The 2,3,7,8-tetrachlorodibenzo-p-dioxin Research Articles

Down-regulation of the detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 by vanadium in Hepa 1c1c7 cells

Recent data suggest that vanadium (V 5+) compounds exert protective effects against chemical-induced carcinogenesis, mainly through modifying various xenobiotic metabolizing enzymes. In fact, we have shown that V 5+ down-regulates the expression of Cyp1a1 at the transcriptional level through an ATP-dependent mechanism. However, incongruously, there is increasing evidence that V 5+ is found in higher amounts in cancer cells and tissues than in normal cells or tissues. Therefore, the current study aims to address the possible effect of this metal on the regulation of expression of an enzyme that helps maintain endogenous antioxidants used to protect tissues/cells from mutagens, carcinogens, and oxidative stress damage, NAD(P)H:quinone oxidoreductase 1 (Nqo1). In an attempt to examine these effects, Hepa 1c1c7 cells and its AhR-deficient version, c12, were treated with increasing concentrations of V 5+ in the presence of two distinct Nqo1 inducers, the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and isothiocyanate sulforaphane (SUL). Our results showed that V 5+ inhibits the TCDD- and SUL-mediated induction of Nqo1 at mRNA, protein, and catalytic activity levels. At transcriptional level, V 5+ was able to decrease the TCDD- and SUL-induced nuclear accumulation of Nrf2 and the subsequent binding to antioxidant responsive element (ARE) without affecting Nrf2 protein levels. Looking at post-transcriptional level; we found that V 5+ did not affect Nqo1 mRNA transcripts turn-over rates. However, at the post-translational level V 5+ increased Nqo1 protein half-life. In conclusion, the present study demonstrates that V 5+ down-regulates Nqo1 at the transcriptional level, possibly through inhibiting the ATP-dependent activation of Nrf2.

Control Strategy of PCDD/Fs in an Industrial Fluidized Bed Incinerator via Activated Carbon Injection

The emissions of a full range of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) from incinerators have become a significant issue in both aspects of environmental and public health protection. Fluidized bed incinerators (FBI) have been widely used as a typical technology for industrial waste treatment in recent years, especially in the petrochemical industry in Japan, Germany, and Taiwan. In response to public concerns, the Environmental Protection Administration (EPA) in Taiwan had proposed strict limits on the emissions of PCDD/Fs for both industrial and municipal incinerators. The emission standard is 0.1 ng/Nm3, expressed as the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalent of 17 specific dioxins and furans congeners. These limits will potentially influence technology selection and design in combustion processes, energy recovery options, and air pollution control devices. Since fluidized bed incinerators with revolving type combustors have been continuously popular in Taiwan for handling industrial sludge, spent oils, and solid wastes, an interesting exercise is to compare the stack concentrations against the proposed standard of concentrations of dioxins and furans (i.e., 0.1 ng/Nm3 TCDD TEQ (toxicity equivalence) and search for a more cost effective way to improve the control strategy. Research findings in this article confirm that the formation and emissions of PCDD/Fs, prior to particulates control device, exhibit a substantial positive correlation with CO concentration in the flue gases. Without using the technique of activated carbon injection to reduce the emissions of PCDD/Fs, the revolving type FBI cannot comply with the emission standards of PCDD/Fs required in Taiwan.

A role for the aryl hydrocarbon receptor and the dioxin TCDD in rheumatoid arthritis

