In Silico and In Vitro multiple analysis approach for screening naturally derived ligands for red seabream aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a key ligand-dependent transcription factor that mediates the toxic effects of compounds such as dioxin. Recently, natural ligands of AHR, including flavonoids, have been attracting physiological and toxicological attention as they have been reported to regulate major biological functions such as inflammation and anti-cancer by reducing the toxic effects of dioxin. Additionally, it is known that natural AHR ligands can accumulate in wildlife tissues, such as fish. However, studies in fish have investigated only a few ligands in experimental fish species, and the AHR response of marine fish to natural AHR ligands of various other structures has not been thoroughly investigated. To explore various natural AHR ligands in marine fish, which make up the most fish, it is necessary to develop new screening methods that consider the specificity of marine fish. In this study, we investigated the response of natural ligands by constructing in vitro and in silico experimental systems using red seabream as a model species. We attempted to develop a new predictive model to screen potential ligands that can induce transcriptional activation of red seabream AHR1 and AHR2 (rsAHR1 and rsAHR2). This was achieved through multiple analyses using in silico/ in vitro data and Tox21 big data. First, we constructed an in vitro reporter gene assay of rsAHR1 and rsAHR2 and measured the response of 10 representatives natural AHR ligands in COS-7 cells. The results showed that FICZ, Genistein, Daidzein, I3C, DIM, Quercetin and Baicalin induced the transcriptional activity of rsAHR1 and rsAHR2, while Resveratrol and Retinol did not induce the transcriptional activity of rsAHR isoforms. Comparing the EC50 values of the respective compounds in rsAHR1 and rsAHR2, FICZ, Genistein, and Daidzein exhibited similar isoform responses, but I3C, Baicalin, DIM and Quercetin show the isoform-specific responses. These results suggest that natural AHR ligands have specific profiling and transcriptional activity for each rsAHR isoform. In silico analysis, we constructed homology models of the ligand binding domains (LBDs) of rsAHR1 and rsAHR2 and calculated the docking energies (U_dock values) of natural ligands with measured in vitro transcriptional activity and dioxins reported in previous studies. The results showed a significant correlation (R2=0.74(rsAHR1), R2=0.83(rsAHR2)) between docking energy and transcriptional activity (EC50) value, suggesting that the homology model of rsAHR1 and rsAHR2 can be utilized to predict the potential transactivation of ligands. To broaden the applicability of the homology model to diverse compound structures and validate the correlation with transcriptional activity, we conducted additional analyses utilizing Tox21 big data. We calculated the docking energy values for 1860 chemicals in both rsAHR1 and rsAHR2, which were tested for transcriptional activation in Tox21 data against human AHR. By comparing the U_dock energy values between 775 active compounds and 1085 inactive compounds, a significant difference (p<0.001) was observed between the U_dock energy values in the two groups, suggesting that the U_dock value can be applied to distinguish the activation of compounds. Furthermore, we observed a significant correlation (R2=0.45) between the AC50 of Tox21 database and U_dock values of human AHR model. In conclusion, we calculated equations to translate the results of an in silico prediction model for ligand screening of rsAHR1 and rsAHR2 transactivation. This ligand screening model can be a powerful tool to quantitatively estimate AHR transactivation of major marine agents to which red seabream may be exposed. The study introduces a new screening approach for potential natural AHR ligands in marine fish, based on homology model-docking energy values of rsAHR1 and rsAHR2, with implications for future agonist development and applications bridging in silico and in vitro data.

Generation and application of a novel transgenic zebrafish line Tg(GAcyp1a:eGFP/Luc) as an in vivo assay to sensitive and specific monitoring of DLCs in the environment.

