Organochlorine exposure and health effects in stranded Steller sea lions (Eumetopias jubatus) and Pacific harbor seals (Phoca vitulina richardii) from Oregon and southern Washington coasts

Organochlorine (OC) pesticides and polychlorinated biphenyls (PCBs) have been detected in a variety of marine mammal species at levels associated with adverse health effects. Little is known about OC levels and impacts on health in pinnipeds with different life histories. We determined the health and levels of 18 OC pesticides and 16 PCB congeners in blubber samples from 20 Steller sea lions and 39 Pacific harbor seals stranded from Oregon and Southern Washington. The most commonly detected OC at the highest concentration was p,p′- dichlorodiphenyldichloroethylene (DDE). PCBs were detected in all samples as well. Hypothesis testing indicated that diseased Steller sea lions (males and females combined) had higher contaminant concentrations than healthy Steller sea lions, and diseased Pacific harbor seals had higher concentrations of total OCs than healthy animals. Differences were also noted between diseased and healthy animals when looking at individual sexes of each species. Diseased Steller sea lions had higher mean contaminant levels than diseased harbor seals and healthy Steller sea lions had higher mean contaminant concentrations than healthy Pacific harbor seals. These results show that species differences exist in both contaminant loads and sensitivity to contaminants, which may be due to differences in life histories and physiology.     

Determination of organochlorine pesticides and polychlorinated biphenyls in human serum using headspace solid-phase microextraction and gas chromatography-electron capture detection

A simple procedure for the determination of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum using headspace solid-phase microextraction (HS-SPME) was developed. The analysis was carried out by gas chromatography (GC) equipped with electron capture detector (ECD). A 2(7-4) Plackett-Burman reduced factorial design for screening and a central composite design for optimizing the significant variables were applied. A 100 microm PDMS fiber, 3/5 headspace ratio (3 ml in 5 ml vial), 85 degrees C extraction temperature, 50 min extraction time, and 1 ml of acidic solution (pH 3) added to 1 ml of diluted serum (1:1) were chosen for the best response in HS extraction mode. The detection limits found were from 1 pg/ml (PCB 167) to 52 pg/ml (beta-HCH), the relative standard deviation for the procedure varied from 3% (PCB 52) to 12% (PCB 189) and the accuracy was checked by using validated solid-phase extraction (SPE) procedure. The method that avoids the use of clean-up steps and the hazardous solvents enabled reliable determinations of the OCPs and the PCBs except beta-HCH. The method was applied to the analysis of 33 human serum samples. The most abundant target compound was p-p'-DDE (range, 0.3-8.0 ng/ml; median value, 2.1 ng/ml). Among the PCBs the prevalent congeners were 138, 153 and 180.     

A simple and fast extraction method for organochlorine pesticides and polychlorinated biphenyls in small volumes of avian serum

A solid-phase extraction (SPE) method was developed using 8M urea to desorb and extract organochlorine pesticides (OCs) and polychlorinated biphenyls (PCBs) from avian serum for analysis by capillary gas chromatography with electron capture detection (GC-ECD). The analytes were efficiently extracted from the denatured serum-lipoprotein-analyte complex by one passage through an Oasis((R)) hydrophilic-lipophilic-balanced (HLB) SPE cartridge. No further clean-up was necessary, the entire extraction procedure and GC-ECD analysis can be accomplished in less than 3h. Serum volumes ranged from 100 microL to 1 mL with absolute recoveries of 90-101% for PCBs and 74% to 101% for the OC pesticides.     

Does Mortality Risk of Cigarette Smoking Depend on Serum Concentrations of Persistent Organic Pollutants? Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) Study

Cigarette smoking is an important cause of preventable death globally, but associations between smoking and mortality vary substantially across country and calendar time. Although methodological biases have been discussed, it is biologically plausible that persistent organic pollutants (POPs) like polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides can affect this association. This study was performed to evaluate if associations of cigarette smoking with mortality were modified by serum concentrations of PCBs and OC pesticides. We evaluated cigarette smoking in 111 total deaths among 986 men and women aged 70 years in the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) with mean follow-up for 7.7 years. The association between cigarette smoking and total mortality depended on serum concentration of PCBs and OC pesticides (P value for interaction = 0.02). Among participants in the highest tertile of the serum POPs summary score, former and current smokers had 3.7 (95% CI, 1.5–9.3) and 6.4 (95% CI, 2.3–17.7) times higher mortality hazard, respectively, than never smokers. In contrast, the association between cigarette smoking and total mortality among participants in the lowest tertile of the serum POPs summary score was much weaker and statistically non-significant. The strong smoking-mortality association observed among elderly people with high POPs was mainly driven by low risk of mortality among never smokers with high POPs. As smoking is increasing in many low-income and middle-income countries and POPs contamination is a continuing problem in these areas, the interactions between these two important health-related issues should be considered in future research.     

