Androgenic steroids in Over-the-Counter dietary Supplements: Analysis for association with adverse health effects.

From 2012 to 2013, approximately 16 New York residents reported vague, nonspecific adverse health effects which included fatigue, loss of scalp hair, and muscle aches. One patient was hospitalized for liver damage. An epidemiological investigation identified a common factor among these patients; the consumption of B-50 vitamin and multimineral supplements from the same supplier. To investigate whether these nutritional supplements might have been responsible for the adverse health effects observed, comprehensive chemical analyses of marketed lots of the supplements were performed. To determine presence of organic components and contaminants, organic extracts of samples were prepared and analyzed using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography high-resolution mass spectrometry (LC-HRMS), and nuclear magnetic resonance (NMR). These analyses revealed the presence of significant levels of methasterone (17ß-hydroxy-2α,17α-dimethyl-5α-androstane-3-one), an androgenic steroid and schedule III-controlled substance; dimethazine, an azine-linked dimer of methasterone; and methylstenbolone (2,17α-dimethyl-17ß-hydroxy-5α-androst-1-en-3-one), a related androgenic steroid. Methasterone and extracts of certain supplement capsules were identified as highly androgenic in luciferase assays by using an androgen receptor promoter construct. This androgenicity persisted for several days after cell exposure to the compounds. The presence of these components in implicated lots were associated with adverse health effects and the hospitalization of one patient and the presentation of symptoms of severe virilization in a child. These findings underscore the need for more rigorous oversight of the nutritional supplement industry.

Rapid method for the detection of rodenticides in contaminated foods.

Rodenticides are toxic chemicals used to control rodent populations and are among the most common household toxicants. Ingestion of foods contaminated with rodenticides may cause severe illness or death in humans and animals. A rapid analytical method was developed for the identification of nine common rodenticides in foods using solid-liquid extraction followed by dispersive-solid phase extraction prior to the analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and UV detection. The method validation on a variety of food matrices including cornmeal, peanut, whole wheat flour and pork liver produced average recoveries between 91.2 and 107% with relative standard deviations between 2.6 and 14% for all studied rodenticides. The method detection limits ranged from 2.7 to 8.2 µg/kg (ppb) for eight rodenticides analyzed by LC-MS/MS and between 0.10 and 0.21 mg/kg (ppm) for bromethalin which was analyzed by LC with UV detection. This method could be useful in preparedness for emergency response situations involving widespread food contamination, terrorist acts or for forensic studies.

Population pharmacokinetics of cefuroxime and uptake into hip and spine bone of patients undergoing orthopaedic surgery.

OBJECTIVES: To reduce the incidence of peri- or postoperative infections in orthopaedic surgery, patients are prophylactically treated with antibiotics. Here, we wanted to know whether effective bone and intervertebral disc concentrations of cefuroxime are reached. METHODS: Patients undergoing surgery of hip (N = 40; 62.5% male) or spine (N = 40; 55% male) were pretreated with 1.5 g of the second-generation cephalosporin cefuroxime before surgery. We studied plasma population kinetics and bone and intervertebral disc (C5/6 till L5/S1) concentrations of cefuroxime using high-performance liquid chromatography. KEY FINDINGS: The plasma kinetics of cefuroxime in 80 patients was analysed using a population approach. The clearance amounted to 7.86 l/h. The peripheral and central volumes of distribution were estimated as 8.45 and 10.4 l, respectively. The concentrations in hip samples amounted to 9.8 ± 0.6 µg/g in cancellous bone and 8.9 ± 0.8 µg/g in cortical bone. Cefuroxime concentrations in vertebral bone and intervertebral discs were calculated as 9.6 ± 1.3 and 8.9 ± 1.1 µg/g, respectively. CONCLUSION: Even if a majority of patients undergoing hip or spine surgery probably achieved adequate concentrations of cefuroxime, not all patients reached bone concentrations of cefuroxime above a recommended breakpoint for susceptible germs at the time of surgery.

Application of dispersive solid phase extraction for trace analysis of toxic chemicals in foods.

