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International Journal of Molecular... Feb 202317α-Methyltestosterone (MT), a synthetic environmental endocrine disruptor with androgenic effects, has been shown to disrupt the reproductive system and inhibit germ...
17α-Methyltestosterone (MT), a synthetic environmental endocrine disruptor with androgenic effects, has been shown to disrupt the reproductive system and inhibit germ cell maturation in . To further investigate the regulation of gonadal development by MT through the hypothalamic-pituitary-gonadal (HPG) axis, were exposed to 0, 25, 50, and 100 ng/L of MT for 7, 14, and 21 days. We analyzed its biological indicators, gonadotropin-releasing hormone (GnRH), gonadotropins, reproduction-related gene expression, and brain tissue transcriptome profiles. We found a significant decrease in the gonadosomatic index (GSI) in males exposed to MT for 21 days compared to the control group. GnRH, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels, as well as the expressions of the , , , , and genes, were significantly reduced in the brains of both male and female fish when exposed to 100 ng/L MT for 14 days compared to the controls. Therefore, we further constructed four RNA-seq libraries from 100 ng/L MT-treated groups of male and female fish, obtaining 2412 and 2509 DEGs in male and female brain tissue, respectively. Three common pathways were observed to be affected in both sexes after exposure to MT, namely, nicotinate and nicotinamide metabolism, focal adhesion, and cell adhesion molecules. Furthermore, we found that MT affected the PI3K/Akt/FoxO3a signaling pathway through the upregulation of and , and the downregulation of and . Therefore, we hypothesize that MT interferes with the levels of gonadotropin-releasing hormone (GnRH, FSH, and LH) in brains through the PI3K/Akt/FoxO3a signaling pathway, and affects the expression of key genes in the hormone production pathway (, and ) to interfere with the stability of the HPG axis, thus leading to abnormal gonadal development. This study provides a multidimensional perspective on the damaging effects of MT on fish and confirms that is a suitable model animal for aquatic toxicology.
Topics: Animals; Female; Male; Methyltestosterone; Transcriptome; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Cyprinidae; Gonadal Steroid Hormones; Cypriniformes; Brain; Gonadotropin-Releasing Hormone; Follicle Stimulating Hormone
PubMed: 36834982
DOI: 10.3390/ijms24043571 -
Biochemical and Biophysical Research... Apr 2023In pharmacokinetics plasma protein binding (PPB) is a well-established parameter impacting drug disposition. The unbound fraction (f) is arguably regarded the effective...
In pharmacokinetics plasma protein binding (PPB) is a well-established parameter impacting drug disposition. The unbound fraction (f) is arguably regarded the effective concentration at the target site. Pharmacology and toxicology, increasingly use in vitro models. The translation of in vitro concentrations to in vivo doses can be supported by toxicokinetic modelling, e.g. physiologically based toxicokinetic models (PBTK). PPB of a test substance is an input parameter for PBTK. We compared three methods to quantify f: rapid equilibrium dialysis (RED), ultrafiltration (UF) and ultracentrifugation (UC) using twelve substances covering a wide range of Log P (-0.1 to 6.8) and molecular weights (151 and 531 g/mol): Acetaminophen, Bisphenol A, Caffeine, Colchicine, Fenarimol, Flutamide, Genistein, Ketoconazole, α-Methyltestosterone, Tamoxifen, Trenbolone and Warfarin. After RED and UF separation, three polar substances (Log P < 2) were largely unbound (f > 70%), while more lipophilic substances were largely bound (f < 33%). Compared to RED or UF, UC resulted in a generally higher f of lipophilic substances. f obtained after RED and UF were more consistent with published data. For half of the substances, UC resulted in f higher than the reference data. UF, RED and both UF and UC, resulted in lower f of Flutamide, Ketoconazole and Colchicine, respectively. For f quantifications, the separation method should be selected according to the test substance's properties. Based on our data, RED is suitable for a broader range of substances while UC and UF are suitable for polar substances.
Topics: Ultrafiltration; Flutamide; Ketoconazole; Renal Dialysis; Protein Binding; Blood Proteins; Ultracentrifugation
PubMed: 36812744
DOI: 10.1016/j.bbrc.2023.02.021 -
Molecules (Basel, Switzerland) Jan 2023Steroid hormone molecules may exhibit very different functionalities based on the associated functional groups and their 3D arrangements in space, i.e., absolute...
