Methyltestosterone Research Articles

Validation of precision-cut liver slices to study drug-induced cholestasis: a transcriptomics approach

Hepatotoxicity is one of the major reasons for withdrawal of drugs from the market. Therefore, there is a need to screen new drugs for hepatotoxicity in humans at an earlier stage. The aim of this study was to validate human precision-cut liver slices (PCLS) as an ex vivo model to predict drug-induced cholestasis and identify the possible mechanisms of cholestasis-induced toxicity using gene expression profiles. Five hepatotoxicants, which are known to induce cholestasis (alpha-naphthyl isothiocyanate, chlorpromazine, cyclosporine, ethinyl estradiol and methyl testosterone) were used at concentrations inducing low (<30 %) and medium (30–50 %) toxicity, based on ATP content. Human PCLS were incubated with the drugs in the presence of a non-toxic concentration (60 µM) of a bile acid mixture (portal vein concentration and composition) as model for bile acid-induced cholestasis. Regulated genes include bile acid transporters and cholesterol transporters. Pathway analysis revealed that hepatic cholestasis was among the top ten regulated pathways, and signaling pathways such as farnesoid X receptor- and liver X receptor-mediated responses, which are known to play a role in cholestasis, were significantly affected by all cholestatic compounds. Other significantly affected pathways include unfolded protein response and protein ubiquitination implicating the role of endoplasmic reticulum stress. This study shows that human PCLS incubated in the presence of a physiological bile acid mixture correctly reflect the pathways affected in drug-induced cholestasis in the human liver. In the future, this human PCLS model can be used to identify cholestatic adverse drug reactions of new chemical entities.

Probing Steroidal Substrate Specificity of Cytochrome P450 BM3 Variants.

M01A82W, M11A82W and M01A82WS72I are three cytochrome P450 BM3 (CYP102A1) variants. They can catalyze the hydroxylation of testosterone (TES) and norethisterone at different positions, thereby making them promising biocatalysts for steroid hydroxylation. With the aim of obtaining more hydroxylated steroid precursors it is necessary to probe the steroidal substrate diversity of these BM3 variants. Here, three purified BM3 variants were first incubated with eight steroids, including testosterone (TES), methyltestosterone (MT), cholesterol, β-sitosterol, dehydroepiandrosterone (DHEA), diosgenin, pregnenolone and ergosterol. The results indicated that the two 3-keto-Δ4-steroids TES and MT can be hydroxylated at various positions by the three BM3 mutants, respectively. On the contrary, the three enzymes displayed no any activity toward the remaining six 3-hydroxy-Δ5-steroids. This result indicates that the BM3 mutants prefer 3-keto-Δ4-steroids as hydroxylation substrates. To further verify this notion, five other substrates, including two 3-hydroxy-Δ5-steroids and three 3-keto-Δ4-steroids, were carefully selected to incubate with the three BM3 variants. The results indicated the three 3-keto-Δ4-steroids can be metabolized to form hydroxysteroids by the three BM3 variants. On the other hand, the two 3-hydroxy-Δ5-steroids cannot be hydroxylated at any position by the BM3 mutants. These results further support the above conclusion, therefore demonstrating the 3-keto-Δ4–steroid substrate preference of BM3 mutants, and laying a foundation for microbial production of more hydroxylated steroid intermediates using BM3 variants.

Low dose of methyltestosterone in ovariectomised rats improves baroreflex sensitivity without geno‐ and cytotoxicity

