Developmental exposure to decabrominated diphenyl ether (BDE‐209): Effects on sperm oxidative stress and chromatin dna damage in mouse offspring

Polybrominated diphenyl ethers (PBDEs) are used as brominated flame retardants and have been found in human milk in recent years. This study investigates whether prenatal exposure to decabrominated diphenyl ether (BDE‐209) induces sperm dysfunction in male offspring. Pregnant CD‐1 mice were gavaged once daily with corn oil (control), 10, 500, and 1500 mg kg^?1 body weight of BDE‐209 from day 0 of gestation to day 17. The outcomes of male reproductive parameters were assessed on postnatal day 71. Anogenital distance, sperm‐head abnormalities, and testicular histopathology were significantly affected in male offspring prenatally exposed to 1500 mg kg^?1. Significant increases in the tendency for sperm DNA denaturation (αT) induction and the DNA fragmentation index (DFI) were found in those exposed to 10, 500, and 1500 mg kg^?1 (P < 0.05). We observed a significant increase of sperm hydrogen peroxide (H₂O₂) generation in the 10 and 1500 mg/kg/day groups compared to the control group (P < 0.05). Although our findings suggested that the mechanisms underlying BDE‐209‐induced sperm DNA damage and H₂O₂ generation might not be represented as a dose‐response relationship, we found that the greater the excess production of sperm H₂O₂, the greater the sperm αT (r = 0.65, P = 0.0155) and DFI (r = 0.53, P = 0.002). In conclusion, developmental exposure to BDE‐209 induced sperm‐head abnormality, oxidative stress, chromatin DNA damage, and testicular histopathological changes. These findings suggest that BDE‐209‐induced male reproductive effects might involve the formation of sperm H₂O₂ which attacks nucleic acids via H₂O₂ generation. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Heating of BDE-209 and BDE-47 in plant oil in presence of o,p′-DDT or iron(III) chloride can produce monochloro–polybromo diphenyl ethers

Heating processes of food can alter the concentrations and composition of organohalogen compounds. In this study the fate of two polybrominated diphenyl ether (PBDE) congeners, decabromodiphenyl ether (BDE-209) and 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) was analyzed when heated in plant oil with and without additional compounds. When the PBDEs were heated in pure plant oil, no transformation was observed. Heating of decabromodiphenyl ether (BDE-209) together with ortho,para′-dichlorodiphenyltrichloroethane (o,p′-DDT) or iron(III) chloride in plant oil resulted in the formation of monochloro–nonabromodiphenyl ethers (Br₉Cl-DEs). Almost 10% of the initial amount of BDE-209 was transferred into Br₉Cl-DEs. Heating BDE-47 in the presence of iron(III) chloride produced two monochlorinated transformation products which were tentatively identified as 2,2′,4-tribromo-4′-chlorodiphenyl ether (4′-Cl-BDE-17) and 2,4,4′-tribromo-2′-chlorodiphenyl ether (2′-Cl-BDE-28). However, the reactivity was smaller and no Br → Cl exchange was observed with o,p′-DDT. The conditions used in our experiments (200 °C; heating 30 min–3 h) indicate that such reactivity may also occur during cooking of fish, meat and other food samples.     

Effects of repeated exposure to decabrominated diphenyl ether (BDE-209) on mice nervous system and its self repair

Decabrominated diphenyl ether (BDE-209) is widely used as brominated flame retardants (BFRs). In order to investigate its direct neurotoxic effects on mice and its self repair, mice were repeatedly exposured to BDE-209. Different concentrations of BDE-209 (0, 0.1, 40, 80 and 160mg/kgbw/d) were administered daily to CD-1 Swiss mice by gavage for 15, 30 and 60 days. Body and brain weights were recorded after the exposure. And the self repair was studied. Levels of acetylcholinesterase (TchE), superoxide dismutase (SOD) and malonic dialdehyde (MDA) in brain were analyzed. Experimental results suggested that the nervous system was permanently damaged by BDE-209 through the cholinergic system enzyme.     