Environmental factors are involved in RA pathogenesis and epidemiological studies have suggested that smoking is an environmental risk factor for RA. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the major toxic components in cigarettes. To clarify the biological effects of smoking in RA, we investigated the role of TCDD in RA pathogenesis. Human synovial tissue was obtained from RA and OA patients and aryl hydrocarbon receptor (AhR) expression in these tissues was evaluated using immunohistochemistry and real-time PCR. Expression of various cytokines was measured by real-time PCR following stimulation of RA synoviocytes with different concentrations of TCDD. To study the role of AhR, we treated RA synoviocytes with alpha-naphthoflavone, a known AhR antagonist. To evaluate which signal transduction pathways were stimulated by the TCDD-AhR interaction, we used inhibitors of nuclear factor-kappaB (NF-kappaB) and extra-cellular stimulus-activated kinase (ERK). Higher AhR mRNA and protein levels were observed in RA synovial tissue than in OA tissue. TCDD up-regulated the expression of IL-1beta, IL-6 and IL-8 through binding to AhR, and this effect was transmitted via the NF-kappaB and ERK signalling cascades. AhR expression in synovial cells was up-regulated by TNF-alpha. TNF-alpha activates AhR expression in RA synovial tissue, and that cigarette smoking and exposure to TCDD enhances RA inflammatory processes. TCDD induces inflammatory cytokines via its association with AhR, resulting in stimulation of the NF-kappaB and ERK signalling cascades. Thus TCDD exposure, such as smoking exacerbates RA pathophysiology.

Curcumin suppresses the transformation of an aryl hydrocarbon receptor through its phosphorylation

Halogenated and polycyclic aromatic hydrocarbons induce diverse biochemical responses through the transformation of a cytosolic aryl hydrocarbon receptor (AhR). In mouse hepatoma Hepa-1c1c7 cells, curcumin, a yellow pigment of Curcuma longa, did not inhibit the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced translocation of the AhR into the nucleus, but rather accelerated it. In the nucleus, curcumin inhibited the TCDD-induced heterodimerization of the AhR with an AhR nuclear translocator (Arnt), an essential partner for the transformation, and also dose-dependently inhibited the TCDD-evoked phosphorylation of both the AhR and Arnt. Moreover, curcumin significantly inhibited the TCDD-induced activation of protein kinase C (PKC), which is involved in the transformation, decreased the TCDD-induced DNA-binding activity of the AhR/Arnt heterodimer, and downregulated CYP1A1 expression. In a cell-free system, curcumin inhibited the binding of 3-methylcholanthrene, an AhR agonist, to the receptor. These results indicate that curcumin is able to bind to the AhR as a ligand, but suppresses its transformation by inhibiting the phosphorylation of AhR and Arnt, probably by PKC.

Differential effect of arecoline on the endogenous dioxin-responsive cytochrome P450 1A1 and on a stably transfected dioxin-responsive element-driven reporter in human hepatoma cells

Dioxin-responsive element-mediated chemical activated luciferase expression (DRE-CALUX) is one of alternative bioassays for the determination of dioxin levels. We have previously established a DRE-CALUX cell line, Huh7-DRE-Luc, by using stable transfection of Huh-7 cells with a reporter plasmid (4xDRE-TATA-Luc) carrying a DRE-driven firefly luciferase gene. It was also shown that arecoline, a major areca nut alkaloid, inhibited the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced cytochrome P450 1A1 (CYP1A1) activation in Huh-7 cells. The TCDD-activated aryl hydrocarbon receptor (AhR) induces the DRE-CALUX activation and CYP1A1 gene expression via binding to DRE in promoter regions of these dioxin-responsive genes. In the present study, the effect of arecoline on the TCDD-induced activation of DRE-CALUX and CYP1A1 enzyme in Huh7-DRE-Luc and Huh-7 cells, respectively, was examined. It was found that arecoline inhibited TCDD-induced CYP1A1 activation and however enhanced TCDD-induced DRE-CALUX activation. This finding indicates the differential effect of arecoline on the endogenous dioxin-responsive CYP1A1 and on a stably transfected DRE-driven reporter in human hepatoma cells. The present study suggests that induction of DRE-CALUX alone does not necessarily parallel with endogenous CYP1A1 gene expression, and that the reporter assay may detect interactions that are not functional in endogenous gene.

