Effects of polystyrene micro/nanoplastics on liver cells based on particle size, surface functionalization, concentration and exposure period.

Micro/nanoplastics (MP/NP) contaminate our food and drinking water but their impact on human health has not been well-documented. The liver is one of the first organs that ingested MP/NP encounter and it has a major role in the clearance of xenobiotics. Therefore, the effects of polystyrene MP/NP on liver HepG2 cells were studied. Cellular responses to particles of various sizes (50-5000 nm) and surface functionalization (aminated, carboxylated or non-functionalized) were determined at different concentrations (0.1-100 µg/mL) and exposure periods (1-24 h). Smaller sized particles were internalized by HepG2 cells more avidly than larger particles regardless of functionalization; the highest uptake being for 50 and 100 nm aminated particles at lower concentrations. Confocal microscopy images of cells corroborated quantitative uptake results. Aminated particles were more toxic to the cells than carboxylated or non-functionalized particles. Among aminated particles smaller particles (50 and 100 nm) were more detrimental to cell viability compared to larger particles (1000 or 5000 nm) with toxicity increasing with concentration. Treatment with the particles for 4 h increased intracellular concentrations of Caspase-3 by 1.5-2.8 fold, but 24 h exposure to the particles attenuated this increase in Caspase-3 concentrations. A slight trend of higher Caspase-3 concentration in cells treated with larger particles (500-5000 nm) compared to smaller particles (50-200 nm) was observed, indicating that larger particles are more likely to direct cells toward apoptotic cell death upon 4 h exposure. Exposure of cells to large PS particles (500-5000 nm) upregulated interleukin-8 and the effect was enhanced at 24 h. Overall, the study demonstrated that smaller aminated particles were most toxic to hepatocytes, but larger particles induced apoptotic cell death or an inflammatory response depending on the length of exposure.

Urinary Concentrations of Triclosan, Bisphenol A, and Brominated Flame Retardants and the Association of Triclosan with Demographic Characteristics and Body Fatness among Women with Newly Diagnosed Breast Cancer.

Background: Triclosan, bisphenol A (BPA), and brominated flame retardants are environmental estrogenic endocrine-disrupting compounds that may influence the prognosis of breast cancer. We examined the urinary concentrations of these compounds and their associations with demographic characteristics and body fatness in a population of women with newly diagnosed breast cancer. Methods: Overnight urine collection and anthropometric measures were obtained from 302 participants. Triclosan, BPA, tetrabromobisphenol A (TBBPA), and tetrabromobenzoic acid (TBBA) concentrations were determined using ultra-performance liquid chromatography−tandem mass spectrometry. Regression analyses were conducted to examine associations of urinary compound concentration with age, menopause, race, ethnicity, educational level, estrogen receptor status, body size, and body composition. Results: Triclosan, BPA, and TBBA were detected in urine samples from 98.3%, 6.0%, and 0.3% of patients, respectively; TBBPA was undetectable. Among patients with quantifiable values, the geometric mean concentrations were 20.74 µg/L (27.04 µg/g creatinine) for triclosan and 0.82 µg/L (1.08 µg/g creatinine) for BPA. Body mass index ≥ 30 vs. <25 kg/m2 was associated with lower creatinine-corrected urinary concentrations of triclosan (−40.00, 95% confidence interval [CI] = −77.19 to −2.81; p = 0.0351). The observed association was predominantly in postmenopausal women (−66.57; 95% CI: −109.18% to −23.96%). Consistent results were found for associations between triclosan levels and fat mass variables. Conclusion: In this study population, women with newly diagnosed breast cancer had triclosan exposure. Assessments of the implications of urinary concentrations of triclosan for women should consider body fatness and menopausal status.

Urinary Excretion of the ß-Adrenergic Feed Additives Ractopamine and Zilpaterol in Breast and Lung Cancer Patients.

