Polycyclic aromatic hydrocarbons in silicone wristbands of Uruguayan children: measurement and exposure source exploration.

Polycyclic aromatic hydrocarbons (PAHs) pose health risks to children, potentially resulting in stunted growth, obesity, and cognitive deficits, but lack of reliable and noninvasive means to measure PAHs results in poor understanding of exposure patterns and sources in this vulnerable population. In this study, 24 children aged ∼7 years (9 boys and 15 girls) from Montevideo, Uruguay wore silicone wristbands for 8 days to monitor the exposure of 27 PAHs. Wristbands were extracted using a modified ethyl acetate tandem solid phase extraction clean up and then analyzed via gas chromatography with tandem mass spectrometry. This analysis has reported LODs for 27 PAHs between 0.05 and 3.91 µg L-1. Eighteen PAHs were detected in >50% of the samples with concentration medians ranging 1.2-16.3 ng g-1 of wristband. Low molecular weight PAHs (2-3 rings) such as naphthalene and its alkyl derivatives were highly correlated (0.7-0.9) in the wristbands, suggesting exposure from related sources. Exposure source exploration focused on secondhand tobacco smoke, potentially through caregivers who reported on smoking habits in an associated survey. A principal components analysis (PCA) was conducted to examine patterns in PAH compounds detected in the wristbands; subsequently, the resulting components were compared according to current smoking among caregivers. The PCA analysis revealed a grouping of participants based on higher exposure of 1-methyl naphthalene, pyrene, fluoranthene, 1-methylphenanthrene, dibenzothiophene and 2-phenyl naphthalene. The derived components did relate with parental smoking, suggesting that some participants experienced exposure to a common source of certain PAHs outside of parental smoking. This is the first study to assess PAH exposure in young children from South America. Using wristbands, our study indicates exposure to multiple, potentially harmful chemicals. Wristbands could provide a comprehensive picture of PAH exposure in children, complementing other non-invasive biomonitoring approaches.

Antioxidant and Antiproliferative Activities of Several Garlic Forms.

It is hypothesized that garlic, Allium sativum, might protect against oxidative stress that causes damage to cells and tissues leading to the development of various health conditions including cancer. However, it is not known whether garlic's potential anticancer benefits differ by form of garlic consumed. This study aimed to quantify and compare the in vitro antioxidant and antiproliferative activity of several garlic forms in water and alcohol extracts including fresh garlic, fresh garlic set aside, heated garlic, heated garlic set aside, garlic powder, black garlic, two commercially available garlic supplements. Antioxidant activity of different garlic forms were measured using three assays: DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay, superoxide assay, and hydroxyl assay. In vitro effects of garlic extracts were investigated against the most common lung cancer subtypes: H520, H1975, and A549 cell lines using the sulforhodamine B (SRB) assay. Among free radical scavenging assays, Garlicin®, a commercially available supplement, displayed high antioxidant activity in water and alcohol extracts (DPPH assay: 2.02 mg AAE (mg ascorbic acid equivalent)/g garlic and 3.53 mg AAE/g garlic, respectively; superoxide assay: 6.73 mg AAE/g garlic and 7.13 mg AAE/g garlic, respectively). In the hydroxyl assay, water extract of fresh garlic crushed and set aside for 10 min showed the highest antioxidant activity. Garlicin® alcohol extract and fresh garlic water extracts strongly inhibited the proliferation of H1975, A549 and H520 cells. Other forms of garlic including garlic powder and black garlic exhibited low antioxidant and antiproliferative activity. Our results demonstrate that the preparation and processing methods of garlic may lead to different antioxidant benefits.

Cellular Lipidome Changes during Retinoic Acid (RA)-Induced Differentiation in SH-SY5Y Cells: A Comprehensive In Vitro Model for Assessing Neurotoxicity of Contaminants.

