Comparative genomic survey and functional analysis of DKKL1 during spermatogenesis in the Chinese soft-shelled turtle (Pelodiscus sinensis).

A feature of the Chinese soft-shelled turtle (Pelodiscus sinensis) is seasonal spermatogenesis; however, the underlying molecular mechanism is not well clarified. Here, we firstly cloned and characterized P. sinensis DKKL1, and then performed comparative genomic studies, expression analysis, and functional validation. P. sinensis DKKL1 had 2 putative N-glycosylation sites and 16 phosphorylation sites. DKKL1 also had classic transmembrane structures that were extracellularly localized. DKKL1's genetic distance was close to turtles, followed by amphibians and mammals, but its genetic distance was far from fishes. DKKL1 genes from different species shared distinct genomic characteristics. Meanwhile, they were also relatively conserved among themselves, at least from the perspective of classes. Notably, the transcription factors associated with spermatogenesis were also identified, containing CTCF, EWSR1, and FOXL2. DKKL1 exhibited sexually dimorphic expression only in adult gonads, which was significantly higher than that in other somatic tissues (P < 0.001), and was barely expressed in embryonic gonads. DKKL1 transcripts showed a strong signal in sperm, while faint signals were detected in other male germ cells. DKKL1 in adult testes progressively increased per month (P < 0.05), displaying a seasonal expression trait. DKKL1 was significantly downregulated in testes cells after the sex hormones (17ß-estradiol and 17α-methyltestosterone) and Wnt/ß-catenin inhibitor treatment (P < 0.05). Likewise, the Wnt/ß-catenin inhibitor treatment dramatically repressed CTCF, EWSR1, and FOXL2 expression. Conversely, they were markedly upregulated after the 17ß-estradiol and 17α-methyltestosterone treatment, suggesting that the three transcription factors might bind to different promoter regions, thereby negatively regulating DKKL1 transcription in response to the changes in the estrogen and androgen pathways, and positively controlling DKKL1 transcription in answer to the alterations in the Wnt/ß-catenin pathway. Knockdown of DKKL1 significantly reduced the relative expression of HMGB2 and SPATS1 (P < 0.01), suggesting that it may be involved in seasonal spermatogenesis of P. sinensis through a positive regulatory interaction with these two genes. Overall, our findings provide novel insights into the genome evolution and potential functions of seasonal spermatogenesis of P. sinensis DKKL1.

The effects of 17α-methyltestosterone on gonadal histology and gene expression along hypothalamic-pituitary-gonadal axis, germ cells, sex determination, and hypothalamus-pituitary-thyroid axis in zebrafish (Danio rerio).

As a synthetic androgen, 17α-methyltestosterone (MT) is widely used in aquaculture to induce sex reversal and may pose a potential risk to aquatic organisms. This ecological risk has attracted the attention of many scholars, but it is not comprehensive enough. Thus, the adverse effects of MT on zebrafish (Danio rerio) were comprehensively evaluated from gonadal histology, as well as the mRNA expression levels of 47 genes related to hypothalamic-pituitary-gonadal (HPG) axis, germ cell differentiation, sex determination, and hypothalamus-pituitary-thyroid (HPT) axis. Adult zebrafish with a female/male ratio of 5:7 were exposed to a solvent control (0.001% dimethyl sulfoxide) and three measured concentrations of MT (5, 51 and 583 ng/L) for 50 days. The results showed that MT had no significant histological effects on the ovaries of females, but the frequency of late-mature oocytes (LMO) showed a downward trend, indicating that MT could induce ovarian suppression to a certain extent. The transcriptional expression of activating transcription factor 4b1 (atf4b1), activating transcription factor 4b2 (atf4b2), calcium/calmodulin-dependent protein kinase II delta 1 (camk2d1), calcium/calmodulin-dependent protein kinase II delta 2 (camk2d2) and calcium/calmodulin-dependent protein kinase II inhibitor 2 (camk2n2) genes in the brain of females increased significantly at all treatment groups of MT, and the mRNA expression of forkhead box L2a (foxl2) and ovarian cytochrome P450 aromatase (cyp19a1a) genes in the ovaries were down-regulated by 5 and 583 ng/L group, which would translate into inhibition of oocyte development. As compared to females, MT had relatively little effects on the reproductive system of males, and only the transcriptional alterations of synaptonemal complex protein 3 (sycp3) and 17-alpha-hydroxylase/17,20-lyase (cyp17) genes were observed in the testes, not enough to affect testicular histology. In addition, MT at all treatments strongly increased corticotropin-releasing hormone (crh) transcript in the brain of females, as well as deiodinase 2 (dio2) transcript in the brain of males. The paired box protein 8 (pax8) gene was significantly decreased at 51 or 583 ng/L of MT in both female and male brains. The above results suggest that MT can pose potential adverse effects on the reproductive and thyroid endocrine system of fish.

