Fungal mediated biotransformation of melengestrol acetate, and T-cell proliferation inhibitory activity of biotransformed compounds.

Glomerella fusaroide, and Rhizopus stolonifer were effectively able to transform the steroidal hormone melengestrol acetate (MGA) (1) into four (4) new metabolites, 17α-acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (2), 17α-acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-1,4,6-triene-3,20-dione (3), 17α-acetoxy-6,7α-epoxy-6ß-methyl-16-methylenepregna-4,6-diene-3,20-dione (4), and 17α-acetoxy-11ß,15ß-dihydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (5). All these compounds were structurally characterized by different spectroscopic techniques. The objective of the current study was to assess the anti-inflammatory potential of melengestrol acetate (1), and its metabolites 2-5. The metabolites and the substrate were assessed for their inhibitory effects on proliferation of T-cells in vitro. The substrate (IC50 = 2.77 ± 0.08 µM) and its metabolites 2 (IC50 = 2.78 ± 0.07 µM), 4 (IC50 = 2.74 ± 0.1 µM), and 5 (IC50 = < 2 µM) exhibited potent T- cell proliferation inhibitory activities, while compound 3 (IC50 = 29.9 ± 0.09 µM) showed a moderate activity in comparison to the standard prednisolone (IC50 = 9.73 ± 0.08 µM). All the metabolites were found to be non-toxic against 3T3 normal cell line. This study thus identifies some potent compounds active against T-cell proliferation. Their anti-inflammatory potential, therefore, deserves to be further investigated.

Sorption and mineral-promoted transformation of synthetic hormone growth promoters in soil systems.

This work examines the fate of synthetic growth promoters (trenbolone acetate, melengestrol acetate, and zeranol) in sterilized soil systems, focusing on their sorption to organic matter and propensity for mineral-promoted reactions. In organic-rich soil matrices (e.g., Pahokee Peat), the extent and reversibility of sorption did not generally correlate with compound hydrophobicity (e.g., K(ow) values), suggesting that specific binding interactions (e.g., potentially hydrogen bonding through C17 hydroxyl groups for the trenbolone and melengestrol families) can also contribute to uptake. In soils with lower organic carbon contents (1-5.9% OC), evidence supports sorption occurring in parallel with surface reaction on inorganic mineral phases. Subsequent experiments with pure mineral phases representative of those naturally abundant in soil (e.g., iron, silica, and manganese oxides) suggest that growth promoters are prone to mineral-promoted oxidation, hydrolysis, and/or nucleophilic (e.g., H2O or OH(-)) addition reactions. Although reaction products remain unidentified, this study shows that synthetic growth promoters can undergo abiotic transformation in soil systems, a previously unidentified fate pathway with implications for their persistence and ecosystem effects in the subsurface.

Fungal transformation and T-cell proliferation inhibitory activity of melengestrol acetate and its metabolite.

Biotransformation of melengestrol acetate (MGA, 17α-acetoxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione) (1) was investigated for the first time by using fungal cultures. Incubation of compound 1 with Cunninghamella blakesleeana yielded a new major metabolite, 17α-acetoxy-11ß-hydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (2). The metabolite 2 was purified by using HPLC, followed by characterization through (1)H- and (13)C-NMR and other spectroscopic techniques. Single crystal X-ray diffraction analysis was used to deduce the three dimensional structures of melengestrol acetate (1) and metabolite 2 for the first time. T-cell proliferation assay was employed to evaluate the immunosuppressant effect of compounds 1 and 2 with IC50=0.5±0.07 and 0.6±0.08µg/mL, respectively. The results indicated that these compounds possess sixfold potent T-cell proliferation inhibitory activity as compared to the standard prednisolone (IC50<3.1µg/mL). Both compounds were found to be non-toxic in a 3T3 (mouse fibroblast) cell-based cytotoxicity assay. This discovery of potent anti-inflammatory activity of compounds 1 and 2 can lead the way to develop new immunosuppressant compounds for clinical application.

Confirmatory method for the determination of various acetylgestagens in animal kidney fat using liquid chromatography-tandem mass spectrometry.

A confirmatory method has been developed and validated that allows for the simultaneous detection of medroxyprogesterone acetate (MPA), megestrol acetate (MGA), melengestrol acetate (MLA), chlormadinone acetate (CMA) and delmadinone acetate (DMA) in animal kidney fat using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The compounds were extracted from kidney fat using acetonitrile, defatted using a hexane wash and subsequent saponification. Extracts were then purified on Isolute CN solid-phase extraction cartridges and analysed by LC-MS/MS. The method was validated in animal kidney fat in accordance with the criteria defined in Commission Decision 2002/657/EC. The decision limit (CCalpha) was calculated to be 0.12, 0.48, 0.40, 0.63 and 0.54 microg kg(-1), respectively, for MPA, MGA, MLA, DMA and CMA, with respective detection capability (CCbeta) values of 0.20, 0.81, 0.68, 1.07 and 0.92 microg kg(-1). The measurement uncertainty of the method was estimated at 16, 16, 19, 27 and 26% for MPA, MGA, MLA, DMA and CMA, respectively. Fortifying kidney fat samples (n = 18) in three separate assays showed the accuracy of the method to be between 98 and 100%. The precision of the method, expressed as % RSD, for within-laboratory reproducibility at three levels of fortification (1, 1.5 and 2 microg kg(-1) for MPA, 5, 7.5 and 10 microg kg(-1) for MGA, MLA, DMA and CMA) was less than 5% for all analytes.