The SH-SY5Y, neuroblastoma cell line, is a common in vitro model used to study physiological neuronal function and the neuronal response to different stimuli, including exposure to toxic chemicals. These cells can be differentiated to neuron-like cells by administration of various reagents, including retinoic acid or phorbol-12-myristate-13-acetate. Despite their common use, there is an incomplete understanding of the molecular changes that occur during differentiation. Therefore, there is a critical need to fully understand the molecular changes that occur during differentiation to properly study neurotoxicity in response to various environmental exposures. Previous studies have investigated the proteome and transcriptome during differentiation; however, the regulation of the cellular lipidome in this process is unexplored. In this work, we conducted liquid chromatography-mass spectrometry (LC-MS)-based untargeted lipidomics in undifferentiated and differentiated SH-SY5Y cells, induced by retinoic acid. We show that there are global differences between the cellular lipidomes of undifferentiated and differentiated cells. Out of thousands of features detected in positive and negative electrospray ionization modes, 44 species were identified that showed significant differences (p-value ≤0.05, fold change ≥2) in differentiated cells. Identification of these features combined with targeted lipidomics highlighted the accumulation of phospholipids, sterols, and sphingolipids during differentiation while triacylglycerols were depleted. These results provide important insights into lipid-related changes that occur during cellular differentiation of SH-5YSY cells and emphasize the need for the detailed characterization of biochemical differences that occur during differentiation while using this in vitro model for assessing ecological impacts of environmental pollutants.

Development of a Liquid Chromatography-Mass Spectrometry-Based In Vitro Assay to Assess Changes in Steroid Hormones Due to Exposure to Per- and Polyfluoroalkyl Substances.

Per- and poly-fluorinated substances (PFASs) are organic pollutants that have been linked to numerous health effects, including diabetes, cancers, and dysregulation of the endocrine system. This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to measure changes in 17 hormones in H295R cell line (a steroid producing adrenocortical cells) upon exposure to PFASs. Due to the challenges in the analysis of steroid hormones using electrospray ionization MS, a chemical derivatization method was employed to achieve 0.07-2 µg/L detection limits in LC-MS/MS. Furthermore, a 10-fold concentration factor through solid-phase extraction (SPE) allows for consistent sub-parts per billion detections. Optimization of the derivatization conditions showed doubly-derivatized products in some hormone analytes, including progesterone, corticosterone, and cortisol, and gave improved ionization efficiency up to 20-fold higher signal than the singly-derivatized product. The use of SPE for sample cleanup to analyze hormones from cellular media using weak anion exchange sorbent yielded 80-100% recovery for the 17 targeted hormones. The method was validated by exposing H295R cells to two known endocrine disruptors, forskolin and prochloraz, which showed expected changes in hormones. An initial exposure of H295R cells with various PFAS standards and their mixtures at 1 µM showed significant increases in progestogens with some PFAS treatments, which include PFBS, PFHxA, PFOS, PFDA, and PFDS. In addition, modest changes in hormone levels were observed in cells treated with other sulfonated or carboxylated headgroup PFASs. This sensitive LC-MS/MS method for hormone analysis in H295R cells will allow for the investigations of the alterations in the hormone production caused by exposure to various environmental insults in cell-based assays and other in vitro models.

Endocrine Therapy-Resistant Breast Cancer Cells Are More Sensitive to Ceramide Kinase Inhibition and Elevated Ceramide Levels Than Therapy-Sensitive Breast Cancer Cells.

ET resistance is a critical problem for estrogen receptor-positive (ER+) breast cancer. In this study, we have investigated how alterations in sphingolipids promote cell survival in ET-resistant breast cancer. We have performed LC-MS-based targeted sphingolipidomics of tamoxifen-sensitive and -resistant MCF-7 breast cancer cell lines. Follow-up studies included treatments of cell lines and patient-derived xenograft organoids (PDxO) with small molecule inhibitors; cytometric analyses to measure cell death, proliferation, and apoptosis; siRNA-mediated knockdown; RT-qPCR and Western blot for gene and protein expression; targeted lipid analysis; and lipid addback experiments. We found that tamoxifen-resistant cells have lower levels of ceramides and hexosylceramides compared to their tamoxifen-sensitive counterpart. Upon perturbing the sphingolipid pathway with small molecule inhibitors of key enzymes, we identified that CERK is essential for tamoxifen-resistant breast cancer cell survival, as well as a fulvestrant-resistant PDxO. CERK inhibition induces ceramide-mediated cell death in tamoxifen-resistant cells. Ceramide-1-phosphate (C1P) partially reverses CERK inhibition-induced cell death in tamoxifen-resistant cells, likely through lowering endogenous ceramide levels. Our findings suggest that ET-resistant breast cancer cells maintain lower ceramide levels as an essential pro-survival mechanism. Consequently, ET-resistant breast cancer models have a unique dependence on CERK as its activity can inhibit de novo ceramide production.