Selection of DNA aptamers against polychlorinated biphenyls as potential biorecognition elements for environmental analysis

Polychlorinated biphenyls (PCBs) have been of major concerns for decades due to their potential toxicity to human health. To trace the PCBs efficiently and sensitively, many detection methods have been developed. Aptamers, a new class of diagnostic tools, are considered to be such additional candidates for detection of pollutants. In the current study, we report the DNA aptamers, isolated by FluMag-SELEX (a modified SELEX [systematic evolution of ligands by exponential enrichment] technology), that recognize PCBs with the dissociation constants (Kd values) down to the micromolar range. Using the selected aptamers, a highly sensitive aptamer-based fluorescent assay for detection of PCBs was established using gold nanoparticles, with a widely linear range from 0.1 to 100 ng/ml. Moreover, our aptamer-based gold nanoprobe displays specificity toward 3,3',4,4'-tetrachlorobiphenyl (PCB77) compared with a few common PCB77 structural analogs. These results open the possibility of using aptamers as biorecognition elements for easy and fast environmental monitoring.     

Sometimes faster can be better: Microneedling IPG strips enables higher throughput for integrative top-down proteomics

Passive rehydration of immobilized pH gradient (IPG) strips for two-dimensional gel electrophoresis (2DE) has, to our knowledge, never been quantitatively evaluated to determine an ideal rehydration time. Seeking to increase throughput without sacrificing analytical rigor, we report that a substantially shorter rehydration time is accomplished when surface area of IPG strips is increased via microneedling. Rehydration for 4 h, post microneedling, provides comparable results to overnight rehydration in final analyses by 2DE, while also shortening the overall protocol by 1 day.     

Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry

Polychlorinated biphenyls (PCBs) are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL) PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR) is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca²⁺ signaling in PC12 cells. We found that ortho-substituted 2,2’,6-trichlorinated biphenyl (PCB19) caused a rapid decline in the Ca²⁺ signaling of bradykinin, a typical G_q- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca²⁺ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca²⁺ entry (SOCE) were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn²⁺ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca²⁺ signaling.     

Stir bar sorptive extraction–thermal desorption–capillary gas chromatography–mass spectrometry applied to the analysis of polychlorinated biphenyls in human sperm

Stir bar sorptive extraction (SBSE) on polydimethylsiloxane (PDMS) was applied to the enrichment of polychlorinated biphenyls (PCBs) from human sperm. The seven Ballschmiter PCBs were used as model compounds. The extracted PCBs were then thermally desorbed from the stir bar and analysed on-line by capillary gas chromatography (CGC) with mass spectrometric detection (MS). Method development started with the analysis of PCBs spiked in water. Methanol had to be added to the samples in order to reduce the influence of glass adsorption on recovery and reproducibility. Recoveries in water for all PCBs varied around 50–60% and were limited for low molecular mass (MM) PCBs by polarity changes in the sample due to methanol addition and for high MM PCBs by non-equilibrium conditions. Matrix suppression by the lipophilic medium lowered the recoveries in the sperm samples proportional with PCB polarity. The method was validated and although limits of detection (LOD) for the individual congeners were in the sub-ppt level (<pg/ml), the limit of quantification (LOQ) was set at 10 ppt (10 pg/ml).     

Polychlorinated biphenyls and organochlorine pesticide levels in tissues of Caretta caretta from the Adriatic Sea

We detected concentrations of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs) in the liver, muscle, and fat of 11 loggerhead sea turtles Caretta caretta from the central and southern Adriatic Sea. All samples contained PCBs at various concentrations, with Congener 138 (28%), 153 (27%), and 180 (32%) dominating the congener composition of the tissues. The dioxin-like congener (118, 13%) was detected in all tissues analyzed. The lower-chlorinated PCBs were not detected. The average of the total PCB concentrations, expressed in nanograms per gram wet weight, was 459.6 ng g(-1) in fat, 82.9 ng g(-1) in liver, and 5.8 ng g(-1) in muscle. Among 13 organochlorine pesticides for which analyses were conducted, 4 were detected: p,p'-DDE (57%); p,p'-DDD (16%); and p,p'-DDT and o,p'-DDT (27%). Spatial differences were found among OC concentrations in loggerheads from the central and southern Adriatic Sea. The only samples containing detectable concentrations of p,p'-DDT and o,p'-DDT were from the southern area.     