Long‐term exposure to high levels of decabrominated diphenyl ether inhibits CD4 T‐cell functions in C57Bl/6 mice

In recent years, the adverse health effects of decabrominated diphenyl ether (BDE‐209) have raised more concerns as a growing number of studies reported its persistence in the environment and abundance in the human population, especially in occupational environmental compartments and exposed personnel. This study applies our previous animal model simulating occupational exposure to BDE‐209 to investigate its potential adverse effects on CD4 T cells. Female C57Bl/6 mice were orally gavaged with BDE‐209 at a dose of 800 mg kg^?1 every 2 days for 10 months and the blood of each mouse was collected for analysis. Kinetic changes of the peripheral immune system were investigated from 1 to 5 months of exposure. The chronic effects on cytokine production, proliferation and the antigen‐specific responses of CD4 T cells were evaluated at 7, 9 and 10 months, respectively. The results have shown that impaired proliferation and cytokine (IFN‐γ, IL‐2 or TNF‐α) production of CD4 T cells were observed in BDE‐209‐exposed mice, accompanied by increased T regulatory cells in the blood. BDE‐209 exposure in vitro also suppressed the reactivity of CD4 T cells at concentrations of 0.01, 0.1, 1 and 10 μM. Furthermore, we observed weaker antigen‐specific CD4 T‐cell responses to Listeria monocytogenes infection in the mice exposed to BDE‐209, suggesting decreased resistance to exogenous pathogens. Taken together, these observations indicate an impaired cellular immunity after long‐term and relative high‐dose exposure to BDE‐209 in adult mice. Copyright © 2015 John Wiley & Sons, Ltd.     

Assessment of the neurotoxic mechanisms of decabrominated diphenyl ether (PBDE-209) in primary cultured neonatal rat hippocampal neurons includes alterations in second messenger signaling and oxidative stress

2′,2′,3′,3′,4′,4′,5′,5′,6′,6′,-decabrominated diphenyl ether (PBDE-209) is the most widely used polybrominated diphenyl ethers (PBDEs) globally. Some animal experiments have found that PBDE-209 caused developmental neurotoxicity. But detailed mechanisms are less well understood. Our experiments were conducted to research the potential neurotoxic mechanisms of PBDE-209 in primary cultured neonatal rat hippocampal neurons by measuring cell viability, apoptotic rate, expression of P38 mitogen-activated protein kinases (MAPKs), calcium ion concentration, oxidative stress, nitrous oxide (NO) content, and global gene DNA methylation levels. The neurons were exposed to different PBDE-209 concentrations (0, 10, 30 and 50 μg/ml). The difference between the experimental groups and control groups was significant (P < 0.05). PBDE-209 increased the rate of apoptosis, expression of P38 MAPK, calcium ion concentration, reactive oxygen species (ROS) level, malondialdehyde (MDA) content and NO content (P < 0.05). In addition, PBDE-209 deceased cell viability, activity of superoxide dismutase (SOD) and the levels of global gene DNA methylation (P < 0.05). The results suggested that PBDE-209 could affect secondary messengers, cause oxidative stress and decrease global gene DNA methylation levels. These actions may contribute to the mechanism of PBDE-209 neurotoxicity.     

Toxicokinetics of methyl tert-butyl ether and its metabolites in humans after oral exposure.