Arecoline inhibits the 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced cytochrome P450 1A1 activation in human hepatoma cells

In the present study, we investigated the effect of arecoline, a major areca nut alkaloid, on the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced activation of cytochrome P4501A1 (CYP1A1) in a human hepatoma cell line Huh-7. We treated Huh-7 cells with 10 nM TCDD in the presence of different concentrations of arecoline (50–300 μM). Our results indicated that arecoline attenuated the TCDD-induced CYP1A1 enzyme activation with an inhibitory effect on cell proliferation. By using real-time RT-PCR, we demonstrated that arecoline inhibited the TCDD-induced activations of CYP1A1 and AhR repressor (AhRR) mRNA expression in a similar pattern. Our results revealed that arecoline inhibited AhR mRNA expression with no direct effect on CYP1A1 enzyme activity. Therefore, in our present study, the observed inhibitory effect of arecoline on CYP1A1 activation was not due to the up-regulation of AhRR or direct inhibitory effect on CYP1A1. Taken together, here we have demonstrated that arecoline attenuates the TCDD-induced CYP1A1 activation mainly via down-regulation of AhR expression in human hepatoma cells, suggesting the possible involvement of arecoline in the AhR-mediated metabolism of environmental toxicants in liver.

Comparison of the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced CYP1A1 gene expression profile in lymphocytes from mice, rats, and humans: Most potent induction in humans

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) exerts its toxicity by binding a transcription factor, the aryl hydrocarbon receptor (AhR). C57BL/6 (C57) mice express AhRs that have high affinity for TCDD, and they strongly express target genes and develop severe toxic effects upon TCDD exposure. By contrast, DBA/2 (DBA) mice have a low-affinity form of AhR, weakly express target genes, and are resistant to TCDD. Although humans express low-affinity AhRs and have been assumed to be refractory to TCDD, their sensitivity to TCDD has yet to be determined. In this study we compared the TCDD-induced CYP1A1 gene expression profiles in lymphocytes from humans, C57 mice, DBA mice, and SD rats to obtain data as a basis for estimating human sensitivity to TCDD. Lymphocyte fractions prepared from the blood of individual humans and animals were cultured with TCDD. Their mRNAs for CYP1A1 and housekeeping genes were measured by RT-PCR or real-time PCR with primers designed for regions that are 100% homologous among each of the genes of all species/strains tested to obtain similar PCR efficiency. TCDD-induced CYP1A1 expression peaked at 2 h in DBA mice and SD rats and at 6 h in C57 mice and humans. At the peak times human lymphocytes showed the most potent CYP1A1 mRNA induction of the four species/strains tested. These results suggest that human lymphocytes are more sensitive to TCDD than the lymphocytes of mice and rats. Since the AhR-dependent gene expression did not reflect the AhR affinity for TCDD, these results also suggest that AhR-dependent gene expression in lymphocytes is modulated by an as yet unidentified mechanism in addition to the AhR affinity.

Quantitative relationship of dioxin-responsive gene expression to dioxin response element in Hep3B and HepG2 human hepatocarcinoma cell lines

Dioxin response element (DRE) is a cis-acting DNA sequence mediating the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced gene expression. The present study was undertaken to elucidate TCDD-responsive gene expression profiles and their relationships to the number of DREs in liver cancer cells. Hep3B and HepG2 human hepatocarcinoma cells were exposed to 50-nM TCDD for 0, 1, 2 and 4 h in culture, after which gene expression profiles were analyzed by the microarray hybridization using a chip containing 24,000 cDNAs prepared from the human liver. The TCDD-responsive expression levels in each gene were calculated by dividing the densitometric values of the hybridization signal for h1, h2 and h4 by that of h0, followed by transformation of the resulting data into a log scale with the base of 2. Up- and down-regulated gene expressions were defined as >0.585 and <−0.585 by the log scale (>1.5 and <1/1.5 arithmetically), respectively, exhibited at any time after h0. Hep3B and HepG2 cells had 27 and 58 TCDD-responsive, up-regulated genes, respectively, of which 78% (21/27) and 62% (36/58) had one or more DREs. Of these 85, 80 genes were up-regulated exclusively in one of the two lines, with CYP1A1 and PPP1R15A being so regulated in both lines. Expression levels of the up-regulated genes at h1, h2 and h4 were correlated with each other ( P < 0.01) and the mean of these regressed to the number of DRE(s) in both lines ( P < 0.01). However, expression of a total of 93 TCDD-responsive, down-regulated genes, of which 46% contained DRE(s), had no relation to the number of DRE(s). In conclusion, results suggest that DREs may cooperatively mediate the expression of TCDD-responsive genes in liver cancer cells.