Simultaneous determination of methyltestosterone and its metabolite in fish by gas chromatography-mass spectrometry.

Methyltestosterone is one of the banned drugs in aquaculture, and it should be monitored in food-producing animals. 17α-Methyl-5ß-androstane-3α,17ß-diol, as the main metabolite of methyltestosterone in vertebrates, could be used as another marker for controlling the administration of methyltestosterone, due to its high residual concentration and slow elimination rate. In this study, an analytical method based on gas chromatography-mass spectrometry (GC-MS) was developed and validated for the simultaneous determination of methyltestosterone and its main metabolite in fish. After pretreatment by liquid-liquid extraction with n-hexane and solid phase extraction with C18 and NH2 columns, the target analytes in the muscle tissues were extracted and concentrated, and the influence of the sample matrix was eliminated. Then, the prepared samples were separated and detected with GC-MS in the selected ion monitoring (SIM) mode. Methyltestosterone-D3 was chosen as the internal standard for quantitation. After optimization, the limits of detection for methyltestosterone and 17α-methyl-5ß-androstane-3α,17ß-diol were 20 µg kg-1 and 15 µg kg-1, respectively. The limits of quantitation were both 50 µg kg-1. The calibration curves showed good linearity in the concentration range from 50.0 ng mL-1 to 500.0 ng mL-1. The correlation coefficients of methyltestosterone and 17α-methyl-5ß-androstane-3α,17ß-diol were more than 0.9990. The recoveries of the analytes in real samples were in the range of 99.7-116.6% with the relative standard deviation of 5.2-8.3%. The established method could meet the demand for simultaneous detection of methyltestosterone and its major metabolite, and it could be used to provide more information on the abuse of methyltestosterone in food-producing animals.

Detection time comparison of non-hydrolysed sulphated metabolites of metenolone, mesterolone and 17α-methyltestosterone analysed by four different mass spectrometric techniques.

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences.

The in vivo metabolism of Jungle Warfare in greyhounds.

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,17ß-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 ß-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.

Molecular Cloning and Expression Responses of Jarid2b to High-Temperature Treatment in Nile Tilapia (Oreochromis niloticus).

Nile tilapia is a GSD + TE (Genetic Sex Determination + Temperature Effect) fish, and high-temperature treatment during critical thermosensitive periods (TSP) can induce the sex reversal of Nile tilapia genetic females, and brain transcriptomes have revealed the upregulation of Jarid2 (Jumonji and AT-rich domain containing 2) expression after 36 °C high-temperature treatment for 12 days during TSP. It was shown that JARID2 forms a complex with polycomb repressive complex 2 (PRC2) that catalyzed H3K27me3, which was strongly associated with transcriptional repression. In this study, Jarid2b was cloned and characterized in Nile tilapia, which was highly conserved among the analyzed fish species. The expression of Jarid2b was upregulated in the gonad of 21 dpf XX genetic females after 12-day high-temperature treatment and reached a similar level to that of males. Similar responses to high-temperature treatment also appeared in the brain, heart, liver, muscle, eye, and skin tissues. Interestingly, Jarid2b expression was only in response to high-temperature treatment, and not to 17α-methyltestosterone (MT) or letrozole treatments; although, these treatments can also induce the sex reversal of genetic Nile tilapia females. Further studies revealed that Jarid2b responded rapidly at the 8th hour after high-temperature treatment. Considering that JARID2 can recruit PRC2 and establish H3K27me3, we speculated that it might be an upstream gene participating in the regulation of Nile tilapia GSD + TE through regulating the H3K27 methylation level at the locus of many sex differentiation-related genes.