Cellular Interactions and Fatty Acid Transporter CD36-Mediated Uptake of Per- and Polyfluorinated Alkyl Substances (PFAS).

Per- and polyfluorinated alkyl substances (PFAS) are a class of widely used compounds in an array of commercial and industrial applications. Due to their extensive use and chemical stability, PFAS persist in the environment and bioaccumulate in humans and wildlife. PFAS exposure have been linked to several negative health effects, including the formation of various cancers, disruption of the endocrine system, and obesity. However, there is a major gap in understanding how structural differences in PFAS impact their interactions within a biological system. In this study, we examined the toxicity of PFAS with differences in chain length, head group, and degree of fluorination in human retinal epithelial cells. We focused on fluorotelomeric and fully fluorinated sulfonates and carboxylates and measured their uptake. Our results showed that sulfonates are taken up at higher levels as compared to their fluorotelomer and carboxylate counterparts. Furthermore, PFAS with 8 and 10 carbons (C8 and C10) are taken up at a higher level compared to those with six carbons (C6). We also investigated the role of the fatty acid transporter CD36 in PFAS uptake and found that increased CD36 levels result in higher levels of PFAS in cells. Overall, our results suggest that the head group structure of PFAS impacts toxicity, with sulfonates inducing a higher decrease in cell viability (∼50%) than carboxylates. Our results also link the activity of CD36 to PFAS uptake into cells.

Types of garlic and their anticancer and antioxidant activity: a review of the epidemiologic and experimental evidence.

Garlic, an Allium vegetable, contains rich flavonoids organosulfur compounds (OSCs) that have potent anticancer properties. The aim of the review is to provide an overview of the different types of garlic, their active compounds, and the potential anticancer benefits with a focus on antioxidant activity. Animal and cell line studies have provided convincing evidence that garlic and its organosulfur compounds inhibit carcinogenesis through a number of events including induction of apoptosis, inhibiting cellular proliferation, scavenging radical oxygen species (ROS), increasing the activities of enzymes such as glutathione S-transferase, and reducing tumor size. Epidemiological studies showed compelling evidence that garlic consumption is associated with decreased risk of colorectal cancer, but inconsistent evidence for stomach, breast, and prostate cancers. Studies also suggest that the presence and potency of garlic OSCs varies with respect to the preparation and form of garlic. Further epidemiological studies with information on garlic form consumed or preparation methods and molecular studies regarding its antioxidant mechanisms, such as increasing enzymatic and nonenzymatic antioxidants levels, are warranted.

Optimized workflow for unknown screening using gas chromatography high-resolution mass spectrometry expands identification of contaminants in silicone personal passive samplers.

RATIONALE: Silicone wristbands have emerged as valuable passive samplers for monitoring of personal exposure to environmental contaminants in the rapidly developing field of exposomics. Once deployed, silicone wristbands collect and hold a wealth of chemical information that can be interrogated using high-resolution mass spectrometry (HRMS) to provide a broad coverage of chemical mixtures. METHODS: Gas chromatography coupled to Orbitrap™ mass spectrometry (GC/Orbitrap™ MS) was used to simultaneously perform suspect screening (using in-house database) and unknown screening (using vendor databases) of extracts from wristbands worn by volunteers. The goal of this study was to optimize a workflow that allows detection of low levels of priority pollutants, with high reliability. In this regard, a data processing workflow for GC/Orbitrap™ MS was developed using a mixture of 123 environmentally relevant standards consisting of pesticides, flame retardants, organophosphate esters, and polycyclic aromatic hydrocarbons as test compounds. RESULTS: The optimized unknown screening workflow using a search index threshold of 750 resulted in positive identification of 70 analytes in validation samples, and a reduction in the number of false positives by over 50%. An average of 26 compounds with high confidence identification, 7 level 1 compounds and 19 level 2 compounds, were observed in worn wristbands. The data were further analyzed via suspect screening and retrospective suspect screening to identify an additional 36 compounds. CONCLUSIONS: This study provides three important findings: (1) a clear evidence of the importance of sample cleanup in addressing complex sample matrices for unknown analysis, (2) a valuable workflow for the identification of unknown contaminants in silicone wristband samplers using electron ionization HRMS data, and (3) a novel application of GC/Orbitrap™ MS for the unknown analysis of organic contaminants that can be used in exposomics studies.

