Tris(1,3-dichloro-2-propyl) phosphate induces endoplasmic reticulum stress and mitochondrial-dependent apoptosis in mouse spermatocyte GC-2 cells.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a frequently detected organophosphorus flame retardants (OPFRs) in various environmental media, and has been evidenced as reproductive toxicity. However, its adverse effects on spermatogenic cells are unknown. In this study, mouse spermatocyte GC-2spd (GC-2) cells were selected as an in vitro model, and the impact of mitochondrial structure and function, endoplasmic reticulum (ER) stress, cell apoptosis and the related molecular mechanisms were investigated. Our study indicated that cell viability was decreased significantly in a dose-dependent manner after TDCIPP treatment with the half lethal concentration (LC50) at 82.8 µM, 50.0 µM and 39.6 µM for 24 h, 48 h and 72 h, respectively. An apoptosis was observed by Annexin V-FITC/PI stain. In addition, fragmentation of mitochondrial structure, an increase of mitochondrial membrane potential (MMP), reduction of cellular adenosine triphosphate (ATP) content, release of cytochrome c and activation of Caspase-3 and Caspase-9 activity implicated that Caspase-3 dependent mitochondrial pathway might play a key role in the process of GC-2 cell apoptosis. Furthermore, ER stress induction was convinced by altered morphology of ER and up-regulation of ER targeting genes, including (Bip, eIF2α, ATF4, XBP1, CHOP, ATF6 and Caspase-12). Taken together, these results demonstrate that both mitochondrial apoptotic pathways and ER stress apoptotic pathways might play important roles in the process of apoptosis in GC-2 cells induced by TDCIPP treatment. Therefore, the potential reproductive toxicity of TDCIPP should not be ignored.

[Determination of 80 pesticide residues in milk using NH_2-MWNTs by liquid chromatography-time of flight mass spectrometry].

OBJECTIVE: A method for the determination of 80 pesticide residues in milk by liquid chromatography-time-of-flight mass spectrometry(LC-QTof-MS) was developed. METHODS: The target compounds in milk were extracted with acetonitrile-methanol(9∶1, V/V) containing 1% acetic acid, and purified by aminated multi-walled carbon nanotubes(NH_2-MWNTs). The chromatographic column was Waters Acquity UPLC BEH C_(18 )(100 mm×2.1 mm, 1.7 µm). The 80 pesticides were detected by liquid chromatography-time-of-flight mass spectrometry and quantified using an external standard method by matrix matched calibration curve. RESULTS: The purification method showed a good linearity(r~2≥ 0.99) over the concentration range from 5 to 100 µg/L for the 80 pesticides in this study. The limits of detection(LODs) and quantification(LOQs) of the 80 pesticides in milk ranged from 0.01 to 0.50 µg/L and 0.03 to 1.50 µg/L, respectively. The mean recoveries of the three spiked levels ranged from 71.5% to 116.9% with the relative standard deviation ranging from 1.2% to 18.1%, indicating that the accuracy and precision of the method were good. Among the milk samples, no residues of the 80 pesticides in this study were found after screening. CONCLUSION: The method has good linearity, good sensitivity, accuracy and precision and is suitable for the simultaneous and rapid determination of 80 pesticide residues in milk.

Evaluation of the cytotoxic activity of triphenyl phosphate on mouse spermatocytes cells.

