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

In Utero Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Affects the Development of Reproductive System in Mouse

PurposeExposure of male reproductive organs to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) has been reported to cause developmental changes. In this study, we evaluated the effects of in utero TCDD exposure on male reproductive development.Materials and MethodsPregnant C57BL/6 mice were administered a single intraperitoneal injection of TCDD (1 µg/kg) on gestation day (GD) 15. The offspring were examined in the immature stage on postnatal day (PND) 30 and in the mature stage on PND 60. The testes were examined for histological changes, androgen receptor (AR), proliferating cell nuclear antigen (PCNA) and apoptosis following the measurement of morphological changes.ResultsAnogenital distance (AGD) and testis weights were reduced by TCDD exposure both on PND 30 and PND 60 while body weights and length of male offspring were not affected by TCDD. The regular sperm developmental stage was impaired with TCDD treatment on PND 30. However, no difference was found between the control group and TCDD groups on PND 60. Simultaneously, the expression of AR was also reduced on PND 30, while it was increased on PND 60 compared with the control group. The expression of PCNA was decreased whereas apoptosis was not affected by TCDD both on PND 30 and PND 60.ConclusionThese results suggest that in utero exposure to TCDD influences the development of testes by inhibiting the expression of AR and PCNA. Moreover, the adverse effects of TCDD on male offspring reduced over time.

Regulation of Aryl Hydrocarbon Receptor Expression and Function by Glucocorticoids in Mouse Hepatoma Cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates most biological responses to 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related aromatic hydrocarbons. Although the role of the AHR in control of drug metabolism and endocrine disruption is partly understood, we know little about the regulation of the AHR itself by endocrine factors. Our work with hypophysectomized rats suggested that hepatic AHR protein level is positively regulated by pituitary-dependent factors. A current hypothesis is that adrenal glucocorticoids elevate AHR expression and enhance responsiveness to AHR agonists. Dexamethasone (DEX) at concentrations that activate the glucocorticoid receptor (GR) increased AHR mRNA, protein, and TCDD-binding by approximately 50% in Hepa-1 mouse hepatoma cells. This response was blocked by the GR antagonist 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl]estra-4,9-dien-3-one (RU486), suggesting GR involvement. This small magnitude increase in AHR levels was functionally significant; pretreatment of Hepa-1 cells with DEX caused a 75% increase in the maximum induction of an AHR-activated luciferase reporter plasmid by TCDD. A luciferase reporter under control of the proximal 2.5 kilobases of the mouse Ahr 5'-flanking region and promoter was induced approximately 2.5-fold by DEX when cotransfected with a mouse GR expression plasmid. This is the first demonstration that glucocorticoids increase AHR levels in hepatoma cells via a GR-dependent transcriptional mechanism, suggesting a novel aspect of cross-talk between the AHR and the GR.

Relative potency based on hepatic enzyme induction predicts immunosuppressive effects of a mixture of PCDDS/PCDFS and PCBS

The toxic equivalency factor (TEF) approach was employed to compare immunotoxic potency of mixtures containing polychlorinated dibenzo- p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls relative to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), using the antibody response to sheep erythrocytes (SRBC). Mixture-1 (MIX-1) contained TCDD, 1,2,3,7,8-pentachlorodibenzo- p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), and 1,2,3,4,6,7,8,9-octachlorodibenzofuran (OCDF). Mixture-2 (MIX-2) contained MIX-1 and the following PCBs, 3,3′,4,4′-tetrachlorobiphenyl (IUPAC No. 77), 3,3′,4,4′,5-pentachlorobiphenyl (126), 3,3′,4,4′,5,5 N-hexachlorobiphenyl (169), 2,3,3′,4,4′-pentachlorobiphenyl (105), 2,3′,4,4′,5-pentachlorobiphenyl (118), and 2,3,3′,4,4′,5-hexachlorobiphenyl (156). The mixture compositions were based on relative chemical concentrations in food and human tissues. TCDD equivalents (TEQ) of the mixture were estimated using relative potency factors from hepatic enzyme induction in mice [DeVito, M.J., Diliberto, J.J., Ross, D.G., Menache, M.G., Birnbaum, L.S., 1997. Dose–response relationships for polyhalogenated dioxins and dibenzofurans following subchronic treatment in mice. I .CYP1A1 and CYP1A2 enzyme activity in liver, lung and skin. Toxicol. Appl. Pharmacol. 130, 197–208; DeVito, M.J., Menache, G., Diliberto, J.J., Ross, D.G., Birnbaum L.S., 2000. Dose–response relationships for induction of CYP1A1 and CYP1A2 enzyme activity in liver, lung, and skin in female mice following subchronic exposure to polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 167, 157–172] Female mice received 0, 1.5, 15, 150 or 450 ng TCDD/kg/day or approximately 0, 1.5, 15, 150 or 450 ng TEQ/kg/day of MIX-1 or MIX-2 by gavage 5 days per week for 13 weeks. Mice were immunized 3 days after the last exposure and 4 days later, body, spleen, thymus, and liver weights were measured, and antibody response to SRBCs was observed. Exposure to TCDD, MIX-1, and MIX-2 suppressed the antibody response in a dose-dependent manner. Two-way ANOVA indicated no differences in the response between TCDD and the mixtures for body weight, spleen/body weight and decreased antibody responses. The results support the use of the TEF methodology and suggest that immune suppression by dioxin-like chemicals may be of concern at or near background human exposures.

