Tetrachlorodibenzo-p-dioxin Research Articles

Phosphodiesterase 2A Forms a Complex with the Co-chaperone XAP2 and Regulates Nuclear Translocation of the Aryl Hydrocarbon Receptor

Phosphodiesterase type 2A (PDE2A) hydrolyzes cyclic nucleotides cAMP and cGMP, thus efficiently controlling cNMP-dependent signaling pathways. PDE2A is composed of an amino-terminal region, two regulatory GAF domains, and a catalytic domain. Cyclic nucleotide hydrolysis is known to be activated by cGMP binding to GAF-B; however, other mechanisms may operate to fine-tune local cyclic nucleotide levels. In a yeast two-hybrid screening we identified XAP2, a crucial component of the aryl hydrocarbon receptor (AhR) complex, as a major PDE2A-interacting protein. We mapped the XAP2 binding site to the GAF-B domain of PDE2A. PDE assays with purified proteins showed that XAP2 binding does not change the enzymatic activity of PDE2A. To analyze whether PDE2A could affect the function of XAP2, we studied nuclear translocation of AhR, i.e. the master transcription factor controlling the expression of multiple detoxification genes. Notably, regulation of AhR target gene expression is initiated by tetrachlorodibenzodioxin (TCDD) binding to AhR and by a poorly understood cAMP-dependent pathway followed by the translocation of AhR from the cytosol into the nucleus. Binding of PDE2A to XAP2 inhibited TCDD- and cAMP-induced nuclear translocation of AhR in Hepa1c1c7 hepatocytes. Furthermore, PDE2A attenuated TCDD-induced transcription in reporter gene assays. We conclude that XAP2 targets PDE2A to the AhR complex, thereby restricting AhR mobility, possibly by a local reduction of cAMP levels. Our results provide first insights into the elusive cAMP-dependent regulation of AhR.

Formation of tetrahalogenated dibenzo- p-dioxins (TXDDs) by pyrolysis of a mixture of 2,4,6-trichlorophenol and 2,4,6-tribromophenol

The microscale pyrolysis of a mixture of 2,4,6-tribromophenol (2,4,6-TBP) and 2,4,6-trichlorophenol (2,4,6-TCP) was investigated. Several pyrolyzed products resulting from the gas phase reactions were analyzed by gas chromatography/mass spectrometry (GC/MS). Some products, including tetrabromo- and tetrachloro-dibenzo- p-dioxins, were identified by comparison of their retention times and mass spectra with those of products obtained from the pyrolysis of the corresponding 2,4,6-TBP and 2,4,6-TCP. 1,3,6,8- and 1,3,7,9-halogen substituted dioxin isomers were produced as major products via the condensation of phenoxyphenol radicals. The condensation of 2,4,6-TBP led to the more rapid production of tetrabromodibenzo- p-dioxins (TBDDs) than of tetrachlorodibenzo- p-dioxins (TCDDs) from 2,3,6-TCP due to the weaker C–Br than C–Cl bond strength. In the study of a mixture of 2,4,6-TCP and 2,4,6-TBP, the most abundant congeners were dibromo-dichlorodibenzo- p-dioxins. On the basis of the pyro-products analysis, the thermal decay of 2,4,6-TCP and 2,4,6-TBP, and the formation of halogenated dioxins as a function of the pyrolysis temperature were also investigated. The increased reactivity can be explained by the hypothesis that “dibromophenoxy radical are formed more easily by debromination of 2,4,6-TBP than that of 2,4,6-TCP, which rapidly reacts with 2,4,6-TCP and 2,4,6-TBP at low temperatures”. The formation distribution of tetra-bromochlorodibenzo- p-dioxins (tetra-BCDDs) indicated that more chlorinated dioxins were produced at low temperatures, but more brominated dioxins were formed at high temperatures.