Evidence that watershed nutrient management practices effectively reduce estrogens in environmental waters.

We evaluate the impacts of different nutrient management strategies on the potential for co-managing estrogens and nutrients in environmental waters of the Potomac watershed of the Chesapeake Bay. These potential co-management approaches represent agricultural and urban runoff, wastewater treatment plant effluent, and combined sewer overflow replacements. Twelve estrogenic compounds and their metabolites were analysed by gas chromatography-mass spectrometry. Estrogenic activity (E2Eq) was measured by in vitro bioassay. We detected estrone E1 (0.05-6.97 ng L-1) and estriol E3 (below detection-8.13 ng L-1) and one conjugated estrogen (estrone-3-sulfate E1-3S; below detection-8.13 ng L-1). E1 was widely distributed and positively correlated with E2Eq, water temperature, and dissolved organic carbon (DOC). Among nonpoint sources, E2Eq, and concentrations of E1, soluble reactive phosphorus (SRP) and total dissolved nitrogen (TDN) decreased by 51-61%, 77-82%, 62-64%, 4-16% in restored urban and agricultural streams with best management practices (BMPs) relative to unrestored streams without BMPs. In a wastewater treatment plant (Blue Plains WWTP), >94% of E1, E1-3S, E3, E2Eq and TDN were removed while SRP increased by 305% during nitrification/denitrification as a part of advanced wastewater treatment. Consequently, E1 and TDN concentrations in WWTP effluents were comparable or even lower than those observed in the receiving stream or river waters, and the effects of wastewater discharges on downstream E1 and TDN concentrations were minor. Highest E2Eq value and concentrations of E1, E3, and TDN were detected in combined sewer overflow (CSO). This study suggests that WWTP upgrades with biological nutrient removal, CSO management, and certain agricultural and urban BMPs for nutrient controls have the potential to remove estrogens from point and nonpoint sources along with other contaminants in streams and rivers.

Spatial distribution of pesticides, organochlorine compounds, PBDEs, and metals in surface marine sediments from Cartagena Bay, Colombia.

Cartagena Bay is an estuarine system located in the Caribbean Sea (Colombia, South America), that receives fresh water from Canal del Dique, which is connected to the Magdalena River, the most important river of Colombia, with some of the most prominent Colombian cities located in its watershed, which has a high sediment yield. An analysis of persistent organic pollutants and heavy metals was carried out on marine sediments from Cartagena Bay. Cartagena Bay sediments deployed the occurrence of total levels of pesticides (thiocarbamates, bromacil, triazines, organochlorines, and organophosphorus), polybrominated diphenyl ethers (PBDEs), and polychlorinated biphenyls (PCBs), in sediments ranging from 0.83-33.67 ng/g dry-weight, 0.05-0.34 ng/g dry-weight, and 0.06-19.58 ng/g dry-weight, respectively. Their concentrations were lower than those reported in NOAA Screening Quick Reference Tables. DDTs and PCBs are banned organochlorine compounds, since, even at low levels, their presence in sediments represents a threat to aquatic organisms and, therefore, to human health through the trophic chain. Sediments showed high concentrations of strontium (50-959.6 mg/kg). All metals evaluated in the marine sediments were found in the S6 sampling point; this was near tannery and hydrocarbon industries (Pb 37.1 mg/kg, Cr 137.2 mg/kg, Cd 1.7 mg/kg, Cu 64.4 mg/kg, As 13.1 mg/kg, Sr 318.9 mg/kg); these results exceeded the accepted values of threshold effect levels (TEL) used as an indicator of their potential risk on marine life.

Catching flame retardants and pesticides in silicone wristbands: Evidence of exposure to current and legacy pollutants in Uruguayan children.