Dioxin Exposure, from Infancy through Puberty, Produces Endocrine Disruption and Affects Human Semen Quality

BackgroundEnvironmental toxicants are allegedly involved in decreasing semen quality in recent decades; however, definitive proof is not yet available. In 1976 an accident exposed residents in Seveso, Italy, to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).ObjectiveThe purpose of this study was to investigate reproductive hormones and sperm quality in exposed males.MethodsWe studied 135 males exposed to TCDD at three age groups, infancy/prepuberty (1–9 years), puberty (10–17 years), and adulthood (18–26 years), and 184 healthy male comparisons using 1976 serum TCDD levels and semen quality and reproductive hormones from samples collected 22 years later.ResultsRelative to comparisons, 71 men (mean age at exposure, 6.2 years; median serum TCDD, 210 ppt) at 22–31 years of age showed reductions in sperm concentration (53.6 vs. 72.5 million/mL; p = 0.025); percent progressive motility (33.2% vs. 40.8%; p < 0.001); total motile sperm count (44.2 vs. 77.5 × 106; p = 0.018); estradiol (76.2 vs. 95.9 pmol/L; p = 0.001); and an increase in follicle-stimulating hormone (FSH; 3.58 vs. 2.98 IU/L; p = 0.055). Forty-four men (mean age at exposure, 13.2 years; median serum TCDD, 164 ppt) at 32–39 years of age showed increased total sperm count (272 vs. 191.9 × 106; p = 0.042), total motile sperm count (105 vs. 64.9 ×106; p = 0.036), FSH (4.1 vs. 3.2 UI/L; p = 0.038), and reduced estradiol (74.4 vs. 92.9 pmol/L; p < 0.001). No effects were observed in 20 men, 40–47 years of age, who were exposed to TCDD (median, 123 ppt) as adults (mean age at exposure, 21.5 years).ConclusionsExposure to TCDD in infancy reduces sperm concentration and motility, and an opposite effect is seen with exposure during puberty. Exposure in either period leads to permanent reduction of estradiol and increased FSH. These effects are permanent and occur at TCDD concentrations < 68 ppt, which is within one order of magnitude of those in the industrialized world in the 1970s and 1980s and may be responsible at least in part for the reported decrease in sperm quality, especially in younger men.

Differential expression of estrogen receptor alpha (ERα) protein in MCF‐7 breast cancer cells chronically exposed to TCDD

Estrogens play a key role in the development and evolution of breast cancer tumors. Estrogen receptor alpha (ERalpha) mediates many of the biological activities of estrogens, and its expression is associated with low invasiveness and good prognosis. Recent epidemiological reports suggest that long-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is implicated in the increased incidence of breast cancer in exposed women. TCDD interferes with the expression of some ERalpha-dependent genes and inhibits estradiol (E2)- dependent growth of breast cancer cells in vitro. However, E2-dependent xenographs of MCF-7 human breast cancer cells resumed growth after a 2-week exposure to TCDD. The mechanisms involved in the resumption of cell growth are not completely understood. In this study, we show that short term-exposure (16 days) to 1 nM TCDD results in the suppression of ERalpha protein expression, while chronic exposure for more than 1 year (LTDX cells) results in the partial re-expression of the receptor. Immunocytochemistry studies showed that re-expression of ERalpha in LTDX cells occurred in some of the cells. Analysis by Western immunoblots indicated that four out of five LTDX clones expressed ERalpha at levels comparable to those in unexposed MCF-7 cells. Removal of TCDD treatment for 16 days restored the expression of ERalpha in the ERalpha-negative clonal cells. These results suggest that MCF-7 cells chronically exposed to TCDD contain at least two cell subpopulations that may respond differently to the ERalpha-mediated effects of TCDD.