Microarray analysis of gene expression in peripheral blood mononuclear cells from dioxin-exposed human subjects

Tetrachlorodibenzo-p-dioxin (TCDD) is classified as a human carcinogen and exerts toxic effects on the skin (chloracne). Effects on reproductive, immunological, and endocrine systems have also been observed in animal models. TCDD acts through the aryl hydrocarbon receptor (AhR) pathway influencing largely unknown gene networks. An industrial accident in Seveso, Italy in 1976 exposed thousands of people to substantial quantities of TCDD. Twenty years after the exposure, this study examines global gene expression in the mononuclear cells of 26 Seveso female never smokers, with similar age, alcohol consumption, use of medications, and background plasma levels of 22 dioxin congeners unrelated to the Seveso accident. Plasma dioxin levels were still elevated in the exposed subjects. We performed analyses in two different comparison groups. The first included high-exposed study subjects compared with individuals with background TCDD levels (average plasma levels 99.4 and 6.7ppt, respectively); the second compared subjects who developed chloracne after the accident, and those who did not develop this disease. Overall, we observed a modest alteration of gene expression based on dioxin levels or on chloracne status. In the comparison between high levels and background levels of TCDD, four histone genes were up-regulated and modified expression of HIST1H3H was confirmed by real-time PCR. In the comparison between chloracne case–control subjects, five hemoglobin genes were up-regulated. Pathway analysis revealed two major networks for each comparison, involving cell proliferation, apoptosis, immunological and hematological disease, and other pathways. Further examination of the role of these genes in dioxin induced-toxicity is warranted.

2,3,7,8-Tetrachlorodibenzo-p-dioxin and Epidermal Growth Factor Cooperatively Suppress Peroxisome Proliferator-Activated Receptor-γ1 Stimulation and Restore Focal Adhesion Complexes during Adipogenesis: Selective Contributions of Src, Rho, and Erk Distinguish These Overlapping Processes in C3H10T1/2 Cells

Stimulation of PPARgamma1 and adipogenesis in multipotential C3H10T1/2 cells by the combination of dexamethasone and 3-isobutyl-1-methylxanthine (DM) is suppressed by 2,3,7,8 tetrachlorodibenzodioxin (TCDD) (10 nM). This suppression requires sustained activation of extracellular signal-regulated kinase (Erk)1/2. We show that it arises from an effect of TCDD on epidermal growth factor (EGF) signaling. DM initiates an early loss of cell adhesion that is reversed by this TCDD/EGF synergy. Src kinase activity was completely essential for adhesion restoration, sustained Erk activation, and suppression of peroxisome proliferator-activated receptor (PPAR)gamma1. MEK/Erk activity did not contribute, however, to TCDD-induced adhesion. Stimulation of adhesion may therefore precede elevation of Erk. Adhesion is produced by interaction of alphabeta integrins with extracellular matrix proteins and subsequent Src-mediated phosphorylation of focal adhesion kinase (FAK, Tyr576/577) and paxillin (Tyr118). TCDD enhanced the steady state Src-mediated phosphorylation of FAK but not of paxillin. Protein tyrosine phosphatase (PTPase) inhibition by orthovanadate (OVA) showed that this Src activity is highly restricted by PTPases. Partial inhibition of PTPases by OVA mimicked TCDD in producing EGF- and Src-dependent effects on cell adhesion and PPARgamma1 suppression. TCDD may therefore induce a protein that enhances Src effectiveness at adhesion sites. Rho kinase (ROCK) inhibition blocked TCDD/EGF stimulation of clustered focal adhesion complexes without affecting either sustained Erk activation or suppression of PPARgamma1. Thus, this ROCK-mediated clustering of integrin complexes is not needed for the effects of TCDD on Erk and PPARgamma1. A minimal cholesterol depletion with beta-methylcyclodextrin attenuated TCDD effects on PPARgamma1 and Erk activation. TCDD intervention is therefore linked to extracellular proteins. It indicates that TCDD-enhanced stimulation of EGF signaling to Erk may derive from the initial alphabeta integrin complexes.

