Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa

The feasibility of the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids, as a novel approach to reduce their production costs, was demonstrated by the cultivation of Pseudomonas aeruginosa IFO3924. Fairly large amounts of PHAs and rhamnolipids were obtained from the bacterial cells and the culture supernatant, respectively. Decanoate was a more suitable carbon source than ethanol and glucose for the simultaneous production, although glucose was suitable for cell growth without an induction period under pH control. The kind of carbon source affected PHA monomer composition markedly and PHA molecular weight slightly. Monorhamnolipids and dirhamnolipids were included in the rhamnolipids extracted from the culture supernatant using decanoate, glucose, or ethanol as the carbon source. Both PHAs and rhamnolipids were synthesized after the growth phase. PHA content in the cell reached a maximum when the carbon source was exhausted. After exhaustion of the carbon source, PHA content decreased rapidly, but rhamnolipid synthesis, which followed PHA synthesis, continued. This resulted in a time lag for the attainment of maximum levels of PHAs and rhamnolipids. The reusability of the cells used in rhamnolipid production was evaluated in the repeated batch culture of P. aeruginosa IFO3924 for the simultaneous production of PHAs and rhamnolipids. High concentrations of rhamnolipids in the culture supernatant were attained at the end of both the first and second batch cultures. High PHA content was achieved in the resting cells that were finally harvested after the second batch. Simultaneous production of PHAs and rhamnolipids will enhance the availability of valuable biocatalysts of bacterial cells, and dispel the common belief that the production cost of PHAs accumulated intracellularly is almost impossible to become lower than that of cells themselves.     

Synthesis and analytics of 2,2,3,4,4-d5-19-nor-5alpha-androsterone--an internal standard in doping analysis

A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed.     

pH-imprinted lipase catalyzed synthesis of dextran fatty acid ester

The application of enzymatic catalysis for the synthesis of polysaccharide-based surfactants was investigated. The polysaccharide dextran, a neutral bacterial polysaccharide consisting of -1,6 linked glucose units, was chemically modified by the attachment of hydrophobic groups through a transesterification reaction with a vinyl decanoate. A screening of commercially available lipases and protease for the synthesis of amphiphilic polysaccharides in DMSO suggested that lipase AY from Candida rugosa modified dextran T-40 with vinyl decanoate at the highest conversion. A pH-adjustment in a phosphate buffer at pH 7.5 prior to use is crucial to make this enzyme active in DMSO. The effect of enzyme concentration and mole ratio of fatty ester to dextran T-40 on the conversion and the rate of reaction were studied. Finally, investigation of the kinetics and regioselectivity of lipase AY-catalyzed modification offer a possibility to regulate the position and the extent of hydrophobic group attached to dextran. These two properties are fundamental for controlling the physico-chemical properties of the final polymeric surfactants.     

Amphetamine-induced aggression is enhanced in rats pre-treated with the anabolic androgenic steroid nandrolone decanoate

Aggression is one of the most commonly reported psychiatric side effects among anabolic-androgenic steroids (AAS) users. Furthermore, anecdotal stories say the aggression is even more profound when a current, or former, AAS-user consumes other drugs of abuse such as amphetamine and alcohol. In the present study, we examined the effect of amphetamine on defensive reactivity and defensive aggression in Sprague-Dawley rats after chronic AAS treatment (daily intramuscular [i.m.] injections with 15 mg/kg nandrolone decanoate [ND] for 14 days). Defensive reactions in rodents occur in response to a real threat, but also to perceived provocation, for example, elicited by innocuous stimuli as reaction towards the experimenter. The defensive reactivity and aggression test employed in this study evaluates each rat's reaction towards four different stimuli (I: approach of a rod; II: startle to an air puff; III: poking with a rod at the flanks, and IV: capturing with a gloved hand) at two different occasions. Immediately following the ND treatment period, no change in the defensive response was found. Nevertheless, an amphetamine challenge given 3 weeks after the last ND or vehicle injection induced a marked increased defensive aggressive response in the ND, compared to vehicle-pre-treated rats. Both ND- and vehicle-pre-treated rats receiving amphetamine were found to be more aggressive than comparable groups receiving a saline injection. It can be concluded that pre-treatment with ND modulates the behavioral response to amphetamine and induces long lasting changes in the behavioral response.     

Gas chromatography–combustion–isotope ratio mass spectrometry analysis of 19-norsteroids: application to the detection of a nandrolone metabolite in urine

Determination of whether the major metabolite of nandrolone in urine, 19-norandrosterone (19-NA), is exogenous or endogenous in origin is one of the most exciting challenges for antidoping laboratories. Gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) can be used to differentiate these two origins by carbon isotopic ratio analysis. A complete method for purification of 19-NA in urine has been established. Acetylated ketosteroids, and in particular 19-NA, are isolated from the urine matrix before analysis after hydrolysis and purification of urine by reversed-phase and normal solid-phase extraction. The limit of detection for 19-NA was about 60 ng with recoveries of 54–60%. Evidence of exogenous administration of 19-NA may be established from isotope ratio determination from the ¹³C/¹²C ratios of several synthetic 19-norsteroids compared to those obtained for endogenous steroids.     