Children are exposed to many potentially toxic compounds in their daily lives and are vulnerable to the harmful effects. To date, very few non-invasive methods are available to quantify children's exposure to environmental chemicals. Due to their ease of implementation, silicone wristbands have emerged as passive samplers to study personal environmental exposures and have the potential to greatly increase our knowledge of chemical exposures in vulnerable population groups. Nevertheless, there is a limited number of studies monitoring children's exposures via silicone wristbands. In this study, we implemented this sampling technique in ongoing research activities in Montevideo, Uruguay which aim to monitor chemical exposures in a cohort of elementary school children. The silicone wristbands were worn by 24 children for 7 days; they were quantitatively analyzed using gas chromatography with tandem mass spectrometry for 45 chemical pollutants, including polychlorinated biphenyls (PCBs), pesticides, polybrominated diphenyl ethers (PBDEs), organophosphorus flame retardants (OPFRs), and novel halogenated flame-retardant chemicals (NHFRs). All classes of chemicals, except NHFRs, were identified in the passive samplers. The average number of analytes detected in each wristband was 13 ±3. OPFRs were consistently the most abundant class of analytes detected. Median concentrations of ΣOPFRs, ΣPBDEs, ΣPCBs, and dichlorodiphenyltrichloroethane (DDT) and its metabolites (dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD)) were 1020, 3.00, 0.52 and 3.79 ng/g wristband, respectively. Two major findings result from this research; differences in trends of two OPFRs (TCPP and TDCPP) are observed between studies in Uruguay and the United States, and the detection of DDT, a chemical banned in several countries, suggests that children's exposure profiles in these settings may differ from other parts of the world. This was the first study to examine children's exposome in South America using silicone wristbands and clearly points to a need for further studies.

A Self-Assembled Iron(II) Metallacage as a Trap for Per- and Polyfluoroalkyl Substances in Water.

An anionic iron(II) tetrahedral molecular cage (FeMOP) was studied for its ability to interact with various per- and polyfluoroalkyl substances (PFASs) in aqueous media. Liquid chromatography tandem mass spectrometry revealed that longer-chain-length (more than six carbons) perfluorocarboxylic, -sulfonic, and fluorotelomers were removed from solution. In contrast, the steric bulk of N-ethyl substituted fluorosulfonamido acetic acid PFASs hindered association with the cage. Solution binding studies in D2O using 19F nuclear magnetic resonance (NMR) titrations and a Job plot show a 1:1 binding stoichiometry for perfluorohexanoic acid (PFHxA) and perfluoroheptanoic acid (PFHpA) with an association constant (Ka) of <103 and thus a favorable free energy of association (ΔG°). Perfluorononanoic acid (PFNA), on the other hand, forms an insoluble host-guest complex with FeMOP with a 1:1 host-guest ratio. Variable temperature (VT) NMR was used to determine the thermodynamic parameters of binding for a 1:1 FeMOP/PFHpA complex in water using a Curie-like model for fast-exchange processes. The extracted parameters suggest a low binding interaction (Ka < 103) driven by an increase in entropy from cage desolvation upon guest binding. The solid-state host-guest complexes formed from solution complexation of PFHxA, PFHpA, and PFNA into the cage were characterized by infrared spectroscopy (FT-IR) and powder X-ray diffraction (PXRD) methods. FT-IR studies suggest an interaction between the fluorocarbon groups of PFASs to the phenylsulfonate functional groups of the ligand. A docking model predicted by computation also indicates this interaction may occur, with the PFASs adsorbing onto the surface of the cage rather than forming a true host-guest complex within the internal cavity. PXRD studies reveal a crystal packing of the complex that is very similar to that of the water-treated FeMOP, with the exception of 1:2 FeMOP/PFNA and 1:1 and 2:1 FeMOP/PFHpA.

Binding of iodinated contrast media (ICM) and their transformation products with hormone receptors: Are ICM the new EDCs?