CYP1A is the most commonly used biomarker and transgenic fish which carrying a cyp1a promoter to drive a reporter gene can be used as reliable way to monitor dioxin/dioxin-like compounds (DLCs) in the environment. Here, we cloned the cyp1a promoter of Gambusia affinis and this promoter showed stronger transcriptional activity than that of zebrafish. Then, a Tg(GAcyp1a:eGFP/Luc) transgenic zebrafish line was first constructed with the G. affinis cyp1a promoter driving eGFP expression using meganuclease I-SceI mediated transgenesis technology. The Tg(GAcyp1a:eGFP/Luc) larvae at 72 h post-fertilization (hpf) were tested by exposing to TCDD for 72 h, and induced GFP was mainly expressed in the liver with low background. The Tg(GAcyp1a:eGFP/Luc) zebrafish showed high sensitivity (limit of detection of 0.322 ng/L TCDD and 0.7 TEQ-ng/L PCDD/Fs) and specificity (insensitive to responses to PAHs and PCBs). In addition, the transgenic line showed a low detection concentration of the DLCs contaminated environmental samples (as low as 1.8 TEQ-ng/L), and the eGFP fluorescence intensity and the chemical-TEQ values were closely correlated. In conclusion, a sensitively and specifically transgenic zebrafish line was established to convenient and effective to detect DLCs in the environment.

New perspective on the regulation of acetylcholinesterase via the aryl hydrocarbon receptor.

Acetylcholinesterase (AChE, EC 3.1.1.7) plays important roles in cholinergic neurotransmission and has been widely recognized as a biomarker for monitoring pollution by organophosphate (OP) and carbamate pesticides. Dioxin is an emerging environmental AChE disruptor and is a typical persistent organic pollutant with multiple toxic effects on the nervous system. Growing evidence has shown that there is a significant link between dioxin exposure and neurodegenerative diseases and neurodevelopmental disorders, most of which involve AChE and cholinergic dysfunctions. Therefore, an in-depth understanding of the effects of dioxin on AChE and the related mechanisms of action might help to shed light on the molecular bases of dioxin impacts on the nervous system. In the past decade, the effects of dioxins on AChE have been revealed in cultured cells of different origins and in rodent animal models. Unlike OP and carbamate pesticides, dioxin-induced AChE disturbance is not due to direct inhibition of enzymatic activity; instead, dioxin causes alterations of AChE expression in certain models. As a widely accepted mechanism for most dioxin effects, the aryl hydrocarbon receptor (AhR)-dependent pathway has become a research focus in studies on the mechanism of action of dioxin-induced AChE dysregulation. In this mini-review, the effects of dioxin on AChE and the diverse roles of the AhR pathway in AChE regulation are summarized. Additionally, the involvement of AhR in AChE regulation during different neurodevelopmental processes is discussed. These AhR-related findings might also provide new insight into AChE regulation triggered by diverse xenobiotics capable of interacting with AhR.

Neurons expressing the aryl hydrocarbon receptor in the locus coeruleus and island of Calleja major are novel targets of dioxin in the mouse brain.

The aryl hydrocarbon receptor (AhR) acts as a receptor that responds to ligands, including dioxin. The AhR-ligand complex translocates from the cytoplasm into the nucleus to induce gene expression. Because dioxin exposure impairs cognitive and neurobehavioral functions, AhR-expressing neurons need to be identified for elucidation of the dioxin neurotoxicity mechanism. Immunohistochemistry was performed to detect AhR-expressing neurons in the mouse brain and confirm the specificity of the anti-AhR antibody using Ahr-/- mice. Intracellular distribution of AhR and expression level of AhR-target genes, Cyp1a1, Cyp1b1, and Ahr repressor (Ahrr), were analyzed by immunohistochemistry and quantitative RT-PCR, respectively, using mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The mouse brains were shown to harbor AhR in neurons of the locus coeruleus (LC) and island of Calleja major (ICjM) during developmental period in Ahr+/+ mice but not in Ahr-/- mice. A significant increase in nuclear AhR of ICjM neurons but not LC neurons was found in 14-day-old mice compared to 5- and 7-day-old mice. AhR was significantly translocated into the nucleus in LC and ICjM neurons of TCDD-exposed adult mice. Additionally, the expression levels of Cyp1a1, Cyp1b1, and Ahrr genes in the brain, a surrogate of TCDD in the tissue, were significantly increased by dioxin exposure, suggesting that dioxin-activated AhR induces gene expression in LC and ICjM neurons. This histochemical study shows the ligand-induced nuclear translocation of AhR at the single-neuron level in vivo. Thus, the neurotoxicological significance of the dioxin-activated AhR in the LC and ICjM warrants further studies.