Steroid hormone molecules may exhibit very different functionalities based on the associated functional groups and their 3D arrangements in space, i.e., absolute configurations and conformations. Infrared (IR) and vibrational circular dichroism (VCD) spectra of four different steroid hormones, namely dehydroepiandrosterone (DHEA), 17-methyltestosterone (MTTT), (16α,17)-epoxyprogesterone (Epoxy-P4), and dehydroepiandrosterone acetate (AcO-DHEA), were measured in deuterated dimethyl sulfoxide and some also in carbon tetrachloride. Extensive conformational searches were carried out using the recent developed conformer-rotamer ensemble sampling tool (CREST) which also accounts for solvent effects using an implicit solvation model. All the CREST conformational candidates were then reoptimized at the B3LYP-D3BJ/def2-TZVPD with the PCM of solvent. The good agreements between the experimental IR and VCD spectra and the theoretical simulations provide a conclusive information about their conformational distribution and absolute configurations. The experimental and theoretical IR and VCD spectra of AcO-DHEA in the carbonyl and alkene stretching region showed some discrepancies, and the possible causes related to solvent effects, large amplitude motions and levels of theory used in the modelling were explored in detail. As part of the investigation, additional calculations at the B3LYP-D3BJ/6-31++G (2d,p) and B3LYP-D3BJ/cc-pVTZ levels, as well as some 'mixed' calculations with the double-hybrid functional B2PLYP-D3 were also carried out. The results indicate that the double-hybrid functional is important for predicting the correct IR band pattern in the carbonyl and alkene stretching region.
PubMed: 36677830
DOI: 10.3390/molecules28020771 -
Ecotoxicology and Environmental Safety Jan 2023Endocrine disruptors (EDs), capable of modulating the sex hormone system of an organism, can exert long-lasting negative effects on reproduction in both humans and the...
Endocrine disruptors (EDs), capable of modulating the sex hormone system of an organism, can exert long-lasting negative effects on reproduction in both humans and the environment. For these reasons, the properties of EDs prevent a substance from being approved for marketing. However, regulatory testing to evaluate endocrine disruption is time-consuming, costly, and animal-intensive. Here, we combined sublethal zebrafish embryo assays with transcriptomics and proteomics for well-characterized endocrine disrupting reference compounds to identify predictive biomarkers for sexual endocrine disruption in this model. Using RNA and protein gene expression fingerprints from two different sublethal exposure concentrations, we identified specific signatures and impaired biological processes induced by ethinylestradiol, tamoxifen, methyltestosterone and flutamide 96 h post fertilization (hpf). Our study promotes vtg1 as well as cyp19a1b, fam20cl, lhb, lpin1, nr1d1, fbp1b, and agxtb as promising biomarker candidates for identifying and differentiating estrogen and androgen receptor agonism and antagonism. Evaluation of these biomarkers for pre-regulatory zebrafish embryo-based bioassays will help identify endocrine disrupting hazards of compounds at the molecular level. Such approaches additionally provide weight-of-evidence for the identification of putative EDs and may contribute significantly to a reduction in animal testing in higher tier studies.
Topics: Animals; Biomarkers; Embryo, Nonmammalian; Endocrine Disruptors; Endocrine System; Estrogens; Gene Expression; Phosphatidate Phosphatase; Water Pollutants, Chemical; Zebrafish
PubMed: 36608563
DOI: 10.1016/j.ecoenv.2023.114514 -
Environmental Toxicology and Chemistry Mar 2023Japanese medaka is specified as a model fish in the test guidelines of the Organisation for Economic Co-operation and Development. Recently, populations of Japanese...
Japanese medaka is specified as a model fish in the test guidelines of the Organisation for Economic Co-operation and Development. Recently, populations of Japanese medaka in Japan were divided into two species, the northern Oryzias sakaizumii and the southern O. latipes. Previously, we reported that induction concentrations for sex reversal by exposure to 17α-methyltestosterone differed significantly between these two species, indicating that they respond differently to endocrine-disrupting chemica. In the present study, we examined the effects of exposure to two more endocrine-disrupting chemicals (bisphenol A and 17β-trenbolone) in O. sakaizumii, and compared the results with those previously reported for O. latipes. Exposure to both bisphenol A and 17β-trenbolone induced testis-ova formation or sex reversal in O. sakaizumii. Exposure to 17β-trenbolone also increased expression of gonadal soma-derived factor (gsdf). Least-observed-effect concentrations for gonadal sex differentiation and gsdf expression were lower for O. latipes than for O. sakaizumii after exposure to bisphenol A, and were lower for O. sakaizumii than for O. latipes after exposure to 17β-trenbolone. These results demonstrate that O. sakaizumii and O. latipes respond differently to androgenic and estrogenic endocrine-disrupting chemicals. Environ Toxicol Chem 2023;42:673-678. © 2022 SETAC.