Plasma mitochondrial derived peptides MOTS-c and SHLP2 positively associate with android and liver fat in people without diabetes.

Mitochondrial-derived peptides (MDPs) are encoded by the mitochondrial genome and hypothesised to form part of a retrograde signalling network that modulates adaptive responses to metabolic stress. To understand how metabolic stress regulates MDPs in humans we assessed the association between circulating MOTS-c and SHLP2 and components of metabolic syndrome (MS), as well as depot-specific fat mass in participants without overt type 2 diabetes or cardiovascular disease. One-hundred and twenty-five Chinese participants (91 male, 34 female) had anthropometry, whole body dual-energy X-ray absorptiometry scans and fasted blood samples analysed. Chinese female participants and an additional 34 European Caucasian female participants also underwent magnetic resonance imaging and spectroscopy (MRI/S) for visceral, pancreatic and liver fat quantification. In Chinese participants (age = 41 ± 1 years, BMI = 27.8 ± 3.9 kg/m2), plasma MOTS-c (315 ± 27 pg/ml) and SHLP2 (1393 ± 82 pg/ml) were elevated in those with MS (n = 26). While multiple components of the MS sequelae positively associated with both MOTS-c and SHLP2, including blood pressure, fasting plasma glucose and triglycerides, the most significant of these was waist circumference (p < 0.0001). Android fat had a greater effect on increasing plasma MOTS-c (p < 0.004) and SHLP2 (p < 0.009) relative to whole body fat. Associations with MRI/S parameters corrected for total body fat mass revealed that liver fat positively associated with plasma MOTS-c and SHLP2 and visceral fat with SHLP2. Consistent with hepatic stress being a driver of circulating MDP concentrations, plasma MOTS-c and SHLP2 were higher in participants with elevated liver damage markers and in male C57Bl/6j mice fed a diet that induces hepatic lipid accumulation and damage. Our findings provide evidence that in the absence of overt type 2 diabetes, components of the MS positively associated with levels of MOTS-c and SHLP2 and that android fat, in particular liver fat, is a primary driver of these associations. MOTS-c and SHLP2 have previously been shown to have cyto- and metabolo-protective properties, therefore we suggest that liver stress may be a mitochondrial peptide signal, and that mitochondrial peptides are part of a hepatic centric-hormetic response intended to restore metabolic balance.

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17ß-methyl-18-nor-5ß-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5ß-metabolite was detected. Additionally, 3α,5ß-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

A further insight into methyltestosterone metabolism: New evidences from in vitro and in vivo experiments.

RATIONALE: Although the metabolism of methyltestosterone (MT) has been extensively studied since the 1950s using different techniques, the aim of this study was to investigate the hydroxylation in positions C2, C4 and C6 after in vitro experiments and in vivo excretion studies using gas chromatography time-of-flight (GC/TOF) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The results could be influenced by the mass spectrometric analyser used. METHODS: Incubations were carried out with human liver microsomes and six enzymes belonging to the cytochrome P450 family using MT as a substrate. The trimethylsilyl derivatives of the samples were analysed using GC/TOF and GC/MS/MS once the correct MS/MS transitions had been selected, mainly for 6-hydroxymethyltestosterone (6-OH-MT) to avoid artefact interferences. A urinary excretion study was then performed after the administration of a 10 mg single oral dose of MT to a volunteer. RESULTS: The formation of hydroxylated metabolites of MT in the C6, C4 and C2 positions after both in vitro and in vivo experiments was observed. Sample evaluation using GC/TOF showed an interference for 6-OH-MT that could only be resolved in GC/MS/MS by monitoring specific transitions. The transitory detection of these hydroxylated metabolites in urine agrees with previous investigations that had described this metabolic route as being of little significance. CONCLUSIONS: In doping analysis, the formation of 4-hydroxymethyltestosterone (oxymesterone) from MT cannot be underestimated. Although it is only detected as a minor and short-term excretion metabolite, it cannot be overlooked as it was found in both in vitro and in vivo experiments. The use of a combination of different mass spectrometric instruments allowed reliable conclusions to be reached, and it was shown that special attention must be given to artefact formation.