Aryl Hydrocarbon Receptor-Mediated Transcription: Ligand-Dependent Recruitment of Estrogen Receptor α to 2,3,7,8-Tetrachlorodibenzo- p-Dioxin-Responsive Promoters

Using chromatin immunoprecipitation assays, we studied the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated recruitment of the aryl hydrocarbon receptor (AhR) and several co-regulators to the CYP1A1 promoter. AhR displayed a time-dependent recruitment, reaching a peak at 75 min and maintaining promoter occupancy for the remainder of the time course. Recruitment of AhR was followed by TIF2/SRC2, which preceded CBP, histone H3 acetylation, and RNA polymerase II (RNAPII). Simultaneous recruitment to the enhancer and the TATA box region suggests the formation of a large multiprotein complex bridging the two promoter regions. Interestingly, estrogen receptor alpha (ERalpha) displayed a TCDD- and time-dependent recruitment to the CYP1A1 promoter, which was increased by co-treatment with estradiol. Transfection in HuH7 human liver cells confirmed previously reported ERalpha enhancement of AhR activity. In contrast, TCDD did not induce the recruitment of ERalpha to the estrogen-responsive pS2 promoter, and after 120 min of co-treatment with estradiol, ERalpha is still present on the CYP1A1 promoter but no longer at pS2. RNA interference studies with T47D cells support a role for ERalpha in TCDD-dependent CYP1A1 expression. Our data suggest that ERalpha acts as a coregulator of AhR-mediated transcriptional activation and that the recruitment of ERalpha by AhR represents a novel mechanism AhR-ERalpha cross talk.

Expression of the arylhydrocarbon receptor in the peri-implantation period of the mouse uterus and the impact of dioxin on mouse implantation

The arylhydrocarbon receptor (AhR) is a nuclear transcription factor mediating toxic effects of chemicals such as dioxins. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a member of polyhalogenated aromatic hydrocarbons family, exerts a wide-variety of toxic effects in a tissue- and species-specific manner including the reproduction process. Recently, AhR-mediated direct effects of TCDD on a cell-specific interaction with ovarian steroids have been shown. However, information regarding the effects of TCDD on the mouse implantation is limited. We therefore examined the expression and localization of AhR in the pregnant mouse uterus from 4 to 10 days of gestation (day 4 to day 10) using immunohistochemistry to investigate the effect of TCDD on uterine tissue during the peri-implantation period. Intense AhR expression was detected in the uterine vasculature throughout the periods examined. We also found that implanted blastocysts and their surrounding luminal epithelia and decidualized stroma expressed AhR on day 5. On days 6 and 7, persistent AhR expression was found in the transitional zone between the invading embryonic tissue and decidual tissue. On days 9 to 10, placental vasculature and spongiotrophoblasts displayed AhR immunoreactivity. The administration of TCDD on day 4 decreased the number of surviving implanted embryos on day 7 in a dose-dependent manner. This effect of TCDD was inhibited by the simultaneous administration of an AhR antagonist, alpha-naphthoflavone (alpha-NF). The spatio-temporal expression of AhR during the peri-implantation phase of the mouse uterus may indicate functional roles of this orphan receptor in fetomaternal interactions as well as substantiate the risk of exposure to chemicals such as dioxins during the reproductive period.