Differential Roles of Three Different Upper Pathway meta Ring Cleavage Product Hydrolases in the Degradation of Dibenzo-p-Dioxin and Dibenzofuran by Sphingomonas wittichii Strain RW1.

Sphingomonas wittichii RW1 grows on the two related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. Previous work by others (P. V. Bunz, R. Falchetto, and A. M. Cook, Biodegradation 4:171-178, 1993, https://doi/org/10.1007/BF00695119) identified two upper pathway meta cleavage product hydrolases (DxnB1 and DxnB2) active on the DBF upper pathway metabolite 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate. We took a physiological approach to determine the role of these two enzymes in the degradation of DBF and DXN by RW1. Single knockouts of either plasmid-located dxnB1 or chromosome-located dxnB2 had no effect on RW1 growth on either DBF or DXN. However, a double-knockout strain lost the ability to grow on DBF but still grew normally on DXN, demonstrating that DxnB1 and DxnB2 are the only hydrolases involved in the DBF upper pathway. Using a transcriptomics-guided approach, we identified a constitutively expressed third hydrolase encoded by the chromosomally located SWIT0910 gene. Knockout of SWIT0910 resulted in a strain that no longer grows on DXN but still grows normally on DBF. Thus, the DxnB1 and DxnB2 hydrolases function in the DBF but not the DXN catabolic pathway, and the SWIT0190 hydrolase functions in the DXN but not the DBF catabolic pathway. IMPORTANCE S. wittichii RW1 is one of only a few strains known to grow on DXN as the sole source of carbon. Much of the work deciphering the related RW1 DXN and DBF catabolic pathways has involved genome gazing, transcriptomics, proteomics, heterologous expression, and enzyme purification and characterization. Very little research has utilized physiological techniques to precisely dissect the genes and enzymes involved in DBF and DXN degradation. Previous work by others identified and extensively characterized two RW1 upper pathway hydrolases. Our present work demonstrates that these two enzymes are involved in DBF but not DXN degradation. In addition, our work identified a third constitutively expressed hydrolase that is involved in DXN but not DBF degradation. Combined with our previous work (T. Y. Mutter and G. J. Zylstra, Appl Environ Microbiol 87:e02464-20, 2021, https://doi.org/10.1128/AEM.02464-20), this means that the RW1 DXN upper pathway involves genes from three very different locations in the genome, including an initial plasmid-encoded dioxygenase and a ring cleavage enzyme and hydrolase encoded on opposite sides of the chromosome.

Separate Upper Pathway Ring Cleavage Dioxygenases Are Required for Growth of Sphingomonas wittichii Strain RW1 on Dibenzofuran and Dibenzo-p-Dioxin.