Modification of mRNA expression after treatment with anabolic agents and the usefulness for gene expression-biomarkers.

With this feasibility study a first step towards a new monitoring system for hormonal treatments was done. Screening of regulation and function of anabolic sex steroids via modified gene expression of mRNA in various tissues could be a new approach to trace treatments with unknown drugs or newly combined cocktails. In the study, uterus, liver and muscle tissue from 24 cycling heifers were taken after the animals were treated either with Melengestrol Acetate (MGA), Finaplix-H (200 mg Trenbolone Acetate) or Ralgro (36 mg Zeranol) for 56 days. In every treatment group always two heifers were given 1-fold, 3-fold and 10-fold doses of the standard preparation, the control group without any treatment consisted of two animals. The different tissue gene expression profiles were investigated via the candidate gene approach. Totally 57 candidate genes were selected according to their functionality by screening the actual literature and composed to functional groups: angiogenesis, apoptosis, cell cycle, endocrine factors, energy metabolism, inflammatory factors, muscle function, oncogenes, protein metabolism and transcription factors. Gene expression was measured using quantitative real-time RT-PCR (qRT-PCR) technology. From 24 tested candidate genes in the liver, 17 showed a significant regulation. Eight genes were influenced by MGA, 9 by Finaplix-H, and 4 by Ralgro. For the muscle tissue 19 genes were tested with the result that in the neck muscle 11 genes were regulated and in the hind limb muscle 8 genes. In the neck 5 genes were affected by MGA, 6 by Finaplix-H and 3 by Ralgro. Only 2 genes were influenced by MGA in the hind limb muscle. Finaplix-H affected 6 and Ralgro 4 genes. In the uterus 29 target genes were tested and 13 were significantly influenced by the anabolic sex steroids. Under Finaplix-H treatment eight target genes were regulated and Ralgro and MGA showed a significant regulation in four target genes. The highest gene expression changes under anabolic treatment were observed in the uterus. The analyzed genes showed significant regulations but further studies, testing different animal husbandry conditions will be needed to identify meaningful expression patterns for the different tissues. With the investigation of the regulation and possible function of anabolic sex steroids via gene expression, a preparatory work for the development of an expression pattern for drug screening was made.

Mobility of the growth promoters trenbolone and melengestrol acetate in agricultural soil: column studies.

There is growing concern about environmentally released man-made chemicals suspected to be responsible for a number of adverse effects on endocrine function in wildlife species and possibly also in humans. Sex hormones are of particular interest due to their regulatory role in developmental processes such as sexual differentiation. Endogenous hormones of human or animal origin as well as exogenous sex steroids used for contraception or as anabolics for farm animals are excreted and reach the environment. We investigated the transport of the synthetic growth promoters trenbolone (TbOH) and melengestrol acetate (MGA) in agricultural soil by means of column experiments with aggregated soil materials (Ap and Bt horizons of a Luvisol). Column effluent concentrations and depth profiles of TbOH and MGA were determined with sensitive enzyme immunoassay systems and HPLC (RP-18), respectively. All procedures were confirmed by liquid chromatography-mass spectrometry. Small amounts of TbOH and MGA passed the columns very quickly. However, both hormones exhibited a high affinity to the organic matter of both horizons leading to a high retardation within the upper layers of the soil columns. Although we cannot deduce whether hormones of animal origin reach the ground water under field conditions, our model experiments show that their transition can be presumed.

Genotoxic potential of xenobiotic growth promoters and their metabolites.

This paper reviews data reported in the literature as well as recent and unpublished studies from our laboratory on the metabolism and genotoxicity of the xenobiotic growth promoters 17beta-trenbolone, melengestrol acetate and zeranol. In our metabolic study, the oxidative in vitro metabolites generated by hepatic microsomes from rats, bovine and humans were analyzed by HPLC and GC/MS. 17beta-Trenbolone gave rise to at least 13 monohydroxylated products, whereas 12 mono- and dihydroxylated metabolites were obtained with melengestrol acetate and at least 5 with zeranol. The genotoxic potential of the parent compounds was studied using the following endpoints: induction of HPRT mutations in cultured V79 cells and of lacI mutations in E. coli; induction of micronuclei in V79 cells; and formation of DNA adducts in cultured primary rat hepatocytes. Negative results were obtained in most of these assay systems. Only the micronucleus induction was marginally positive with 17beta-trenbolone and zeranol at near-cytotoxic concentrations. Commercial melengestrol acetate was found to contain an impurity causing apoptosis in V79 cells. The genotoxic potential of the numerous oxidative metabolites of the xenobiotic growth promoters remains to be studied.