ß2-Adrenergic agonists (ß-agonists) have been legally used in the U.S. for almost two decades to increase lean muscle mass in meat animals. Despite a cardiotoxic effect after high-dose exposure, there has been limited research on human ß-agonist exposures related to meat consumption. We quantified urinary concentrations of ractopamine and zilpaterol, two FDA-approved ß-agonist feed additives, and examined the extent to which the concentrations were associated with estimated usual meat intake levels. Overnight urine samples from 324 newly diagnosed breast cancer patients and spot urine samples from 46 lung cancer patients at the time of diagnosis, prior to treatment, were collected during 2006-2010 and 2014-2015, respectively. Urinary ractopamine and zilpaterol concentrations were measured by LC-MS/MS. Ractopamine and zilpaterol, respectively, were detected in 8.1% and 3.0% of the urine samples collected (n = 370). Only 1.1% (n = 4) of the urine samples had zilpaterol concentrations above the limit of quantification, with the mean value of 0.07 ng/mL in urine. The presence of detectable ractopamine and zilpaterol levels were not associated with meat consumption estimated from a food frequency questionnaire, including total meat (P = 0.13 and 0.74, respectively), total red meat (P = 0.72 and 0.74), unprocessed red meat (P = 0.74 and 0.73), processed red meat (P = 0.72 and 0.15), and poultry intake (P = 0.67 for ractopamine). Our data suggest that the amount of meat-related exposure of ß-agonists was low.

Simultaneous development of both competitive and noncompetitive immunoassays for 2,2',4,4'-tetrabromodiphenyl ether using phage-displayed peptides.

Twenty-five phages that selectively bind to a monoclonal antibody (Mab) 1H2 specific to 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in the absence or presence of BDE47 have been selected from phage-display libraries containing cyclic 7-mer, linear 7-mer, and linear 12-mer randomized peptides. Competitive and noncompetitive enzyme-linked immunosorbent assays (ELISA) for BDE47 were developed by using a clone C7-1 specific to the BDE47-free Mab 1H2 and a clone XC7-8 specific to the BDE47-bound Mab 1H2, respectively. The half-maximum signal inhibition concentration (IC50) of the competitive phage ELISA and the half-maximum signal enhancement concentration (EC50) of the noncompetitive phage ELISA for BDE47 were 6.8 ng mL(-1) and 4.2 ng mL(-1), respectively. The noncompetitive phage ELISA showed higher cross-reactivity with BDE28, BDE99, and BDE100 than the competitive one, ranging between 1.3 and 6.5 % versus 0.3 and 0.8 %. Recoveries of the competitive and the noncompetitive phage ELISAs for BDE47 in sewage sludge and fillet samples were 96-124 % and 97-120 %, respectively. The results of the two types of phage ELISAs for BDE47 in the real-world samples agreed well with a gas chromatography/electron capture detector-ion trap mass spectrometer method.

Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.

Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17ß-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC(50) and calculated limit of detection (LOD) are 0.32 µg L(-1) and 0.022 µg L(-1) for E2, 37.2 µg L(-1) and 24.5 µg L(-1) for BaP, and 31.6 µg L(-1) and 2.8 µg L(-1) for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368-375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1.

The wheat pathogen Stagonospora nodorum produces multiple necrotrophic effectors (also called host-selective toxins) that promote disease by interacting with corresponding host sensitivity gene products. SnTox1 was the first necrotrophic effector identified in S. nodorum, and was shown to induce necrosis on wheat lines carrying Snn1. Here, we report the molecular cloning and validation of SnTox1 as well as the preliminary characterization of the mechanism underlying the SnTox1-Snn1 interaction which leads to susceptibility. SnTox1 was identified using bioinformatics tools and verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid protein with the first 17 amino acids predicted as a signal peptide, and strikingly, the mature protein contains 16 cysteine residues, a common feature for some avirulence effectors. The transformation of SnTox1 into an avirulent S. nodorum isolate was sufficient to make the strain pathogenic. Additionally, the deletion of SnTox1 in virulent isolates rendered the SnTox1 mutated strains avirulent on the Snn1 differential wheat line. SnTox1 was present in 85% of a global collection of S. nodorum isolates. We identified a total of 11 protein isoforms and found evidence for strong diversifying selection operating on SnTox1. The SnTox1-Snn1 interaction results in an oxidative burst, DNA laddering, and pathogenesis related (PR) gene expression, all hallmarks of a defense response. In the absence of light, the development of SnTox1-induced necrosis and disease symptoms were completely blocked. By comparing the infection processes of a GFP-tagged avirulent isolate and the same isolate transformed with SnTox1, we conclude that SnTox1 may play a critical role during fungal penetration. This research further demonstrates that necrotrophic fungal pathogens utilize small effector proteins to exploit plant resistance pathways for their colonization, which provides important insights into the molecular basis of the wheat-S. nodorum interaction, an emerging model for necrotrophic pathosystems.

