Androstadienedione Research Articles

Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ1 -dehydrogenase and catalase in Bacillus subtilis.

3-ketosteroid-Δ1 -dehydrogenase (KSDD), a flavin adenine dinucleotide (FAD)-dependent enzyme involved in sterol metabolism, specifically catalyses the conversion of androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). However, the low KSDD activity and the toxic effects of hydrogen peroxide (H2 O2 ) generated during the biotransformation of AD to ADD with FAD regeneration hinder its application on AD conversion. The aim of this work was to improve KSDD activity and eliminate the toxic effects of the generated H2 O2 to enhance ADD production. The ksdd gene obtained from Mycobacterium neoaurum JC-12 was codon-optimized to increase its expression level in Bacillus subtilis, and the KSDD activity reached 12·3Umg-1 , which was sevenfold of that of codon-unoptimized gene. To improve AD conversion, catalase was co-expressed with KSDD in B. subtilis 168/pMA5-ksddopt -katA to eliminate the toxic effects of H2 O2 generated during AD conversion. Finally, under optimized bioconversion conditions, fed-batch strategy was carried out and the ADD yield improved to 8·76gl-1 . This work demonstrates the potential to improve enzyme activity by codon-optimization and eliminate the toxic effects of H2 O2 by co-expressing catalase. This study showed the highest ADD productivity ever reported and provides a promising strain for efficient ADD production in the pharmaceutical industry.

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Background: 3-Ketosteroid-Δ 1 -dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen as a new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd ( ksdd II ) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The K m and V max values of purified KSDD were 8.91 μM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADD from AD efficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production. Normal 0 21 false false false ES X-NONE X-NONE MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Calibri,sans-serif;}

Utilization of phytosterol-containing vegetable oils as a substrate for production of androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione by using Mycobacterium sp.

Abstract Several kinds of phytosterol-containing vegetable oils were screened for use as substrate for production of androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) by Mycobacterium sp. DSM-2966 and Mycobacterium sp. DSM-2967. The results indicated that canola oil gave a higher of total desired products in aqueous media with yields of 6.48and 7.92 mg/100 mL by Mycobacterium sp. DSM-2966 and Mycobacterium sp. DSM-2967, respectively. In contrast, the extremely low products were obtained when coconut and palm olein oils were processed by both strains. The main product from Mycobacterium sp. DSM-2966 was AD, meanwhile Mycobacterium sp. DSM-2967 gave ADD superior to AD. The molecular structure of phytosterol substrates affected the bioconversion efficiency. The use of lower concentration and ratio of vegetable oil in the media gave more capability of biotransformation. The low cost of vegetable oils, their biocompatible to microorganism and the use of substrate without extraction process propose the potential for large-scale production of AD and ADD intermediates.

Enhanced intracellular soluble production of 3‐ketosteroid‐Δ1‐dehydrogenase from Mycobacterium neoaurum in Escherichia coli and its application in the androst‐1,4‐diene‐3,17‐dione production

AbstractBACKGROUNDThe 3‐ketosteroid‐Δ1‐dehydrogenase (KSDD) expressed in Escherichia coli was mainly in the form of inclusion bodies and the KSDD enzyme activity was at a low level. Therefore, the regulation of process conditions and supplementation of natural osmolytes were carried out to improve the soluble expression of KSDD in E. coli.RESULTSEffects of temperature, inducer concentration, and osmolytes (K‐glutamate, proline, and betaine) supplementation on cell growth and soluble KSDD production in recombinant Escherichia coli were investigated. With the decrease of cultivation temperature, lower isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) concentration, and betaine addition, high intracellular soluble KSDD production was realized in the recombinant E. coli BL21/pET28a‐ksdd and the KSDD enzyme activity reached 11.7 U mg−1, which is the highest KSDD activity ever reported. In addition, the whole‐cell biocatalyst of this recombinant strain was used for bioconversion of androst‐4‐ene‐3,17‐dione (AD) to androst‐1,4‐diene‐3,17‐dione (ADD). By optimization of the transformation conditions and applying a fed‐batch strategy, a final ADD yield of 5.7 g L−1 was achieved with a productivity of 0.158 g (L h)−1, which is the highest reported productivity using a microbial process. More importantly, no by‐products were detected during the whole bioconversion process.CONCLUSIONThese results provide the basis for industrial scale production of ADD. © 2016 Society of Chemical Industry

A mutant form of 3-ketosteroid-Δ(1)-dehydrogenase gives altered androst-1,4-diene-3, 17-dione/androst-4-ene-3,17-dione molar ratios in steroid biotransformations by Mycobacterium neoaurum ST-095.