Circulating levels of persistent organic pollutants in relation to visceral and subcutaneous adipose tissue by abdominal MRI

Objective:

We and others have shown relationships between circulating levels of persistent organic pollutants (POPs) and different measures of obesity in both cross‐sectional and prospective studies. Since viscerally located fat seems to be the most harmful type, we investigated whether plasma POP levels were more closely related to visceral adipose tissue (VAT) than to subcutaneous adipose tissue (SAT).

Design and Methods:

Thousand hundred and sixteen subjects aged 70 years were investigated in the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study; 23 POPs were analyzed using high‐resolution gas chromatography/high‐resolution mass spectrometry. Abdominal magnetic resonance imaging, measuring VAT and SAT, respectively, was performed in a representative subsample of 287 subjects.

Results:

The less chlorinated polychlorinated biphenyl (PCB) congeners (105 and 118), and the pesticides dichlorodiphenyldichloroethylene (DDE), hexachlorobenzene (HCB), and trans‐nonachlordane (TNC) were positively related to both VAT and SAT, whereas the more highly chlorinated PCBs (153, 156, 157, 169, 170, 180, 194, 206, and 209) were inversely related to both VAT and SAT. PCB189 was related to the VAT/SAT ratio in an inverted U‐shaped manner (P = 0.0008).

Conclusions:

In conclusion, the results were in accordance with our previous studies using waist circumference and fat mass as obesity measure. However, the novel finding that PCB189 was related to the VAT/SAT ratio deserves further investigation since exposure to this PCB congener, which has previously been linked to diabetes development, might thereby play a role in the distribution of abdominal adipose tissue.     

Quantification of dialkylphosphate metabolites of organophosphorus insecticides in human urine using 96-well plate sample preparation and high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry

Organophosphorus (OP) pesticides kill by disrupting a targeted pest's brain and nervous systems. But if humans and other animals are sufficiently exposed, OP pesticides can have the same effect on them. We developed a fast and accurate high-performance liquid chromatography–tandem mass spectrometry method for the quantitative measurement of the following six common dialkylphosphate (DAP) metabolites of organophosphorus insecticides: dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate, (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP). The general sample preparation included 96-well plate solid phase extraction using weak anion exchange cartridges. The analytical separation was performed by high-performance liquid chromatography with a HILIC column. Detection involved a triple quadrupole mass spectrometer with an ESI probe in negative ion mode using multiple reaction monitoring. Repeated analyses of urine samples spiked at 150, 90 and 32 ng/mL with the analytes gave relative standard deviations of less than 22%. The extraction efficiency ranged from 40% to 98%. The limits of detection were in the range of 0.04–1.5 ng/mL. The throughput is 1152 samples per week, effectively quadrupling our previous throughput. The method is safe, quick, and sensitive enough to be used in environmental and emergency biological monitoring of occupational and nonoccupational exposure to organophosphates.     

High throughput method for the determination of organochlorine pesticides and polychlorinated biphenyls in human serum

An improved method for the determination of selected organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum was developed. The method requires low volume of serum (500 microl) and 48-96 samples per day can be prepared by one analyst without special automatic equipment. Initial extraction was performed using 96-well solid-phase extraction disk plates and was followed by a clean-up with silica gel/sulfuric acid. Different denaturation, elution and clean-up conditions were tested. Quantification was carried out by gas chromatography equipped with electron capture detector (GC-ECD) or mass spectrometer (GC-MS). Recoveries of PCB congeners 28, 52, 101, 118, 138, 153 and 180 and OCPs HCB, beta-HCH, p,p'-DDE and p,p'-DDT at two spiking levels (n=8) varied from 57 to 120%, and intra-day relative standard deviation from 1 to 11%, both depending on spiking level and compound. Inter-day relative standard deviation was <15% in all cases. Limit of quantification (LOQ) for these PCBs ranged from 0.08 to 0.13 ng/ml and for these OCPs from 0.16 to 0.40 ng/ml. The optimized method was applied to the analysis of 1000 serum samples from different places of Spain.     