Analyzing Toxicity Through Electrophilicity

The toxicological structure-activity relationships are investigated using conceptual DFT based descriptors like global and local electrophilicities. In the present work the usefulness of electrophilicity in predicting toxicity of several polyaromatic hydrocarbons (PAH) is assessed. The toxicity is expressed through biological activity data (pIC50) defined as molar concentration of those chemicals necessary to displace 50% of radiolabeled tetrachlorodibenzo-p-dioxin (TCDD) from the arylhydrocarbon (Ah) receptor. The experimental toxicity values (pIC50) for the electron acceptor toxin like polychlorinated dibenzofurans (PCDF) are taken as dependent variables and the DFT based global descriptor electrophilicity index (omega) is taken as independent variable in the training set. The same model is then tested on a test set of polychlorinated biphenyls (PCB). A good correlation is obtained which vindicates the importance of these descriptors in the QSAR studies on toxins. These toxins act as electron acceptors in the presence of biomolecules whereas aliphatic amines behave as electron donors some of which are also taken into account for the present work. The toxicity values of the aliphatic amines in terms of the 50% inhibitory growth concentration (IGC50) towards ciliate fresh-water protozoa Tetrahymena pyriformis are considered. Since there is no global nucleophilicity we apply local nucleophilicity (omegamax+) as the descriptor in this case of training set. The same regression model is then applied to a test set of amino alcohols. Although the correlation is very good the statistical analysis reflects some cross validation problem. As a further check the amines and amino alcohols are used together to form both the training and the test sets to provide good correlation. It is demonstrated that the toxicity of several toxins (both electron donors and acceptors) in the gas and solution phases can be adequately explained in terms of global and local electrophilicities. Amount of charge transfer between the toxin and the biosystem, simulated as nucleic acid bases and DNA base pairs, indicates the importance of charge transfer in the observed toxicity. The major strength of the present analysis vis-à-vis the existing ones rests on the fact that it requires only one descriptor having a direct relationship with toxicity to provide a better correlation. Importance of using the information from both the toxin and the biosystem is also analyzed.

Developmental differences in elimination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during Xenopus laevis development

Although 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is a potent developmental toxicant in most vertebrates, several frog species are insensitive to TCDD, especially during early life stages. Previous experiments with ranid frogs suggest that TCDD insensitivity results largely from rapid elimination. Recent studies in the African clawed frog (Xenopus laevis; family Pipidae) link low TCDD toxicity with the low binding affinity of aryl hydrocarbon receptors, which mediate the toxic effects of dioxin-like compounds. The present study sought to examine TCDD elimination in X. laevis embryos and tadpoles, enabling an integrated assessment of the relative roles of TCDD elimination and AHR-related mechanisms in TCDD insensitivity within a single frog species. Using tadpoles (stage 52-55; approximately 1 month old) exposed to [3H]TCDD, we observed that TCDD has a relatively short half life of 102.6 h, consistent with other frogs and much faster than reported clearance rates in developing fish. In contrast, TCDD elimination is much slower during early development. Embryos exposed during primary organogenesis (from stage 31-41, beginning approximately 36 h after fertilization) exhibited little TCDD elimination during the subsequent 96 h. Enhanced TCDD clearance in later developmental stages may follow the appearance of a functional digestive tract and the onset of feeding. These results suggest that rapid elimination is unlikely to contribute mechanistically to TCDD insensitivity during development of the cardiovascular system, which is significantly perturbed by TCDD in fish embryos.

Molecular Mechanisms of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Cardiovascular Embryotoxicity

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons are widespread environmental contaminants and potent developmental toxicants. Hallmarks of embryonic exposure include edema, hemorrhage, and mortality. Recent studies in zebrafish and chicken have revealed direct impairment of cardiac muscle growth that may underlie these overt symptoms. TCDD toxicity is mediated by the aryl hydrocarbon receptor, but downstream targets remain unclear. Oxidative stress and growth factor modulation have been implicated in TCDD cardiovascular toxicity. Gene expression profiling is elucidating additional pathways by which TCDD might act. We review our understanding of the mechanism of TCDD embryotoxicity at morphological and molecular levels.

Atmospheric Deposition of Polychlorinated Dibenzo-p-dioxins (PCDDs) and Polychlorinated Dibenzofurans (PCDFs) in Kanazawa, Japan

The deposition processes of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in Kanazawa, Japan were studied by examination of the concentrations of PCDDs and PCDFs (PCDD/DFs) and their tetra- to octa-chlorinated homologues in the rain. The weighted mean deposition flux was 360 pg/m2/day [7.7 pg-toxicity equivalency quantity (TEQ)/m2/day]. Deposition fluxes obtained in this study were lower than the average value of Japan, suggesting that Kanazawa is a less polluted area. The seasons with the highest and next highest deposition fluxes were winter and spring, respectively, possibly due to the presence of an inversion layer in winter and spring that reduced the atmospheric dilution of pollutants. In addition, the ratio of PCDFs to total deposition flux in winter was larger than the ratios in the other seasons, possibly due to the burning of fossil fuels for residential heating. Deposition flux of each tetra- to octa-chlorinated homologue of PCDD/DFs was negatively correlated with surface temperature. Other meteorological parameters were positively correlated with almost all tetra- to octa-chlorinated homologues, except for heptachloro dibenzo-p-dioxins (HpCDDs) and octachloro dibenzo-p-dioxin (OCDD), possibly as a result of the photochemical reaction of pentachlorophenol (PCP), which produced mainly OCDD and traces of HpCDDs. In ambient air, the two most dominant homologues were tetrachloro dibenzo-p-dioxins (TeCDDs) and OCDD, while, in soil, the two most dominant homologues were OCDD and TeCDDs. The washout ratios increased with the increase in chlorine substitution. Thus, the difference in washout ratios between homologues might be one of the reasons for the difference of the homologue profile between the air and soil.