The objectives of this study were to develop and validate a method for the identification of toxic organic chemicals, including groups of controlled substances, alkaloids and pesticides that are highly toxic and considered threats to public health. This project aims to ensure our laboratory's readiness to respond to emergencies involving our food supply in cooperation with the Food Emergency Response Network (FERN) program. The food matrices were homogenized in a blender or food processor prior to extraction with an acetonitrile-water mixture using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure. The extracts were then analyzed by either gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-electrospray tandem mass spectrometry (LC-ESI/MS/MS). Method validation was performed on a variety of food matrices including lettuce, grapes, milk, chicken, pork and beef. MDLs for the toxic compounds ranged from 0.01 to 0.66 mg/kg (ppm). The findings in this study will provide a valuable resource for the determination of toxic chemicals in food matrices for emergency response situations.

Determination of trace amounts of ethylene glycol and its analogs in water matrixes by liquid chromatography/tandem mass spectrometry.

Contamination of drinking water by ethylene glycol (EG) is a public health concern. EG causes adverse health effects in humans and animals, including cardiopulmonary and acute renal failure. EG and other glycols, such as propylene glycol (PG) are major components in antifreeze liquids, which may be the main source of contamination of ground water. A sensitive LC/electrospray ionization (ESI)-MS/MS method was developed to measure trace amounts of EG, diethylene glycol, and 1,2- and 1,3-PG in several water sources, including municipal tap, lake, river, and salinated water. In this method, glycols in water samples were derivatized with benzoyl chloride by the Schotten-Baumann reaction, followed by liquid-liquid extraction using pentane as the organic solvent prior to the LC/ESI-MS/MS determination. QC included analysis of a method blank and samples fortified at low and high levels. Analytical data showed excellent linear calibration for all observed glycols, with good precision and accuracy. The method detection limits for the studied glycols ranged from 1.9 to 6.1 ng/mL across the water matrixes tested. This method is suitable to help assess water quality in areas that may be prone to glycol contamination.

Polychlorinated biphenyls and polybrominated diphenyl ethers alter striatal dopamine neurochemistry in synaptosomes from developing rats in an additive manner.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are widespread environmental contaminants associated with changes in behavior and neurochemical function in laboratory animals and behavioral deficits in children. PCBs and PBDEs are found in food, especially in seafood and dairy products, and coexposure to these contaminants is likely. We examined the effects of an environmentally relevant mixture of PCBs (Fox River Mix [FRM]) and a PBDE mixture (DE-71) alone and in combination on synaptosomal and medium dopamine (DA) levels and the levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in striatal synaptosomes derived from postnatal days (PND) 7, PND14, or PND21 rats. FRM elevated medium DA and reduced synaptosomal DA concentrations with greater potency than equimolar concentrations of DE-71. The effects of FRM, but not DE-71, were dependent on the age of the animals from which the synaptosomes were derived, with greater effects observed in synaptosomes from the youngest animals. We used Bliss' model of independence to assess the possible interaction(s) of a 1:1 mixture of FRM and DE-71 on synaptosomal DA function and found that the effects of the FRM/DE-71 mixture were additive. Furthermore, as for FRM alone, the effects of the FRM/DE71 mixture were greater in synaptosomes prepared from PND7 rats than in synaptosomes from PND14 and PND21 rats. Because the effects of these contaminants are additive, it is necessary to take into account the cumulative exposure to organohalogen contaminants such as PCBs and PBDEs during risk assessment.

Use of methanol for the efficient extraction and analysis of melamine and cyanuric acid residues in dairy products and pet foods.

The recent worldwide shortage of acetonitrile has prompted the development of a new method using methanol as an alternative organic solvent in the extraction and liquid chromatographic analysis of melamine and cyanuric acid that may be present as contaminants in dairy products and pet foods. A simple extraction of melamine and cyanuric acid residues in fortified samples was successfully achieved, using a methanol-water mixture and analysis by isotopic dilution high-performance liquid chromatography-triple-quadrupole mass spectrometry (HPLC-MS/MS). A two-step centrifugation procedure was employed to remove matrix components from extracts. The separation of melamine and cyanuric acid was carried out on a Dionex Acclaim Trinity P1 column, with a methanol and ammonium acetate buffer used as the mobile phase. Excellent linearity was achieved for both the melamine and cyanuric acid calibrations. A variety of dairy products and pet foods were fortified with melamine and cyanuric acid at three levels, 1, 2.5, and 10 microg/g, producing recovery yields of 101-119% for melamine and 84-123% for cyanuric acid. The lower limit of quantification (LLOQ) of melamine was 0.03 microg/g for liquid milk and 0.05 microg/g for dry infant milk formula. The quantitative results were comparable with those derived from previous methods that have been proposed by the U.S. Food and Drug Administration for the screening of melamine and its analogues in foods.