Mycobacterium neoaurum ST-095 and its mutant M. neoaurum JC-12, capable of transforming phytosterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AD), produce very different molar ratios of ADD/AD. The distinct differences were related to the enzyme activity of 3-ketosteroid-Δ(1)-dehydrogenase (KSDD), which catalyzes the C1,2 dehydrogenation of AD to ADD specifically. In this study, by analyzing the primary structure of KSDDI (from M. neoaurum ST-095) and KSDDII (from M. neoaurum JC-12), we found the only difference between KSDDI and KSDDII was the mutation of Val(366) to Ser(366). This mutation directly affected KSDD enzyme activity, and this result was confirmed by heterologous expression of these two enzymes in Bacillus subtilis. Assay of the purified recombinant enzymes showed that KSDDII has a higher C1,2 dehydrogenation activity than KSDDI. The functional difference between KSDDI and KSDDII in phytosterol biotransformation was revealed by gene disruption and complementation. Phytosterol transformation results demonstrated that ksdd I and ksdd II gene disrupted strains showed similar ADD/AD molar ratios, while the ADD/AD molar ratios of the ksdd I and ksdd II complemented strains were restored to their original levels. These results proved that the different ADD/AD molar ratios of these two M. neoaurum strains were due to the differences in KSDD. Finally, KSDD structure analysis revealed that the Val(366)Ser mutation could possibly play an important role in stabilizing the active center and enhancing the interaction of AD and KSDD. This study provides a reliable theoretical basis for understanding the structure and catalytic mechanism of the Mycobacteria KSDD enzyme.

English

The steroid metabolites 9-hydroxy-androstenedione (9-OH-AD), androstadienedione (ADD) and androstenedione (AD) are important steroidal pharmaceuticals. In order to raise the production of steroid metabolites, an efficient resting cell phytosterol bioconversion process was developed to produce 9-OH-AD in the presence of hydroxypropyl-b-cyclodextrin (HP-b-CD). Cell growth medium containing phytosterol as an inducer positively improved cell activity. Under aerobic conditions, bioconversion proceeded at 70 g L –1 phytosterol in the presence of HP-b-CD (the optimized molar ratio of HP-b-CD/phytosterol was 1:1) with 30 g L –1 resting Mycobacterium neoaurum NwIB-yV cells (cell dry mass) in a 5-L bioreactor, where 9-OH-AD production and space-time yield reached 36.4 g L –1 and 9.1 g L –1 d –1 , respectively. The recycling of cells and HP-b-CD enables cost-saving and industrial applications. This bioprocess was also applied for the production of ADD and AD. The production of these steroid metabolites was much higher than that reported in previous studies.

Detection of selected corticosteroids and anabolic steroids in calf milk replacers by liquid chromatography–electrospray ionisation – Tandem mass spectrometry

The use of corticosteroids and anabolic steroids in food producing animals is regulated or banned in the European Union (EU). However, their use as growth promoters cannot be excluded. Milk replacers, considered by EU legislation as feeds, may be a good way of administration of these compounds. In order to improve the control of growth promoter utilization in animal husbandry and preventing possible consequences to animal welfare, we developed a method for multiresidue analysis of prednisolone, prednisone, dexamethasone, cortisone, cortisol, 17α- and 17β-boldenone and their precursor androstadienedione (ADD), testosterone, epitestosterone, 17α- and 17β-nandrolone, and trenbolone in powdered milk for calves. All analytes were extracted, after a common deproteinization and defatting sample pre-treatment, by a unique immunoaffinity column and analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS) in both positive and negative electrospray ionization (ESI) modes. The method was validated according to the criteria of the Commission Decision 2002/657/CE. The analytical limits were from 0.39 to 0.73 ng mL−1 for the decision limit (CCα) and 0.46–0.99 ng mL−1 for detection capability (CCβ). The analysis of 50 samples of milk replacers for calves, always revealed the presence of cortisol and cortisone (average concentrations 2.56 and 1.06 ng mL−1, respectively), frequently testosterone and epitestosterone (1.24 and 0.63 ng mL−1, respectively), occasionally β-nandrolone (0.82 ng mL−1) and prednisolone (0.41 ng mL−1). The other anabolic steroids were never found.