Triphenyl phosphate (TPhP) is one of the most commonly found organophosphorus flame retardants (OPFRs) in the environment and the general population. Continuous daily exposure to TPhP may adversely impact male reproductive health. However, few researches were conducted to investigate the direct effects of TPhP on the progress of sperm growth and development. In this study, mouse spermatocyte GC-2spd (GC-2) cells were selected as an in vitro model, the impact of oxidative stress, mitochondrial impairment, DNA damage, cell apoptosis and the related molecular mechanisms were investigated using high content screening (HCS) system. Our study indicated that cell viability was decreased significantly in a dose-dependent manner after TPhP treatment with the half lethal concentration (LC50) at 105.8, 61.61 and 53.23 µM for 24, 48 and 72 h. A concentration-related apoptosis occurrence was observed in GC-2 cells after TPhP exposure for 48 h. In addition, the elevated intracellular reactive oxygen species (ROS) and the total antioxidant capacity (T-AOC) also observed after exposing to 6, 30 and 60 µM of TPhP. Furthermore, based on the enhancement of pH2AX protein and alteration of nuclear morphology or DNA content, DNA damage might be induced by higher concentration of TPhP treatment. Simultaneously, alteration of mitochondrial structure, enhancement of mitochondrial membrane potential (MMP), reduction of cellular adenosine triphosphate (ATP) content, altered expression of Bcl-2 family proteins, release of cytochrome c and increase of caspase-3 and caspase-9 activity demonstrated that caspase-3 dependent mitochondrial pathway might play a key role in the process of GC-2 cell apoptosis. Taken together, these results showed that TPhP was a mitochondrial toxicant and apoptotic inducer, which might trigger alike responses in human spermatogenic cells. Therefore, the potential reproductive toxicity of TPhP should not be ignored.

Emerging Brominated Flame Retardants 2-Ethylhexyl-2,3,4,5-tetrabromobenzoate (EHTBB) and Bis(2-ethylhexyl)-tetrabromophthalate (BEH-TEBP) in Chinese Food and Their Health Implications.

2-Ethylhexyl-2,3,4,5-tetrabromobenzoate (EHTBB) and bis(2-ethylhexyl) tetrabromophthalate (BEH-TEBP) have been frequently detected in the environment, whereas studies in food are scare. The European Food Safety Authority has requested data for their risk assessment. Herein, dietary exposure and hazard quotient (HQ) were studied based on the 5th (2009-2012) and 6th (2015-2018) Chinese total diet studies (TDSs). EHTBB was found in 61.1 and 75.9% of the two TDS sample sets, respectively. The concentrations of EHTBB in animal-derived food were higher than those in plant-derived food. The estimated daily intakes (EDIs) were 1.33 and 0.97 ng/kg bw/day, and vegetables contributed to 48.5 and 39.2% of the EDIs based on the 5th and 6th TDS, respectively. The dietary exposure to EHTBB was similar to that to hexabromocyclododecane, brominated diphenyl ether-209, and tetrabromobisphenol A (TBBPA). The HQ for EHTBB was similar to that for decabromodiphenyl ethane and surpassed that for TBBPA. Therefore, EHTBB warrants further study in food.

Total flavonoid contents in bamboo diets and reproductive hormones in captive pandas: exploring the potential effects on the female giant panda (Ailuropoda melanoleuca).

Phytoestrogens have been shown to affect the reproductive hormone levels in both humans and animals. As the main category of phytoestrogens, total flavonoids have a particularly important impact on female animals. To investigate the potential relationship between the total flavonoids in bamboo and the reproductive hormones in female giant pandas, urinary samples and dietary bamboo samples were collected from three main breeding locations (Beijing, Shaanxi and Sichuan). The chemical constituents of the total flavonoids in the bamboo were analysed and quantified using high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). Estradiol (E2), progestin (P), testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) were measured via radioimmunoassay (RIA). The results revealed that the total flavonoids in the bamboo from Sichuan were significantly higher than those in the bamboo from Beijing and Shaanxi, and the concentration in bamboo from Shaanxi was higher than that from Beijing (P < 0.05). The urinary E2, P, T, FSH and LH levels in pandas from Beijing were significantly lower than those in pandas from Sichuan and Shaanxi (P < 0.05). The concentrations of six reproductive hormones were positively associated with the total flavonoid contents in bamboo. In addition, the birth rate of pandas in Sichuan was significantly higher than the birth rate of pandas in Beijing and Shaanxi (P < 0.05). Thus, the flavonoids of bamboo may be related to reproduction and giant pandas might retain a sensitive adaptation to phytoestrogens from bamboo. The total flavonoids of bamboo may play a distinct role in the reproductive success of giant pandas.