AhR-Agonist-Induced Transcriptional Changes of Genes Involved in Thyroid Function in Primary Porcine Thyrocytes

The Ah receptor (AhR) is a ligand transcription factor mediating toxic effects of chemicals such as dioxins. The 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and the coplanar polychlorinated biphenyl 126 (PCB 126) are member of the polyhalogenated aromatic hydrocarbons family exerting a variety of toxic effects in a tissue-specific and species-specific manner including thyroid function. In the present study, we aimed to investigate the effects of TCDD (1 and 10 nM) and dioxin-like PCB 126 (306 nM) on the AhR signaling pathway and on the gene expression profiles of key factors involved in thyroid function, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium iodide symporter (NIS), TSH receptor (TSHR), and cathepsins (Cat B and L), using a primary porcine thyrocyte culture as the experimental model. AhR and ARNT expression was detected both as mRNA and on the protein level. Expression did not vary upon treatment with either TCDD or PCB 126. However, treatment with TCDD and PCB 126 induced an AhR signaling response, as indicated by the expression of the AhR-target gene cytochrome P-450 1A1 (CYP1A1). Both 10 nM TCDD and PCB 126 treatment induced a significant downregulation in the expression of NIS and cathepsin B without affecting any of the other parameters investigated. In conclusion, these data indicate that (a) thyrocytes are targets of TCDD and TCDD-like compounds and (b) there is evidence for two independent most likely AhR-mediated molecular mechanisms, by which these compounds negatively interfere with thyroid function.

Role of the carboxy-terminal transactivation domain and active transcription in the ligand-induced and ligand-independent degradation of the mouse Ah b−1 receptor

To assess the importance of transactivation domains (TAD), DNA binding and transcription on the degradation of the AH receptor (AHR), Hepa-1 cells were pre-treated with actinomycin D (AD) or cycloheximide (CHX) and exposed to 2,3,7,8 tetrachlorodibenzo- p-dioxin (TCDD). AD or CHX did not affect nuclear localization or DNA binding of the AHR but inhibited ligand-induced degradation. In contrast, AD or CHX did not inhibit geldanamycin (GA) induced degradation of the AHR. To assess the role of the COOH-terminal TAD in AHR degradation, stop codons were placed at nucleotide 1501 and 1921 of the Ah b−1 AHR coding region to generate AHR 500 and AHR 640. Stable cell lines were generated and exposed to TCDD. Cells expressing AHR 500 did not induce CYP1A1 protein, but exhibited significant degradation of AHR 500. Cells expressing AHR 640 induced CYP1A1 protein to 50% of the level of cells expressing wild type AHR and exhibited significant degradation of AHR 640. Importantly, AD and CHX did not inhibit the TCDD-induced degradation of either AHR 500 and AHR 640 and these receptors showed a more rapid profile of ligand-induced degradation compared to cells expressing wild type AHR. TCDD exposure to Hepa-1 cells with reduced aryl hydrocarbon receptor nuclear translocator (ARNT), showed ligand-induced degradation of the AHR that was not blocked by AD. However, AD inhibited TCDD-induced degradation when ARNT expression was restored. These results show that multiple mechanisms exist for the ligand and GA-induced degradation of the AHR and suggest that ligand-induced degradation can switch between two mechanisms depending on the presence of a functional TAD and the binding to DNA.