Sphingomonas wittichii RW1 is one of a few strains known to grow on the related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. Previous work by others (B. Happe, L. D. Eltis, H. Poth, R. Hedderich, and K. N. Timmis, J Bacteriol 175:7313-7320, 1993, https://doi.org/10.1128/jb.175.22.7313-7320.1993) showed that purified DbfB had significant ring cleavage activity against the DBF metabolite trihydroxybiphenyl but little activity against the DXN metabolite trihydroxybiphenylether. We took a physiological approach to positively identify ring cleavage enzymes involved in the DBF and DXN pathways. Knockout of dbfB on the RW1 megaplasmid pSWIT02 results in a strain that grows slowly on DBF but normally on DXN, confirming that DbfB is not involved in DXN degradation. Knockout of SWIT3046 on the RW1 chromosome results in a strain that grows normally on DBF but that does not grow on DXN, demonstrating that SWIT3046 is required for DXN degradation. A double-knockout strain does not grow on either DBF or DXN, demonstrating that these are the only ring cleavage enzymes involved in RW1 DBF and DXN degradation. The replacement of dbfB by SWIT3046 results in a strain that grows normally (equal to the wild type) on both DBF and DXN, showing that promoter strength is important for SWIT3046 to take the place of DbfB in DBF degradation. Thus, both dbfB- and SWIT3046-encoded enzymes are involved in DBF degradation, but only the SWIT3046-encoded enzyme is involved in DXN degradation.IMPORTANCES. wittichii RW1 has been the subject of numerous investigations, because it is one of only a few strains known to grow on DXN as the sole carbon and energy source. However, while the genome has been sequenced and several DBF pathway enzymes have been purified, there has been very little research using physiological techniques to precisely identify the genes and enzymes involved in the RW1 DBF and DXN catabolic pathways. Using knockout and gene replacement mutagenesis, our work identifies separate upper pathway ring cleavage enzymes involved in the related catabolic pathways for DBF and DXN degradation. The identification of a new enzyme involved in DXN biodegradation explains why the pathway of DBF degradation on the RW1 megaplasmid pSWIT02 is inefficient for DXN degradation. In addition, our work demonstrates that both plasmid- and chromosomally encoded enzymes are necessary for DXN degradation, suggesting that the DXN pathway has only recently evolved.

Pathology of salivary gland dysfunction and restoration of function.

In this review, the author shows that simultaneous multiple disorders caused by reactivation of Epstein-Barr virus can lead to salivary gland disorders as part of Sjogren's syndrome (SS). Therefore, clinicians must differentiate SS from other diseases when diagnosing and treating salivary gland disorders. In particular, the author explains how microbial infection in SS overcomes immunological tolerance, leading to pathological changes, and how cytokine overexpression and endocrine disrupters contribute to glandular tissue injury. Also, the author suggests that involvement of reactive oxygen species is a common pathogenesis of salivary gland disorders and SS, so regulation of oxidative stress is an effective treatment for both. The results of clinical studies on restoring salivary gland function and regenerating salivary glands with tissue stem cells may provide clues on elucidating the cause of SS.

Pathways and influential factors study on the formation of PBDD/Fs during co-processing BDE-209 in cement kiln simulation system.

The thermal processes of cement kilns are sources of polybrominated dibenzofurans and dioxins (PBDD/Fs); however, when co-processing decabromodiphenyl ether (BDE-209) soil in cement kilns, very few reports have investigated the mechanism of PBDD/Fs formation from BDE-209. Therefore, the pathways and factors that influence the formation of PBDD/Fs were investigated using Box-Behnken design (BBD) of the response surface methodology (RSM) at lab-scale. The PBDEs, HBr/Br2 and PBDD/Fs emissions in flue gas from the simulated thermal process were analyzed using gas chromatography/mass spectroscopy (GC/MS), high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS), and ion chromatography (IC). Density functional theory (DFT) was also used to further discuss the formation of PBDD/Fs. The major products of BDE-209 thermal decomposition in flue gas were 97.1% HBr/Br2 (a.v. 26.6%/70.6%) > 2.7% PBDEs >0.2% PBDD/Fs. Formation of precursors were the main pathways for PBDD/Fs, and those precursors were dominated by higher-brominated PBDEs (heptã deca-BDEs); debromination of BDE-209 was also a crucial pathway for the formation of PBDD/Fs throughout the thermal process. Interestingly, it was easier to form HpBDD/Fs from OBDD/Fs than from PBDEs. The O2 percentage and interaction factors of O2 percentage, temperature, and CaCO3 percentage have the largest influence on PBDD/Fs emissions and formation.

Dioxin-like compounds in paired maternal serum and breast milk under long sampling intervals.