Protein kinase C δ is activated in mouse ovarian surface epithelial cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Interactions between the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and protein kinase C (PKC) signaling pathways are governed in cell and tissue-specific manners, albeit the physiological significance of which is unclear. This research sought to define the effects of TCDD on the PKC pathway using a mouse ovarian surface epithelial cancer cell line (ID8). Phorbol-12-myristate-13-acetate (PMA) potentiated (1 nM) TCDD-induced 7-ethoxyresorufin-O-deethylase (EROD) activity after 24h of treatment, and pre-treatment with (1 microM) of either a general PKC inhibitor (BisI) or PKCdelta-specific inhibitor (Rotterlin) abolished the potentiation indicating that activation of PKC enhances TCDD signal transduction. Western blot analysis revealed that unstimulated ID8 cells express PKCalpha, beta, epsilon, tau, lambda and RACK1. PKCgamma, eta, theta and DGKtheta were not detected. TCDD (1 nM) increased PKCdelta protein approximately eight-fold after 24h of treatment and this effect was dose-dependent (0.1-100 nM); other PKC isoforms and related signaling proteins tested were unaffected by TCDD treatment. Immunofluorescent microscopy revealed that TCDD (1 nM) promoted the subcellular redistribution of PKCdelta, from the cytoplasm and the nucleus to the perinuclear area after 2h of treatment, however, after 24h of treatment PKCdelta was observed in nuclear structures that resembled nucleoli. TCDD (1 nM) also increased total PKC and PKCdelta-specific kinase activities in biphasic time-responsive manners. Total PKC and PKCdelta-specific activities increased after 1-2h of treatment. Then TCDD increased the total PKC activity again after 12h of treatment, whereas, PKCdelta-specific activity resurged at 24h and remained elevated at 48 h after treatment. The results indicate that TCDD preferentially induces PKCdelta protein expression and phosphotransferase activity, and its membrane translocation, indicating a potential intracellular role for PKCdelta as an effector molecule for TCDD-mediated biological events in this ovarian cancer cell line.

Constitutive and TCDD-Induced Expression of Ah Receptor-Responsive Genes in the Pituitary

The 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and related substances cause a wide variety of pathological alterations, with the most severe being progressive anorexia and body weight loss. These features suggest a possible involvement of the nervous system and endocrine organs, including the pituitary gland. TCDD-related toxicity is considered mainly to be mediated by the aryl hydrocarbon receptor (AHR) protein, which binds TCDD, and heterodimerizes with its partner protein, the aryl hydrocarbon receptor nuclear translocator (ARNT), and binds to xenobiotica responsive elements (XREs) in the promoter regions of biotransformation genes as well as genes involved in growth, differentiation and cellular homeostasis. In the present study, we have investigated the expression of AHR responsive genes in the pituitary of untreated and TCDD treated 129/SV/C57BL/6 mice in vivo and in pituitary cells in vitro. After TCDD or beta-naphthoflavone (βNF) treatment, the relative levels of cytochrome P4501A1 (CYP1A1) mRNA and protein were dramatically increased in pituitary cells. The AHR repressor (AHRR) mRNA level was induced 7–13-fold by TCDD and βNF. Furthermore, the expression of the adrenocorticotrophic hormone (ACTH) precursor, the proopiomelanocortin ( POMC) gene, was investigated. A three-fold increase in POMC mRNA was observed in the pituitary of TCDD treated mice. POMC mRNA level was also increased in the pituitary cell line AtT-20 after TCDD treatment. The proteins encoded by POMC translational products, ACTH and β-endorphin, were found with immunocytochemistry staining to be increased in AtT-20 cells after TCDD exposure. The presence of several XRE sequences in the promoter region and in the first intron of the human POMC gene suggest that the up-regulation of POMC expression in the pituitary may play a role in the endocrine alterations induced by TCDD. All together, the results point to the pituitary gland being a direct target for TCDD.