Methylmercury inhibits dopaminergic function in rat pup synaptosomes in an age-dependent manner.

Methylmercury (MeHg) is an environmental neurotoxicant that is especially harmful during brain development. Previously, we found greater sensitivity to MeHg-induced oxidative stress and greater loss of mitochondrial membrane potential in synaptosomes from early postnatal rats than in synaptosomes from older rat pups and adults. Here, we determine whether MeHg exposure also leads to greater changes in dopamine (DA) levels and dopamine transporter (DAT) function in synaptosomes from early postnatal rats. We report that MeHg exposure leads to DAT inhibition, and increases the levels of released DA compared to control; further, the effects are much greater in synaptosomes prepared from postnatal day (PND) 7 rats than in synaptosomes from PND 14 or PND 21 animals. In addition to the effects of MeHg in young rats, we observed age-dependent differences in dopaminergic function in unexposed synaptosomes: synaptosomal DA levels increased with age, whereas medium (released) DA levels were high at PND 7 and were lower in PND 14 and PND 21 synaptosomes. DAT activity increased slightly from PND 7 to PND 14 and then increased more strongly to PND 21, suggesting that higher DA release, in addition to the lower DAT activity seen in PND 7 animals, was responsible for the age differences in levels of released DA. These results demonstrate that MeHg affects the dopaminergic system during early development; it thus may contribute to the neurobehavioral effects seen in MeHg-exposed children.

Coplanar PCB congeners increase uterine weight and frontal cortical dopamine in the developing rat: implications for developmental neurotoxicity.

We show that developmental exposure of the laboratory rat to the coplanar polychlorinated biphenyl (PCB) congener 3,4,3',4'-tetrachlorobiphenyl (TCB) and the structurally similar congener 3,4,5,3',4'-pentachlorobiphenyl (PtCB) elevates dopamine (DA) concentrations in the prefrontal cortex (PFC). To determine whether these coplanar congeners are estrogenic, and may thus contribute to the elevations in PFC DA, we measured uterine wet weight (UWW) in prepubertal rats exposed to TCB or PtCB. For comparison, additional animals were exposed to either the ortho-substituted congener 2,4,2',4'-tetrachlorobiphenyl (o-TCB) or 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), a coplanar congener highly resistant to metabolism. Both TCB and PtCB increased UWW, but this effect was blocked after exposure to the anti-estrogen ICI 182,780. Neither o-TCB nor HCB altered UWW. These results demonstrate that certain coplanar PCB congeners and/or their metabolites, are estrogenic, and suggest that exposure during critical periods of neuronal development may increase central DA concentrations, and by inference, alter behavior.

Polychlorinated biphenyls alter extraneuronal but not tissue dopamine concentrations in adult rat striatum: an in vivo microdialysis study.

Polychlorinated biphenyls (PCBs) reduce tissue dopamine (DA) concentrations and increase media DA concentrations in both in vitro preparations of bovine adrenal chromaffin cells and adult rat striatal tissue. To determine whether these changes also occur in the intact animal, we used in vivo microdialysis to determine changes in concentrations of DA in striatal dialysates from freely moving adult male rats after exposure to 25 mg/kg/day Aroclor 1254 for varying periods of time. We also determined DA concentrations in striatal tissue obtained postmortem from similarly treated animals. The effects of PCBs on dialysate DA concentrations depended on the length of exposure; DA concentrations were significantly elevated after 3 days of exposure and were significantly reduced after exposure for periods of 1 week or longer. On the other hand, striatal tissue concentrations of DA, determined postmortem in rats exposed to PCBs for the same periods of time, were not significantly altered. We suggest that these time-dependent alterations in dialysate DA concentrations a) reflect PCB-induced alterations of both plasma membrane and vesicular DA transporter function; b) provide a more sensitive index of altered central DA function after exposure to PCBs than does measurement of postmortem tissue DA concentrations; and c) play an important role in mediating some PCB-mediated changes in behavior.