In vitro assessment of effects of persistent organic pollutants on the transactivation of estrogen receptor α and ß (ERα and ERß) from the Baikal seal (Pusa sibirica).

To assess the effect of exposure to persistent organic pollutants (POPs) on the estrogen receptor (ER) signaling pathway in Baikal seals (Pusa sibirica), we investigated the molecular characterizations and functions of two Baikal seal ER (bsER) isoforms, bsERα and bsERß. The bsERα and bsERß cDNA clones isolated have an open reading frame of 595 and 530 amino acid residues, respectively. The tissue distribution analyses of bsER mRNAs showed that bsERα transcripts were primarily found in the ovary and uterus, and bsERß in the muscle in wild Baikal seals. The immunofluorescence staining assay showed that 17ß-estradiol (E2) treatment promoted the nuclear translocation of in vitro-expressed bsERα. Transient transfection of bsERα in U2OS cells enhanced the transcription of pS2, an ER target gene of E2. We then measured bsER-mediated transactivation potencies of POPs in an in vitro reporter gene assay system, in which a bsERα or bsERß expression vector was transfected into COS-1 cells. For comparison, transactivation potencies of POPs on mouse ERs (mERα and mERß) were also evaluated in the same manner. Results showed significant dose-dependent responses of bsERs and mERs when treated with p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE). bsERs and mERs showed no response when exposed to polychlorinated biphenyls (PCBs) or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Comparison of the dose-response curves of DDTs across species (bsERs vs. mERs) showed that bsERα had a response similar to mERα, but bsERß was less sensitive than mERß. Comparing the lowest observable effective concentrations of p,p'-DDT (2.8 µM) and p,p'-DDE (10 µM) for in vitro bsERα-mediated transactivation with their hepatic concentrations in wild Baikal seals indicated that some individuals accumulated these compounds at levels comparable to the effective concentrations, suggesting the potential disruption of the bsERα signaling pathway in the wild population by these compounds. Co-transfection experiments with bsER and the aryl hydrocarbon receptor (AHR) suggested that high accumulation of estrogenic compounds exerts a synergistic effect with dioxin-like congeners on ER signaling through AHR activation in the wild seal population.

Dioxin and Related Compound Detection: Perspectives for Optical Monitoring.

Dioxins and related compounds are environmental xenobiotics that are dangerous to human life, due to the accumulation and persistence in the environment and in the food chain. Cancer, reproductive and developmental issues, and damage to the immune system and endocrine system are only a few examples of the impact of such substances in everyday life. For these reasons, it is fundamental to detect and monitor these molecules in biological samples. The consolidated technique for analytical evaluation is gas chromatography combined with high-resolution mass spectrometry. Nowadays, the development of mid-infrared optical components like broadband laser sources, optical frequency combs, high performance Fourier-transform infrared spectroscopy, and plasmonic sensors open the way to new techniques for detection and real time monitoring of these organic pollutants in gaseous or liquid phase, with sufficient sensitivity and selectivity, and in short time periods. In this review, we report the latest techniques for the detection of dioxins, furans and related compounds based on optical and spectroscopic methods, looking at future perspectives.

A clean-up method for determination of multi-classes of persistent organic pollutants in sediment and biota samples with an aliquot sample.

In the present study, we developed a novel method to simultaneously detect eight classes of persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs), polybrominated diphenyl ethers (PBDEs), polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs), polychlorinated naphthalenes (PCNs), organochlorine pesticides (OCPs), short-chain chlorinated paraffins (SCCPs) and Dechlorane Plus (DP), in sediment and fish tissue samples. The samples were extracted by accelerated solvent extraction (ASE) system, and the extracts were purified and fractionated into fraction1 and fraction2 with multi-layer silica gel column, followed by further fractionation using a basic alumina column (fraction1) and a Florisil column (fraction2), respectively. Finally, fractions were analyzed with different chromatographic columns and different detectors according to the properties of a chemical compound. The limit of detection for eight classes of POPs ranged from 1 to 30 pg/gdw in fish tissue samples, and from 1 to 33 pg/gdw in sediment samples, except for SCCPs (from 5.6 to 7.6 ng/g in fish tissue samples, and from 1.2 to 8.3 ng/gdw in sediment samples). The recoveries of eight classes of POPs spiked in fish tissue and sediment samples ranged from 43% to 120%, and from 45% to 115%, respectively. The relative standard deviation (RSD) was less than 25%. The accuracy of method was satisfactorily demonstrated by analysis of standard reference materials NIST SRM 1649b and WMF-01. In addition, the newly developed method was successfully applied for analysis of sediment and fish samples collected from the Baiyangdian Lake.