Enhanced Production of Androst-1,4-Diene-3,17-Dione by Mycobacterium neoaurum JC-12 Using Three-Stage Fermentation Strategy.

To improve the androst-1,4-diene-3,17-dione (ADD) production from phytosterol by Mycobacterium neoaurum JC-12, fructose was firstly found favorable as the initial carbon source to increase the biomass and eliminate the lag phase of M. neoaurum JC-12 in the phytosterol transformation process. Based on this phenomenon, two-stage fermentation by using fructose as the initial carbon source and feeding glucose to maintain strain metabolism was designed. By applying this strategy, the fermentation duration was decreased from 168 h to 120 h with the ADD productivity increased from 0.071 g/(L·h) to 0.108 g/(L·h). Further, three-stage fermentation by adding phytosterol to improve ADD production at the end of the two-stage fermentation was carried out and the final ADD production reached 18.6 g/L, which is the highest reported ADD production using phytosterol as substrate. Thus, this strategy provides a possible way in enhancing the ADD production in pharmaceutical industry.

In vitro simulation of the equine hindgut as a tool to study the influence of phytosterol consumption on the excretion of anabolic–androgenic steroids in horses

Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that β-Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including β-Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. The functionality of the in vitro model was confirmed by monitoring the formation of short-chain fatty acids and the consumption of amino acids and carbohydrates throughout the digestion process. In vitro digestion samples were analyzed with a validated UHPLC–MS/MS method. The addition of β-Bol gave rise to the formation of ADD (androsta-1,4-diene-3,17-dione) or αT. Upon addition of ADD to the in vitro digestions, the transformation of ADD to β-Bol was observed and this for all eight horses’ inocula, in line with previously obtained in vivo results, again confirming the functionality of the in vitro model. The transformation ratio proved to be inoculum and thus horse dependent. The addition of pure phytosterols (50% β-sitosterol) or phytosterol-rich herbal supplements on the other hand, did not induce the detection of β-Bol, only low concentrations of AED, a testosterone precursor, could be found (0.1ng/mL). As such, the digestive transformation of ADD could be linked to the detection of β-Bol, and the consumption of phytosterols to low concentrations of AED, but there is no direct link between phytosterols and β-Bol.

Genetic differences in ksdD influence on the ADD/AD ratio of Mycobacterium neoaurum.

Mycobacterium neoaurum TCCC 11028 (MNR) and M. neoaurum TCCC 11028 M3 (MNR M3) significantly differ in the ratio of androst-1,4-diene-3,17-dione (ADD) to androst-4-ene-3,17-dione (AD) produced. The large fluctuations are related to the dehydrogenation activity of 3-ketosteroid-Δ(1)-dehydrogenase (KsdD). Analysis of the primary structure of KsdD showed that the Ser-138 of KsdD-MNR changed to Leu-138 of KsdD-MNR M3 because of C413T in the ksdD gene. This phenomenon directly affected KsdD activity. The effect of the primary structure of KsdD on dehydrogenation activity was confirmed through exogenous expression. Whole-cell transformation initially revealed that KsdD-MNR showed a higher dehydrogenation activity than KsdD-MNR M3. Then, ksdD gene replacement strain was constructed by homologous recombination. The results of steroid transformation experiments showed that the ability of the MNR M3ΔksdD::ksdD-MNR strain to produce ADD was improved and it returned to the similar level of the MNR strain. This result indicated that the ADD/AD ratio of the two M. neoaurum strains was influenced by the difference in ksdD. The mechanism by which residue mutations alter enzyme activity may be connected with the crystal structure of KsdD from Rhodococcus erythropolis SQ1. As a key amino acid residue in the active center position, Ser-138 played an important role in maintaining the active center in the hydrophobic environment of KsdD. This study may serve as a basis for future studies on the structural analysis and catalytic mechanism of dehydrogenase.