Application of α- and ß-naphthoflavones as monooxygenase inhibitors of Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651 in transformation of 17α-methyltestosterone.

In this work, 17α-methyltestosterone was effectively hydroxylated by Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651. A. coerulea KCh 93 afforded 6ß-, 12ß-, 7α-, 11α-, 15α-hydroxy derivatives with 44%, 29%, 6%, 5% and 9% yields, respectively. S. racemosum KCh 105 afforded 7α-, 15α- and 11α-hydroxy derivatives with yields of 45%, 19% and 17%, respectively. Chaetomium sp. KCh 6651 afforded 15α-, 11α-, 7α-, 6ß-, 9α-, 14α-hydroxy and 6ß,14α-dihydroxy derivatives with yields of 31%, 20%, 16%, 7%, 5%, 7% and 4%, respectively. 14α-Hydroxy and 6ß,14α-dihydroxy derivatives were determined as new compounds. Effect of various sources of nitrogen and carbon in the media on biotransformations were tested, however did not affect the degree of substrate conversion or the composition of the products formed. The addition of α- or ß-naphthoflavones inhibited 17α-methyltestosterone hydroxylation but did not change the percentage composition of the resulting products.

Isaria fumosorosea KCh J2 Entomopathogenic Strain as an Effective Biocatalyst for Steroid Compound Transformations.

The catalytic activity of enzymes produced by an entomopathogenic filamentous fungus (Isaria fumosorosea KCh J2) towards selected steroid compounds (androstenedione, adrenosterone, progesterone, 17α-methyltestosterone and dehydroepiandrosterone) was investigated. All tested substrates were efficiently transformed. The structure of the substrate has a crucial impact on regio- and stereoselectivity of hydroxylation since it affects binding to the active site of the enzyme. Androstenedione was hydroxylated in the 7α-position to give a key intermediate in the synthesis of the diuretic-7α-hydroxyandrost-4-ene-3,17-dione with 82% conversion. Adrenosterone and 17α-methyltestosterone were hydroxylated in the 6ß-position. Hydroxylated derivatives such as 15ß-hydroxy-17α-methyltestosterone and 6ß,12ß-dihydroxy-17α-methyltestosterone were also observed. In the culture of Isaria fumosorosea KCh J2, DHEA was effectively hydroxylated in the C-7 position and then oxidized to give 7-oxo-DHEA, 3ß,7α- and 3ß,7ß-dihydroxy-17a-oxa-d-homo-androst-5-ene-17-one. We obtained 7ß-OH-DHEA lactone with 82% yield during 3 days transformation of highly concentrated (5 g/L) DHEA.

The extent to which immunity, apoptosis and detoxification gene expression interact with 17 alpha-methyltestosterone.

Innate immunity is the first line of defence against invasion by foreign pathogens. One widely used synthetic androgen for the production of all-male fish, particularly commercially valuable Nile tilapia, Oreochromis niloticus, is 17 alpha-methyltestosterone (MT). The present study investigates the effect of MT on innate immunity, cellular apoptosis and detoxification and the mortality rate, during and after the feeding of fry with 0-, 40-and 60-mg MT/kg. Expression analysis was completed on interleukin 1 beta (il1ß), interleukin 8 (il8), tumour necrosis factor alpha (tnfα), CXC2- and CC-chemokines, interferon (ifn), myxovirus resistance (mx), toll-like receptor 7 (tlr7), immunoglobulin M heavy chain (IgM heavy chain), vitellogenin (vtg), cellular apoptosis susceptibility (cas) and glutathione S-transferase α1 (gstα1). Expression analysis revealed that MT had a significant impact on these genes, and this impact varied from induction to repression during and after the treatment. Linear regression analysis showed a significant association between the majority of the tested gene transcript levels and mortality rates on the 7th and 21st days of hormonal treatment and 2 weeks following hormonal cessation. The results are thoroughly discussed in this article. This is the first report concerning the hazardous effect of MT on a series of genes involved in immunity, apoptosis and detoxification in the Nile tilapia fry.