Mechanism of bisphenol AF-induced progesterone inhibition in human chorionic gonadotrophin-stimulated mouse Leydig tumor cell line (mLTC-1) cells.

Bisphenol AF (BPAF) has been shown to inhibit testicular steroidogenesis in male rats. However, the precise mechanisms related to the toxic effects of BPAF on reproduction remain poorly understood. In the present study, a mouse Leydig tumor cell line (mLTC-1) was used as a model to investigate the mechanism of steroidogenic inhibition and to identify the molecular target of BPAF. Levels of progesterone and the concentration of cyclic adenosine monophosphate (cAMP) in cells exposed to BPAF were detected, and expression of key genes and proteins in steroid biosynthesis was assessed. The results showed that BPAF exposure decreased human chorionic gonadotrophin (hCG)-stimulated progesterone production in a dose-dependent manner. The 24-h IC50 (half maximal inhibitory concentration) value for BPAF regarding progesterone production was 70.2 µM. A dramatic decrease in cellular cAMP concentration was also observed. Furthermore, BPAF exposure inhibited expression of genes and proteins involved in cholesterol transport and progesterone biosynthesis. Conversely, the protein levels of steroidogenic acute regulatory protein (StAR) were not altered, and those of progesterone were still decreased upon 22R-hydroxycholesterol treatment of cells exposed to higher doses of BPAF. Together, these data indicate that BPAF exposure inhibits progesterone secretion in hCG-stimulated mLTC-1 cells by reducing expression of scavenger receptor class B type I (SR-B1) and cytochrome P450 (P450scc) due to the adverse effects of cAMP. However, StAR might not be the molecular target in this process.

Effects of Ambient Environmental Factors on the Stereotypic Behaviors of Giant Pandas (Ailuropoda melanoleuca).

Stereotypies are commonly observed in zoo animals, and it is necessary to better understand whether ambient environmental factors contribute to stereotypy and how to affect animal welfare in zoo settings. This study investigated the relationships between stereotypic behaviors and environmental factors including ambient temperatures, humidity, light intensity, sound intensity and number of visitors. Seven giant pandas were observed in three indoor enclosures and three outdoor enclosures. Environmental factors were measured for both indoor and outdoor enclosures and the effect they had on stereotypical behaviors was investigated. Our research found that light intensity significantly correlated with all stereotypies behaviors. Higher environmental temperature reduced the duration of pacing but increased the frequency of pacing, the duration and frequency of door-directed, meanwhile the duration of head-toss. However, we found no noticeable effect of humidity on stereotypic behaviors except for the frequency of head-toss. We also found that sound intensity was not correlated with stereotypies. Finally, the growth of visitors was negatively associated with the duration of door-directed. These results demonstrated that various environmental factors can have significant effects on stereotypic behaviors causing the expression of various stereotypies. Thus, stereotypies in zoo animals may not simply represent suboptimal welfare, but rather might be adopted as a means of coping with an aversive environment.

Bisphenol A and nonylphenol in foodstuffs: Chinese dietary exposure from the 2007 total diet study and infant health risk from formulas.

Concentrations of bisphenol A (BPA) and nonylphenol (NP) were measured in food samples from the 2007 Chinese total diet study (TDS). BPA and NP were detected in 72 and 143 of 144 TDS samples, respectively, with concentrations ranging from 20 ng/kg to 267 µg/kg and 30 ng/kg to 1,268 µg/kg, respectively. BPA was highest in Hebei (HB) province for most food groups. NP was much higher in milk than in other foods across all the provinces. Mean exposures to BPA and NP were estimated to be 43 ng/kg bw/day and 520 ng/kg bw/day for a Chinese adult, respectively, well below the tolerable daily intake (TDI). Mean exposure to NP from formula for infants aged 0-6 years ranged from 300 ng/kg bw/day to 17 µg/kg bw/day. For infants aged 0-1 year, intakes ranged from 5 to 17 µg/kg bw/day, which is above the TDI.

Development of a simple liquid chromatography-tandem mass spectrometry method for multiresidue determination of antifungal drugs in chicken tissues.