Occurrence and spatial distribution of chemical contaminants in edible fish species collected from UK and proximate marine waters.

The occurrence of a range of regulated and emerging organic environmental contaminants was investigated in 182 samples of edible marine fish sampled mainly from UK marine regions, but extending northerly to the coast of Norway and south to the Algarve. These species (sprats, mackerel, turbot, halibut, herring, grey mullet, sea bass, grey mullet, sardines, etc.) are among those considered to be at the highest risk of contamination with regulated contaminants such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs, dioxins), and polychlorinated biphenyls (PCBs), but the occurrence of polybrominated diphenylethers (PBDEs) and polybrominated biphenyls (PBBs) was also investigated. Sub-sets of samples (50-75) were also analysed for emerging contaminants: polychlorinated naphthalenes (PCNs), polybrominated and mixed halogenated dibenzo-p-dioxins, dibenzofurans and biphenyls (PBDD/Fs, PXDD/Fs and PXBs) and perfluoroalkyl substances (PFAS). Contaminant occurrence varied with species and location, but all measured contaminants were detected, with sprats, sea bass, sardines, mackerel, and herring showing higher tissue concentrations. The concentrations of the different contaminants in the various samples were mapped utilising the GPS coordinate data of the capture locations to visualise spatial distribution levels. In terms of catch location, fish sampled from the coasts of southern Britain, north-western France and the Irish Sea appeared to contain proportionately higher levels of some contaminants - e.g. samples from the Irish Sea tended to show higher PCN concentrations, whereas higher levels of PCBs were observed in some fish sampled off the coasts of northern France. Similarly, samples of mullet from the southeast coast of UK showed much higher concentrations of BDE-99 than the other regions. In terms of occurrence trends, PCDD/F and PCB concentrations show a modest decline over the last decade but where limited background data is available for emerging contaminants, there is no evidence of downward trends.

Adipose Tissue as a Site of Toxin Accumulation.

We examine the role of adipose tissue, typically considered an energy storage site, as a potential site of toxicant accumulation. Although the production of most persistent organic pollutants (POPs) was banned years ago, these toxicants persist in the environment due to their resistance to biodegradation and widespread distribution in various environmental forms (e.g., vapor, sediment, and water). As a result, human exposure to these toxicants is inevitable. Largely due to their lipophilicity, POPs bioaccumulate in adipose tissue, resulting in greater body burdens of these environmental toxicants with obesity. POPs of major concern include polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and furans (PCDDs/PCDFs), and polybrominated biphenyls and diphenyl ethers (PBBs/PBDEs), among other organic compounds. In this review, we (i) highlight the physical characteristics of toxicants that enable them to partition into and remain stored in adipose tissue, (ii) discuss the specific mechanisms of action by which these toxicants act to influence adipocyte function, and (iii) review associations between POP exposures and the development of obesity and diabetes. An area of controversy relates to the relative potential beneficial versus hazardous health effects of toxicant sequestration in adipose tissue. © 2017 American Physiological Society. Compr Physiol 7:1085-1135, 2017.

Molecular interactions of dioxins and DLCs with the xenosensors (PXR and CAR): An in silico risk assessment approach.

Dioxins and dioxin-like compounds (DLCs) are known to cause endocrine disruption in humans and animals. Being lipophilic xenobiotic chemicals, they can be easily absorbed into the biological system from the surrounding environments, thereby causing various health dysfunctions. In the present study, a total of 100 dioxins and DLCs were taken, and their binding pattern was assessed with the xenosensors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in comparison with the corresponding known inhibitors and a well-studied endocrine disrupting xenobiotic, bisphenol A (BPA). The nuclear receptors CAR and PXR are known to play a significant role in handling potential toxins by coordinating cellular transport and metabolic functions of the same. Among different endocrine-disrupting chemicals used in the present study, DLCs (PCDFs and PCBs) elicited better interactions in comparison with the parent dioxin (polychlorinated dibenzodioxins) compounds. On comparing D scores of all the compounds against both the receptors, PCDF 8-hydroxy-3,4-dichlorodibenzofuran (8-OH-DCDF) and PCB tetrachlorobenzyltoluene (TCBT) exhibited significant molecular interactions against PXR (-7.633 kcal mol-1 ) and CAR (-8.389 kcal mol-1 ), respectively. Predominant interactions were found to be H-bonding, π-π stacking, hydrophobic, polar, and van der Waals. By contrast, BPA and some natural ligands tested in this study showed lower binding affinities with these receptors than certain DLCs reported herein, ie, certain DLCs might be more toxic than the proven toxic agent, BPA. Such studies play a pivotal role in the risk assessment of exposure to dioxins and DLCs on human health.