Development of indirect competitive fluorescence immunoassay for 2,2',4,4'-tetrabromodiphenyl ether using DNA/dye conjugate as antibody multiple labels.

An indirect competitive fluorescence immunoassay using a DNA/dye conjugate as antibody multiple labels was developed on 96-well plates for the identification and quantification of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in aqueous samples. A hapten, 2,4,2'-tribromodiphenyl ether-4'-aldehyde, was synthesized, and was conjugated to bovine serum albumin to form a coating antigen. Specific recognition of the antigen by anti-PBDE antiserum was confirmed by a surface plasmon resonance measurement. In the immunoassay, the coating antigen was adsorbed on a 96-well plate first, and a sample, antiserum and biotinylated goat anti-rabbit secondary antibody were then added and reacted sequentially. A biotinylated, double-stranded DNA with 219 base pairs was attached to the secondary antibody by using streptavidin as a molecular bridge. In situ multiple labeling of the antibody was accomplished after addition of a DNA-binding fluorescent dye, SYBR Green I. The working range of the immunoassay for the BDE-47 standard was 3.1-390 microg/L, with an IC50 value of 15.6 microg/L. The calculated LOD of the immunoassay is 0.73 microg/L. The immunoassay demonstrated relatively high selectivity for BDE-47, showing very low cross-reactivity (< 3%) with BDE-15, BDE-153 and BDE-209. With a spiked river water sample containing 50 microg/L BDE-47, quantification by the immunoassay was 41.9 microg/L, which compared well with the standard GC-ECD method (45.7 microg/L). The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.

Multiplex screening of persistent organic pollutants in fish using spectrally encoded microspheres.

Persistent organic pollutants (POPs) are environmental and food-related contaminants of global public health concern and known to be carcinogenic and endocrine disruptors. Their monitoring is essential, and an easy-to-use, rapid, and affordable multianalyte screening method with simplified sample preparation can be a valuable tool prior to instrumental analysis. For this purpose, a flow cytometric immunoassay (FCIA), based on a spectrally encoded microbeads technology, was developed for the multiplex detection of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (BDEs) in buffer and fish extracts. The sensitivities of the assays in the three-plex FCIA format were similar to the individual FCIAs for the marker compounds benzo[a]pyrene (BaP), 3,3',4,4'-tetrachlorobiphenyl (PCB77), and 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in buffer with IC(50) values of 0.4, 20, and 2 µg L(-1), respectively. Apart from the three markers, we could detect at least 14 other POPs. Extracts of fish with different fat content, prepared with a simplified extraction and cleanup procedure, had an insignificant influence on the overall three-plex FCIA performance, with the exception of some impact on the PAHs detection. The performance of the three-plex FCIA, in combination with the simple extraction procedure, is adequate for regulatory control in accordance with the required limits.

Development of a monoclonal antibody-based, congener-specific and solvent-tolerable direct enzyme-linked immunosorbent assay for the detection of 2,2',4,4'-tetrabromodiphenyl ether in environmental samples.

A sensitive direct enzyme-linked immunosorbent assay (ELISA) for the specific detection of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in environmental samples was developed. A hapten mimicking BDE-47 was synthesized by introducing a butyric acid spacer into 5-hydroxy-BDE-47 and coupled to keyhole limpet hemocyanin to form an immunogen for the production of monoclonal antibodies (Mabs) against BDE-47. The most sensitive direct ELISA was formatted with a Mab, designated as 4F2, in combination with 5-(2,4-dibromophenoxy)pentanoic acid peroxidase as a tracer. The inhibition half-maximum concentrations and limit of detection of BDE-47 in phosphate buffered saline with 25% DMSO were 1.4 ± 0.05 and 0.1 ng mL(-1), respectively. Cross-reactivity values of the ELISA with the tested BDE congeners and metabolites were ≤5.8%. This assay was used to determine BDE-47 in soil, sediment and house dust samples after ultrasonic extraction, simple cleanup and concentration steps. The average recoveries, repeatabilities (intraday extractions and analyses), and intra-laboratory reproducibilities (interday extractions and analyses) were in a range of 92-126%, 8-19% and 9-25%, respectively. Applied to 44 real samples, the results of this assay displayed a statistically significant correlation with those of a gas chromatography-mass spectrometry method (R(2)=0.79-0.85), indicating this ELISA is a suitable tool for environmental analyses of BDE-47.