This study evaluated the effects of the isolated use of a low dose of methyltestosterone (MT) on cardiovascular reflexes and hormonal levels and its geno- and cytotoxic safety in ovariectomized rats. Female Wistar rats were divided into four groups (n = 6), respectively: SHAM (received vehicle methylcellulose 0.5%), SHAM + MT (received MT 0.05 mg/kg), OVX (received vehicle), and OVX + MT (received MT). Twenty-one days after ovariectomy, treatment was given orally daily for 28 days. The Bezold-Jarisch reflex (BJR) was analyzed by measuring the bradycardic and hypotensive responses elicited by phenylbiguanide (PBG) administration. The baroreflex sensitivity (BRS) was evaluated by phenylephrine and sodium nitroprussite. Myocyte hypertrophy was determined by morphometric analysis of H&E stained slides. Biochemical data were analyzed, as well as micronucleus assay. MT improved BRS and increased testosterone values, but did not change estradiol in the OVX group. MT did not promote changes in mean arterial pressure, heart rate, BJR, serum concentrations of troponin I, weight and histopathology of the heart. MT was able to restore the BRS in OVX rats. The geno- and cytotoxic safety of the MT was demonstrated by the absence of an increase in the micronucleus (PCEMN) or change in the ratio between normochromatic erythrocytes and polychromatic erythrocytes (NCE/PCE).

22. PENGARUH UMUR LARVA IKAN NILA (Oreochromis niloticus) TERHADAP PENINGKATAN PENJANTANAN MENGGUNAKAN HORMON METHYL TESTOSTERONE (MT) ALAMI Effect of Tilapia (Oreochromis niloticus) Age in Masculinization Process Using Methyl Testosterone (MT) Natural Hormones

This research aims to know the influence of larvae of nile tilapia (Oreochromis niloticus) age on masculinization process by the administration of methyl testosterone (MT) natural hormones. This research used 270 nile tilapia larvae divided into 3 age of groups: 5 days (PI), 10 days (PII), and 20 days (PIII), with three repetitions. Larvae were dipped in MT natural hormone for 20 days then reared until age of 60 days, followed by observations of fish sex. The average percentage of male-sex of tilapia fish after dipping with methyl testosterone natural hormone on group PI, PII, and PIII were 68.07%, 66.47%, and 57.29%, respectively. It can be concluded that the age of larvae do not affect the percentage of masculinization process of nile tilapia fish. ____________________________________________________________________________________________________________________ Key words: larvae tilapia, masculinization, methyl testosterone natural hormone

14. PENGARUH LAMA PERENDAMAN LARVA DALAM HORMON METIL TESTOSTERON ALAMI TERHADAP PENJANTANAN IKAN LELE DUMBO (Clarias gariepinus) The Effect of Soaking Time of African Catfish Larvae (Clarias gariepinus) in Natural Methyl Testosterone Hormone on Masculinization

The purpose of this study was to determine the effect of immersion time on African catfish (Clarias gariepinus) larvae in natural methyl testosterone hormone on masculinization. This study was conducted in Center of Fish Culture Lamnyong, Banda Aceh. Experimental animals used in this study were 270 African catfish larvae aged 10 days. The study was designed using completely randomized design (CRD) with 3 treatments and 3 replications. African catfish larvae aged 10 days in group P1, P2, and P3 were immersed in natural methyl testosterone hormone for 10 hours, 20 hours, and 30 hours, respectively. Observations were carried out until fish reach 50 days old. Data were analyzed by analysis of variance (Anova) followed by least significant difference test (LSD). Percentage of male catfish observed in P1, P2, and P3 were 64.24 %, 93.11%, dan 59.94% respectively. As a conclusion, immersion with natural methyl testosterone hormone can change the sex of catfish to be male. ____________________________________________________________________________________________________________________ Key words: african catfish, methyl testosterone natural, masculinization

Male-typical courtship, spawning behavior, and olfactory sensitivity are induced to different extents by androgens in the goldfish suggesting they are controlled by different neuroendocrine mechanisms