Suppression of CYP1A1 expression by 4-nonylphenol in murine Hepa-1c1c7 cells

This study investigated the effects that 4-nonylphenol (NP) has on CYP1A1 expression in Hepa-1c1c7 cell cultures. NP alone did not affect CYP1A1-specific 7-ethoxyresorufin- O-deethylase (EROD) activity. In contrast, the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-inducible EROD activities were markedly reduced upon concomitant treatment with TCDD and NP in a dose-dependent manner. Treatment with tamoxifen, an anti-estrogen that acts through the estrogen receptor, did not affect the suppressive effects that NP has on TCDD-inducible EROD activity. The TCDD-inducible CYP1A1 mRNA levels were markedly suppressed upon concomitant treatment with TCDD and NP that is consistent with their effects on EROD activity. A transient transfection assay using dioxin-response element (DRE)-linked luciferase and an electrophoretic mobility shift assay revealed that NP reduced the transformation of the aryl hydrocarbon (Ah) receptor to a form capable of binding specifically to the DRE sequence of the CYP1A1 gene promoter. These results suggest that the down-regulation of CYP1A1 gene expression by NP in Hepa-1c1c7 cells might be an antagonism of the DRE-binding potential of the nuclear Ah receptor, but is not mediated through the estradiol receptor.

Inductive and inhibitory effects of non- ortho-substituted polychlorinated biphenyls on estrogen metabolism and human cytochromes P450 1A1 and 1B1

The effects of a series of non- ortho-substituted polychlorinated biphenyls (PCBs) on human cytochrome P450 1A1 (CYP1A1), a 17β-estradiol (E 2) 2-hydroxylase, and P450 1B1 (CYP1B1), an E 2 4-hydroxylase, were investigated in HepG2 and MCF-7 cells. Elevated rates of 2- and 4-methoxyestradiol (2- and 4-MeOE 2) formation in PCB-treated cultures were measured as activities of CYP1A1 and CYP1B1, respectively. Of the congeners investigated, 3,4,4′,5-tetrachlorobiphenyl (PCB 81), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), and 3,4′,5-trichlorobiphenyl (PCB 39) caused marked stimulation of E 2 metabolism in both cell lines. Northern blot analyses confirmed that exposure of MCF-7 cells to PCBs 81, 126, and 39 caused highly elevated levels of the CYP1A1 and CYP1B1 mRNAs. Exposure of MCF-7 cells to 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169) resulted in elevated levels of the CYP1A1 and CYP1B1 mRNAs, but did not cause elevated rates of E 2 metabolism; rather, 4-MeOE 2 production was depressed to below control levels in PCB 169-treated cultures. PCB 169 also inhibited the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced 4-MeOE 2 and, to a lesser extent, 2-MeOE 2 production in MCF-7 cells, as did PCB 126 and several other congeners. In microsomal assays, inhibition of cDNA-expressed human CYP1B1 by PCBs 169 and 126 was demonstrated. These studies with one subgroup of PCBs, the non- ortho-substituted congeners, underscore the complexity and diversity of effects of PCBs, as individual congeners were found both to induce expression and to inhibit activity of human CYP1B1 and CYP1A1.

Mammalian and teleost cell line bioassay and chemically derived 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin equivalent concentrations in lake trout (Salvelinus namaycush) from Lake Superior and Lake Ontario, North America