PCDD/F and dioxin-like PCB minimization: A 13-year experimental study along the flue gas cleaning system of a secondary aluminium refining plant.

A 13-years study shows that a careful design of the flue gas cleaning system of a full scale secondary aluminium refining plant results in a minimized and very stable emission of Polychlorinated Dibenzo-p-Dioxins (PCDD), Polychlorinated Dibenzo Furans (PCDF) and dioxin-like Polychlorinated Biphenyls (PCB). The value of equivalent toxicity of PCDD/F in the emission was definitely of an order of magnitude less than the regulation limit. In the initial flue gas cleaning system, the PCB mean fingerprint after the slow cooling of the flue gas was typical of de novo synthesis. Instead, in the presence of quenching, there was evidence that the fast cooling of flue gas prevented the PCB de novo synthesis. In fact, the PCB profile was similar to that in the air collected from the aspiration hoods for the quenching. The gas-phase and solid-phase partitioning of PCBs, before and after the fabric filters, highlights the predominant role of the vapor phase with respect to the total removal efficiency. The polycyclic aromatic hydrocarbons breakdown could be an additional de novo formation pathway even in industrial plants.

The association of peripubertal serum concentrations of organochlorine chemicals and blood lead with growth and pubertal development in a longitudinal cohort of boys: a review of published results from the Russian Children's Study.

Organochlorine chemicals and lead are environmental exposures that have endocrine disrupting properties (EDCs) which interfere with many aspects of hormone action. Childhood and adolescence are windows of susceptibility for adverse health effects of EDCs. Our ongoing study, the Russian Children's Study (RCS), is one of the few longitudinal studies investigating the impact of EDCs on growth and puberty in boys. It is conducted in the historically contaminated city of Chapaevsk, in the Samara region. The study focuses on evaluating the associations of persistent organochlorine chemicals and lead with growth and pubertal timing. At enrollment in 2003-2005, we collected blood from 516 boys at ages 8-9 years to measure dioxins, furans, polychlorinated biphenyls (PCBs), chlorinated pesticides and lead. At enrollment and at annual visits through the ages of 18-19 years, a physician performed physical examinations that included pubertal staging and testicular volume measurements. We review the history of Chapaevsk as a research site and summarize published RCS data on the association of peripubertal serum concentrations of organochlorines and blood lead levels with growth, pubertal onset and sexual maturity. Overall, we found that persistent organochlorines and lead negatively affected growth during puberty. Our results also suggest that total toxic equivalents (TEQs), dioxin-like compounds, organochlorine pesticides and lead may delay, while nondioxin-like-PCBs may advance, the timing of male puberty. These findings promoted remediation programs in Chapaevsk, with improvement in health indicators, resulting in Chapaevsk being designated a member of the World Health Organization (WHO) network "Healthy Cities" in 2015.

Dioxin-like rather than non-dioxin-like PCBs promote the development of endometriosis through stimulation of endocrine-inflammation interactions.

Polychlorinated biphenyls (PCBs) contain 209 congeners with various structure-activities. Exposure to PCBs was related to disorders of female reproduction. Endometriosis (EM) is an estrogen- and inflammation-dependent disease with high prevalence and severe health outcomes. Epidemiological studies have shown the effects of PCBs exposure on EM in regard to various structures of PCBs. However, little evidence is available from the toxicology considering the structure of PCBs. In the study, environmentally relevant concentrations of PCBs were used to treat primary cultured endometrial cells and an EM mouse model. Dioxin-like CB126, but not non-dioxin-like CB153, significantly enhanced 17ß-estradiol (E2) biosynthesis in a dose-dependent manner. Among the genes related to estrogen metabolism, the level of 17ß-hydroxysteroid dehydrogenase 7 (HSD17B7) showed significant increase following CB126 exposure. We further found that CB126 exposure decreased the methylation of the HSD17B7 promoter. Elevated expression of HSD17B7 was observed in the eutopic endometrium of EM patients. CB126 rather than CB153 triggered the inflammatory response by directly stimulating the secretion of inflammatory factors and indirectly reducing the level of lipoxin A4 (LXA4). Furthermore, the inflammation enhanced the expression of HSD17B7. Antagonism of the aryl hydrocarbon receptor (AhR) diminished the effects induced by CB126. In vivo, the PCB-treated EM mouse model confirmed that CB126 rather than CB153 increased the levels of both E2 and inflammatory factors in peritoneal fluid and promoted the development of endometriotic lesions. In all, CB126, but not CB153, triggered EM development by stimulating estrogen biosynthesis, inflammation and their interactions and that these effects were mediated by the AhR receptor.