A validated UHPLC-MS/MS method to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses.

Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to the Association of Official Racing Chemists (AORC) and European Commission (EC) guidelines, proving the power of this new method. Low limits of detection (0.2 ng/mL), good reproducibility (percentage of standard deviation (%RSD) < 10%), high recovery (94.6 to 117.1%), selectivity and specificity, and a linear response (confirmed with R(2) > 0.99 and lack-of-fit analysis) were obtained for all included AASs. With this method, urine samples of 105 guaranteed untreated horses (47 geldings, 53 mares, and 5 stallions serving as a control) were screened for β-boldenone and five related natural steroids: androstadienedione (ADD), androstenedione (AED), alpha-testosterone (αT), beta-testosterone (βT), and progesterone (P). Progesterone, β-testosterone, and α-testosterone were detected in more than half of the horses at low concentrations (<2 ng/mL). Occasionally, not only testosterone and progesterone but also low concentrations of AED, ADD, and boldenone (Bol) were found (0.5-5 ng/mL). Graphical Abstract A sensitive, new and fully validated UHPLC-MS/MS method has been developed that is able to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses (mares and geldings).

Determination of α- and β-boldenone sulfate, glucuronide and free forms, and androstadienedione in bovine urine using immunoaffinity columns clean-up and liquid chromatography tandem mass spectrometry analysis

The debate about the origins of boldenone in bovine urine is ongoing for two decades in Europe. Despite the fact that its use as a growth promoter has been banned in the European Union (EU) since 1981, its detection in bovine urine, in the form of α-boldenone conjugate, is considered fully compliant up to 2ngmL−1. The conjugated form of β-boldenone must be absent. In recent years, the literature about boldenone has focused on the identification of biomarkers that can indicate an illicit treatment. β-boldenone sulfate is a candidate molecule, even if the only studies currently available have taken place in small populations. In this study, a method for the determination of sulfate and glucuronate conjugates of β-boldenone was developed and validated according to the European Commission Decision 2002/657/EC and applied to α-boldenone sulfate and glucuronide, α- and β-boldenone free forms and androstadienedione (ADD), too. The clean-up with immunoaffinity columns enabled the direct determination of the conjugates and free forms and allowed specific and sensitive analyses of urine samples randomly selected to verify this method. The decision limits (CCα) ranged between 0.07 and 0.08ngmL−1, the detection capabilities (CCβ) between 0.08 and 0.1ngmL−1. Recovery was higher than 92% for all the analytes. Intra-day repeatability was between 5.8% and 17.2%, and inter-day repeatability was between 6.0% and 21.8% for the studied free and conjugated forms. This method has been developed as a powerful tool with the aim to study the origin of boldenone in a trial on a significant number of animals.

Diurnal variations of progesterone, testosterone, and androsta-1,4-diene-3,17-dione in the rumen and in vitro progesterone transformation by mixed rumen microorganisms of lactating dairy cows