Superantigen-primed T cells exposed to 2,3,7,8–tetrachlorodibenzo-p-dioxin (TCDD) replicate poorly following recall encounter

The current study investigated the effect of tetrachlorodibenzo-p-dioxin (TCDD) on the ability of staphylococcal enterotoxin A (SEA)-primed T cells to divide by dual-labeling the cells with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and antibodies against the specific T cell receptors. C57BL/6 wild-type mice were injected ip with TCDD (10 microg/kg body weight) followed by hind footpad injections of SEA (10 microg/footpad). The draining popliteal lymph nodes (PLN) were harvested 1-4 days posttreatment, labeled with CFSE and cultured for 1-4 days without further stimulation or in the presence of the recall antigen. TCDD-exposed SEA-reactive Vbeta3+ and Vbeta11+ T cells showed decreased cell divisions upon in vitro culture in the absence of any stimulation, which correlated with increased levels of apoptosis. The recall cell-division response was also defective in SEA-reactive T cells isolated from TCDD-exposed mice. However, during the recall response, cells from TCDD-exposed mice did not exhibit a defect in apoptosis, suggesting the defective recall response may result from a state of anergy rather than increased apoptosis. Using AhR knockout (KO) mice, we found AhR involvement in the regulation of defective cell division and apoptosis induced by TCDD. Together, these data demonstrate, while TCDD-induced apoptosis may account for the decreased primary T cell proliferative response, that the reduced cell division seen during subsequent exposure to recall antigen may result from a state of anergy. The study also demonstrates that a combined use of superantigen and CFSE may offer a simple and useful tool to monitor the ability of immunotoxicants to alter the proliferative responsiveness of antigen-specific T cells.

Arsenite-Induced Aryl Hydrocarbon Receptor Nuclear Translocation Results in Additive Induction of Phase I Genes and Synergistic Induction of Phase II Genes

Complex mixtures of carcinogenic metalloids, such as arsenic, and polycyclic aromatic hydrocarbons or halogenated aromatic hydrocarbons are common environmental contaminants. The biological consequences of exposure to these mixtures are unpredictable and, although the health effects of individual chemicals may be known, the toxicity of environmental mixtures is largely unexplored. Arsenic, not a potent mutagen by itself, is co-mutagenic with many DNA-damaging agents. Mixtures of arsenite plus benzo[a]pyrene (B[a]P) augment B[a]P mutagenicity, suggesting that arsenite might uncouple expression of phase I and II genes responsible for detoxification. We have studied the effects of arsenite exposure on the activation of the aryl hydrocarbon receptor (AHR) and its subsequent role in gene transactivation. Treatment of mouse Hepa-1 cells with arsenite induces AHR nuclear translocation and binding to the Cyp1a1 gene promoter with the same efficiency as tetrachlorodibenzo-p-dioxin (TCDD), the most potent ligand of the AHR; however, TCDD and B[a]P are an order of magnitude more potent than arsenite in up-regulating Cyp1a1 transcription. Global profiling analyses of cells treated with arsenite plus B[a]P indicate that several phase I and II detoxification genes are in some cases additively and in others synergistically deregulated by the mixtures. Real-time reverse transcription-polymerase chain reaction analyses of mouse embryonic fibroblasts showed that the mixtures had an additive effect on the mRNA levels of Cyp1b1, a prototypical phase I detoxification gene, and an AHR-dependent synergistic effect on the corresponding levels of Nqo1, a prototypical phase II gene. We conclude that exposure to arsenite/B[a]P mixtures causes regulatory changes in the expression of detoxification genes that ultimately affect the metabolic activation and disposition of toxicants.

Neurotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells

An environmental pollutant, tetrachloro dibenzo dioxin (TCDD) is known to illicit the cognitive disability and motor dysfunction in the developing brain. TCDD induced effects leading to neurodevelopmental and neurobehavioral deficit may have been defined, however underlying molecular mechanism and possible intracellular targets remain to be elucidated. In this study, we attempted to analyze TCDD-induced neurotoxic effects in the granule cells from cerebellum where certain cognitive abilities and motor function command are known to be excuted. [3H]PDBu, (phorbol 12,13-dibutyrate) binding assay indicated that TCDD induced a dose-dependent increase of total PKC activity and its induction was the aryl hydrocarbon receptor (AhR) dependent and N-methyl-D-aspartate receptor (NMDAR) independent. TCDD also caused the translocation of both PKC-alpha and -epsilon in a dose-dependent manner but associated with different receptors; PKC-alpha via AhR but not PKC-epsilon indicating an isozyme-specific pattern of the induction. Increase of the ROS formation was also observed in the cells treated with TCDD in a dose-dependent and an AhR-dependent manner. The treatment of the cells with the diamino dicyano-bis(2-aminophenylthio) butadiene (U0126, MEK-1/2 inhibitor), dizocilpine maleate (MK-801, non-competitive N-methyl-D-aspartate glutamate receptor antagonist) and vitamin E attenuated the TCDD-induced ROS production indicating that TCDD-induced ROS formation may be associated with activation of ERK-1/2 in the MAP kinase pathway or the NMDA receptor. TCDD also increased [Ca2+]i, which is associated with ROS formation and PKC activation in the cerebellar granule cells. It is suggested that TCDD activates the NMDA receptor, which may induce a sustained increase of [Ca2+]i in neurons followed by the ROS formation. Our findings may contribute to understanding the mechanism of TCDD-related neurotoxicity, thereby improving the health risk assessment of neurotoxic compounds in humans.