TCDD deregulates contact inhibition in rat liver oval cells via Ah receptor, JunD and cyclin A

The aryl hydrocarbon receptor (AhR) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Activation of the AhR by TCDD leads to its dimerization with aryl hydrocarbon nuclear translocator (ARNT) and transcriptional activation of several phase I and II metabolizing enzymes. However, this classical signalling pathway so far failed to explain the pleiotropic hazardous effects of TCDD, such as developmental toxicity and tumour promotion. Thus, there is an urgent need to define genetic programmes orchestrated by AhR to unravel its role in physiology and toxicology. Here we show that TCDD treatment of rat liver oval cells leads to induction of the transcription factor JunD, resulting in transcriptional upregulation of the proto-oncogene cyclin A which finally triggers a release from contact inhibition. Ectopic expression of cyclin A in confluent cultures overcomes G(1) arrest, indicating that increased cyclin A levels are indeed sufficient to bypass contact inhibition. Functional interference with AhR-, but not with ARNT, abolished TCDD-induced increase in JunD and cyclin A and prevented loss of contact inhibition. In summary, we have discovered a novel AhR-dependent and probably ARNT-independent signalling pathway involving JunD and cyclin A, which mediates TCDD-induced deregulation of cell cycle control.

AH receptor agonist activity in human blood measured with a cell-based bioassay: Evidence for naturally occurring AH receptor ligands in vivo

In the present study, an aryl hydrocarbon receptor (AHR)-driven reporter gene bioassay was used to measure the activity, measured as an induction equivalent (IEQ) as compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or IEQ concentration in human blood samples from 10 volunteers under different dietary regimens. Blood concentrations of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and polychlorinated biphenyls (PCBs), as determined by analytical chemistry (HR-GC/MS), and expressed as toxic equivalents (TEQs) with the use of TCDD equivalency factors (TEFs), were within a range that has been reported in the general US population, ranging from 0.022 to 0.119 ppt (whole blood basis). However, the human blood IEQ measured directly via bioassay ranged from 13.4 to 218 ppt (whole blood basis). These order of magnitude greater IEQs compared to the TEQs for dioxins, furans, and certain PCBs suggests that human blood contains a relatively high level of AHR agonists able to activate the CYP1A1 dioxin response element (DRE)-linked reporter gene bioassay and that this AHR activity is not accounted for by PCDDs/Fs and dioxin-like PCBs based on standard HR-GC/MS and TEF analysis. When study participants switched from a "baseline" to a high-vegetable diet, increases in bioassay IEQ were observed that were statistically significant (P<0.05). In addition, IEQ activity was elevated above levels observed following dietary intervention in two subjects given indole-3-carbinol (I3C) supplements. We conclude that a substantial portion of the IEQ activity occurred as a result of the increased intake of natural AHR agonists (NAHRAs) present in many fruits, vegetables. and herbs. Our findings also suggest that dietary NAHRAs constitute a substantial daily dietary intake of AHR-active compounds, and these NAHRAs could influence AHR status in humans and play a role in a basal level of AHR activation.

Dioxin Exposure and Time to Pregnancy in Seveso

ISEE-687 Objective: Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant, has been associated with reduced fertility in multiple animal species at doses below toxic levels. To date, no epidemiologic studies have examined the association of TCDD exposure with infertility in women. On July 10, 1976, as a result of a chemical explosion, residents of Seveso, Italy, experienced the highest levels of TCDD in a human population. Twenty years later, we initiated the Seveso Women's Health Study (SWHS), a retrospective cohort study of the reproductive health of the women. We examined the association between TCDD exposure and fertility as measured by time to pregnancy (TTP). Materials and Methods: The SWHS cohort comprises 981 women who were 0 to 40 years in 1976, resided in the most contaminated areas, and had adequate archived sera collected soon after the explosion. Individual serum TCDD exposure was measured by high-resolution mass spectrometry. We examined the relation of serum TCDD with TTP or the number of months to become pregnant, using a discrete-time analogue of the Cox proportional hazards model. TTP (months) was treated as a continuous variable and censored at 13 months if women had reported attempting to conceive for a period of 12 months or longer. Results: Of 475 women who reported a post-explosion pregnancy, 340 had a planned or noncontracepted pregnancy resulting in a live birth. The median TTP was 3.0 months. Preliminary results suggest that women with higher serum TCDD levels took longer to become pregnant. Adjusting for parity, and parental smoking and age, a 10-fold increase in TCDD was associated with a 32% reduced odds of achieving pregnancy (adj-fOR = 0.76, 95% CI: 0.61, 0.94). Conclusions: These results suggest TCDD exposure may be associated with reduced fertility in the Seveso cohort. The results will be interpreted in light of study advantages and limitations.