Five Holstein lactating dairy cows fed 5 total mixed rations (TMR) with different forage combinations were used in a 5 × 5 Latin square design to investigate diurnal variations of progesterone (P4), testosterone, and androsta-1,4-diene-3,17-dione (ADD) concentrations in the rumen. Meanwhile, different P4 inclusion levels [0 (control), 2, 20, 40, 80, and 100ng/mL in culture fluids] were incubated in vitro for 6, 12, 24, 36, 48, and 72h together with rumen mixed microorganisms grown on a maize-rich feed mixture (maize meal:Chinese ryegrass hay = :1) with an aim to determine microbial P4 transformation into testosterone and ADD. Ruminal P4, testosterone, and ADD concentrations of lactating dairy cows were greater in the TMR with forage combination of corn silage plus alfalfa hay or Chinese wild ryegrass hay than the TMR with the corn stover-based forage combination. The diurnal fluctuation pattern showed that P4, testosterone, and ADD concentrations in the rumen were greater at nighttime than daytime and peaked 6h after feeding in the morning or afternoon. The in vitro batch cultures showed that the P4 elimination rate was highest at the P4 addition of 20ng/mL and declined with the further increased addition of P4. The treatments after dosing P4 exhibited a shorter time than the control group until half of the initial P4 inclusion was eliminated (i.e., half time), and the lowest half time (1.46h) occurred at the P4 addition of 20ng/mL. In summary, the ruminal steroids concentration was affected by forage type and quality, and the rumen microorganisms exhibited great ability to transform P4 into testosterone and ADD, depending on incubation time and initial P4 addition level, suggesting that the host might affect the metabolism of its rumen microorganisms via the endogenous steroids.

Bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione by recombinant Bacillus subtilis expressing ksdd gene encoding 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum JC-12

The enzyme 3-ketosteroid-Δ1-dehydrogenase (KSDD), involved in steroid metabolism, catalyzes the transformation of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD) specifically. Its coding gene was obtained from Mycobacterium neoaurum JC-12 and expressed on the plasmid pMA5 in Bacillus subtilis 168. The successfully expressed KSDD was analyzed by native-PAGE. The activities of the recombinant enzyme in B. subtilis were 1.75U/mg, which was about 5-fold that of the wild type in M. neoaurum. When using the whole-cells as catalysts, the products were analyzed by tin-layer chromatography and high-performance liquid chromatography. The recombinant B. subtilis catalyzed the biotransformation of AD to ADD in a percent conversion of 65.7% and showed about 18 folds higher than M. neoaurum JC-12. The time required for transformation of AD to ADD was about 10h by the recombinant B. subtilis, much shorter than that of the wild-type strain and other reported strains. Thus, the efficiency of ADD production could be improved immensely. For industrial applications, the recombinant B. subtilis containing KSDD provides a new pathway of producing steroid medicines.

Influence of hydroxypropyl-β-cyclodextrin on phytosterol biotransformation by different strains of Mycobacterium neoaurum

Cyclodextrins (CDs) can improve productivity in the biotransformation of steroids by increasing conversion rate, conversion ratio, or substrate concentration. However, little is known of the proportion of products formed by multi-catabolic enzymes, e.g., via sterol side chain cleavage. Using three strains with different androst-1,4-diene-3,17-dione (ADD) to androst-4-ene-3,17-dione (AD) ratios, Mycobacterium neoaurum TCCC 11028 (MNR), M. neoaurum TCCC 11028 M1 (MNR M1), and M. neoaurum TCCC 11028 M3 (MNR M3), we found that hydroxypropyl-β-cyclodextrin (HP-β-CD) can appreciably increase the ratio of ADD to AD, the reaction rate, and the molar conversion. In the presence of HP-β-CD, conversion of 0.5 g/L of phytosterol (PS) was 2.4, 2.4, and 2.3 times higher in the MNR, MNR M1, and MNR M3 systems, respectively, than in the controls. The ADD proportion increased by 38.4, 61.5, and 5.9 % compared with the control experiment, which resulted in a strong shift in the ADD/AD ratio in the ADD direction. Our results imply that the three PS-biotransforming strains cause efficient side chain degradation of PS, and the increased conversion of PS when using HP-β-CD may be associated with the higher PS concentration in each case. A similar solubilizing effect may not induce a prominent influence on the ADD/AD ratio. However, the different activities of the Δ¹-dehydrogenase of PS-biotransforming strains result in different incremental percentage yields of ADD and ADD/AD ratio in the presence of HP-β-CD.