New biosourced chiral molecularly imprinted polymer: Synthesis, characterization, and evaluation of the recognition capacity of methyltestosterone.

New biosourced chiral cross-linkers were reported for the first time in the synthesis of methyltestosterone (MT) chiral molecularly imprinted polymers (cMIPs). Isosorbide and isomannide, known as 1,4:3,6-dianhydrohexitols, were selected as starting diols. The cMIPs were synthesized following a noncovalent approach via thermal radical polymerization and monitored by Raman spectroscopy. These cross-linkers were fully characterized by 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry. The cross-polarization magic angle spinning 13 C NMR, Fourier transform infrared spectroscopy, scanning electron microscopy, and specific surface areas following the Brunauer-Emmett-Teller (BET) method were used to characterize the cMIPs. The effect of stereochemistry of cross-linkers on the reactivity of polymerization, morphology, and adsorption-recognition properties of the MIP was evaluated. The results showed that the cMIP exhibited an obvious improvement in terms of rebinding capacity for MT as compared with the nonimprinted polymer (NIP). The highest binding capacity was observed for cMIP-Is (27.298 mg g-1 ) for high concentrations (500 mg L-1 ). However, the isomannide homologue cMIP-Im showed higher recovery-up to 65% and capacity for low concentrations (15 mg L-1 ). The experimental data were properly fitted by the Freundlich adsorption isothermal model.

Methyltestosterone alters sex determination in the American alligator (Alligator mississippiensis).

Effects of xenobiotics can be organizational, permanently affecting anatomy during embryonic development, and/or activational, influencing transitory actions during adulthood. The organizational influence of endocrine-disrupting contaminants (EDC's) produces a wide variety of reproductive abnormalities among vertebrates that exhibit temperature-dependent sex determination (TSD). Typically, such influences result in subsequent activational malfunction, some of which are beneficial in aquaculture. For example, 17-αmethyltestosterone (MT), a synthetic androgen, is utilized in tilapia farming to bias sex ratio towards males because they are more profitable. A heavily male-biased hatchling sex ratio is reported from a crocodile population near one such tilapia operation in Guanacaste, Costa Rica. In this study we test the effects of MT on sexual differentiation in American alligators, which we used as a surrogate for all crocodilians. Experimentally, alligators were exposed to MT in ovo at standard ecotoxicological concentrations. Sexual differentiation was determined by examination of primary and secondary sex organs post hatching. We find that MT is capable of producing male embryos at temperatures known to produce females and demonstrate a dose-dependent gradient of masculinization. Embryonic exposure to MT results in hermaphroditic primary sex organs, delayed renal development and masculinization of the clitero-penis (CTP).

Properties of PLA/PCL particles as vehicles for oral delivery of the androgen hormone 17α-methyltestosterone.

The aim of this study was to produce PLA (poly(lactic acid)) and PCL (polycaprolactone) oral carriers through the precipitation of the polymer solutions using supercritical CO2 as an antisolvent for the controlled release of the hydrophobic model drug 17α-methyltestosterone (MT). Such drug is a steroidal hormone used orally to develop and sustain primary and secondary male sex characteristics, e.g. for female Nile tilapia sex reversal in aquaculture. The influence of hormone, PLA and PCL concentrations on particle formation was analyzed, showing that high PCL concentrations produced particles with rougher surfaces and greater mean diameters. The incorporation efficiency of MT ranged from 20 to 51%, and its addition resulted in increases in particle mean diameter from 23 to 54 µm. Aggregation was observed for particles incorporating or not MT and high concentrations of MT led to the formation of more amorphous structures, changing the thermal behavior of the particles. The exposure of the PLA/PCL particles to pH conditions simulating gastrointestinal fish conditions showed that hormone release fraction at acidic pH ranged from 8 to 63% (over 2h), while in the basic pH the proportion released varied from 23 to 60% (over 10h), reaching levels adequate for the desired in vivo activity.

Synthesis and chemical reactions of the steroidal hormone 17α-methyltestosterone.