Methyl tert-butyl ether (MTBE) is widely used as an additive to gasoline, to increase oxygen content and reduce tailpipe emission of pollutants. Widespread human exposure to MTBE may occur due to leakage of gasoline storage tanks and a high stability and mobility of MTBE in ground water. To compare disposition of MTBE after different routes of exposure, its biotransformation was studied in humans after oral administration in water. Human volunteers (3 males and 3 females, identical individuals, exposures were performed 4 weeks apart) were exposed to 5 and 15 mg 13C-MTBE dissolved in 100 ml of water. Urine samples from the volunteers were collected for 96 h after administration in 6-h intervals and blood samples were taken in intervals for 24 h. In urine, MTBE and the MTBE-metabolites tert-butanol (t-butanol), 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were quantified, MTBE and t-butanol were determined in blood samples and in exhaled air in a limited study of 3 male volunteers given 15 mg MTBE in 100 ml of water. MTBE blood concentrations were 0.69 +/- 0.25 microM after 15 mg MTBE and 0.10 +/- 0.03 microM after 5 mg MTBE. MTBE was rapidly cleared from blood with terminal half-lives of 3.7 +/- 0.9 h (15 mg MTBE) and 8.1 +/- 3.0 h (5 mg MTBE). The blood concentrations of t-butanol were 1.82 +/- 0.63 microM after 15 mg MTBE and 0.45 +/- 0.13 microM after 5 mg MTBE. Approximately 30% of the MTBE dose was cleared by exhalation as unchanged MTBE and as t-butanol. MTBE exhalation was rapid and maximal MTBE concentrations (100 nmol/l) in exhaled air were achieved within 10-20 min. Clearance of MTBE by exhalation paralleled clearance of MTBE from blood. T-butanol was cleared from blood with half-lives of 8.5 +/- 2.4 h (15 mg MTBE) and 8.1 +/- 1.6 h (5 mg MTBE). In urine samples, 2-hydroxyisobutyrate was recovered as major excretory product, t-butanol and 2-methyl-1,2-propane diol were minor metabolites. Elimination half-lives for the different urinary metabolites of MTBE were between 7.7 and 17.8 h. Approximately 50% of the administered MTBE was recovered in urine of the volunteers after both exposures, another 30% was recovered in exhaled air as unchanged MTBE and t-butanol. The obtained data indicate that MTBE-biotransformation and excretion after oral exposure is similar to inhalation exposure and suggest the absence of a significant first-pass metabolism of MTBE in the liver after oral administration.     

Neurotoxicity of the pentabrominated diphenyl ether mixture, DE-71, and hexabromocyclododecane (HBCD) in rat cerebellar granule cells in vitro

Polybrominated diphenyl ethers (PBDE) and hexabromocyclododecane (HBCD) are compounds used as additive flame retardants in plastics, electronic equipment, and textiles. The aim of the present study was to investigate the in vitro effects of the pentabrominated diphenyl ether mixture, DE-71, and HBCD on cerebellar granule cells (CGC). Both DE-71 and HBCD induced death of CGC in low micromolar concentrations. The NMDA receptor antagonist MK801 (3 μM), and the antioxidant α-tocopherol (50 μM) significantly reduced the cell death. Incubation of the compounds together with the rat liver post-mitochondrial (S9) fraction reduced cell death by 58 and 64% for DE-71 and HBCD, respectively. No ROS formation and no elevation in intracellular calcium were observed. We further demonstrated apoptotic morphology (Hoechst straining) after exposure to low levels of the two brominated flame retardants and signs of DNA laddering were found after DE-71 exposure. However, other hallmarks of apoptosis, like caspase activity, were absent indicating an atypical form of apoptosis induced by DE-71. After intraperitoneal injection of the two compounds both DE-71 and HBCD were found in significant amounts in brain (559 ± 194 and 49 ± 13 μg/kg, respectively) and liver (4,010 ± 2,437 and 1,248 ± 505 μg/kg, respectively) 72 h after injection. Our results indicate that the lower brominated PBDEs have a higher potency of bioaccumulation than HBCD, and that both compounds have a neurotoxic potential in vitro.     

Toxicologic and immunologic effects of perinatal exposure to the brominated diphenyl ether (BDE) mixture DE‐71 in the Sprague‐Dawley rat