Trace analysis of 28 steroids in surface water, wastewater and sludge samples by rapid resolution liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive rapid resolution liquid chromatography–tandem mass spectrometry (RRLC–MS/MS) method, combined with solid-phase extraction, ultrasonic extraction and silica gel cartridge cleanup, was developed for 28 steroids including 4 estrogens (estrone (E1), 17β-estradiol (E2), 17α-ethynyl estradiol (EE2), diethylstilbestrol (DES)), 14 androgens (androsta-1,4-diene-3,17-dione (ADD), 17α-trenbolone, 17β-trenbolone, 4-androstene-3,17-dione, 19-nortestoserone, 17β-boldenone, 17α-boldenone, testosterone (T), epi-androsterone (EADR), methyltestosterone (MT), 4-hydroxy-androst-4-ene-17-dione (4-OHA), 5α-dihydrotestosterone (5α-DHT), androsterone (ADR), stanozolol (S)), 5 progestagens (progesterone (P), ethynyl testosterone (ET), 19-norethindrone, norgestrel, medroxyprogesterone (MP)), and 5 glucocorticoids (cortisol, cortisone, prednisone, prednisolone, dexamethasone) in surface water, wastewater and sludge samples. The recoveries of surface water, influents, effluents and sludge samples were 90.6–119.0% (except 5α-DHT was 143%), 44.0–200%, 60.7–123% and 62.6–138%, respectively. The method detection limits for the 28 analytes in surface water, influents, effluents and freeze-dried sludge samples were 0.01–0.24 ng/L, 0.02–1.44 ng/L, 0.01–0.49 ng/L and 0.08–2.06 ng/g, respectively. This method was applied in the determination of the residual steroidal hormones in two surface water of Danshui River, 12 wastewater and 8 sludge samples from two wastewater treatment plants (Meihu and Huiyang WWTPs) in Guangdong (China). Ten analytes were detected in surface water samples with concentrations ranging between 0.4 ng/L (17β-boldenone) and 55.3 ng/L (5α-DHT); twenty analytes in the wastewater samples with concentrations ranging between 0.3 ng/L (P) and 621 ng/L (5α-DHT); and 12 analytes in the sludge samples with concentrations ranging between 1.6 ng/g (E1) and 372 ng/g (EADR).

Biotransformation of AD and ADD using Nostoc muscorum

The potential of microalgae for steroid transformation has been studied less than other microorganisms. They have been applied for the years in the treatment of environmental pollutions and preparation of nutrients. At the first time, Abul-Hajj and Qian indicated the conversion of androstendione (AD) to testosterone with eleven different strains of microalgae. Previtera’s group showed the transformations of progesterone and prednisolone in several microalgal cultures within four studies. In other investigations, steroid substrates were 5?-androstane-3, 17-dione, adrenosterone, androsta-1, 4-diene-3, 17-dione (ADD) and 17?-hydoxy-17?-methylandrosta-1, 4-dien-3-one. Hydrocortisone biotransformation using Nostoc muscorum was also reported with Tabatabie group. In this study, the ability of Nostoc muscorum, blue green algae, for biotransformation of AD and ADD were investigated. This potential has not been previously examined. The selected microalgae was isolated during a screening program from soil samples collected from paddy field of north of Iran. The fermentation media were included BG-11 medium together with AD or ADD 0.05%, separately, as steroid substrates. The experiment was illuminated continuously with fluorescent lamps at 40 µEm-2S-1, and incubated at a temperature of 25±2°C without shaking for ten days. The chloroform extract was loaded on silica gel plates with acetone/hexan (1:1) solvent system. The obtained fractions were further purified by re-chromatography, followed by crystallization. Then, the metabolites and unconverted substrates from each experiment were identified by melting points and spectral methods including 13C NMR, 1H NMR, FTIR and MS. The fermentation reaction of AD with Nostoc muscorum led to accumulation of testosterone in the broth medium. 1-Dehydrotestosterone was the product of ADD bioconversion in the microalgal culture. In both cases, the bioconversion characteristics observed were 17-carbonyl reduction. Thus, the microorganism represented to act selectively at the ketone group on the D-ring of both androstane like steroids.