Dioxin Alters Gene Expression in Mouse Embryonic Tooth Explants

Dioxins are ubiquitous environmental poisons that cause disturbances in developing organs, including the teeth. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at the cap stage leads to reduced tooth size and deformation of cuspal morphology. Our hypothesis was that TCDD affects the expression of genes specific for tooth development, which leads to these aberrations. Mouse embryonic E14 tooth germs were cultured for 24 hrs with/without 1 microM TCDD. Analysis of total RNA on Affymetrix arrays showed that TCDD altered the expression of 31 known genes by a fold factor of at least 2. Genes implied in tooth development expressed only slight changes. Genes active at the cap stage were selected for quantitative PCR analysis. Of these, the most highly up-regulated were Follistatin and Runx2, while TGFbeta1 and p21 were the most down-regulated genes. Incomplete tooth morphogenesis caused by TCDD may thus result from modified expression of developmentally regulated genes.

Effects of maternal exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin on fetal brain growth and motor and behavioral development in offspring rats

The effects of maternal exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) during pregnancy on fetal brain growth and neurobehavioral development in early developmental stages were investigated using rat offspring. TCDD in corn-oil (0.1 μg/kg) was orally administrated to the dams from the 9th to 19th gestational day. When TCDD effects on the fetal brain weight were analyzed on the 19th gestational day, weight ratio of the brain to the whole body, and that of the forebrain without the cerebral cortex to the whole brain were larger in the exposed group than those of the control group, suggesting premature fetal brain development. TCDD effects on motor functions were investigated using newborns in an inclined plane task. Motor development assessed by righting response on an inclination was delayed in the exposed offspring in the 8th–12th postnatal day, especially in male. Also, TCDD effects on active avoidance behavior in a shuttle box were investigated using the offspring after weaning. Latency in the active avoidance learning was longer, and locomotor activity was reduced in the exposed male offspring in the 41st–44th postnatal day. The results demonstrated that maternal TCDD exposure delayed fetal brain growth and neurodevelopment of the offspring in early stage, especially in male rats.

Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Developing Male Wistar(Han) Rat. II: Chronic Dosing Causes Developmental Delay

We have investigated whether fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat using chronically exposed rats to ensure continuous exposure of the fetus. Five- to six-week-old rats were exposed to control diet, or diet containing TCDD, to attain an average dose of 2.4, 8, and 46 ng TCDD/kg/day for 12 weeks, whereupon the rats were mated and allowed to litter; rats were switched to control diet after parturition. Male offsprings were allowed to develop until kills on PND70 (25 per group) or PND120 (all remaining animals). Offspring from the high-dose group showed an increase in total litter loss, and the number of animals alive on postnatal day (PND)4 in the high-dose group was approximately 26% less than control. The high and medium dose offsprings showed decreased weights at various ages. Balano-preputial separation (BPS) was significantly delayed in all three dose groups compared to control. There were no significant effects of maternal treatment when the offsprings were subjected to a functional observational battery or learning tests, with the exception that the high-dose group showed a deficit in motor activity. Twenty rats per group were mated to females, and there were no significant effects of maternal treatment on the fertility of these rats or on the F1 or F2 sex ratio. Sperm parameters at PND70 and 120 showed no significant effect of maternal treatment, with the exception that there was an increase in the proportion of abnormal sperm in the high-dose group at PND70; this is associated with the developmental delay in puberty in this dose group. There were no remarkable findings of maternal treatment on organ weights, with the exception that testis weights were reduced by approximately 10% at PND70 (but not PND120), and although the experiment was sufficiently powered to detect small changes, ventral prostate weight was not reduced. There were no significant effects of maternal treatment upon histopathological comparison of high-dose and control group organs. These data confirm that developmental exposure to TCDD shows no potent effect on adult sperm parameters or accessory sexual organs, but show that delay in BPS occurs after exposure to low doses of TCDD, and this is dependent upon whether TCDD is administered acutely or chronically.