Brominated diphenyl ethers (BDEs) are persistent environmental contaminants found in human blood, tissues, and milk. To assess the impact of the commercial BDE mixture DE‐71 on the developing immune system in relation to hepatic and thyroid changes, adult (F0) rats were exposed to DE‐71 by gavage at doses of 0, 0.5, 5, or 25 mg/kg body weight (bw)/d for 21 weeks. F0 rats were bred and exposure continued through gestation, lactation and postweaning. F1 pups were weaned and exposed to DE‐71 by gavage from postnatal day (PND) 22 to 42. On PND 42, half of the F1 rats were assessed for toxicologic changes. The remaining F1 rats were challenged with the T‐dependent antigen keyhole limpet hemocyanin (KLH) and immune function was assessed on PND 56. Dose‐dependent increases in total BDE concentrations were detected in the liver and adipose of all F0 and F1 rats. In F0 rats, increased liver weight, hepatocellular hypertrophy, and decreased serum thyroxine (T4) were characteristic of DE‐71 exposure. In F1 rats perinatal DE‐71 exposure caused a nondose‐dependent increase in body weight and dose‐dependent increases in liver weight and hepatocellular hypertrophy. Serum T3 and T4 levels were decreased. In spleen from DE‐71 exposed rats the area occupied by B cells declined while the area occupied by T cells increased; however, cellular and humoral immune responses to KLH challenge were not altered. Thus hepatic and thyroid changes in rats exposed perinatally to DE‐71 were associated with altered splenic lymphocyte populations, an effect which has been linked to hypothyroidism. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Dietary exposure to polybrominated diphenyl ether 47 (BDE-47) inhibits development and alters thyroid hormone-related gene expression in the brain of Xenopus laevis tadpoles

Few studies have investigated the thyroid-disrupting effects of polybrominated diphenyl ethers (PBDEs) across multiple levels of biological organization in anurans, despite their suitability for the screening of thyroid disruptors. Therefore, the present study evaluated the effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on development, thyroid histology and thyroid hormone-related gene expression in Xenopus laevis exposed to 0 (control), 50 (low), 500 (medium) or 5000 μg BDE-47/g food (high) for 21 days. Only the high dose of BDE-47 hindered growth and development; however, thyroid hormone-associated gene expression was downregulated in the brains of tadpoles regardless of dose. These results show that BDE-47 disrupts thyroid hormone signaling at the molecular and whole-organism levels and suggest that gene expression in the brain is a more sensitive endpoint than metamorphosis. Furthermore, the altered gene expression patterns among BDE-47-exposed tadpoles provide insight into the mechanisms of PBDE-induced thyroid disruption and highlight the potential for PBDEs to act as neurodevelopmental toxicants.     

Effects of decabromodiphenyl ether (BDE‐209) on mRNA transcription of thyroid hormone pathway and spermatogenesis associated genes in Chinese rare minnow (Gobiocypris rarus)

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants, which are ubiquitous environmental contaminant found in both abiotic and biotic environmental samples. Deca‐BDE (BDE‐209) is the principal component, which is currently used worldwide. In this study, the effect of BDE‐209 on the mRNA levels of thyroid hormone (TH) related genes and spermatogenesis associated genes were determined from larvae and adult rare minnow (Gobiocypris rarus) exposed to concentrations 0.01, 0.1, 1, and 10 μg/L for 21 days. The results showed that the type II deiodinase (dio2) and sodium iodide symporter (nis) mRNA levels were significantly up‐regulated in the larvae at 10 μg/L treatment. In adult, histopathological observations showed that liver of female fish were degenerated at 10 μg/L treatment, and inhibition of spermatogenesis were observed in testis of male fish. In addition, the thyroid hormone receptor α (trα), dio2, and nis mRNA levels in the liver of male and female fish were significantly up‐regulated, whereas dio2 and nis mRNA levels were significantly down‐regulated in the brain. These results indicate that exposure to BDE‐209 could result in tissue‐specific alternations of TH‐related genes expression in adults. Moreover, the mRNA levels of the testis‐specific apoptosis genes, the spermatogenesis‐associated 4 (spata4) and spermatogenesis‐associated 17 (spata17), were down‐regulated at 10 μg/L treatment in testis of male fish. Our results suggest that BDE‐209 may pose threat to normal thyroid and reproductive function in fish. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 1–9, 2014.     