Easy stereoselective synthesis of 5α-estrane-3β,17α-diol, the major metabolite of nandrolone in the horse

5α-Estrane-3β,17α-diol is the major metabolite of nandrolone in horse urine. The presence of 5α-estrane-3β,17α-diol in female and gelding urines is prohibited by Racing Rules and its natural presence in male urine led regulation authorities to establish a concentration threshold of 45 ng/mL. This paper describes a rapid, simple and stereoselective synthesis of 5α-estrane-3β,17α-diol, providing horseracing laboratories with an essential reference material for their antidoping performance.     

The impact of nandrolone decanoate administration on ovarian and uterine tissues in rat: Luteinizing hormone profile,histopathological and morphometric assessment

The study had been conducted to evaluate the effects of nandrolone decanoate (abused repeated doses) on female rat’s ovary and uterus during administration and withdrawal. The study included 18 rats that were divided into control group (n = 6) and treated group (n = 12). The treated group was injected intramuscular (IM) with nandrolone decanoate (7 mg/kg body weight) for three consecutive days, for two weeks. The study stated that nandrolone decanoate increases the weights of body, ovary, and uterus. Moreover, it has a tendency of bringing upon modifications in the biochemical, histopathological, and morphological makeup of the female reproductive aspects. In conclusion, nandrolone decanoate has been identified as deleterious element for the female rats, and it is suggested that keen observations must be made on the human abusers to control and manage the possible pathologies.     

Dose‐Dependent Effects and Reversibility of the Injuries Caused by Nandrolone Decanoate in Uterine Tissue and Fertility of Rats

This study is the first to investigate the effects of different doses of nandrolone decanoate (ND) upon uterine tissue and fertility, and if the reproductive alterations can be restored after cessation of the treatment. Wistar female rats were treated with ND at doses of 1.87, 3.75, 7.5, and 15 mg/kg body weight, diluted in vehicle (n = 30/group), or received only mineral oil (control group, n = 45). The animals were divided into three periods of study: ND‐treated receiving a daily subcutaneous injection for 15 consecutive days (1), and treatment with ND followed by 30‐day recovery (2), and 60‐day recovery (3). At the end of each period, five females per group were induced to death to histopathological analysis and the others were allowed to fertility evaluation (at 19th gestational day). Animals that received ND followed by 30‐day recovery exhibited persistent diestrous and marked suppression of reproductive capacity. Conversely, after 60‐day recovery, only lowest doses females (1.87 and 3.75 mg/kg) exhibited restoration of normal estrous cyclicity. Uterine weights were increased after ND treatment similarly to that of the controls after 60‐day recovery. The ND‐treated groups showed histopathological changes in the endometrium, myometrium, and perimetrium, and an increase in the thickness of both muscular and serous layers. Notably, the recovery of uterine tissue after ND treatment was dose‐ and period‐dependent. We reported that administration of ND promoted damage in uterine tissue and fertility of rats, and the recovery periods were insufficient to restore all of the side effects caused by ND under a dose‐dependent response     

Determination of nandrolone and metabolites in urine samples from sedentary persons and sportsmen

Metabolites of nandrolone were determined in the urine of several sportsmen, sedentary and post-menopausal women by capillary gas chromatography–mass spectrometry quadrupole (GC–MS) and capillary gas chromatography mass–mass spectrometry ion trap (GC–MS–MS) methods. The method employed was GC–EI-MS with 17α-methyltestosterone as internal standard with ethyl ether extraction prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405 and 420 for the nandrolone metabolites, and 418 and 403 for nandrolone derivative. Recovery for nandrolone, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) was 97.20, 94.17 and 95.54%, respectively. Detection limits for nandrolone, 19-NA and 19-NE were 0.03, 0.01 and 0.06 ng/ml. Metabolites of nandrolone (19-NA and 19-NE) were found in 12.5% (n=40) of sportsmen and 40% (n=10) of post-menopausal women.     

Long-term effects of pubertal anabolic-androgenic steroid exposure on reproductive and aggressive behaviors in male rats

The current study examined acute and long-term effects of anabolic-androgenic steroid (AAS) exposure during puberty on copulation, vocalizations, scent marking, and intermale aggression, both with and without tail pinch, in intact male rats. Animals received 5 mg/kg of testosterone, nandrolone, stanozolol, or vehicle, beginning at puberty. After 5 weeks, behavior tests were performed while continuing AAS injections. AAS treatment was then discontinued. Behaviors were tested during 3-5 weeks, 9-11 weeks, and 15-17 weeks of withdrawal. During AAS administration, stanozolol males showed significant reductions in all behaviors compared with controls, except aggression with tail pinch. Nandrolone treatment significantly reduced vocalizations and scent marking, and testosterone had no significant effect on behavior. During withdrawal, behaviors in stanozolol males recovered to control levels at variable rates: aggression at 4 weeks; mounts, vocalizations, and scent marking at 9 weeks; and ejaculations at 15 weeks of withdrawal. Stanozolol males showed significantly higher levels of tail pinch-induced aggression during every withdrawal test. Nandrolone-treated males scent-marked at control levels by 9 weeks withdrawal but displayed significantly fewer vocalizations and significantly more tail pinch-induced aggression than controls for the entire study. Testosterone-treated males scent-marked significantly below controls at 3 weeks withdrawal and showed significantly more tail pinch-induced aggression at 5 weeks withdrawal. All three AAS significantly increased tail pinch-induced aggression compared with corresponding nontail pinch tests, even at study endpoint. These results suggest that alterations in androgen-dependent behaviors by pubertal AAS exposure can persist long after drug exposure, and some effects may even be permanent.