Tissue distribution of polychlorinated biphenyls and organochlorine pesticides and potential toxicity to Alaskan northern fur seals assessed using PCBs congener specific mode of action schemes.

The fur seal (Callorhinus ursinus) population has decreased in their primary breeding grounds in the Bering Sea; contamination is among suspected causes. Our goal was to better understand the extent of contamination of seal tissues with certain organochlorine compounds by measuring the concentrations of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) in fur seal tissues from St. Paul Island, to gain a better perspective of tissue congener distribution and to evaluate the observed PCB levels against toxicologically significant levels for modes of action. Concentrations of 145 PCB congeners (Sigma(145)PCBs) and 12 OCPs were measured with gas chromatography-ion trap mass spectrometry in 8 different tissues of 10 male northern fur seals. The mean concentrations of SigmaOCPs [in ng/g lipid weight (lw)] were 1180 in blubber, 985 in the heart, 1007 in the liver, 817 in the kidney, 941 in muscle, 660 in reproductive tissues, 204 in the brain, and 322 in the lung. The mean concentrations of Sigma(145)PCBs (in ng/g lw) were 823 in blubber, 777 in the liver, 732 in the heart, 646 in reproductive tissues, 638 in muscle, 587 in the kidney, 128 in the lung, and 74.3 in brain tissues. Concentrations of PCBs affecting the aryl hydrocarbon receptor expressed as total PCB toxic equivalents (SigmaPCB-TEQs) ranged from 0.3 to 545 pg/g lw for the various tissues. The major contributors to SigmaPCB-TEQs are CB-118 in muscle, brain, lung, kidney, and liver, CB-126 in blubber, and CB-118 and CB-126 equally in the heart and reproductive tissues. Concentrations of PCBs affecting Ca(2+) homeostatsis expressed as the neurotoxic equivalent (NEQ) showed SigmaPCB-NEQs ranged from 17.7 to 215 ng/g lw in all tissues. Although no composite measure of perturbation of thyroid function is available, sufficient amounts of congeners with high binding to the thyroxine transport system were present to warrant consideration of this mode of action in future studies. Analyses of 145 PCBs and mode of action evaluation suggest that PCB contamination could potentially exert an effect on the Alaskan northern fur seal population although the PCB concentrations have been decreasing in the fur seals over the last decade.

Suitability of a magnetic particle immunoassay for the analysis of PBDEs in Hawaiian euryhaline fish and crabs in comparison with gas chromatography/electron capture detection-ion trap mass spectrometry.

A gas chromatograph/electron capture detector-ion trap mass spectrometer (GC/ECD-ITMS) was used for the determination of polybrominated diphenyl ethers (PBDEs) in euryhaline fish and crabs. GC/ECD-ITMS results showed that average recoveries from the spiked fish samples are in a range of 58-123% with relative standard deviations (RSDs) of 5-19%. PBDE concentrations obtained from GC/ECD-ITMS ranged from 28 ng/g to 1845 ng/g lipid weight (lw) in all aquatic species collected from Hawaiian brackish waters. The general BDE congener concentration profile observed in this study is BDE-47>BDE-100>BDE-154>BDE-99>BDE-153>BDE-28>BDE-183. The ELISA results expressed as BDE-47 equivalents correlated well with those of GC/ECD-ITMS, with a correlation coefficient (R(2)=0.68) and regression coefficient (slope=0.82). Comparison of ELISA with GC/ECD-ITMS results demonstrated that ELISA provides a timely and cost-effective method to screen PBDEs in fish and crab samples.

Development of a magnetic particle immunoassay for polybrominated diphenyl ethers and application to environmental and food matrices.