Embryotoxic potential of N-methyl-pyrrolidone (NMP) and three of its metabolites using the rat whole embryo culture system

N-methyl-2-pyrrolidone (NMP), which undergoes extensive biotransformation, has been shown in vivo to cause developmental toxicity and, especially after oral treatment, malformations in rats and rabbits. Data are lacking as to whether the original compound or one of its main metabolites is responsible for the toxic effects observed. Therefore, the relative embryotoxicity of the parent compound and its metabolites was evaluated using rat whole embryo culture (WEC) and the balb/c 3T3 cytotoxicity test. The resulting data were evaluated using two strategies; namely, one based on using all endpoints determined in the WEC and the other including endpoints from both the WEC and the cytotoxicity test. On basis of the first analysis, the substance with the highest embryotoxic potential is NMP, followed by 5-hydroxy-N-methyl-pyrrolidone (5-HNMP), 2-hydroxy-N-methylsuccinimide (2-HMSI) and N-methylsuccinimide (MSI). Specific dysmorphogeneses induced by NMP and 5-HNMP were aberrations in the head region of the embryos, abnormal development of the second visceral arches and open neural pores. The second evaluation strategy used only two endpoints of the WEC, i.e. the no observed adverse effect concentration (NOAEC_WEC) and the lowest concentration leading to dysmorphogenesis in 100% of the cultured embryos (IC_{Max WEC}). In addition to these WEC endpoints the IC_{50 3T3} from the cytotoxicity test (balb/c 3T3 fibroblasts) was included in the evaluation scheme. These three endpoints were applied to a prediction model developed during a validation study of the European Centre for the Validation of Alternative Methods (ECVAM) allowing the classification of the embryotoxic potential of each compound into three classes (non-, weakly- and strongly embryotoxic). Consistent results from both evaluation strategies were observed, whereby NMP and its metabolites revealed a direct embryotoxic potential. Hereby, only NMP and 5-HNMP induced specific embryotoxic effects and were classified as weakly embryotoxic, whereas the other two metabolites, 2-HMSI and MSI, were determined to be non-embryotoxic.     

海星甾醇琥珀酸酯(A1998)脂肪乳静脉给药后在大鼠体内的组织分布

目的:研究海星甾醇琥珀酸酯(3β-羟基雄甾-5烯-17酮琥珀酸酯,A1998)脂肪乳经静脉给药后在大鼠体内的组织分布经时变化规律.方法:大鼠单次静脉注射20 mg·kg-1 A1998脂肪乳后,分别于不同时间点剖取各组织脏器,采用HPLC柱前衍生法测定大鼠各组织脏器药物含量.结果:A1998在大鼠体内主要分布于肺、肝、脾等器官,给药后15 min,测得肺脏药物含量最高(32.73±3.87)μg·g-1,脾脏(23.47±8.04)μg·g-1及肝脏(14.87±1.63)μg·g-1次之,均远高于同时间点大鼠血药浓度.A1998在上述3脏器中药物浓度维持时间亦久,至给药36 h后仍能检测出较高浓度,分别为(4.37±2.74),(4.69±2.37)及(8.30±5.96)μg·g-1.静注A1998后心脏及肾脏中药物浓度的经时变化与同时间点血药浓度相似.在胃、小肠、子宫、卵巢、体脂中未检测到药物分布.结论:大鼠单次静脉注射20 mg·kg-1 A1998脂肪乳后,药物在血浆中消除迅速,并快速分布于各组织.A1998在大鼠体内的组织分布具有较强的选择性,各组织中药物浓度经时变化结果提示对肺、肝、脾等脏器具有高度的亲和力.     

大鼠心肌多胺代谢限速酶ODC、SSAT活性分析

目的建立大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性分析方法。方法以Langendorff离体灌流心肌为实验材料,制备心肌组织匀浆;分别以dl[114C]Ornithine及[114C]acetylCoenzymeA为底物,以液体闪烁计数仪记录生成的14CO2及[14C]acetylspermidine的放射活度,并以其代表ODC,SSAT的活性;计算大鼠心肌ODC、SSAT的酶促反应动力学参数,筛选出适宜的底物浓度;同时观察一氧化氮(NO)供体硝普钠(SNP)对酶活性的影响。结果①大鼠心肌ODC、SSAT基础活性分别为:(9.67±3.09)nmol·mg-1Pro·h-1;(3.59±0.91)nmol·mg-1Pro·min-1。②ODC催化LOrnithine的酶促反应动力学参数Km=(54.95±8.14)μmol·L-1;Vmax=(2.364±0.37)nmol·mg-1·h-1;SSAT催化AcetylCoenzymeA的酶促反应动力学参数Km=(12.87±1.88)μmol·L-1;Vmax=(0.50±0.07)nmol·mg-1·min-1。③大鼠心肌ODC、SSAT活性检测的底物浓度分别为:90μmol·L-1(18.5kBq)DL[114C]Ornithine及36μmol·L-1(2.96kBq)[114C]acetylCoA。④SNP呈浓度依赖性地抑制ODC的活性、诱导SSAT的活性。结论建立了大鼠心肌多胺代谢限速酶鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰转移酶(SSAT)活性的分析方法,该方法简便易行;根据Km值确定测定大鼠心肌ODC及SSAT?     