Topics: Animals; Male; Gonads; Oryzias; Testis; Trenbolone Acetate; Female
PubMed: 36582147
DOI: 10.1002/etc.5552 -
Molecular Biology Reports Mar 2023Paris polyphylla var. yunnanensis is an important medicinal plant, and the main active ingredient of the plant is polyphyllin, which is a steroid saponin with...
BACKGROUND
Paris polyphylla var. yunnanensis is an important medicinal plant, and the main active ingredient of the plant is polyphyllin, which is a steroid saponin with pharmacological activities. The central enzyme genes participating in the biosynthesis of polyphyllin are increasingly being uncovered; however, UGTs are rarely illustrated.
METHODS AND RESULTS
In this study, we cloned a new sterol glycosyltransferase from Paris polyphylla var. yunnanensis and identified its catalytic function in vitro. PpUGT6 showed the ability to catalyse the C-3 glycosylation of pennogenin sapogenin of polyphyllin, and PpUGT6 showed catalytic promiscuity towards steroids at the C-17 position of testosterone and methyltestosterone and the triterpene at the C-3 position of glycyrrhetinic acid. Homology modelling of the PpUGT6 protein and virtual molecular docking of PpUGT6 with sugar acceptors and donors were performed, and we predicted the key residues interacting with ligands.
CONCLUSIONS
Here, PpUGT6, a novel sterol glycosyltransferase related to the biosynthesis of polyphyllin from P. polyphylla, was characterized. PpUGT6 catalysed C-3 glycosylation to pennogenin sapogenin of polyphyllin, which is the first glycosylation step of the biosynthetic pathway of polyphyllins. Interestingly, PpUGT6 demonstrated glycodiversification to testosterone and methyltestosterone at C-17 and triterpene of glycyrrhetinic acid at the C-3 position. The virtual molecular docking of PpUGT6 protein with ligands predicted the key residues interacting with them. This work characterized a novel SGT glycosylating pennogenin sapogenin at C-3 of polyphyllin from P. polyphylla and provided a reference for further elucidation of the phytosterol glycosyltransferases in catalytic promiscuity and key residues interacting with substrates.
Topics: Sterols; Sapogenins; Glycosyltransferases; Methyltestosterone; Ligands; Molecular Docking Simulation; Steroids; Liliaceae; Glycyrrhetinic Acid
PubMed: 36562935
DOI: 10.1007/s11033-022-08199-y -
Heliyon Dec 2022The 17α-methyltestosterone is the most common synthetic hormone used in male mono-sex production of Nile tilapia, . The current research aimed at finding out the most...
The 17α-methyltestosterone is the most common synthetic hormone used in male mono-sex production of Nile tilapia, . The current research aimed at finding out the most effective dose of 17α-methyltestosterone to produce quality Nile tilapia fry. Soon after absorbing the yolk sac, Nile tilapia fry was fed with a mixture of commercial fish feed and 17α-methyltestosterone for 28 days. Five doses of 17α-methyltestosterone, i.e., 0 mg, 50 mg, 60 mg, 70 mg, and 80 mg per kg feed, were used to treat tilapia that has been reared for additional 90 days to compare sex reversal, development, and survival rates. Both gonad histology and Squash test were performed to expose the sex percentage of accurately. The highest male 94.44% was obtained at 60 mg 17α-MT/kg feed dose followed by 91.67%, 88.89%, 86.11%, and 47.22% at 70, 80, 50, and 0 mg 17α-MT/kg feed dose. The groups treated with 17α-methyltestosterone hormone showed superior growth performance in comparison to the control group. The highest weight (14.62 ± 0.59 g) and length (92.18 ± 3.01 mm) were found at 60 mg dose whereas the lowest weight (8.64 ± 0.38 g) and length (70.17 ± 3.75 mm) were in the control group. The group given 60 mg 17α-MT feed represented the highest survival rate (84.10%) among other hormone-treated groups. The study disclosed that 60 mg 17α-MT/kg feed might be treated as the optimal dose for producing quality mono-sex male tilapia in the commercial hatchery.
PubMed: 36536906
DOI: 10.1016/j.heliyon.2022.e12252 -
Steroids Feb 2023Δ6-Methyltestosterone was reported as the main active ingredient of the purported "dietary supplement" Jungle Warfare. This compound is structurally similar to...