AbstractFor livers of lake trout from three Great Lakes sites, the 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) equivalent concentrations (TEQs) were determined in four ways. Bioassay‐derived (BD) TEQs were measured with rat (H4IIE) and rainbow trout (RTL‐W1) cell lines, and chemistry‐derived (CD) TEQs were calculated with TCDD equivalency factors (TEFs) derived with H4IIE and RTL‐W1. Generally, BD‐TEQs and CD‐TEQs for individual samples did not differ significantly for either H4IIE or RTL‐W1, indicating that all of the AhR‐active compounds in a sample were accounted for by the congener analysis and that these compounds acted in an additive fashion in both mammalian and piscine systems. However, contributions of individual chemicals to overall CD‐TEQs differed with H4IIE and RTL‐W1 TEFs, and for some individual samples, TEQs did depend on the method used. Hepatic TEQs and ethoxyresorufin‐O‐deethylase (EROD) levels differed significantly between sites. For TEQs, the order was Glenora &gt; Jackfish Bay &gt; Black Bay; for EROD activity, the order was Jackfish Bay &gt; Glenora = Black Bay. No correlation was found between hepatic TEQs and EROD activity, which suggests that the two measurements are evaluating different but related consequences of contaminant exposure.

Enzyme Induction and Acute Endocrine Effects in Prepubertal Female Rats Receiving Environmental PCB/PCDF/PCDD Mixtures

Air, subsurface soil, and superficial dust from a National Priorities List landfill located in southern Illinois were sampled to determine their potential toxicities. The major components of these landfill extracts were polychlorinated biphenyls (PCBs), with significant amounts of polychlorinated dibenzofurans (PCDFs) and small amounts of polychlorinated dibenzodioxins (PCDDs). The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalency factor approach has been proposed to estimate the toxic potency of complex mixtures of chlorinated aromatics for environmental risk assessment. However, most components of environmental residues are nonplanar and do not act as aryl hydrocarbon (Ah) receptor agonists, so there is a great risk of not identifying adverse responses that are not dioxinlike. We used a 2-day prepubertal female rat bioassay to examine multiple biological responses, including both dioxinlike and nondioxinlike effects from these landfill extracts. As expected, both types of effects were detected. The soil and dust extracts produced similar dose-response relationships for 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufin O-depentylase, 7-benzyloxyresorufin O-debenzylase, and 4-nitrophenol UDP-glucuronyltransferase induction; the dose response for the air extract deviated from the other two extracts. Soil, dust, and air extracts effectively reduced serum total thyroxine (T4) with similar dose-response relationships, despite the significantly different TCDD toxic equivalent (TEQ) values of these three extracts. Both soil (346 mg PCB/kg) and air (175 mg PCB/kg) extracts caused a greater than 30% increase in uterine wet weight. This study suggests that a more comprehensive approach is required to improve current risk assessment of environmental mixtures. TCDD TEQs reflect only a portion of effects and may especially underpredict effects on T4.

Enzyme induction and acute endocrine effects in prepubertal female rats receiving environmental PCB/PCDF/PCDD mixtures.

Air, subsurface soil, and superficial dust from a National Priorities List landfill located in southern Illinois were sampled to determine their potential toxicities. The major components of these landfill extracts were polychlorinated biphenyls (PCBs), with significant amounts of polychlorinated dibenzofurans (PCDFs) and small amounts of polychlorinated dibenzodioxins (PCDDs). The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalency factor approach has been proposed to estimate the toxic potency of complex mixtures of chlorinated aromatics for environmental risk assessment. However, most components of environmental residues are nonplanar and do not act as aryl hydrocarbon (Ah) receptor agonists, so there is a great risk of not identifying adverse responses that are not dioxinlike. We used a 2-day prepubertal female rat bioassay to examine multiple biological responses, including both dioxinlike and nondioxinlike effects from these landfill extracts. As expected, both types of effects were detected. The soil and dust extracts produced similar dose-response relationships for 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufin O-depentylase, 7-benzyloxyresorufin O-debenzylase, and 4-nitrophenol UDP-glucuronyltransferase induction; the dose response for the air extract deviated from the other two extracts. Soil, dust, and air extracts effectively reduced serum total thyroxine (T4) with similar dose-response relationships, despite the significantly different TCDD toxic equivalent (TEQ) values of these three extracts. Both soil (346 mg PCB/kg) and air (175 mg PCB/kg) extracts caused a greater than 30% increase in uterine wet weight. This study suggests that a more comprehensive approach is required to improve current risk assessment of environmental mixtures. TCDD TEQs reflect only a portion of effects and may especially underpredict effects on T4.