Structural modifications of natural products with complex structures like steroids require great synthetic effort. A review of literature is presented on the chemistry of the steroidal hormone 17α-methyltestosterone that is approved by Food and Drug Administration (FDA) in the United States as an androgen for estrogen-androgen hormone replacement therapy treatment. The analog also offers special possibilities for the prevention/treatment of hormone-sensitive cancers. The testosterone skeleton has important functionalities in the molecule that can act as a carbonyl component, an active methylene compound, α,ß-unsaturated enone and tertiary hydroxyl group in various chemical reactions to access stereoisomeric steroidal compounds with potent activity. In addition, microbiological methods of synthesis and transformation of this hormone are presented.

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.

Prediction of Android and Gynoid Body Adiposity via a Three-dimensional Stereovision Body Imaging System and Dual-Energy X-ray Absorptiometry.

OBJECTIVE: Current methods for measuring regional body fat are expensive and inconvenient compared to the relative cost-effectiveness and ease of use of a stereovision body imaging (SBI) system. The primary goal of this research is to develop prediction models for android and gynoid fat by body measurements assessed via SBI and dual-energy x-ray absorptiometry (DXA). Subsequently, mathematical equations for prediction of total and regional (trunk, leg) body adiposity were established via parameters measured by SBI and DXA. METHODS: A total of 121 participants were randomly assigned into primary and cross-validation groups. Body measurements were obtained via traditional anthropometrics, SBI, and DXA. Multiple regression analysis was conducted to develop mathematical equations by demographics and SBI assessed body measurements as independent variables and body adiposity (fat mass and percentage fat) as dependent variables. The validity of the prediction models was evaluated by a split sample method and Bland-Altman analysis. RESULTS: The R(2) of the prediction equations for fat mass and percentage body fat were 93.2% and 76.4% for android and 91.4% and 66.5% for gynoid, respectively. The limits of agreement for the fat mass and percentage fat were -0.06 ± 0.87 kg and -0.11% ± 1.97% for android and -0.04 ± 1.58 kg and -0.19% ± 4.27% for gynoid. Prediction values for fat mass and percentage fat were 94.6% and 88.9% for total body, 93.9% and 71.0% for trunk, and 92.4% and 64.1% for leg, respectively. CONCLUSIONS: The three-dimensional (3D) SBI produces reliable parameters that can predict android and gynoid as well as total and regional (trunk, leg) fat mass.

Effects of methyltestosterone, letrozole, triphenyltin and fenarimol on histology of reproductive organs of the copepod Acartia tonsa.

The marine calanoid copepod Acartia tonsa was exposed to methyltestosterone (MET, 1.6-126 µg L(-1)), letrozole (LET, 10-1000 µg L(-1)), triphenyltin chloride (TPT, 0.0014-0.0088 µg L(-1) TPT-Sn) and fenarimol (FEN, 2.8-105 µg L(-1)) for 21 d covering a full life-cycle. All four compounds investigated are known to act as androgens in vertebrates. The digestive tract, musculature, nervous system, reproductive organs, gonad and accessory sexual glands were examined by light microscopy after routine staining and immune-labelling for detection of apoptosis and determination of proliferation activities. MET induced an inhibition of oogenesis, oocyte maturation and yolk formation, respectively, which was most pronounced at the lowest concentrations tested. In LET exposed males, spermatogenesis was enhanced with very prominent gamete stages; in some stages apoptosis occurred. The spermatophore was hypertrophied and displayed deformations. In females, LET induced a disorder of oogenesis and disturbances in yolk synthesis. TPT stimulated the male reproductive system at 0.0014 and 0.0035 µg TPT-SnL(-1), whereas inhibiting effects were observed in the female gonad at 0.0088 µg TPT-SnL(-1). In FEN exposed females proliferation of gametes was reduced and yolk formation showed irregular features at 2.8-105 µgL(-1). In FEN exposed males an elevated proliferation activity was observed. No pathological alterations in other organ systems, e.g. the digestive tract including the hindgut acting as respiratory organ, the nervous system, or the musculature were seen. This indicates that the effects on gonads might be caused rather by disturbance of endocrine signalling or interference with hormone metabolism than by general toxicity.