Serum and breast milk are both important biological samples to evaluate body burden of dioxin-like compounds which include polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs). We collected maternal serum at early pregnancy, and breast milk at 3-8 weeks after delivery from 55 mothers living in Beijing, China, and measured 29 dioxin-like compounds in these samples. The sampling intervals in this study were extended up to 10 months to analyze differences of contents between serum and breast milk under long sampling intervals. The results showed that mean TEq level of PCDD/Fs in serum (9.8 pg TEq g-1 lipid) was 1.7 times higher than that in milk (4.5 pg TEq g-1 lipid), while the TEq concentrations of dl-PCBs in serum (1.2 pg TEq g-1 lipid) was significantly lower than that in milk (2.0 pg TEq g-1 lipid). There were only two congeners, OCDD (r = 0.32) and PCB105 (r = 0.33), the correlations of which between serum and milk were significant. The differences in distributions of congeners in serum and milk might be influenced by number of chlorine substituents and structures of congeners. In addition, maternal age and BMI were positively and negatively correlated with mass concentrations of dioxin-like compounds in milk and serum respectively. These results suggest that, compared with serum, it is limited to use breast milk to assess long-term exposure for the wider population.

The aryl hydrocarbon receptor 2 potentially mediates cytochrome P450 1A induction in the jungle crow (Corvus macrorhynchos).

To understand the role of aryl hydrocarbon receptor (AHR) isoforms in avian species, we investigated the functional characteristics of two AHR isoforms (designated as jcAHR1 and jcAHR2) of the jungle crow (Corvus macrorhynchos). Two amino acid residues corresponding to Ile324 and Ser380 (high sensitive type) in chicken AHR1 that are known to determine dioxin sensitivity were Ile325 and Ala381 (moderate sensitive type) in jcAHR1 and Val306 and Ala362 (low sensitive type) in jcAHR2. The quantitative comparison of the two jcAHR mRNA expression levels in a Tokyo jungle crow population showed that jcAHR2 accounted for 92.4% in the liver, while jcAHR1 accounted for only 7.6%. Both in vitro-expressed jcAHR1 and jcAHR2 proteins exhibited a specific binding to [3H]-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transactivation potencies for jcAHR1 and jcAHR2 in in vitro reporter gene assays were measured in jcAHR-expressed cells exposed to 16 dioxins and related compounds (DRCs). Both jcAHR1 and jcAHR2 were activated in a congener- and an isoform-specific manner. EC50 value of TCDD for jcAHR2 (0.61 nM) was six-fold higher than that for jcAHR1 (0.098 nM), but jcAHR2 had higher transactivation efficacy than jcAHR1 in terms of the magnitude of response. The high transactivation efficacy of jcAHR2 in DRCs is in contrast to that of AHR2s in other avian species with low transactivation efficacy. Molecular docking simulations of TCDD with in silico jcAHR1 and jcAHR2 homology models showed that the two sensitivity-decisive amino acids indirectly controlled TCDD-binding modes through their surrounding amino acids. Deletion assays of jcAHR2 revealed that 736-805 amino acid residues in the C-terminal region were critical for its transactivation. We suggest that jcAHR2 plays a critical role in regulating the AHR signaling pathway, at least in its highly expressed organs.

Multigenerational and Transgenerational Effects of Dioxins.

Dioxins are ubiquitous and persistent environmental contaminants whose background levels are still reason for concern. There is mounting evidence from both epidemiological and experimental studies that paternal exposure to the most potent congener of dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), can lower the male/female ratio of offspring. Moreover, in laboratory rodents and zebrafish, TCDD exposure of parent animals has been reported to result in reduced reproductive performance along with other adverse effects in subsequent generations, foremost through the paternal but also via the maternal germline. These impacts have been accompanied by epigenetic alterations in placenta and/or sperm cells, including changes in methylation patterns of imprinted genes. Here, we review recent key studies in this field with an attempt to provide an up-to-date picture of the present state of knowledge to the reader. These studies provide biological plausibility for the potential of dioxin exposure at a critical time-window to induce epigenetic alterations across multiple generations and the significance of aryl hydrocarbon receptor (AHR) in mediating these effects. Currently available data do not allow to accurately estimate the human health implications of these findings, although epidemiological evidence on lowered male/female ratio suggests that this effect may take place at realistic human exposure levels.

A novel ß-propeller phytase from the dioxin-degrading bacterium Sphingomonas wittichii RW-1.