Δ6-Methyltestosterone was reported as the main active ingredient of the purported "dietary supplement" Jungle Warfare. This compound is structurally similar to 17α-methyltestosterone, containing an additional Δ6 double bond, and is reported to possess notable androgenic activity, raising concerns over the potential for abuse of Jungle Warfare in sport. The in vivo metabolism of Δ6-methyltestosterone in greyhounds was investigated. Urinary phase I (unconjugated) and phase II (glucuronide) metabolites were detected following oral administration using liquid chromatography-mass spectrometry. No phase II sulfate metabolites were detected. The major phase I metabolite was confirmed as 16α,17β-dihydroxy-17α-methylandrosta-4,6-dien-3-one by comparison with a synthetically-derived reference material. Minor amounts of the parent drug were also confirmed. Glucuronide conjugated metabolites were also observed, but were found to be resistant to hydrolysis using the Escherichia coli β-glucuronidase enzyme. Qualitative excretion profiles, limits of detection, and extraction recoveries were determined for the parent drug and the major phase I metabolite. These results provide a method for the detection of Jungle Warfare abuse in greyhounds suitable for incorporation into routine screening methods conducted by anti-doping laboratories.
Topics: Animals; Dogs; Methyltestosterone; Gas Chromatography-Mass Spectrometry; Glucuronides; Androgens; Mass Spectrometry; Doping in Sports; Anabolic Agents; Substance Abuse Detection
PubMed: 36511323
DOI: 10.1016/j.steroids.2022.109150 -
Comparative Biochemistry and... Feb 2023This study aimed to establish zebrafish-based in vivo and in silico assay systems to evaluate the antiandrogenic potential of environmental chemicals. Zebrafish embryos...
This study aimed to establish zebrafish-based in vivo and in silico assay systems to evaluate the antiandrogenic potential of environmental chemicals. Zebrafish embryos were exposed to 17α-methyltestosterone (TES) alone or coexposed to TES and representative antiandrogens including flutamide, p,p'-DDE, vinclozolin, fenitrothion, and linuron. We assessed the transcript expression of the androgen-responsive gene sulfotransferase family 2, cytosolic sulfotransferase 3 (sult2st3). The expression of sult2st3 was significantly induced by TES in the later stages of embryonic development. However, the TES-induced expression of sult2st3 was inhibited by flutamide in a concentration-dependent manner (IC: 5.7 μM), suggesting that the androgen receptor (AR) plays a role in sult2st3 induction. Similarly, p,p'-DDE, vinclozolin, and linuron repressed the TES-induced expression of sult2st3 (IC: 0.35, 3.9, and 52 μM, respectively). At the highest concentration tested (100 μM), fenitrothion also suppressed sult2st3 expression almost completely. Notably, p,p'-DDE and linuron did not inhibit sult2st3 induction due to higher concentrations of TES; instead, they potentiated TES-induced sult2st3 expression. Fenitrothion and linuron, which had relatively low antiandrogenic potentials in terms of sult2st3 inhibition, induced broader toxicities in zebrafish embryos; thus, the relationship between developmental toxicities and antiandrogenic potency was unclear. Additionally, an in silico docking simulation showed that all five chemicals interact with the zebrafish AR at relatively low interaction energies and with Arg702 as a key amino acid in ligand binding. Our findings suggest that a combination of zebrafish-based in vivo and in silico assessments represents a promising tool to assess the antiandrogenic potentials of environmental chemicals.
Topics: Animals; Flutamide; Zebrafish; Dichlorodiphenyl Dichloroethylene; Fenitrothion; Linuron
PubMed: 36442599
DOI: 10.1016/j.cbpc.2022.109513 -
Journal of Morphology Jan 2023There are few detailed descriptions of the morphology of the male external genitalia, the urogenital papilla (UGP), of the Black Rockfish (Sebastes schlegelii...
There are few detailed descriptions of the morphology of the male external genitalia, the urogenital papilla (UGP), of the Black Rockfish (Sebastes schlegelii Hilgendorf, 1880). The purpose of this study was to evaluate this organ histologically and to determine the time of development of the UGP in Black Rockfish. Twelve adult males, three adult females and around 500 juveniles were used in the experiment. The juveniles were divided into normal developmental and androgen groups. The androgen group was exposed to methyltestosterone (100 μg/L) for 2 h daily for 38 days. Samples (N = 10 per sampling) were randomly selected for analysis every 5 days from 30 to 116 days after birth. Parameters assessed included the type of epithelium, composition of connective tissue, muscular tissue, and the timing of UGP development. Differences in these parameters between normal developmental and androgen groups were evaluated. The results indicated that the UGP of the adult fish contains the sperm duct and ureter, which have the function of transporting sperm and urine, respectively. The androgen-treated juvenile fish developed the UGP earlier than the normal development group. This study provides a reference for understanding the external genitalia of other viviparous fishes by studying the UGP of the male Black Rockfish.
Topics: Female; Male; Animals; Androgens; Phylogeny; Semen; Perciformes; Fishes; Fertilization
PubMed: 36373244
DOI: 10.1002/jmor.21534