Iodinated contrast media (ICM) have been detected at high concentrations (as high as about 3 µg/L) in surface water systems, and recently in fish brains and gonad. The mismatch between the polarity of ICM and the high lipid content of brain raises questions on whether their bioaccumulation is receptor-mediated. Furthermore, the structural similarity of ICM to the natural thyroid hormones thyroxine and triiodothyronine suggest potential binding of ICM to nuclear receptors in the endocrine system. Therefore, an in silico approach based on Surflex-Dock module of SYBYL was used to investigate the molecular docking of selected ICM (diatrizoic acid, iohexol, iopamidol, and iopromide). These ICM showed interaction with nuclear receptors that play key roles in endocrine regulation, including the androgen and estrogen receptors. Furthermore, the results indicate peroxisome proliferator-activated receptor gamma (PPARg) as one of the viable targets in the endocrine disrupting potential of ICM with higher Cscores for the ICM and iopromide transformation products than the reference ligand for the receptor. The data obtained from in silico calculations showed stronger binding of iohexol to the transthyretin-binding pocket compared to the natural hormones, thyroxine and triiodothyronine, suggesting the potential of ICM to act as endocrine disrupting chemicals (EDCs) in the environment.

GMDTC Chelating Agent Attenuates Cisplatin-Induced Systemic Toxicity without Affecting Antitumor Efficacy.

Cisplatin is a platinum-based chemotherapeutic drug widely used in the treatment of various cancers such as testicular, ovarian, lung, bladder, and cervical cancers. However, its use and the dosage range applied have been limited by severe side effects (e.g., nephrotoxicity and ototoxicity) and by the development of resistance to cisplatin in patients during treatment. Metal chelators have shown promising potential in overcoming these problems often associated with platinum drugs. Previously, a new chelating agent, sodium (S)-2-(dithiocarboxylato((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-4(methylthio)butanoate (GMDTC), was developed. In this study, we examined the effect of GMDTC in modifying cisplatin-induced toxicities following in vitro and in vivo exposures. GMDTC treatment dramatically reduced cisplatin-induced apoptosis and cytotoxicity in HK2 cells by decreasing the amount of intracellular platinum. In the 4T1 breast cancer mouse model, GMDTC reduced cisplatin-induced nephrotoxicity by reducing cisplatin deposition in the kidney. GMDTC attenuated cisplatin-induced elevations in blood urea nitrogen and plasma creatinine, ameliorated renal tubular dilation and vacuolation, and prevented necrosis of glomeruli and renal tubular cells. GMDTC also inhibited cisplatin-induced ototoxicity as shown by improved hearing loss which was assessed using the auditory brainstem response test. Furthermore, GMDTC attenuated cisplatin-induced hematotoxicity and hepatotoxicity. Importantly, co-treatment of cisplatin with GMDTC did not affect cisplatin antitumor efficacy. Tumor growth, size, and metastasis were all comparable between the cisplatin only and cisplatin-GMDTC co-treatment groups. In conclusion, the current study suggests that GMDTC reduces cisplatin-induced systemic toxicity by preventing the accumulation and assisting in the removal of intracellular cisplatin, without compromising cisplatin therapeutic activity. These results support the development of GMDTC as a chemotherapy protector and rescue agent to overcome the toxicity of and resistance to platinum-based antineoplastic drugs.

Mass spectrometry based detection of common vitellogenin peptides across fish species for assessing exposure to estrogenic compounds in aquatic environments.

The identification of myriad of chemicals in the environment that mimic hormones and affect the endocrine functions of exposed organism is a daunting analytical challenge for environmental scientists and engineers. Many of these endocrine disrupting chemicals (EDCs) are present at very low concentrations in the aquatic systems, but yet affect the metabolic, developmental, and reproductive functions in exposed fish and wildlife. Vitellogenin (VTG) protein is a widely used biomarker in fish for assessing exposure to EDCs, and is commonly measured using species-specific immunochemical techniques. In this study, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that can measure common peptides from digested VTG in multiple fish species. In the initial experiments using high resolution mass spectrometry, two peptides (ALHPELR and FIELIQLLR) were identified as common fragments in the digested VTG protein isolated from three different fish species (Pimephales promelas, Micropterus salmoides, and Fundulus heteroclitus). Then, a quantitative analysis using LC-MS/MS under selected reaction monitoring mode was developed for the detection of these two peptides in trypsin-digested plasma from female fish (positive control), estrogen-exposed male fish (test sample), and unexposed male fish (negative control) using two of the same species used for identifying the common peptides (P. promelas, and M. salmoides) and one new species (Ameiurus nebulosus) that was not included during the selection of peptides. Results from this study demonstrate the potential of LC-MS/MS as an effective cross-species method to detect VTG in fish, which can be an alternative analytical technique for assessing endocrine disruption in multiple fish species.