Atrazine and PCB 153 and their effects on the proteome of subcellular fractions of human MCF-7 cells

Several man-made organic pollutants including polychlorinated biphenyls (PCBs) and several pesticides may exhibit endocrine disrupting (ED) properties. These ED molecules can be comparatively persistent in the environment, and have shown to perturb hormonal activity and several physiological functions. The objective of this investigation was to study the impact of PCB 153 and atrazine on human MCF-7 cells, and to search for marker proteins of their exposure. Cells were exposed to environmentally high but relevant concentrations of atrazine (200ppb), PCB 153 (500ppb), 17-β estradiol (positive control, 10nM) and DMSO (0.1%, negative control) for t=36h (n=3 replicates/exposure group). Proteins from cell membrane and cytosol were isolated, and studied by 2D-DiGE. Differentially regulated proteins were trypsin-digested and identified by MALDI-ToF-ToF and NCBInr database. A total of 36 differentially regulated proteins (>|1.5| fold change, P<0.05) were identified in the membrane fraction and 22 in the cytosol, and were mainly involved in cell structure and in stress response, but also in xenobiotic metabolism. 67% (membrane) and 50% (cytosol) of differentially regulated proteins were more abundant following atrazine exposure whereas nearly 100% (membrane) and 45% (cytosol) were less abundant following PCB 153 exposure. Western blots of selected proteins (HSBP1, FKBP4, STMN1) confirmed 2D-DiGE results. This study emphasizes the numerous potential effects that ED compounds could have on exposed humans.     

Genetic mutation analysis at early stages of cell line development using next generation sequencing

A central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. This objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. Recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development. However, one quality attribute has, until recently, been absent from the standard battery of analytical tests facilitating informed choices early in cell line selection: genetic sequence confirmation. Techniques historically used for mutation analysis, such as detailed mass spectrometry, have limitations on the sample number and turnaround times making it less attractive at early stages. Thus, we explored the utility of Next‐Generation Sequencing (NGS) as a solution to address these limitations. Amplicon sequencing is one such NGS technique that is robust, rapid, sensitive, and amenable to multiplexing, all of which are essential attributes for our purposes. Here we report a NGS method based upon amplicon sequencing that has been successfully incorporated into our cell line development workflow alongside other high‐throughput protein analytical assays. The NGS method has demonstrated its value by identifying at least one Chinese hamster ovary (CHO) clone expressing a variant form of the biotherapeutic in each of the four clinical programs in which it has been utilized. We believe this sequence confirmation method is essential to safely accelerating the time to clinical proof of concept of biotherapeutics, and guard against delays related to sequence mutations. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:813–817, 2016     

Metabolomic analysis of the effects of polychlorinated biphenyls in nonalcoholic fatty liver disease

Polychlorinated biphenyls (PCBs) are persistent organic pollutants and have been associated with abnormal liver enzymes and suspected nonalcoholic fatty liver disease (NAFLD), obesity, and the metabolic syndrome in epidemiological studies. In epidemiological surveys of human PCB exposure, PCB 153 has the highest serum levels among PCB congeners. To determine the hepatic effects of PCB 153 in mice, C57BL/6J mice were fed either a control diet (CD) or a high fat diet (HFD) for 12 weeks, with or without PCB 153 coexposure. The metabolite extracts from mouse livers were analyzed using linear trap quadrupole-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTICR MS) via direct infusion nanoelectrospray ionization (DI-nESI) mass spectrometry. The metabolomics analysis indicated no difference in the metabolic profile between mice fed the control diet with PCB 153 exposure (CD+PCB 153) and mice fed the control diet (CD) without PCB 153 exposure. However, compared with CD group, levels of 10 metabolites were increased and 15 metabolites were reduced in mice fed HFD. Moreover, compared to CD+PCB 153 group, the abundances of 6 metabolites were increased and 18 metabolites were decreased in the mice fed high fat diet with PCB 153 exposure (HFD+PCB 153). Compared with HFD group, the abundances of 2 metabolites were increased and of 12 metabolites were reduced in HFD+PCB 153 group. These observations agree with the histological results and indicate that the metabolic effects of PCB 153 were highly dependent on macronutrient interactions with HFD. Antioxidant depletion is likely to be an important consequence of this interaction, as this metabolic disturbance has previously been implicated in obesity and NAFLD.     