PCDD, PCDF, dl-PCB and organochlorine pesticides monitoring in São Paulo City using passive air sampler as part of the Global Monitoring Plan.

The persistent organic pollutants (POPs), such as organochlorine pesticides and PCBs, are ordinarily monitored in the aquatic environment or in soil in the environmental quality monitoring programs in São Paulo, Brazil. One of the core matrices proposed in the POPs Global Monitoring Plan (GMP) from the Stockholm Convention list is the ambient air, which is not a usual matrix for POPs monitoring in the country. In this study POP levels were evaluated in the air samples from an urban site in São Paulo City over five years, starting in 2010 as a capacity building project for Latin America and the Caribbean region for POP monitoring in ambient air using passive samplers. Furthermore, after the end of the Project in 2012, the monitoring continued in the same sampling site as means to improving the analytical capacity building and contribute to the GMP data. The POPs monitored were 17 congeners of 2,3,7,8 chloro-substituted PCDDs and PCDFs, dioxin-like PCBs, indicator PCBs, organochlorine pesticides and toxaphene. The results show a slight decrease in PCDD/F, dl-PCBs and indicator PCBs levels along the five years. The organochlorine pesticide endosulfan was present at its highest concentration at the beginning of the monitoring period, but it was below detection level in the last year of the monitoring. Some other organochlorine pesticides were detected close to or below quantitation limits. The compounds identified were dieldrin, chlordane, α-HCH, γ-HCH, heptachlor, heptachlor epoxide, hexachlorobenzene and DDTs. Toxaphene congeners were not detected. These results have confirmed the efficacy of passive sampling for POP monitoring and the capacity building for POP analysis and monitoring was established. However more needs to be done, including expansion of sampling sites, new POPs and studies on sampling rates to be considered in calculating the concentration of POPs in ambient air using a passive sampler.

Comparative liver accumulation of dioxin-like compounds in sheep and cattle: Possible role of AhR-mediated xenobiotic metabolizing enzymes.

PCDDs, PCDFs, and PCBs are persistent organic pollutants (POPs) that accumulate in animal products and may pose serious health problems. Those able to bind the aryl hydrocarbon receptor (AhR), eliciting a plethora of toxic responses, are defined dioxin-like (DL) compounds, while the remainders are called non-DL (NDL). An EFSA opinion has highlighted the tendency of ovine liver to specifically accumulate DL-compounds to a greater extent than any other farmed ruminant species. To examine the possible role in such an accumulation of xenobiotic metabolizing enzymes (XME) involved in DL-compound biotransformation, liver samples were collected from ewes and cows reared in an area known for low dioxin contamination. A related paper reported that sheep livers had about 5-fold higher DL-compound concentrations than cattle livers, while the content of the six marker NDL-PCBs did not differ between species. Specimens from the same animals were subjected to gene expression analysis for AhR, AhR nuclear translocator (ARNT) and AhR-dependent oxidative and conjugative pathways; XME protein expression and activities were also investigated. Both AhR and ARNT mRNA levels were about 2-fold lower in ovine samples and the same occurred for CYP1A1 and CYP1A2, being approximately 3- and 9-fold less expressed in sheep compared to cattle, while CYP1B1 could be detectable in cattle only. The results of the immunoblotting and catalytic activity (most notably EROD) measurements of the CYP1A family enzymes were in line with the gene expression data. By contrast, phase II enzyme expression and activities in sheep were higher (UGT1A) or similar (GSTA1, NQO1) to those recorded in cattle. The overall low expression of CYP1 family enzymes in the sheep is in line with the observed liver accumulation of DL-compounds and is expected to affect the kinetics and the dynamics of other POPs such as many polycyclic aromatic hydrocarbons, as well as of toxins (e.g. aflatoxins) or drugs (e.g. benzimidazole anthelmintics) known to be metabolized by those enzymes.