A method involving LC coupled with MS/MS (LC/MS/MS) was designed for simultaneous quantification of 10 antifungal drugs (voriconazole, griseofulvin, clotrimazole, bifonazole, econazole, ketoconazole, itraconazole, miconazole, terconazole, and fluconazole) in the liver and muscles of chickens. Homogenized tissue samples were extracted with acetonitrile and subsequently underwent freezing-delipidation. A Waters Acquity Ultra Performance LC BEH C18 column was used to separate the analytes, coupled with MS/MS using an electrospray ionization source. The accuracy of the method was confirmed with a mean recovery of 71-121%, and acceptable coefficients of variation (4-23%, n = 6). The detection capability of these compounds in two different matrixes was 0.50-2.82 microg/kg. This method can be applied for the screening and confirmation of target antifungal drugs in chicken tissues.

Identification of biomarkers for melamine-induced nephrolithiasis in young children based on ultra high performance liquid chromatography coupled to time-of-flight mass spectrometry (U-HPLC-Q-TOF/MS).

Milk products contaminated with melamine caused renal disease in young children in mainland China in 2008. The present study was designed to identify potential markers and assess the underlying metabolomic mechanisms of melamine-induced nephrolithiasis in young children. Urine samples were collected from healthy children (n=74) and from children diagnosed with nephrolithiasis (n=73) with either a positive (n=40) or a negative (n=33) history of melamine exposure. Ultra high-performance liquid chromatography coupled to time of flight mass spectrometry (U-HPLC-MS/MS) was applied to profile the abundances of metabolites. Partial least squares-discriminant analysis (PLS-DA) was used to discriminate between the samples. Seven compounds were found to highly discriminate between healthy controls and nephrolithiasis patients with a history of melamine exposure. The critical markers such as proline and 5C-aglycone were the predominant markers in the control group and detected only rarely in nephrolithiasis patients with a history of melamine exposure. In contrast, hypoxanthine at was the most significant compound that distinguished nephrolithiasis patients with a history of melamine exposure. It was increased to 116.12±23.34 µg/L (mean±S.D.) in the melamine-induced nephrolithiasis group, whereas the non-melamine group was at the level of 67.47±9.33 µg/L (p<0.001). The biomarkers for melamine-induced nephrolithiasis identified by this study may have clinical application in determining the aetiology of renal disease in young children.

Simultaneous analysis of synthetic musks and triclosan in human breast milk by gas chromatography tandem mass spectrometry.

A comprehensive method was developed for the simultaneous analysis in human breast milk of 12 synthetic musks, five nitro musks, six polycyclic muks and one macrocyclic musk; as well as one musk metabolite and triclosan. The target analytes were freeze dried and extracted using the accelerated solvent extraction (ASE) procedure. The extracts were further purified by gel permeation chromatography (GPC) and florisil solid-phase extraction (SPE) and then analyzed by gas chromatography tandem mass spectrometry (GC-MS/MS). Recoveries of the analytes based on the isotopic internal standard correction ranged from 82.4% to 112%, with relative standard derivations less than 20%. The method quantification limits (MQLs) were 0.6-5.4 ng/g lipid. The analytes were detected in human breast milk samples and ranged from 11.7 to 308.6 ng/g lipid.

Analysis of hormone antagonists in clinical and municipal wastewater by isotopic dilution liquid chromatography tandem mass spectrometry.

A comprehensive method was developed for the simultaneous trace analysis of ten hormone antagonist pharmaceuticals (raloxifene, exemestane, letrozole, anastrozole, mifepristone, finastride, tamoxifen, N-desmethyltamoxifen, clomiphene, and toremifene) in municipal sewage and hospital wastewater samples. The target compounds were firstly extracted using an Oasis HLB cartridge, followed by purification by an aminopropyl cartridge, and were then analyzed by liquid chromatography electrospray ionization tandem mass spectrometry in positive ion mode. The recoveries for the analytes based on internal standard calibration in different test matrices ranged from 67.6 to 118.6% (with the exception of mifepristone in clinical wastewater samples), with relative standard deviations less than 20%. The method quantification limits of the ten pharmaceuticals were in the range 0.10-2.0 ng/L. Excluding exemestane and N-desmethyltamoxifen, eight drugs were detected at 0.20-195.0 ng/L in hospital wastewater and municipal wastewater samples from Beijing.

