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.

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.

Basis of melengestrol acetate action as a progestin.

To provide insight into potential mechanisms contributing to the various biological responses of cattle to treatment with progesterone, norgestomet, and melengestrol acetate (MGA), MCF-7 cells were utilized to determine the relative binding affinity of the progesterone receptor for MGA, norgestomet, progesterone, and a progesterone agonist (R5020), and to determine if progesterone, MGA, or norgestomet have estrogenic and/or anti-estrogenic activities. The progesterone receptor had greater affinity (P<0.05) for MGA, R5020, and norgestomet than for progesterone; and the affinity for norgestomet exceeded (P<0.05) that of MGA and R5020. Estrogen stimulates proliferation of MCF-7 cells; therefore these cells have been utilized as a bioassay to detect estrogenic and anti-estrogenic activity. Progesterone (10(-11) to 10(-5)M) did not promote cellular proliferation. However, MGA (10(-8), 10(-7), and 10(-6)M) increased (P<0.05) cell proliferation compared to the control group (10(-11) to 10(-9) and 10(-5)M MGA did not stimulate cell proliferation), and MGA-induced cell proliferation (10(-8)M) was reduced (P=0.095) by an estradiol antagonist (ICI 182,780; ICI). Cellular proliferation increased (P<0.05) with norgestomet (10(-5)M) compared to the control group (10(-11) to 10(-6)M norgestomet did not stimulate cell proliferation) and the increased proliferation was decreased (P<0.05) by ICI. Neither progesterone nor MGA demonstrated anti-estrogenic activity. Norgestomet (10(-10) to 10(-6)M) did reduce (P<0.05) maximal estrogen-stimulated cell proliferation, but not to basal levels. In summary, the affinities of the progesterone receptor for norgestomet, MGA, and progesterone are consistent with their effective dose to inhibit ovulation in vivo, but their progestin and their estrogenic/anti-estrogenic activities cannot fully explain why progesterone and norgestomet are more capable of reprogramming the reproductive axis in anestrous postpartum cows compared to MGA.

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.

Characterisation of the affinity of different anabolics and synthetic hormones to the human androgen receptor, human sex hormone binding globulin and to the bovine progestin receptor.

For the steroidal growth promoters trenbolone acetate (TBA) and melengestrol acetate (MGA) neither the complete spectrum of biological activities nor the potential endocrine disrupting activity of their excreted metabolites in the environment is fully understood. The potency of these substances in [3H]dihydrotestosterone ([3H]-DHT) displacement from the recombinant human androgen receptor (rhAR) and from human sex-hormone binding globulin (hSHBG) was evaluated. In addition, the potency for [3H]-ORG2058 displacement from the bovine uterine progestin receptor (bPR) was tested. For comparison, different anabolics and synthetic hormones were also tested for their binding affinities. For 17beta-trenbolone (17beta-TbOH), the active compound after TBA administration, an affinity the rhAR similar to dihydrotestosterone (DHT) and a slightly higher affinity to the bPR than progesterone were demonstrated. The affinity of the two major metabolites, 17alpha-trenbolone and trendione, was reduced to less than 5% of the 17beta-TbOH-value. The affinity of these three compounds and of MGA to the hSHBG was much lower compared with DHT. MGA showed a 5.3-fold higher affinity than progesterone to the bPR but only a weak affinity to the rhAR. The major MGA metabolites have an affinity to the bPR between 85% and 28% of the affinity of progesterone. In consequence, MGA and TBA metabolites may be hormonally active substances, which will be present in edible tissues and in manure. We conclude that detailed investigations on biodegradation, distribution and bio-efficacy of these substances are necessary.

Recycling and degradation of anabolic agents in animal excreta.

A variety of anabolic agents are currently added to animal feeds to increase growth rate and improve feed efficiency. These compounds and their metabolites are largely excreted. Prior to the use of anabolic agents as feed additives and the advent of confined livestock production, natural recycling occurred which generally resulted in benefit to the animal with no known adverse effects on consumer health. However, the current interest in the use of animal excreta in livestock feed and the possible presence of anabolic agents and their metabolites from this practice has created an additional need for information on the occurrence of anabolic agent residues in consumer products. This report will consider the definition of anabolic agents in its broadest sense and discuss the research on hormones used in animal feed that may be found in animal excreta. In addition to feed additive residues, endogenous compounds may also be found in animal excreta. Endogenous estrogens and androgens have been detected in excreta from domestic livestock and poultry. Research results suggest that substantial estrogenic and androgenic activity may be detected in fresh animal excreta. However, little is known about the effects of various processing methods of excreta such as heat drying and fermentation on its hormonal activity. The effects of feed additive residues and endogenous hormones in excreta used for feed will be discussed relative to their impact on animal health and occurrence in animal products.