced higher cytotoxicity (cell viability inhibition, intracellular oxidative stress, and cell membrane injury) and pulmonary toxicity (inflammatory response (IR) and oxidative stress (OS)) than the samples from industry site, while the PM2.5 from industry site exhibited higher EDP (estrogenic and anti-androgenic activity). The cytotoxicity and pulmonary toxicity of PM2.5 in the winter were higher than those in the autumn, while no seasonal difference in the endocrine-disrupting potential was observed (p > 0.05). The Pearson correlation analysis between the biological effects and particulate chemicals revealed that the PM2.5-induced inflammatory response and oxidative stress were closely associated with the particulate PAHs and heavy metals (Pearson correlation coefficients: rIR, PAHs = 0.822-0.988, rIR, heavy metals = 0.895-0.971, rOS, PAHs = 0.843-0.986, and rOS, heavy metals = 0.887-0.933), while particulate di (2-ethylhexyl)phthalate (DEHP) substantially contributed to the EDP of PM2.5 (rEDP, DEHP = 0.981). This study indicated that the toxicity and EDP of PM2.5 could vary with the surrounding environment and season, which was closely associated with the variations of particulate chemicals. Further studies are needed to clarify the associations between the harmful effects of PM2.5 and other contributing factors.