Efficient biotransformation of cholesterol to androsta-1,4-diene-3,17-dione by a newly isolated actinomycete Gordonia neofelifaecis

A newly isolated actinomycete, Gordonia neofelifaecis (NRRL B-59395) from the faeces of Neofelis nebulosa, was used to selectively degrade the side-chain of cholesterol. The intermediates were purified and characterized. Quantitative analysis of the accumulated metabolites from cholesterol side-chain cleavage was conducted during the biotransformation. The results showed that the profile of accumulated intermediates was different from those of other reported microorganisms. Among the five metabolites, androsta-1,4-diene-3,17-dione (ADD) was the main product of the side-chain degradation, with a high conversion rate (87.2%), indicating its potential for industrial production of ADD. At the end of transformation, the substrate cholesterol was completely consumed. The effect of some factors on the bioconversion was also investigated. To our best knowledge, this is the first report regarding cholesterol side-chain cleavage using bacteria belonging to Gordonia.

Degradation Kinetics of Testosterone by Manure‐Borne Bacteria: Influence of Temperature, pH, Glucose Amendments, and Dissolved Oxygen

Land application of manure may contribute endocrine disrupting compounds (EDCs) such as steroid hormones to the environment. Little attention has been paid to the potential for degradation of steroid hormones by manure-borne bacteria and their degradation kinetics and pathways. In a laboratory study, the potential for biodegradation of testosterone, 17beta-estradiol (E2) and progesterone by swine (Sus scrofa) manure-borne bacteria was examined. In addition, the impact of temperature, pH (6, 7, and 7.5), glucose amendments (0, 3, and 22 mmol L(-1)), and presence of oxygen on testosterone degradation kinetics was determined. Testosterone, 17beta-estradiol and progesterone were biodegraded within 25 h of reaction initiation under aerobic conditions. The degradation of testosterone followed pseudo first-order and zero-order reaction kinetics under aerobic and anaerobic conditions, respectively, in tryptic soy broth (TSB) pre-enriched systems. The half-life (t1/2) for the degradation of testosterone under anaerobic conditions was six times longer than aerobic conditions. Testosterone degradation was found to significantly increase (- 17%) when incubated at 37 degrees C vs. 22 degrees C. The impact of pH (t1/2 ranged from 4.4-4.9 h) and glucose amendments (t1/2 ranged from 4.6-5.1 h) on the testosterone degradation rate were found to be small. Testosterone was transformed to dehydrotestosterone (DHT) (major degradation product), androstenedione (AD), and androstadienedione (ADD) under aerobic conditions as revealed by liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS). These results indicate that testosterone is rapidly degraded by manure-borne bacteria under a wide range of environmentally relevant conditions. However, the formed degradation products are still of potential concern due to their endocrine disrupting potential.

Characterisation of steroids in wooden crates of veal calves by accelerated solvent extraction (ASE®) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-QqQ-MS-MS)

Illegal steroid administration to enhance growth performance in veal calves has long been, and still is, a serious issue facing regulatory agencies. Over the last years, stating undisputable markers of illegal treatment has become complex because of the endogenous origin of several anabolic steroids. Knowledge on the origin of an analyte is therefore of paramount importance. The present study shows the presence of steroid analytes in wooden crates used for housing veal calves. For this purpose, an analytical procedure using accelerated solvent extraction (ASE(R)), solid-phase extraction (SPE) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-MS-MS) is developed for the characterisation of androstadienedione (ADD), boldenone (bBol), androstenedione (AED), beta-testosterone (bT), alpha-testosterone (aT), progesterone (P) and 17alpha-hydroxy-progesterone (OH-P) in wood samples. In samples of wooden crates used for housing veal calves, ADD, AED, aT and P could be identified. Using the standard addition approach concentrations of these analytes were determined ranging from 20 +/- 4 ppb to 32 +/- 4 ppb for ADD, from 19 +/- 5 ppb to 44 +/- 17 ppb for AED, from 11 +/- 6 ppb to 30 +/- 2 ppb for aT and from 14 +/- 1 ppb to 42 +/- 27 ppb for P, depending on the sample type. As exposure of veal calves to steroid hormones in their housing facilities might complicate decision-making on illegal hormone administration, inequitable slaughter of animals remains possible. Therefore, complete prohibition of wooden calf accommodation should be considered.