ß-propeller phytase-like sequences (BPP-like sequences) are widespread in the microbial world and have been found in the sequenced genomes of aquatic, soil, and plant bacteria. Exploring NCBI microbial genome database for putative genes encoding phytase, a BPP-like sequence from Sphingomonas wittichii RW-1 (Sequence ID: CP000699.1), known for its capacity of degrading polychlorinated dibenzo-p-dioxins and dibenzofurans, was recognized. The putative phytase gene (phySw) was amplified with specific primers, cloned, and overexpressed in Escherichia coli and the catalytic properties of the recombinant PhySw protein were analyzed. The results show that phySw encodes an enzyme with the properties of ß-propeller phytases: it requires the presence of Ca2+ ions, it is optimally active at 55 °C, and it has a pH optimum of 6.0 with good activity in the range 6.0-8.0. Furthermore, the enzyme exhibits a good thermostability, recovering 68% of its original activity after treatment at 80 °C for 10 min, and shows a good substrate specificity for phytic acid. These properties render this enzyme a candidate as an animal feed additive (e.g., for aquaculture industry). The isolation of phytases from a hydrocarbon-utilizing microorganism also opens new scenarios for their possible application in combating oil pollution.

Comparative study of persistent organic pollutant (POP) (chlorinated pesticides, PCBs, and dioxins/furans) concentrations in cancer-affected human organs with those of healthy organs.

The concentrations of POPs (persistent organic pollutants) including 16 compounds of OCPs, 12 dioxin-like PCBs congeners, and 17 PCDDs/Fs congeners were determined in 46 human adipose tissue samples gathered from Jordanian citizens. Thirteen adipose tissue samples of healthy people were collected from Jordan University Hospital and 33 adipose tissue samples of cancer-affected patients were collected from King Hussein Cancer Center. All samples were extracted, cleaned-up, and analyzed using GC/MS. In the healthy person's samples, among the OCP compounds, the highest concentration was found for heptachlor-oxo-epoxide (5696.71 µg/kg), while among the PCB congeners, the non-ortho PCB 126 shows the highest TEQ concentrations (5554.5 µg TEQ/kg) and among the PCDDs/Fs congeners, the highest TEQ value was found for the congener 2,3,4,7,8-PeCDFs (5.93 µg TEQ/kg). For the cancer-affected patient's samples, the highest concentration among the OCP compounds was found for o,p-DDT (638.7 µg/kg), while among the PCBs congeners, the highest TEQ value was found for the non-ortho-PCB 126 (3366.24 µg TEQ/kg) and among the PCDDs/Fs congeners, the highest TEQ value was found for the congener 1,2,3,7,8-PeCDDs (20.64 µg TEQ/kg). OCP concentration level in adipose tissue samples for healthy people was 32 times higher than for cancer patient persons, while the TEQ values for dioxin-like PCB concentrations in adipose tissue samples of healthy people was 2.2 times higher than in the samples of cancer-affected patient and the TEQ values for PCDDs/Fs in adipose tissue samples of cancer-affected patient was 3 times higher than in the samples of healthy people.

Catbirds are the New Chickens: High Sensitivity to a Dioxin-like Compound in a Wildlife Species.

Dioxins and dioxin-like compounds (DLCs) are highly toxic and persistent global pollutants with extremely large differences in sensitivity across taxonomic groups. The chicken has long been considered uniquely sensitive to DLCs among avian species; but DLC toxicity in nondomesticated birds is largely untested, and the relevance of the chicken as an ecological model is uncertain. New approaches that use genotyping of the AHR1 ligand binding domain to screen for DLC sensitivity among avian species predicted that the gray catbird, a relevant wildlife species, is also highly sensitive. We tested this prediction using egg injections of a dioxin-like PCB (PCB-126) and found that the catbird is at least as sensitive as the chicken to DLCs, based on both embryotoxicity and mRNA induction of phase I metabolizing enzymes (CYP1A4/5). This study is the first to confirm that there are wildlife species as sensitive as the chicken and demonstrates how using predictive genotyping methods and targeted bioassays can focus toxicity assessments on ecologically relevant species.