Male-typical reproductive behaviors vary greatly between different species of fishes with androgens playing a variety of roles that appear especially important in the gonochorist cypriniform fishes. The goldfish is an important model for the cypriniformes and while it is clear that male goldfish are fully feminized by prostaglandin F2α(PGF2α), it is not clear whether females will exhibit normal levels of male-typical reproductive behaviors as well as olfactory function when treated with androgens. To answer this question, we exposed sexually-regressed adult female goldfish to several types of androgen and monitored their tendencies to court (inspect females) and mate (spawn, or attempt to release gametes) while monitoring their olfactory sensitivity until changes in these attributes were maximized. Untreated adult males (intact) were included to determine the extent of masculinization. Treatments included the natural androgens, 11-ketotestosterone and testosterone (KT and T), administered via capsules (KT+T-implanted fish); the artificial androgen, methyltestosterone (MT), administered via capsules (MT-C); and MT administered in the fishes’ water (MT-B). Male-typical olfactory sensitivity to a pheromone (15keto-PGF2α) increased in all androgen-treated groups and by week 6 was fully equivalent to that of males. Male-typical courtship behavior increased in all androgen-treated groups although slowly, and only MT-B females came to exhibit levels equivalent to those of males after 18weeks. In contrast, male-typical mating activity increased only slightly, with MT-B females reaching levels one-third that of males after 30weeks. We conclude that while androgens fully masculinize olfactory sensitivity and courtship behavior in goldfish, mating behavior is controlled by a different neuroendocrine mechanism(s) that has yet to be fully elucidated.

PENGARUH UMUR TERHADAP PENJANTANAN LARVA IKAN LELE DUMBO (Clarias gariepinus) YANG DIRENDAM MENGGUNAKAN HORMON METIL TESTOTERON ALAMI (The Influence of Age on Masculinization of Clarias gariepinus Larvae Immersed by Methyl Testosterone Natural Hormone)

This research aims to examines the influence of age on masculinization of Clarias gariepinus larvae immersed by methyl testosterone (MT) natural hormone. This research used 270 catfish larvae divided into 3 groups of age those are 5, 10, and 20 days. The research implemented complete random design (CRD). Immersion was performed using methyl testosterone hormone at the dose of 0.25 g/L for 20 hours. Observation of fish was done after 60 day-old fish. Data obtained was analyzed using one-way analysis of variance The average percentages of male-sex catfish after immersing in natural MT on group I, II, and III were 97.67, 91.07, and 86.67%, respectively. The results showed that the age of the larvae did not affect significantly (P&gt;0.05) on the percentage of male sex of Clarias gariepinus. The average percentage of male sex in 5 days age group was higher than 10 and 20 days age group. In conclusion, the masculinization of Clarias gariepinus larva immersed by MT natural hormone is not influenced by age of fish before dipping.

Oxidative Stress Induction in Monosex Nile Tilapia (Oreochromis niloticus, Linnaeus, 1758): A Field Study on the Side Effects of Methyltestosterone

In this survey, fishes were obtained from four localities: Assiut as a control and Beheira, Alexandria and Kafr EL-Sheikh; three farms from each governate as farmed monosex produced using Methyltestosterone (MT). Serum MT, total antioxidant capacity (TAC), malondialdehyde (MDA), and total peroxides (TPX) were estimated, followed by calculation of oxidative stress index (OSI). MT concentration in the serum of fishes farmed at Assiut showed no detectable levels of hormonal residues, while the monosex farms showed high levels of MT concentration in the serum of the sampled fishes. In comparison with control fishes of Assiut farms, serum TAC levels of monosex fishes collected from farms of Beheira and Alexandria were significantly lower. Serum TPX content of the monosex fishes obtained from Alexandria farms were significantly higher than the wild fishes obtained from Assiut farms. Calculated from ratio of serum TPX content and TAC concentration, OSI illustrated a significant difference between control fishes collected from Assiut and monosex fishes collected from Beheira. Although few significant changes were found in the examined oxidative stress endpoints, the results of this work put our foot in the beginning of road to link hormonally sex reversal practice with oxidative stress induction, and provocative for other researchers to invade this field of research more deeply utilizing more specific relevant markers and cutting-edge techniques

Cloning, expression of, and evidence of positive selection for, the prolactin receptor gene in Chinese giant salamander (Andrias davidianus).