Toxicity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Developing Male Wistar(Han) Rat. I: No Decrease in Epididymal Sperm Count after a Single Acute Dose

It has been reported that fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat. We set out to replicate and extend these effects using a robust experimental design. Groups of 75 (control vehicle) or 55 (50, 200, or 1000 ng of TCDD/kg bodyweight) female Wistar(Han) rats were exposed to TCDD on gestational day (GD)15, then allowed to litter. The high-dose group dams showed no sustained weight loss compared to control, but four animals had total litter loss. Pups in the high-dose group showed reduced body weight up till day 21, and pups in the medium dose group showed reduced body weight in the first week postpartum. Balano-preputial separation was significantly delayed in the high-dose group male offspring. There were no significant effects of treatment when the offspring were subjected to a functional observational battery or mated with females to assess reproductive capability. Twenty-five males per group were killed on postnatal day (PND) 70, and approximately 60 animals per group (approximately 30 for the high-dose group) on PND120 to assess seminology and other end points. At PND120, the two highest dose groups showed a statistically significant elevation of sperm counts, compared to control; however, this effect was small (approximately 30%), within the normal range of sperm counts for this strain of rat, was not reflected in testicular spermatid counts nor PND70 data, and is therefore postulated to have no biological significance. Although there was an increase in the proportion of abnormal sperm at PND70, seminology parameters were otherwise unremarkable. Testis weights in the high-dose group were slightly decreased at PND70 and 120, and at PND120, brain weights were decreased in the high-dose group, liver to body weight ratios were increased for all three dose groups, with an increase in inflammatory cell foci in the epididymis in the high-dose group. These data show that TCDD is a potent developmental toxin after exposure of the developing fetus but that acute developmental exposure to TCDD on GD15 caused no decrease in sperm counts.

Serum Dioxin Concentrations and Risk of Uterine Leiomyoma in the Seveso Women's Health Study

Uterine leiomyomata (fibroids), benign neoplasms of the smooth muscle, are a major cause of hysterectomy. Exposure to hormonally active chemicals may play an etiologic role. The authors investigated the risk of uterine leiomyoma associated with exposure to 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) for women who resided near Seveso, Italy, in 1976 at the time of a chemical explosion. Twenty years later, women enrolled in the Seveso Women's Health Study were asked about history of fibroids, medical records were obtained, and vaginal ultrasonography was performed for a subset. Serum collected soon after the explosion was analyzed for TCDD. A likelihood-based method that combines both historical and current status (ultrasound) data was adapted to estimate the hazard ratio. Of 956 eligible women, 251 (26.3%) had fibroids. Compared with that for women with TCDD levels of < or = 20 parts per trillion, the age-adjusted hazard ratios were 0.58 (95% confidence interval: 0.41, 0.81) for women with levels of 20.1-75.0 parts per trillion and 0.62 (95% confidence interval: 0.44, 0.89) for women with levels of >75.0 parts per trillion. This finding suggests that TCDD may have antiestrogenic effects in the uterine myometrium, in contrast to apparently estrogenic effects previously found in the breast of Seveso Women's Health Study women.

Glucocorticoids enhance aryl hydrocarbon receptor expression and function in mouse hepatoma cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates most biological responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Although the AHR’s role in cont...

Quantitative structure—activity relationships for the prediction of relative in vitro potencies (REPs) for chloronaphthalenes