2,3,7,8 Tetrachlorodibenzo-p-Dioxin (TCDD) Directly Enhances the Maturation and Apoptosis of Dendritic Cells In Vitro

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) suppresses adaptive immune responses and modulates the function of numerous cells involved in these responses. Our laboratory has shown that dendritic cells (DCs), which are important for the initiation of T-cell-dependent immunity, from TCDD-exposed mice exhibited reduced cell numbers and had altered expression of costimulatory molecules that are critical for the activation of T-cells. To further characterize the effects of TCDD on DCs and to elucidate a potential mechanism of toxicity, we investigated the direct effects of TCDD on DC maturation and survival in vitro. DCs, derived from bone marrow cells, were exposed to TCDD and then treated with TNFα to induced maturation. Apoptosis of bone marrow derived DCs (bmDCs) was induced by activating CD95 on the surface of the cells and was measured by annexin V staining. The TCDD-mediated changes in the expression of genes associated with apoptosis were examined using a pathway-specific c-DNA microarray. The results demonstrate that TCDD-treatment of bmDCs enhanced TNFα-induced maturation, measured as an increase in the expression of major histocompatibility complex class II, CD86, CD40, and CD54. In addition, TCDD exposure significantly augmented CD95-mediated death of bmDCs and altered the transcription of several genes involved in apoptosis. These findings confirm and extend the in vivo effects of TCDD on DC activation, and suggest that TCDD induces these changes, at least in part, via direct effects on the DC.

Sex-specific alterations of cerebral cortical cell size in rats exposed prenatally to dioxin

Sex-specific patterns of cerebral cortical lateralization have been documented consistently in both the human and animal brain. Male rats tend to exhibit pronounced right hemisphere dominance compared with females, while females typically exhibit more diffuse lateralization patterns and greater left hemisphere bias compared with males. Prenatal TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin) exposure produces demasculinization of male offspring sexual behavior, suggesting interference with sexual differentiation of the brain. In previous studies, a reversal of cortical thickness patterns in rats was shown after prenatal TCDD exposure on gestational day 8 (GD 8). The current study, based on the same brain sections, attempted to define changes in the number of cortical cells and cell size distributions in brains of offspring from TCDD-treated dams. Pregnant females were given a single oral dose of 0 or 180 ng kg(-1) TCDD on GD 8. Cell counts and sizes were determined in 3-month-old offspring. Areas 17 and 18a at bregma -3.8 were analysed using digitized, enhanced images of brain sections produced by a photomicroscope fitted with a high-resolution digital camera. Prenatal TCDD exposure altered the relative proportions of smaller and larger cell sizes in male, but not in female offspring. Both exposed males and females, however, exhibited a significant reversal of hemispheric dominance based on cell number. These findings demonstrate that prenatal exposure to TCDD alters the normal patterns of cortical cell asymmetry in a manner consistent with our previous data on thickness patterns.

DR-CALUX ® screening of food samples: evaluation of the quantitative approach to measure dioxin, furans and dioxin-like PCBs