Coplanar polychlorinated biphenyl-induced CYP1A1 is regulated through caveolae signaling in vascular endothelial cells

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that can induce inflammatory processes in the vascular endothelium. We hypothesize that the plasma membrane microdomains called caveolae are critical in endothelial activation and toxicity induced by PCBs. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We focused on the role of caveolae and their major protein component, caveolin-1 (Cav-1), on aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 1A1 (CYP1A1) by coplanar PCBs. Endothelial cell exposure to PCB77 increased both caveolin-1 and CYP1A1 levels in a time-dependent manner in total cell lysates, with a maximum increase at 6h. Furthermore, PCB77 accumulated mainly in the caveolae-rich fraction, as determined by gas chromatograph-mass spectrometry. Immunoprecipitation analysis revealed that PCB77 increased AhR binding to caveolin-1. Silencing of caveolin-1 significantly attenuated PCB77-mediated induction of CYP1A1 and oxidative stress. Similar effects were observed in caveolin-1 null mice treated with PCB77. These data suggest that caveolae may play a role in regulating vascular toxicity induced by persistent environmental pollutants such as coplanar PCBs. This may have implications in understanding mechanisms of inflammatory diseases induced by environmental pollutants.     

Distribution,Sources and Risk Assessment of Polychlorinated Biphenyls in Soils from the Midway Atoll,North Pacific Ocean

Concentrations of 28 polychlorinated biphenyls (PCBs) were assessed in soils from the Midway Atoll in the central North Pacific Ocean. The analytical procedure involved the application of accelerated solvent extraction (ASE) and gas chromatography coupled with ion trap mass spectrometric detection (GC/ITMS) for identification and quantification. Among the 28 PCB congeners studied, 26 of them, except CB195 and CB209, were detected in the analyzed samples at different frequencies. The total concentrations of 28 indicator PCBs (ΣPCBs) ranged from 2.6 to 148.8 ng g⁻¹ with an average value of 50.7 ng g⁻¹ and median of 39.5 ng g⁻¹. Sources and congeners’ pattern of PCB were investigated in the soil of Midway Atoll. The principal component analysis indicated that the compositions of PCBs in most of the soil samples were similar. The total concentrations of PCBs were used to assess the cancer risk probabilities in humans via ingestion, dermal contact and inhalation of soil particles. Very low cancer risk was found in all soil samples caused by ΣPCBs.     

Levels of Polychlorinated Biphenyls (PCBs) and Three Organochlorine Pesticides in Fish from the Aleutian Islands of Alaska

Background

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs) and chlorinated pesticides, have been shown to have many adverse human health effects. These contaminants therefore may pose a risk to Alaska Natives that follow a traditional diet high in marine mammals and fish, in which POPs bioaccumulate.

Methods and Findings

This study examined the levels of PCBs and three pesticides [p, p′-DDE, mirex, and hexachlorobenzene (HCB)] in muscle tissue from nine fish species from several locations around the Aleutian Islands of Alaska. The highest median PCB level was found in rock sole (Lepidopsetta bilineata, 285 ppb, wet weight), while the lowest level was found in rock greenling (Hexagrammos lagocephalus, 104 ppb, wet weight). Lipid adjusted PCB values were also calculated and significant interspecies differences were found. Again, rock sole had the highest level (68,536 ppb, lipid weight). Concerning the PCB congener patterns, the more highly chlorinated congeners were most common as would be expected due to their greater persistence. Among the pesticides, p, p′-DDE generally dominated, and the highest level was found in sockeye salmon (Oncorhynchus nerka, 6.9 ppb, wet weight). The methodology developed by U.S. Environmental Protection Agency (USEPA) was used to calculate risk-based consumption limits for the analyzed fish species. For cancer health endpoints for PCBs, all species would trigger strict advisories of between two and six meals per year, depending upon species. For noncancer effects by PCBs, advisories of between seven and twenty-two meals per year were triggered. None of the pesticides triggered consumption limits.

Conclusion

The fish analyzed, mainly from Adak, contain significant concentrations of POPs, in particular PCBs, which raises the question whether these fish are safe to eat, particularly for sensitive populations. However when assessing any risk of the traditional diet, one must also consider the many health and cultural benefits from eating fish.