Chloronaphthalenes (CNs), due to their structural similarities to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the other “dioxin-like” compounds, can bind to the aryl hydrocarbon receptor (AhR) and induce a wide range of pleotrophic effects. Relative potency of individual dioxin analogues can be measured relative to that of TCDD. Relative effects potencies (REP) can be based on many responses, including in vivo and in vitro responses. Both in vivo and in vitro tests, based on either indigenous responses such as the induction of ethoxyresorufin O-deethylase (EROD) or exogenous reporter genes under the control of the AhR such as luciferase can be used to determine REP values. Here we used measured REP values determined for CNs in two assays. Both assays are based on H4IIE rat hepatoma cells. The H4IIE assay is based on expression of the endogenous reporter gene (CYP-1 A) that codes for the expression of EROD and the H4IIE-luc assay which is based on the exogenous reporter gene (luciferase) transfected into the H4IIE cell line. Experimentally determined REP were available for only 17 and 18 of the 75 possible choronaphthalene congeners, for the H4IIE and H4IIE-luc assays, respectively. For this reason computational models were developed to allow prediction of the relative potencies of the other CN congeners. Predictive relationships were based on quantum chemical descriptors obtained from Density Functional Theory (DFT) calculations (B3LYP/6–311++G**). The final models were found by means of a hybrid method combining a genetic algorithm and artificial neural networks. REP values estimated for individual CNs based on the H4IIE assay ranged from 4.3 × 10− 9 to 3.2 × 10− 2 while those based on the H4IIE-luc assay ranged from 4.0 × 10− 8 to 1.8 × 10− 3. CN congeners nos. 66, 67, 70 and 73 were exhibited the greatest REP values in both assays. The 1,2,3,5,6,8-hexaCN congener (no. 68) had a REP value that was 10-fold less. The remaining congeners had REP values that were less or did not cause sufficient up-regulation of the monitored genes to allow for the calculation of a REP. Interactions of CNs with the AhR could be affected by three possible factors: molecular size, steric interactions and electrostatic interactions. These findings are discussed relative to the use of consensus TCDD equivalency factors' (TEFs) for use in risk assessments of CNs for regulatory purposes.

Suppression of Endogenous Antioxidant Enzymes by 2,3,7,8-Tetrachlorodibenzo-p-dioxin–Induced Oxidative Stress in Chicken Liver During Development

Domestic chickens (Gallus gallus) are an excellent model in which to evaluate developmental toxicity and oxidative stress because of their high sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The goal of this study was to measure the effects of environmentally relevant doses of TCDD on endogenous hepatic antioxidant enzyme activity in hatchling chickens. The vehicle (sunflower oil) or 2, 20, or 200 pg/g TCDD was injected into chicken eggs before incubation. On hatching, livers were harvested and quickly frozen. The changes in activity of antioxidant enzymes, including glutathione peroxidase (GPx), glutathione reductase (GRx), copper zinc superoxide dismutase (SOD), and catalase (CAT) were determined as indicators of oxidative stress. TCDD exposure was associated with a significant suppression of the activities of the protective endogenous enzymes GPx, GRx, and SOD in the liver, even at the lowest dose. CAT activity was also suppressed, but not significantly. The measured decreases were 37% to 63% for GPx, 50% to 58% for GRx, 30% to 40% for SOD, and 16% to 24% for CAT. Noncomplex dose-response relationships were evident in GPx and GRx, whereas SOD and CAT curves were U-shaped. These results demonstrate that a decreased ability to scavenge reactive oxygen species may result from developmental TCDD exposure at very low doses, contributing to oxidative stress and thus to the embryotoxicity of TCDD.

Neurological and neurophysiological examinations on workers with chronic poisoning by 2,3,7,8‐TCDD: follow‐up 35 years after exposure

Between 1965 and 1968, about 350 workers were accidentally exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a chemical plant, which was producing herbicides based on the trichlorophenoxyacetic acid. About 80 workers developed signs of poisoning. The estimated mean concentration of TCDD at the time of exposure was about 5000 pg/g of plasma fat. Only 15 subjects from the original cohort remained available for the recent follow-up in 2004. All were men, mean age 60 years. The mean current TCDD plasma concentration was 128 pg/g. Neurological examination revealed some CNS impairment in eight subjects. Signs of polyneuropathy were found in nine subjects, confirmed by NCV studies in three cases. Electroencephalography was abnormal in three cases; Visual-evoked potential in five cases. Acquired dyschromatopsia was detected in six patients. SPECT showed focal reduction of perfusion in various brain locations in all but one patient. Two neuropsychological variables and the frequency of abnormal neurophysiological tests in individual patients correlated with TCDD plasma level. The findings support the hypothesis that TCDD can damage the nervous system. In addition to a direct neurotoxic effect of TCDD, changes secondary to vasculopathy should be considered, in the pathophysiology of the damage, because of the high frequency of lipid metabolism disorders and their complications.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure of normal human dermal fibroblasts results in AhR-dependent and -independent changes in gene expression