Prolactin receptor (PRLR) is a protein associated with reproduction in mammals and with osmoregulation in fish. In this study, the complete length of Chinese giant salamander Andrias davidianus prolactin receptor (AD-prlr) was cloned. Andrias davidianus prlr expression was high in the kidney, pituitary, and ovary and low in other examined tissues. The AD-prlr levels were higher in ovary than in testis, and increased in ovaries with age from 1 to 6 years. To determine effect of exogenous androgen and aromatase inhibitor on AD-prlr expression, methyltestosterone (MT) and letrozole (LE) were injected, resulting in decreased AD-prlr in both brain and ovary, with MT repressing prlr transcription more rapidly than did LE. The molecular evolution of prlr was assessed, and found to have undergone a complex evolution process. The obranch-site test detected four positively selected sites in ancestral lineages prior to the separation of mammals and birds. Fourteen sites underwent positive selection in ancestral lineages of birds and six were positively selected in amphibians. The site model showed that 16, 7, and 30 sites underwent positive selection in extant mammals, amphibians, and birds, respectively. The positively selected sites in amphibians were located outside the transmembrane domain, with four in the extracellular and three in the intracellular domain, indicating that the transmembrane region might be conserved and essential for protein function. Our findings provide a basis for further studies of AD-prlr function and molecular evolution in Chinese giant salamander. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 707-719, 2015. © 2015 Wiley Periodicals, Inc.

Effect of cold shock and hormone on growth and male morphotypes of freshwater prawn,Macrobrachium rosenbergii(de Man)

A study was carried out to determine the effects of cold shock and 17α-methyl testosterone hormone on growth in male morphotypes of giant freshwater prawn Macrobrachium rosenbergii. Juvenile males were exposed at 18 ± 0.25°C for 24 h with normal feed and cultured at 27 ± 0.7°C for 70 days as well as administrated feed with hormone separately at 400 mg kg−1 feed for the first 30 days of culture period (70 days). The growth (Mean ± SD) of male juveniles at harvest was closely homogenous in the cold shock (CLS) treated group; while it was heterogeneous in hormone-treated (MH) and control (CO) groups. Although total body weight of blue claw (BC) males was not significantly different in the controls and the treatments, the 2nd pereiopods (claw) weight and length of the BC was significantly lower in CLS (0.52 ± 0.05 g, 10.02 ± 0.65 cm) than that in the CO (0.71 ± 0.16 g, 12.53 ± 0.57 cm) and MH (0.75 ± 0.08 g, 12.85 ± 1.18 cm) treatments. Survival of prawns was similar in treatments. The morphotypes of male prawn were significantly different with 59% of BC males with small-sized claws in CLS and, 45% and 30% of BC males with large-sized claws in MH and CO respectively. Cold shock adversely affected male morphotypes of the prawn so that numbers of BC males with small-sized claw was the highest in CLS tank. However, the CLS treatment resulted in more uniform marketable prawns and suggested a high production of BC males in an all-male culture system.

Tentative Structural Assignment of a Glucuronide Metabolite of Methyltestosterone in Tilapia Bile by Liquid Chromatography–Quadrupole-Time-of-Flight Mass Spectrometry

Methyltestosterone (MT), a strong androgenic steroid, is not approved for use in fish aquaculture in the United States. It is used in the U.S. under an investigational new animal drug exemption (INAD) only during the early life stages of fish. There is a possibility that farmers feed fish with MT to enhance production for economic gains. Therefore, there is a need to develop methods for the detection of MT and its metabolite residues in fish tissue for monitoring purposes. Previously, our laboratory developed a liquid chromatography-quadrupole time-of-flight (LC-QTOF) method for characterization of 17-O-glucuronide metabolite (MT-glu) in bile of tilapia dosed with MT. The system used was an Agilent 6530 Q-TOF equipped with electrospray jet stream technology, operating in positive ion mode. Retrospective analysis of the data generated in that experiment by a feature-finding algorithm, combined with a search against an in-house library of possible MT-metabolites, resulted in the discovery of a major glucuronide metabolite of MT in the bile extracts. Preliminary data indicate it to be a glucuronide of a hydroxylated MT (OHMT-glu) which persists in tilapia bile for at least 2 weeks after dosing. We present the tentative structural assignment of the OHMT-glu in tilapia bile and time course of development. This glucuronide can serve as a marker to monitor illegal use of MT in tilapia culture.

Sensitive, fast, and specific immunoassays for methyltestosterone detection.