Emissions of PCDD/Fs, PBDD/Fs, dioxin like-PCBs and PAHs from a cement plant using a long-term monitoring system.

The aim of the present work was to assess the emission of different persistent organic pollutants from a cement plant over a period of one year, under normal operational conditions. Thus, a long-term sampling device was installed in the clinker kiln stack of the cement plant. The factory uses petroleum coke as primary fuel, but also alternative fuels such as solid recovered fuel (SRF), automotive shredder residue (ASR), sewage sludge, waste tires, and meat and bone meal (MBM) wastes, with an energy substitution level of about 40%. Both PCDD/Fs (together with dl-PCBs) and PBDD/Fs were continuously sampled, with a total of ten samples collected in 2-4week periods. Also, PAHs were sampled during one-week periods, in order to evaluate their emissions in three different samples. The emission levels throughout the year were much lower than the set legal limits in all substances, being <10pgI-TEQ/Nm(3) in the case of PCDD/Fs. The data obtained allowed calculation of updated emission factors for the cement sector, which were 8.5ng I-TEQ/ton clinker for PCDD/Fs and 3.2ng WHO-TEQ/ton clinker for PCBs. With respect to the congener distribution, 2,3,7,8-TCDF accounts for 60 to 68% of the total toxicity for PCDD/Fs, and in PBDD/F emissions, a clear predominance of octa-substituted species (both dioxin and furan) was found.

Evaluation of relative effect potencies (REPs) for dioxin-like compounds to derive systemic or human-specific TEFs to improve human risk assessment.

Toxic equivalency factors (TEFs) are generally applied for estimating human risk of dioxins and dioxin-like compounds using systemic (e.g., blood) levels, even though these TEFs are established based on intake doses in rodent studies. This review shows that systemic relative effect potencies (REPs) can deviate substantially from intake REPs, but are similar to in vitro-derived REPs. Interestingly, the in vitro REPs for 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) and 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF) are up to one order of magnitude higher than their in vivo REPs and WHO-TEFs, based on oral intake. In addition, clear species-differences in in vitro REPs were apparent for some congeners. Especially the human-derived REP for polychlorinated biphenyl 126 is one to two orders of magnitude lower than rodent REPs and its current WHO-TEF. Next, suggested adapted systemic or human-specific TEFs for these congeners were applied to calculate changes in systemic TEQ concentrations in studies from the USA, Germany and Japan and compared with either the JECFA TDI or USEPA RfD of TCDD. Overall, the effect of such TEF changes for these three congeners on total TEQ roughly balances each other out in the general population. However, results may be different for situations in which a specific group of congeners dominates. For those congeners that show a distinct deviation between either intake and systemic REPs or between rodent- and human-based in vitro REPs, we propose that especially REPs derived from human-based in vitro models are weighted more heavily in establishing systemic or human-specific TEF values to improve human health risk assessment.

Relative sensitivities among avian species to individual and mixtures of aryl hydrocarbon receptor-active compounds.

Dioxins and dioxin-like compounds (DLCs) are potent toxicants to most vertebrates. Sensitivities to DLCs vary among species. In the present study, the sensitivities of avian species (chicken [Gallus gallus], ring-necked pheasant [Phasianus colchicus], and Japanese quail [Coturnix japonica]) to some polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) were determined by using species-specific, in vitro, transactivation assays based on a luciferase reporter gene under control of species-specific aryl hydrocarbon receptors. In ring-necked pheasant and Japanese quail, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was not the most potent inducer of toxic effects. Especially for Japanese quail, the relative potency values of most of 9 PCDD/Fs tested were greater than for TCDD. The rank order of avian species sensitivities to DLCs was chicken > ring-necked pheasant > Japanese quail. Effects of binary mixtures of TCDD, 2,3,7,8-tetrachlorodibenzofuran, and 2,3,4,7,8-pentachlorodibenzofuran were strictly additive. Moreover, we also found that the primary DLCs that were responsible for most of the potency of the DLC mixtures can be deduced by using ordination in a multidimensional space defined by the avian species sensitivities. Overall, the relative potency and the species sensitivities of these chemicals could guide risk assessments to wild species when exposure to mixtures of DLCs in the environment.