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of lesions in mammals including severe skin lesions. The majority of TCDD's biological effects are mediated through activation of the aryl hydrocarbon receptor (AhR). We have chosen to examine the effect of TCDD and the AhR pathway on dermal fibroblasts because this cell type plays an integral role in skin homeostasis through the production of cytokines and other factors that regulate epidermal proliferation and differentiation. Our data show that normal human dermal fibroblasts (NHDFs) are responsive to TCDD, as demonstrated by induction of cytochrome p450 1B1 (CYP1B1) expression. Further, our data demonstrate that TCDD treatment of NHDFs results in significant (75-90%) decrease in expression of Id-1 and Id-3, proteins that are involved in regulation of cell proliferation and differentiation. The Id (Inhibitor of DNA binding) proteins are transcriptional inhibitors that function by forming inactive heterodimers with other HLH proteins. TCDD-repression of Id-1 and -3 is independent of de novo protein synthesis; co-treatment with cycloheximide has no effect on TCDD inhibition of Id-1 and Id-3. Co-treatment with the AhR antagonist alpha-naphthoflavone also does not block inhibition of Id-1 and Id-3 by TCDD, suggesting that TCDD inhibition of Id-1 and Id-3 is, at least in part, mediated independently of the AhR pathway. Our data also show that TCDD inhibits expression of the cell cycle regulatory gene p16(ink4a), which is often linked to Id expression. TCDD-induced reduction of p16(ink4a) expression is also independent of protein synthesis and the AhR pathway.

Exposure to 3,3′,4,4′,5‐pentachlorobiphenyl during embryonic development has a minimal effect on the cytochrome P4501A response to 2,3,7,8‐tetrachlorodibenzo‐P‐dioxin in cultured chicken embryo hepatocytes

Concentrations of dioxin-like compounds in avian eggs can vary substantially between individuals, species, and collection sites. Although the inducibility of cytochrome P4501A (CYPIA) in hepatocyte cultures is commonly used to predict species sensitivity to environmental contaminants, it is not known how exposure to dioxin-like compounds during embryonic development might alter this biomarker response. To investigate this question, we injected vehicle or 0.4, 0.8, or 1.6 microg/kg of 3,3',4,4',5-pentachlorobiphenyl (PCB 126) into the air cell of fertilized chicken eggs before incubation, and we measured CYP1A endpoints in cultured hepatocytes of day-19 embryos from each treatment group. The CYP1A response to PCB 126 also was assessed in whole liver tissue. Embryonic exposure to the most environmentally relevant treatment, 0.4 microg/kg of PCB 126, increased CYPIA4 mRNA expression 29-fold compared to control values in whole liver tissue but only twofold compared to control values in cultured hepatocytes. The CYPIA response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was not altered in hepatocytes cultured from embryos treated with 0.4 microg/kg of PCB 126, but at 0.8 and 1.6 microg/kg of PCB 126, several concentration-dependent effects were observed. For example, embryos exposed to higher concentrations of PCB 126 in ovo were more responsive to CYP1A mRNA induction by TCDD in vitro. These findings suggest that exposure to environmental levels of dioxin-like compounds during incubation is not likely to alter species-sensitivity estimates derived from in vitro CYP1A data.

Ovarian Endocrine Disruption Underlies Premature Reproductive Senescence Following Environmentally Relevant Chronic Exposure to the Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-Dioxin1

The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. While previous work has focused on mechanisms of short-term endocrine disruption and ovotoxicity in response to AHR ligands, we have shown recently that chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces premature reproductive senescence in female rats without depletion of ovarian follicular reserves. In the current study, premature reproductive senescence was induced using a range of low-dose exposure to TCDD (0, 1, 5, 50, and 200 ng kg(-1) wk(-1)) beginning in utero and continuing until the transition to reproductive senescence. Doses of 50 and 200 ng TCDD kg(-1) wk(-1) delayed the age at vaginal opening and accelerated the loss of normal reproductive cyclicity with age without depletion of follicular reserves. Serum estradiol concentrations were decreased in a dose-dependent fashion (> or = 5 ng kg(-1) wk(-1)) across the estrous cycle in perisenescent rats still displaying normal cyclic vaginal cytology. Serum FSH, LH, and progesterone profiles were unchanged by TCDD. The loss of reproductive cyclicity following chronic exposure to TCDD was not accompanied by decreased responsiveness to GnRH. Ovarian endocrine disruption is the predominant functional change preceding the premature reproductive senescence induced by chronic exposure to low doses of the AHR-specific ligand TCDD.