The effect of manganese exposure in Atp13a2-deficient mice.

Loss of function mutations in the P5-ATPase ATP13A2 are associated with Kufor-Rakeb Syndrome and Neuronal Ceroid Lipofuscinosis. While the function of ATP13A2 is unclear, in vitro studies suggest it is a lysosomal protein that interacts with the metals manganese (Mn) and zinc and the presynaptic protein alpha-synuclein. Loss of ATP13A2 function in mice causes sensorimotor deficits, enhanced autofluorescent storage material, and accumulation of alpha-synuclein. The present study sought to determine the effect of Mn administration on these same outcomes in ATP13A2-deficient mice. Wildtype and ATP13A2-deficient mice received saline or Mn at 5-9 or 12-19 months for 45days. Sensorimotor function was assessed starting at day 30. Autofluorescence was quantified in multiple brain regions and alpha-synuclein protein levels were determined in the ventral midbrain. Brain Mn, iron, zinc, and copper concentrations were measured in 5-9 month old mice. The results show Mn enhanced sensorimotor function, increased autofluorescence in the substantia nigra, and increased insoluble alpha-synuclein in the ventral midbrain in older ATP13A2-deficient mice. In addition, the Mn regimen used increased Mn concentration in the brain and levels were higher in Mn-treated mutants than controls. These results indicate loss of ATP13A2 function leads to increased sensitivity to Mn in vivo.

In Vitro and In Vivo Assessment of Aqueously Extractable Estrogens in Poultry Manure after Pilot-scale Composting.

Poultry manure contains free and conjugated forms of the natural estrogens 17ß-estradiol and estrone, which can be transported to receiving waters via runoff when land-applied. Previous studies have demonstrated estrogens in runoff from poultry manure-amended fields but have not tracked changes in estrogenicity within this water over time. Microbial conversion of conjugated estrogens (a major portion of water-extractable estrogens) to parent forms may result in temporary increases in estrogenicity in natural water bodies. The present study created 80-L batches of simulated poultry manure runoff, which were investigated over 10 d for estrogenicity by bioluminescent yeast estrogen screen assay and fathead minnow () vitellogenin induction model. The efficacy of different compost conditions (in-vessel aeration ± turning, and piling) on reduction/elimination of aqueously extractable estrogens in poultry manure was also investigated. Results indicate 3- to 10-fold increases in estrogenicity in various poultry manure mixtures during 10-d observations. Estrogenicity returned to low levels in postcompost treatments but remained elevated in the precompost treatment. Aerated compost resulted in >75% reductions in initial, peak, and 10-d mean estrogenicity in aqueous mixtures (0.3, 0.8, and 0.5 ng 17ß-estradiol equivalents [EEQ] L, respectively) compared with the precompost mixture (1.4, 4.8, and 2.1 ng EEQ L, respectively). Estrogenicity was significantly higher in the aqueous extract from the piled treatment than the aerated treatment, and 10-d exposure of male fish to the piled treatment resulted in statistically significant vitellogenin induction. Collectively, our results suggest a need to investigate estrogenicity in surface waters for several days after receiving manure-influenced runoff.

Chemical and biological assessment of endocrine disrupting chemicals in a full scale dairy manure anaerobic digester with thermal pretreatment.

Concentrated animal feeding operations are important sources of estrogens and their conjugates, which are introduced into the environment through manure land application. In this study, concentrations of estrogens were measured in an anaerobic co-digestion system with thermal pasteurization pretreatment. Free estrogens (estrone (E1), 17α-estradiol (E2α), 17ß-estradiol (E2ß), estriol (E3)) were analyzed by gas chromatography with mass spectrometry (GC/MS), and conjugated estrogens (sulfate- and glucuronide-conjugates) were analyzed by liquid chromatography with tandem mass spectrometry (LC/MS/MS). Additionally, yeast estrogen screen assay was used to determine the estrogenic potential of the manure. The total hormone concentrations (mainly E1, E2α, E2ß, and sulfated estrogens) were observed at concentrations up to a total of 7100ng/L in the liquid fraction, while free estrogen levels were 630ng/kg in the solid fraction of the untreated manure. The total hormone concentration did not decrease significantly during digestion, however, the relative composition of the estrogens changed from E2α (65%) being the predominant species before digestion to mostly E1 (72%) after digestion. This conversion process has important implications because E1 is more estrogenic than E2α. Total E2 equivalents associated with E1, E2α and E2ß concentrations as determined by GC/MS indicate that E1 is the most important contributor to the endocrine-disruption activity of the treated manure.