European legislation laid down maximum tolerable levels of dioxin in feed and food as well as analytical method requirements. In order to face with large monitoring programs, it was foreseen in the EU strategy to integrate screening methods, using either a qualitative (screening) approach, or a quantitative approach. In this study, dioxin results obtained using the Dioxin Responsive Chemical-Activated LUciferase gene eXpression (DR-CALUX ®) cell-based assay (quantitative approach), were compared with gas chromatography–high resolution mass spectrometry (GC–HRMS) analyses data. Instead of using World Health Organization–toxic equivalent factor (WHO–TEF), the comparison was based on the assessment of relative effective potencies (REPs) for each congener of the 17 toxic 2,3,7,8-polychlorodibenzo- p-dioxins/furans (PCDD/Fs) and 12 dioxin-like polychlorobiphenyls (DL-PCBs). According to published data, DR-CALUX ®-REP evaluated here appear similar to WHO–TEF for PCDD/Fs while lower values were observed for DL-PCBs. We analyzed two “home made” contaminated fat samples, displaying both the same WHO–toxic equivalent quantities (WHO–TEQ) concentration (12 pg WHO–TEQ g −1). They were spiked with either a low or a high amount of DL-PCBs. In both cases, the DR-CALUX ® measured concentration (picogram 2,3,7,8 tetrachlorodibenzo- p-dioxin (TCDD) eq. g −1) corresponded to the PCDD/Fs WHO–TEQ concentration only. A good agreement was nevertheless found between the DR-CALUX ® measurements and the recalculated DR-CALUX ®-TEQ contents (using DR-CALUX ®-REP instead of WHO–TEF), demonstrating that the observed response was due, in both cases, to the addition of the responses of the standards added to the fat. By contrast, in real contaminated samples (feed or cod liver samples), DR-CALUX ® measured concentrations were similar to WHO–TEQ GC–HRMS measured concentrations. But, depending on the PCDD/Fs and DL-PCBs congener content, the DR-CALUX ® measured concentrations were either lower or higher than calculated DR-CALUX ®-TEQ contents, demonstrating that possible co-extracted contaminants contributed to the CALUX response. Owing to these divergences, the quantitative determination of dioxin-like content in food and feed using CALUX as screening method is questionable, except for samples displaying constant congener patterns, in which cases, correction factors could be applied.

PTV-LV-GC/MS/MS as screening and complementary method to HRMS for the monitoring of dioxin levels in food and feed

Recent developments in trapping efficiency inside ion trap mass spectrometer permitted to lower instrument detection limit (IDL). An IDL of 200 fg μl −1 injected with a signal-to-noise ratio of 5:1 for tetrachlorodibenzo- p-dioxin (TCDD) was obtained by gas chromatography coupled to a quadrupole ion storage mass spectrometer in tandem mode (GC/MS/MS). Coupling large volume programmable temperature vaporizer (PTV-LV) injection to GC/MS/MS provides an alternative and complementary method to classical splitless-GC injection connected to high-resolution mass spectrometry (splitless-GC/HRMS) method for dioxin monitoring in food and feed. An injection volume of 10 μl was found to be the best compromise between the sensitivity requirements and the robustness required for a high throughput method. PTV-LV-GC/MS/MS and Splitless-GC/HRMS were compared by performing analysis on five different matrices such as beef fat, yolk eggs, milk powder, animal feed and serum samples covering a concentration range of two orders of magnitude (i.e. 0.2–25 ng WHO-TEQ kg −1). An analysis of variance (ANOVA) was carried out. Fisher tests pointed out that the method effect for all the 2,3,7,8 congeners was not significant, indicating that the null hypothesis (H 0: μ 1= μ 2=…= μ n ) was not rejected. Moreover, the interaction effects between methods and matrices were not significant for most of the 2,3,7,8 congeners. However, three congeners (2,3,7,8-TCDF; 1,2,3,4,7,8-HxCDD and 1,2,3,4,6,7,8-HpCDD) were characterized by P-values lower than the significance level ( α=0.05). In toxic equivalence (TEQ), the study showed that no significant bias was observed between the two methods. Consequently, PTV-LV-GC/MS/MS is an attractive technique and can be used as a cost effective complementary method to HRMS for dioxin levels monitoring in food and feed.

Comparison of Compositions of Polychlorinated Dibenzo-p-dioxins (PCDDs) and Dibenzofurans (PCDFs) in Air and Soil Samples Collected in Ishikawa