WHO/UNEP global surveys of PCDDs, PCDFs, PCBs and DDTs in human milk and benefit-risk evaluation of breastfeeding.

Since 1987, the World Health Organization (WHO) carried out global surveys on polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) in human milk. This study presents a review of the three most recent surveys from 2000 to 2010, including DDT. The objective was to identify global quantitative differences and provide baseline information for 52 countries or provide time-trends for countries with previous data. Individual human milk samples were collected following a WHO-designed procedure and combined to form a national pooled sample. Here, we report global levels for PCDDs, PCDFs, PCBs and the sum of o,p'-DDT, p,p'-DDT, o,p'-DDE, p,p'-DDE, o,p'-DDD and p,p'-DDD (ΣDDTs). A concise risk-benefit evaluation related to human milk contamination with these persistent organic pollutants (POPs) was also done. Large global and regional differences were observed. Levels of PCDDs and PCDFs were highest in India and some European and African countries. PCB levels were highest in East and West Europe. The highest levels of ΣDDTs were found in less industrialized countries. A temporal downward trend for PCDDs, PCDFs and PCBs is indicated. A risk-benefit assessment indicates that human milk levels of PCDDs, PCDFs and PCBs are still significantly above those considered toxicologically safe, while ΣDDTs are below or around those considered safe. With respect to potential adverse health effects, a more dominant role of in utero exposure versus lactational exposure is indicated. If potential adverse effects are balanced against positive health aspects for (breastfed) infants, the advantages of breastfeeding far outweigh the possible disadvantages. Our observations provide a strong argument to plea for further global source-directed measures to reduce human exposure further to dioxin-like compounds.

Evaluating Public and Regulatory Acceptance for Urban Soil Management Approaches.

Regulators ( = 14) and the public ( = 30) were surveyed to compare how they perceived contaminated soil management strategies, including bioavailability assessments, using a mental models approach. Both groups proposed similar soil contamination definitions and agreed laboratory tests were needed to identify contaminants. When responding to open-ended questions about management options, regulators emphasized the risk assessment process, whereas the public noted specific treatment options. The majority of the public (68%) and regulators (86%) were concerned about particular contaminants. The public emphasized general contaminant categories, such as petroleum products and chemicals. Regulators listed specific compounds, including arsenic and dioxin. Both groups mentioned lead. Public and regulators had similar levels of agreement for soil removal ( = 0.96) and allowing soils with low bioavailability to remain in place ( = 0.66). The public were most opposed (43% disagree or strongly disagree) to using soil capping. Both groups were willing to consider using bioavailability assessments for contaminated soils. All regulators had heard of bioavailability, whereas 21 of the 24 (88%) public had not heard of this concept. Across all soil management options, the public tended to have higher rates of strongly disagree/disagree and neutral responses compared with regulators ( = 0.01). The neutral responses may indicate public ambivalence or insufficient information to respond about treatment options. Communication and public education efforts should emphasize the analytical process used to justify site-specific treatments. Additional surveys should evaluate public and regulator definitions of successful soil management and contaminant remediation in specific situations (i.e., case studies with specific contaminants and receptors of interest).

Aerobic Biodegradation of OCDD by P. Mendocina NSYSU: Effectiveness and Gene Inducement Studies.

The goals of this study were to assess the effectiveness of (1) enhancing octachlorinated dibenzo-p-dioxin (OCDD) biodegradation under aerobic conditions by Pseudomonas mendocina NSYSU (P. Mendocina NSYSU) with the addition of lecithin, and (2) inducing OCDD ring-cleavage genes by pentachlorophenol (PCP) and OCDD addition. P. Mendocina NSYSU could biodegrade OCDD via aerobic cometabolism and lecithin was used as a primary substrate. Approximately 74 and 67% of OCDD biodegradation was observed after 60 days of incubation with lecithin and glucose supplement, respectively. Lecithin was also used as the solubilization additive resulting in OCDD solubilization and enhanced bioavailability of OCDD to P. Mendocina NSYSU. Two intradiol and extradiol ring-cleavage dioxygenase genes (Pmen_0474 and Pmen_2526) were identified from gene analyses. Gene concentration was significantly enhanced after the inducement by PCP and OCDD. Higher gene inducement efficiency was obtained using PCP as the inducer, and Pmen_2526 played a more important role in OCDD biodegradation.