"One-shot" analysis of polybrominated diphenyl ethers and their hydroxylated and methoxylated analogs in human breast milk and serum using gas chromatography-tandem mass spectrometry.

The presence of polybrominated diphenyl ethers (PBDEs) and their hydroxylated (OH-BDE) and methoxylated (MeO-BDE) analogs in humans is an area of high interest to scientists and the public due to their neurotoxic and endocrine disrupting effects. Consequently, there is a rise in the investigation of the occurrence of these three classes of compounds together in environmental matrices and in humans in order to understand their bioaccumulation patterns. Analysis of PBDEs, OH-BDEs, and MeO-BDEs using liquid chromatography-mass spectrometry (LC-MS) can be accomplished simultaneously, but detection limits for PBDEs and MeO-BDEs in LC-MS is insufficient for trace level quantification. Therefore, fractionation steps of the phenolic (OH-BDEs) and neutral (PBDEs and MeO-BDEs) compounds during sample preparation are typically performed so that different analytical techniques can be used to achieve the needed sensitivities. However, this approach involves multiple injections, ultimately increasing analysis time. In this study, an analytical method was developed for a "one-shot" analysis of 12 PBDEs, 12 OH-BDEs, and 13 MeO-BDEs using gas chromatography with tandem mass spectrometry (GC-MS/MS). This overall method includes simultaneous extraction of all analytes via pressurized liquid extraction followed by lipid removal steps to reduce matrix interferences. The OH-BDEs were derivatized using N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS-MTFA), producing OH-TBDMS derivatives that can be analyzed together with PBDEs and MeO-BDEs by GC-MS/MS in "one shot" within a 25-min run time. The overall recoveries were generally higher than 65%, and the limits of detection ranged from 2 to 14 pg in both breast milk and serum matrices. The applicability of the method was successfully validated on four paired human breast milk and serum samples. The mean concentrations of total PBDEs, OH-BDEs, and MeO-BDEs in breast milk were 59, 2.2, and 0.57 ng g(-1) lipid, respectively. In serum, the mean total concentrations were 79, 38, and 0.96 ng g(-1) lipid, respectively, exhibiting different distribution profiles from the levels detected in breast milk. This "one-shot" GC-MS/MS method will prove useful and cost-effective in large-scale studies needed to further understand the partitioning behavior, and ultimately the adverse health effects, of these important classes of brominated flame retardants in humans.

Interactive Effects of N6AMT1 and As3MT in Arsenic Biomethylation.

In humans, arsenic is primarily metabolized by arsenic (+3 oxidation state) methyltransferase (As3MT) to yield both trivalent and pentavalent methylated metabolites. We recently reported that the putative N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) can biotransform monomethylarsonous acid (MMA(III)) to dimethylarsinic acid, conferring resistance of human cells to arsenic exposure. To further decipher the role of N6AMT1 and its interaction with As3MT in arsenic biomethylation, we examined the relative contribution of N6AMT1 and As3MT in metabolizing arsenic using several newly modified UROtsa human urothelial cells, ie, UROtsa cells with either a constant level of N6AMT1 or As3MT in combination with an inducible level of As3MT or N6AMT1, respectively. Our analysis confirmed the involvement of N6AMT1 in MMA(III) biomethylation but not for inorganic arsenic. In a comparable level of N6AMT1 and As3MT, the effect of N6AMT1 mediated MMA(III) biomethylation was obscured by the action of As3MT. Furthermore, we showed that the levels of N6AMT1 and As3MT proteins varied among and within human normal and cancerous tissues. Overall, the data showed that N6AMT1 has a role in MMA(III) biomethylation, but its effect is relatively minor and limited compared with As3MT. In addition, the varied levels and distributions of N6AMT1 and As3MT among human tissues may potentially contribute to the tissue specificity and susceptibility to arsenic toxicity and carcinogenicity.