Increasing exposure levels cause an abrupt change in the absorption and metabolism of acutely inhaled benzo(a)pyrene in the isolated, ventilated, and perfused lung of the rat.

The carcinogenic polycyclic aromatic hydrocarbons (PAHs) are active primarily at the site of entry to the body. Lung cancer following inhalation of PAH-containing aerosols such as tobacco smoke is one likely example. A suggested mechanism for this site preference is a slow passage of the highly lipophilic PAHs through the thicker epithelia of the conducting airways, accompanied by substantial local metabolism in airway epithelium. However, it is likely that the airway epithelium will become saturated with PAHs at surprisingly low exposure levels. The purpose of this research was to quantify the level of saturation for inhaled benzo(a)pyrene (BaP) in the isolated, perfused lung (IPL) of the rat. BaP was coated onto carrier particles of silica 3.5 microm diameter at three different levels. The DustGun aerosol generator was then used to deliver respectively 2.2, 36, and 8400 ng of BaP to the IPL with the carrier particles in less than 1 min. For 77 min after the exposure, single-pass perfusate was collected from the lungs. Lungs were then removed and, with the perfusate, analyzed for BaP and metabolites. Results show that the absorption and metabolism of inhaled BaP in the lungs was highly dose dependent. At low exposure levels absorption of BaP in the mucosa was proportional to the concentration in the air/blood barrier and proceeded with substantial local metabolism. At higher exposure levels the capacity of the epithelium to dissolve and metabolize BaP became saturated, and the absorption rate remained constant until crystalline BaP had dissolved, and the process proceeded with much smaller fractions of BaP metabolites produced in the mucosa. This phenomenon may explain the well-known difficulties of inducing lung cancer in laboratory animals with inhalants containing carcinogenic PAHs, where similar lifespan exposures are used as humans may experience but with much higher dose rates.     

Distribution and biomarker of carbon‐14 labeled fullerene C60 ([14C(U)]C60) in pregnant and lactating rats and their offspring after maternal intravenous exposure

A comprehensive distribution study was conducted in pregnant and lactating rats exposed to a suspension of uniformly carbon‐14 labeled C₆₀ ([¹⁴C(U)]C₆₀). Rats were administered [¹⁴C(U)]C₆₀ (~0.2 mg [¹⁴C(U)]C₆₀ kg^–1 body weight) or 5% polyvinylpyrrolidone (PVP)‐saline vehicle via a single tail vein injection. Pregnant rats were injected on gestation day (GD) 11 (terminated with fetuses after either 24 h or 8 days), GD15 (terminated after 24 h or 4 days), or GD18 (terminated after 24 h). Lactating rats were injected on postnatal day 8 and terminated after 24 h, 3 or 11 days. The distribution of radioactivity in pregnant dams was influenced by both the state of pregnancy and time of termination after exposure. The percentage of recovered radioactivity in pregnant and lactating rats was highest in the liver and lungs. Radioactivity was quantitated in over 20 tissues. Radioactivity was found in the placenta and in fetuses of pregnant dams, and in the milk of lactating rats and in pups. Elimination of radioactivity was < 2% in urine and feces at each time point. Radioactivity remained in blood circulation up to 11 days after [¹⁴C(U)]C₆₀ exposure. Biomarkers of inflammation, cardiovascular injury and oxidative stress were measured to study the biological impacts of [¹⁴C(U)]C₆₀ exposure. Oxidative stress was elevated in female pups of exposed dams. Metabolomics analysis of urine showed that [¹⁴C(U)]C₆₀ exposure to pregnant rats impacted the pathways of vitamin B, regulation of lipid and sugar metabolism and aminoacyl‐tRNA biosynthesis. This study demonstrated that [¹⁴C(U)]C₆₀ crosses the placenta at all stages of pregnancy examined, and is transferred to pups via milk. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method after chiral derivatization for the simultaneous stereoselective determination of methylenedioxy‐methamphetamine (MDMA) and its phase I and II metabolites in human blood plasma