Urban Endocrine Disruptors Targeting Breast Cancer Proteins.

Humans are exposed to a huge amount of environmental pollutants called endocrine disrupting chemicals (EDCs). These molecules interfere with the homeostasis of the body, usually through mimicking natural hormones leading to activation or blocking of their receptors. Many of these compounds have been associated with a broad range of diseases including the development or increased susceptibility to breast cancer, the most prevalent cancer in women worldwide, according to the World Health Organization. Thus, this article presents a virtual high-throughput screening (vHTS) to evaluate the affinity of proteins related to breast cancer, such as ESR1, ERBB2, PGR, BCRA1, and SHBG, among others, with EDCs from urban sources. A blind docking strategy was employed to screen each protein-ligand pair in triplicate in AutoDock Vina 2.0, using the computed binding affinities as ranking criteria. The three-dimensional structures were previously obtained from EDCs DataBank and Protein Data Bank, prepared and optimized by SYBYL X-2.0. Some of the chemicals that exhibited the best affinity scores for breast cancer proteins in each category were 1,3,7,8-tetrachlorodibenzo-p-dioxin, bisphenol A derivatives, perfluorooctanesulfonic acid, and benzo(a)pyrene, for catalase, several proteins, sex hormone-binding globulin, and cytochrome P450 1A2, respectively. An experimental validation of this approach was performed with a complex that gave a moderate binding affinity in silico, the sex hormone binding globulin (SHBG), and bisphenol A (BPA) complex. The protein was obtained using DNA recombinant technology and the physical interaction with BPA assessed through spectroscopic techniques. BPA binds on the recombinant SHBG, and this results in an increase of its α helix content. In short, this work shows the potential of several EDCs to bind breast cancer associated proteins as a tool to prioritize compounds to perform in vitro analysis to benefit the regulation or exposure prevention by the general population.

Guiana Dolphins (Sotalia guianensis) and DR-CALUX for Screening Coastal Brazilian Environments for Dioxins and Related Compounds.

Guiana dolphin is the top predator of highest toxicological concern in Brazil and many studies on levels of persistent, bioaccumulative, and toxicant (PBT) pollutants have been performed on the species. However, due to high costs of the analyses, only one investigation comprised the determination of dioxins and related compounds (DRCs) in Guiana dolphin tissues. The dioxin responsive-chemically activated luciferase gene expression (DR-CALUX(®)) cell bioassay was used in the present study for the analyses of hepatic samples from 28 male Guiana dolphins in order to screen estuarine environments for DRCs, comprising three regions (Northeastern, Southeastern, and Southern) and four states [Paraná (PR), Rio de Janeiro (RJ), Espírito Santo (ES), and Ceará (CE)] of Brazil. High bioanalytical equivalent (BEQ) concentrations [dioxins (pg BEQ/g lipid)] were found, varying from 1.94 to 15.6 pg BEQ/g. A significant negative correlation between BEQ concentrations and total length was found in Guiana dolphins from Brazil (all analysed dolphins). This pattern also was verified for RJ state, pointing to (1) chemically induced developmental disruption or to (2) increasing efficiency of the detoxifying activity with the growth of the animal. Comparison was performed with literature data and significantly higher BEQ levels were found in Brazilian Guiana dolphins than in those reported for North Sea harbour porpoises. Higher levels were found in Southeastern (the most PBT-contaminated area of the country) than in Southern region. However, it is not possible to affirm that Guiana dolphins are more contaminated by DRCs in SE than in S region, because individuals were lengthier in S than in SE region. Our results seem to have mirrored dolphin exposure to PCBs in Brazil according to the literature. Further studies are required for investigating the hypotheses 1 and 2 mentioned above.