An indirect competitive enzyme-linked immunosorbent assay (icELISA) and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT) residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%–100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed.

Preliminary Report on the use of Androgen for the Production of All-male &lt;i&gt;Oreochromis niloticus&lt;/i&gt;

Fresh hatchlings of Oreochromis niloticus was fed with 17 α methyltestosterone (MT) at 30 µg/g diet for 28 days. Comparative analysis of hormone treated fish with non hormone treated fish indicated improved growth in the former by 44.3% in six months of rearing in outdoor concrete tanks. In both indoor and outdoor rearing conditions, hormone treatment did not have any negative significant effect on survival. Sex ratio in the hormone treated group was 12 males: 1female, and 1.5 males: 1 female in the untreated group. There is a significant difference in the expected 1:1 ratio of sexes in MT treated group which indicated that sex reversal of genetic females to males was successful using 17 α methyltestosterone at 30 µg/g diet for 28 days.

COMPARATIVE GROWTH PERFORMANCE OF MALE MONOSEX AND MIXED SEX NILE TILAPIA (Oreochromis niloticus L.) REARED IN EARTHEN PONDS

Comparative growth performance of male monosex and mixed sex Nile tilapia (Oreochromis niloticus ) was conducted in six earthen ponds each measuring 150 m 2 at the Kenya Marine & Fisheries Research Institute (KMFRI), Kegati Aquaculture Research Station. Male monosex and mixed sex of O. niloticus juveniles at an average weight of 12.2 ± 0.02 g and average length of 7.6 ± 0.02 cm were collected from nursery production ponds. The male monosex juveniles were obtained by feeding newly hatched juveniles with feed dosed with 17 α methyl testosterone hormone for a period of one month. Stocking was at 3 juvenile

Effects of spray-drying on w/o/w multiple emulsions prepared from a stearic acid matrix.

The goal of this study was to explore the effects of spray-drying on w/o/w double emulsions of methyltestosterone (MT) loaded in a stearic acid matrix. MT-loaded nanoparticles were formulated by a water-in-oil-in-water emulsion technique using 50, 75, and 100 mg of stearic acid, 2% and 3% w/v polyvinyl alcohol, 5% w/v lactose, and 0.2% w/v chitosan. The emulsions were immediately spray-dried based on an optimized model of inlet temperature and pump rate, and characterized for optimized responses with regard to particle size, polydispersity index, and zeta potential, for both emulsion and powder samples. Dynamic light scattering analysis shown that the nanoparticles increased in size with increasing concentrations of polyvinyl alcohol and stearic acid. Scanning electron microscopy indicated that the MT-loaded nanoparticles were spherical in shape, had a smooth surface, and were in an amorphous state, which was confirmed by differential scanning calorimetry. These MT-loaded nanoparticles are a promising candidate carrier for the delivery of MT; however, further studies are needed in order to establish the stability of the system and the cargo release profile under normal conditions of use.

Highly sensitive “signal on” plasmonic ELISA for small molecules by the naked eye

Based on the H2O2-mediated generation of gold nanoparticles, a colorimetric catalase nanosensor was developed and utilized as a signal output in a catalase-labelled immunoassay. Methyltestosterone (MT), which is used illegally as a growth promoter during the cattle production cycle, was selected as a small molecule model in this new ELISA development. As a result, a “signal on” plasmonic ELISA for MT was established. This plasmonic ELISA can quantitatively detect MT by spectrophotometry with a 50 times higher sensitivity than the traditional ELISA. In addition, it allows the highly sensitive and easily qualitative detection of MT by the naked eye. This plasmonic direct competitive ELISA can be utilized for developing cost-effective and sensitive ELISA against various small molecules by the naked eye.