A sensitive magnetic particle enzyme-linked immunoassay (ELISA) was developed to analyze polybrominated diphenyl ethers (PBDEs) in water, milk, fish, and soil samples. The assay was rapid and can be used to analyze fifty samples in about 1h after sample cleanup. The assay has a limit of detection (LOD) below 0.1 ppb towards the following brominated diphenyl ether (BDE) congeners: BDE-47, BDE-99, BDE-28, BDE-100, and BDE-153, with the LOD approximately the same as GC-NCI-MS. The congeners most readily recognized in the ELISA were BDE-47 and BDE-99 with the cross-reactivities of BDE-28, BDE-100, and BDE-153 being less than 15% relative to BDE-47. As anticipated, the sensitivities are proportional to the similarities between the hapten structure and the BDE congener structure. Some oxygenated congeners with structural similarity to the hapten showed high to moderate cross-reactivities. Very low cross-reactivity was observed for other PBDEs or chlorinated environmental contaminants. The assay gave good recoveries of PBDEs from spiked water samples and a very small within and between day variance. Comparison with GC-NCI-MS demonstrated the ELISA method showed equivalent precision and sensitivity, with better recovery. The lower recovery of the GC-NCI-MS method could be caused by the use of an internal standard other than an isotopically substituted material that could not be used because of the fragmentation pattern observed by this method. The cleanup methods prior to ELISA were matrix dependent, no pretreatment was needed for environmental water samples, while fish, milk, and soil samples required various degrees of cleanup. Analysis of this wide variety of environmental samples by both ELISA and GC-MS demonstrated ELISA provides a timely and cost-effective method to screen for PBDEs in a variety of samples.

Simultaneous use of gas chromatography/ion trap mass spectrometry - electron capture detection to improve the analysis of bromodiphenyl ethers in biological and environmental samples.

Bromodiphenyl ethers (BDEs) are a class of synthetic flame retardants and are widely present in the environment. Analysis of higher BDE congeners has proven to be a challenge. We report the development of a method that enhances their analysis by splitting the eluent of a gas chromatograph (GC) between an electron capture detector (ECD) and an ion trap mass spectrometer (ITMS): 1:10, ECD:ITMS. This allowed the quantitation of the lower molecular weight (MW) BDE congeners (Br1-Br7) with the ITMS and of the higher MW BDEs (Br8-Br10) with the highly sensitive ECD. The IT temperature, ionization mode, and MS/MS parameters (excitation amplitude and stability parameter) were optimized. This method took the advantages of the best detector for the different BDE homologues and was suitable for the analysis of BDEs in environmental and biological samples. Average recoveries were 52-112% for BDEs from spiked sand samples and 57-126% from spiked lard samples after accelerated solvent extraction followed by silica gel and alumina column clean-up. Average recoveries ranged from 51% to 130% for 13C-labeled BDEs spiked in the real and in matrix samples. The method detection limits for specific congeners were 0.18-120 pg/g of the BDEs in animal tissue samples, and 0.05-40 pg/g in soil and indoor dust samples. The utility of the method was demonstrated by analyzing actual harbor seal blubber, indoor dust and soil samples. The concentration of each BDE ranged from non-detectable (nd) to 41 ng/g in the dry soil sample, nd to 1042 ng/g in the indoor dust, nd to 15 ng/g wet weight in the Alaskan harbor seal blubber sample, and 0.02 to 11 ng/microL of the identified 23 of the 42 breakdown products from BDE-209 after zerovalent iron treatment. Finally, an interlaboratory comparison showed high correspondence between the GC/ITMS-ECD method and a GC high-resolution MS system for the analysis of BDEs in soil samples.

Characterization of a U.S. Isolate of Beet black scorch virus.

ABSTRACT The first reported U.S. isolate of Beet black scorch necrovirus (BBSV) was obtained and characterized. Host range of the virus for localized and occasionally systemic infection included the Chenopodiaceae and Tetragonia expansa; Nicotiana benthamiana supported symptomless systemic infection by the virus. The complete nucleotide sequence of the genomic RNA of the virus, designated BBSV-Co, exhibits 93% similarity to the genome of the 'Ningxia' isolate of BBSV from China. Amino acid sequence similarity in predicted genes ranged from 95% in the p4 gene to 97% in the p82 and coat protein genes. A potential additional gene exists within the U.S. isolate of BBSV that is absent from Chinese isolates of BBSV due to nucleotide differences between these isolates within the coat protein gene. Coat protein analysis by isoelectric focusing and by mass spectroscopy indicated the presence of phosphorylated residues. Using primer extension analysis of the 5' end of the genome and site-directed mutants of genomic clones of BBSV-Co from which infectious RNA was produced, the native 5' end of the BBSV-Co genome was determined to be 5'-GAAACCTAACC...3', lacking the two terminal adenosine nucleotides in the published sequences of BBSV from China.