Dioxins are present as impurities in agrochemicals applied to the soil. To examine the possibility that dioxins end up in the atmosphere, we compare the homologue composition and some characteristic isomer distributions of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in air and soil samples collected at sites in Ishikawa Prefecture, Japan. Tetrachloro dibenzo-p-dioxin (TeCDD) was the most abundant PCDD homologue in the air samples, while octachloro dibenzo-p-dioxin (OCDD) was the most abundant PCDD homologue in the soil samples. However, at several site, the most predominant homologue in the soil sample was not OCDD but TeCDD. The mean ratio of TeCDDs to PCDDs in the air samples was significantly smaller in winter than in other seasons, but no such seasonal variation was observed in the distribution of PCDF homologues. The abundance of PCDF homologues was inversely related to the number of chlorine substitutions in the air samples. The contribution of each toxic isomer (2,3,7,8-chlorine-substituted) to the total toxicity equivalency quantity (TEQ) in the air samples tended to be intermediate between their contributions to the TEQs in the soil and cinder samples. This result suggested that the air samples were influenced by both soil/dust suspension and combustion. In order to clarify the contributions of agrochemicals to dioxins in the air, we focused on the ratios of several characteristic isomers that are indicators of agrochemicals (chloronitrophen: CNP and pentachlorophenol: PCP) to their homologues. Significant decreases in the ratios of the isomers to their homologues in the air samples were observed only in winter, probably as a result of suppression of dust suspension by the snow cover. Therefore, the contribution of combustion to the dioxin concentration in the air was thought to be relatively large in winter.

Embryonic exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin in chickens: effects of dose and embryonic stage on hatchability and growth

Chicken embryos ( Gallus domesticus) were injected with 0, 8, 20 or 50 ng tetrachlorodibenzo- p-dioxin (TCDD) per egg at embryonic day (ED) 4, 8 or 12 to investigate the effects of differential periods of sensitivity to TCDD exposure. At hatch, all chicks were weighed, sexed and examined macroscopically to identify possible malformations. Liver, bursa, heart and spleen masses were recorded from a number of chicks. The remaining chicks were raised until 6 weeks of age and body and organ masses, plasma concentrations of thyroid hormones, triglycerides and glucose were measured. Dose and stage during embryonic development at which injection was performed affected hatchability. Fifty nanogram of TCDD was highly toxic for 4-day-old chicken embryos. TCDD was less toxic for chicken embryos of 8- and especially 12-days old. One-day-old chick and organ weights were not different between TCDD doses at all injection days. However, injection performed at ED4 or ED8 with 20 and 50 ng, respectively, significantly depressed post-hatch body mass gain. Moreover, body mass gain in males was more depressed than in females. The delayed growth in TCDD treated chickens was accompanied by changes in T 3/T 4 ratio that at some ages were significantly higher compared to control animals. No pronounced changes in plasma triglycerides or glucose concentrations during postnatal life were observed. Absolute and relative organ masses of 6-week-old chickens showed no remarkable changes.

Contaminant exposure and effects in pinnipeds: implications for Steller sea lion declines in Alaska

After nearly 3 decades of decline, the western stock of Steller sea lions (SSL; Eumetopias jubatus) was listed as an endangered species in 1997. While the cause of the decline in the 1970s and 1980s has been attributed to nutritional stress, recent declines are unexplained and may result from other factors including the presence of environmental contaminants. SSL tissues show accumulation of butyltins, mercury, PCBs, DDTs, chlordanes and hexachlorobenzene. SSL habitats and prey are contaminated with additional chemicals including mirex, endrin, dieldrin, hexachlorocyclohexanes, tetrachlorodibenzo- p-dioxin (TCDD) and related compounds, cadmium and lead. In addition, many SSL haulouts and rookeries are located near other hazards including radioactivity, solvents, ordnance and chemical weapon dumps. PCB and DDT concentrations measured in a few SSL during the 1980s were the highest recorded for any Alaskan pinniped. Some contaminant exposures in SSL appear to be elevated in the Gulf of Alaska and Bering Sea compared to southeast Alaska, but there are insufficient data to evaluate geospatial relationships with any certainty. Based on very limited blubber data, current levels of PCBs may not pose a risk to SSL based on comparison to immunotoxicity tissue benchmarks, but SSL may have been at risk from pre-1990 PCB exposures. While exposure to PCBs and DDTs may be declining, SSL are likely exposed to a multitude of other contaminants that have not been monitored. The impacts of these exposures on SSL remain unknown because causal effects have not been established. Field studies with SSL have been limited in scope and have not yet linked contaminant exposures to adverse animal health or population effects. Several biomarkers may prove useful for monitoring exposure and additional research is needed to evaluate their utility in SSL. We conclude that there are insufficient data to reject the hypothesis that contaminants play a role in the continued decline of SSL, and suggest that a coordinated monitoring program be developed which can be related to key biological, ecological and laboratory toxicity data.