Masculinization of Tilapia (Oreochromis niloticus) Using Methyl Testosterone Natural Hormone

This study aimed to determine the effect of methyl testosterone natural hormoneon the rate of masculinization of tilapia (Oreochromis niloticus). The study used 270 tilapia larvae with the age of 7 days as experimental animals. Tilapia larvae were divided into 3 treatments; eachtreatment consisted of 30 larvae fish. This study was an experimental study using completely randomized design with 3 treatments and 3replications. Treatment I as control group was fed with pellets ad libitum, treatment II was given feed containing methyl testosterone naturalhormone, and treatment group III was given hormone treatment by soaking the fish larvae with methyl testosterone natural hormone for 20hours. Larvae were raised in accordance with applicable procedures. Observation was conducted when the larvae fish were at 60 days old andthe sex of fish can be observed. Parameter examined was the percentage of the male sex. The average (± SD) percentage of male tilapia in groupPI, PII, and PIII were 36.39±3.97, 66.72±6.13, and 82.37±6.87%, respectively. The results showed that the percentage of male sex of tilapia fedwith natural hormone of methyl testosterone through the diet and dipping was significantly different (P0.05) from control fish. In conclusion,the administration of natural hormone of methyl testosterone increased the percentage of the male sex of tilapia).____________________________________________________________________________________________________________________Key words: tilapia larvae, natural hormone methyl testosterone, the male sex percentage

Induction of Primary Male in Juvenile Red Spotted Grouper Epinephelus akaara by Immersion of 17α-Methyltestosterone

We investigated the androgenic effects of 17α- methyltestosterone (MT) on gonadal sex reversal in juvenile red spotted grouper Epinephelus akaara. The fish were immersed in 17α-MT at 1 and 5 mg/L. Treatment method of 17α-MT was once weekly for 4 and 8 weeks. Fish were sampled at 12 months after end of the treatment period in order to histological analysis. At the initiation of an experiment (70 day after hatching), juvenile red spotted grouper have the paired primordial gonads with somatic cells bellow kidney in the posterior portion of the body cavity. Formation of ovarian cavity indicates that the ovarian differentiation beginning at 70 DAH in red spotted grouper. At 12 months after end of the treatment period, control group, 17α-MT 1 mg/L treatment group for 4 and 8 weeks, and 17α-MT 5 mg/L treatment group for 4 weeks were all female. However, sex-changed males without ovarian cavity were observed in the 17α-MT 5 mg/L treatment group for 8 weeks. In grouper, we firstly reported that the red spotted grouper be able to induce the primary males by hormone treatment prior to gonadal sex differentiation.

Effect of Dietary Natural Phytochemicals on Sex-reversal, Growth Performance, Feed Utilization and Body Composition of Nile Tilapia (Oreochromis niloticus) Fry

a feeding study was conducted to evaluate the effect of different natural phytochemicals as alternative sex-reversal agents instead of 17α-methyl testosterone (MT). Newly hatched Nile tilapia (Oreochromis niloticus) fry were treated with MT (60 mg/kg), ginseng extract (GE; 0.2 or 0.4 g/kg), Tribulus terrestris extract (TTE; 0.6 or 1.2 g/kg) and date palm pollen (DPP; 3 or 6 g/kg) in diet. The treatment period lasted for 28 days for MT and 84 days for other phytochemicals. The results showed that the maximum male percentage recorded with MT (92.30%), followed by TTE (1.2 g/kg; 64.48%), TTE (0.6 g/kg; 57.76%) and DPP (6 g/kg; 56.67%), while the male percentage of the control was 48.36%. All studied treatments showed normal survival rates along the experimental period, which proves that the used phytochemicals are nontoxic. Fish treated with phytochemicals exhibited successful growth acceleration compared to the control group especially with TTE treatments. The same manner was observed with feed conversion ratio. Carcass composition showed that MT treatment has significantly increased moisture and decreased ether extract than other treatments. The effects of phytochemicals showed a slight improvement of protein and ether extract content compared to the control diet. Consequently, it can be concluded that the use of phytochemicals in fish diets as growth promoters is more effective than sex-reversal agents. It is recommended to use TTE in Nile tilapia fry diets.

Tilapia Sex Reversal Using Methyl Testosterone (MT) and its Effect on Fish, Man and Environment

Tilapia Sex Reversal Using Methyl Testosterone (MT) and its Effect on Fish, Man and Environment