Assessment of experimental data on PCB‐induced reproduction inhibition in mink, based on an isomer‐ and congener‐specific approach using 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin toxic equivalency

AbstractThis paper describes an attempt to derive a median effect level (EC50) of PCBs for reproduction of mink based on experimental literature data. Unfortunately, the conditions of the mink studies carried out during the last two decades vary widely, which makes it difficult to establish unequivocal dose‐effect relationships. This study describes an attempt to correct for the differences in exposure time using a one‐compartment bioaccumulation model. This model estimates the whole‐body concentration of PCBs in mink. Two approaches are tested. First, the whole‐body concentration of 10 isomer groups of PCBs in mink were estimated and compared with reproduction data to calculate an EC50 value. Alternatively, estimates for the whole‐body concentration in mink of 11 individual biologically active PCB congeners were made. With these, the toxic equivalent concentration (TEC) in mink was estimated using the 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) equivalent factor (TEF) approach. Whole‐body dose‐effect relationships were estimated and EC50 values for litter size and kit survival were calculated. The application of the one‐compartment bioaccumulation model of the first approach resulted in a significant improvement in the dose‐effect relationship in comparison to the raw data set. A further improvement in this relationship was achieved using the congener‐specific bioaccumulation model in combination with the TEF approach. This study proposes a critical body residue (EC50) for mink litter size of 1.2 μg/g (total PCBs/wet weight). From the second approach a critical body residue (EC50) expressed in TCDD equivalency for litter size of 160 pg/g (TCDD equivalence/wet weight) and 200 ng/g (TCDD equivalence/wet weight) for kit survival is proposed.

Correlation between bioassay-derived P4501A1 induction activity and chemical analysis of clam ( Laternula elliptica) extracts from McMurdo Sound, Antarctica

Variable levels of halogenated aromatic hydrocarbons were measured in clams ( Laternula elliptica) collected from McMurdo Sound, Antarctica. Clams collected in and near Winter Quarters Bay contained high levels of organochlorine compounds, particularly polychlorinated biphenyls (PCBs). A strong gradient has been documented in Winter Quarters Bay that been linked to human activities at McMurdo Station. The activity of clam extracts as inducers of P4501A1-dependent ethoxyresorufin O-deethylase (EROD) activity was determined using in vitro bioassays utilizing rat hepatoma H4IIE cells. The extracts which exhibited the highest induction activities were those derived from clams collected in contaminated areas. Additionally, there was an excellent linear correlation between induced EROD activity versus total PCB levels (r 2=0.96). The complimentary nature of both the analytical and bioanalytical data confirms the utility of the latter assay and provides a method for estimating the 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) toxic equivalents in extracts from marine biota.

Superinduction of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible expression of aldehyde dehydrogenase by the inhibition of protein synthesis

Inhibition of protein synthesis by cycloheximide or puromycin super-induced the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible isoform of aldehyde dehydrogenase (ALDH-3) in rat hepatoma cells. Treatment with cycloheximide did not affect the basal expression but markedly enhanced the TCDD-inducible expression of ALDH-3 mRNA. The co-treatment of cycloheximide and TCDD for 24 h caused a 10-fold greater accumulation of ALDH-3 mRNA than did TCDD alone. The transcription rate of the ALDH-3 gene in the co-treated cells was also 4- to 5-fold higher than that in the cells treated with TCDD alone. The superinduction of ALDH-3 mRNA was observed only when cycloheximide was added prior to or simultaneously with the addition of TCDD. These results suggest that the mechanism of TCDD-inducible transcription of the ALDH-3 gene involves a labile protein that modulates or represses the action of TCDD.