[Pre-warning model of bacterial foodborne illness based on performance of principal component analysis combined with support vector machine].

OBJECTIVE: Based on the historical data of bacterial foodborne illness, the scoring system applied on the pre-warning model system was established in this study according to the rated harm factors. It could build up effectively the predictive model in the analysis of foodborne illness accidents. METHODS: Extracting the useful information, the principal component analysis was performed on the normalized raw data to reduce the dimension. The result was split into 70% data randomly for training set into the regression model of support vector machine that was used to predict the remaining 30% . RESULTS: Through reducing the dimensions for selecting the optional PCs, it could optimize the calibration and improve the efficiency. The combining method of principal component analysis (PCA) and support vector machine (SVM) could provide the reliable results in the pre-warning model, especially for the high-dimensional data with the limited sample populations. Furthermore, it could achieve 80% accuracy with the optimized parameters. CONCLUSION: The pre-warning model of bacterial foodborne illness could give the assessment of the poisoning accidents and provided the scientific theory for reducing the incidence of bacterial of food poisoning.

Simultaneous detection of residues of beta-adrenergic receptor blockers and sedatives in animal tissues by high-performance liquid chromatography/tandem mass spectrometry.

A method using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was developed to screen and confirm residues of multi-class veterinary drugs in animal tissues (porcine kidney, liver, muscle; bovine muscle). Thirty target drugs (19 beta-blockers, 11 sedatives) were determined simultaneously in a single run. Homogenized tissue samples were extracted with acetonitrile and purified using a NH2 solid-phase extraction cartridge. An Acquity UPLC BEH C18 column was used to separate the analytes, followed by tandem mass spectrometry using an electrospray ionization source in positive mode. Recovery studies were done at three fortification levels. Overall average recoveries in pig muscle, kidney, and liver fortified at three levels from 76.4% to 118.6% based on matrix-fortified calibration with coefficients of variation from 2.2% to 19.9% (n=6). The limit of quantification of these compounds in different matrices was 0.5-2.0 microg/kg. This method was successfully applied in screening and confirming target drugs in >200 samples.

Determination of the residues of 50 anabolic hormones in muscle, milk and liver by very-high-pressure liquid chromatography-electrospray ionization tandem mass spectrometry.

A sensitive and specific method for determination of the residues of 50 anabolic hormones in muscle (pork, beef, shrimp), milk and pig liver was developed. Analytes were separated and acquired by liquid chromatography coupled with an electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS). Target compounds were simultaneously extracted with methanol after enzyme hydrolysis, and purified using a graphitized carbon-black solid-phase extraction (SPE) and followed by NH(2) SPE cartridge. Limits of quantification were 0.04-2.0microgkg(-1); average recoveries were 76.9-121.3%; and the relative standard deviation was 2.4-21.2%. This method has been successfully applied in real samples.

Simultaneous analysis of thirteen diuretics residues in bovine milk by ultra-performance liquid chromatography/tandem mass spectrometry.

A simple, rapid, sensitive and specific method used to screen and confirm multi-class diuretics residues in whole bovine milk is described. Thirteen drugs of four different classes including carbonic anhydrase inhibitors, loop, thiazide and potassium-sparing diuretics were extracted from whole milk by acetonitrile followed by further purification with hexane. The analytes were separated using an ACQUITY UPLC BEH C18 column and detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS). MS data acquisition was performed by a time-scheduled multiple reaction monitoring program, selecting two ion transitions for each target compound. The overall average recoveries based on matrix-fortified curves fortified with diuretics at three levels ranged from 80.6 to 108.8% with the coefficients of variation ranging from 2.6 to 19.7% (n = 6). The limits of quantitation (LOQs) of diuretics in bovine milk were 5.0 microg/kg for spironolactone and 0.5 microg/kg for other analytes, respectively.