3,4‐Methylenedioxymethamphetamine (MDMA, ecstasy) is a racemic drug of abuse and its two enantiomers are known to differ in their dose‐response curves. The S‐enantiomer was shown to be eliminated at a higher rate than the R‐enantiomer. The most likely explanation for this is a stereoselective metabolism also claimed in in vitro studies. Urinary excretion studies showed that the main metabolites in humans are 4‐hydroxy 3‐methoxymethamphetamine (HMMA) 4‐sulfate, HMMA 4‐glucuronide and 3,4‐dihydroxymethamphetamine (DHMA) 3‐sulfate. For stereoselective pharmacokinetic analysis of phase I and phase II metabolites in human blood plasma useful analytical methods are needed. Therefore the aim of the presented study was the development and validation of a stereoselective liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the simultaneous quantification of MDMA, 3,4‐methylenedioxyamphetamine, DHMA, DHMA 3‐sulfate, HMMA, HMMA 4‐glucuronide, HMMA 4‐sulfate, and 4‐hydroxy 3‐methoxyamphetamine in blood plasma for evaluation of the stereoselective pharmacokinetics in humans. Blood plasma samples were prepared by simple protein precipitation and afterwards all analytes were derivatized using N‐(2,4‐dinitro‐5‐fluorophenyl) L‐valinamide resulting in the formation of diastereomers which were easily separable on standard reverse phase stationary phases. This simple and fast method was validated according to international guidelines including specificity, recovery, matrix effects, accuracy and precision, stabilities, and limits of quantification. The method proved to be selective, sensitive, accurate and precise for all tested analytes except for DHMA. Copyright © 2014 John Wiley & Sons, Ltd.     

Distribution and biomarkers of carbon‐14‐labeled fullerene C60 ([14C(U)]C60) in female rats and mice for up to 30 days after intravenous exposure

A comprehensive distribution study was conducted in female rats and mice exposed to a suspension of uniformly carbon‐14‐labeled C₆₀ ([¹⁴C(U)]C₆₀). Rodents were administered [¹⁴C(U)]C₆₀ (~0.9 mg kg^?1 body weight) or 5% polyvinylpyrrolidone‐saline vehicle alone via a single tail vein injection. Tissues were collected at 1 h and 1, 7, 14 and 30 days after administration. A separate group of rodents received five daily injections of suspensions of either [¹⁴C(U)]C₆₀ or vehicle with tissue collection 14 days post exposure. Radioactivity was detected in over 20 tissues at all time points. The highest concentration of radioactivity in rodents at each time point was in liver, lungs and spleen. Elimination of [¹⁴C(U)]C₆₀ was < 2% in urine and feces at any 24 h time points. [¹⁴C(U)]C₆₀ and [¹⁴C(U)]C₆₀‐retinol were detected in liver of rats and together accounted for ~99% and ~56% of the total recovered at 1 and 30 days postexposure, respectively. The blood radioactivity at 1 h after [¹⁴C(U)]C₆₀ exposure was fourfold higher in rats than in mice; blood radioactivity was still in circulation at 30 days post [¹⁴C(U)]C₆₀ exposure in both species (<1%). Levels of oxidative stress markers increased by 5 days after exposure and remained elevated, while levels of inflammation markers initially increased and then returned to control values. The level of cardiovascular marker von Willebrand factor, increased in rats, but remained at control levels in mice. This study demonstrates that [¹⁴C(U)]C₆₀ is retained in female rodents with little elimination by 30 days after i.v. exposure, and leads to systemic oxidative stress. Copyright © 2015 John Wiley & Sons, Ltd.