Effects Of 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.

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.

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.

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.

Sex ratio of the offspring of Sprague–Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero and lactationally in a three-generation study

Reports of a decreased male/female sex ratio in children born to males exposed to TCDD in Seveso, Italy, at a young age have sparked examinations of this endpoint in other populations exposed to TCDD or related compounds. Overall, the male/female sex ratio results reported in these studies, with slightly different age-exposed male populations, have shown mixed results. Experimental studies of the effects of in utero exposure to TCDD in laboratory animals have reported no effect on the f(1) sex ratio and mixed results for the sex ratio of the f(2) generation. In order to better understand the potential effects of TCDD on second generation sex ratio, we retrieved archived data from a comprehensive three-generation feeding study of TCDD in rats that was conducted and published in the 1970s, but which did not publish data on sex ratio of the offspring [Murray, F.J., Smith, F.A., Nitschke, K.D., Humiston, C.G., Kociba, R.J., Schwetz, B.A., 1979. Three-generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet. Toxicol. Appl. Pharmacol. 50, 241-252]. A re-examination of the original Murray et al. data found no statistically significant treatment-related changes in postnatal day 1 sex ratio in any generation of treated animals, consistent with one other relatively large study reporting on this endpoint. We discuss mechanistic data underlying a potential effect of TCDD on this endpoint. We conclude that the inconsistency in findings on sex ratio of the offspring of male rats exposed to TCDD in utero is likely due to random variation associated with a relatively small sample size, although differences between studies in strain of rat, dose regimen, and day of ascertainment of sex ratio cannot be ruled out.

Suppressive Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vascular Remodeling That Takes Place in the Normal Labyrinth Zone of Rat Placenta during Late Gestation

The maintenance of the placental vasculature is essential for sustaining normal fetal growth. On the basis of our previous observation that fetal death was accompanied by placental hypoxia upon exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (R. Ishimura et al., 2002a, Toxicol. Appl. Pharmacol. 185, 197-206), we here investigated the effects of TCDD on the placenta, focusing on the development of the labyrinth zone. Holtzman rats were administered a single oral dose of 1.6 mug of TCDD/kg body weight or an equivalent volume of vehicle (control) on gestation day (GD) 15, and placental tissues were analyzed on GD20. Immunohistochemical staining showed that the exposure to TCDD decreased the size of maternal blood sinusoids and caused the constriction of fetal capillaries in the placenta. In contrast, we found that vascular remodeling occurred in the labyrinth zone of normal rat placenta; that is, the vascular development in the normal labyrinth zone during the late gestation (from GD16 to GD20) showed dilated maternal blood sinusoids and fetal capillaries accompanied by a decrease in thickness and the apoptosis of trophoblasts. The present results demonstrate that this remodeling is suppressed by TCDD, which is further supported by the decreased expression level of Tie2 mRNA, the gene which is associated with vascular remodeling, and also by the decrease in the number of apoptotic trophoblasts in TCDD-exposed rats. The present study provided a new finding on the development of the vasculature in the labyrinth zone during the late gestation under normal conditions and showed the inhibition of vascular remodeling in TCDD-exposed rats.

Adverse Health Effects in Humans Exposed to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD)

The environmental contaminant 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) belongs to the category of highly toxic, persistent organic pollutants that accumulate in animal fat and plant tissues. Today, background TCDD levels in human fat are showing a decreasing trend. The food chain is the main source of exposure in the human population. TCDD regulates the expression of a wide range of drug-metabolizing enzymes and has an impact on a large number of biological systems. The most pronounced effects have occurred in occupational settings following the uncontrolled formation of TCDD after industrial accidents, as well as in rare intentional intoxications. Although the acute effects of TCDD exposure are well described in the literature, the long-term consequences have been underevaluated. The most well-known symptoms of severe acute intoxication are chloracne, porphyria, transient hepatotoxicity, and peripheral and central neurotoxicity. Because of the long-term persistence of TCDD in the human body, atherosclerosis, hypertension, diabetes, vascular ocular changes, and signs of neural system damage, including neuropsychological impairment, can be present several decades after massive exposure. Such chronic effects are nonspecific, multifactorial, and may be causally linked to TCDD only in heavily intoxicated subjects. This opinion is supported by the dose-dependent effect of TCDD found in exposed workers and by experimental animal studies.

The effects of maternal exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin on testicular steroidogenesis in infantile male rats

Exposure of adult male animals to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreases serum androgen concentrations. Reduction in androgen levels after maternal exposure has also been reported, but these results have not been reproduced. We have earlier shown that TCDD stimulates rather than inhibits testosterone synthesis in the prenatal rat testis. The aim of the present study was to elucidate in utero-induced effects of TCDD on testicular steroidogenesis in the 14-day-old infant rats. At that time the foetal Leydig cell population is still the prevailing source of androgens. Pregnant Sprague-Dawley dams were given a single oral dose of TCDD (0, 0.04, 0.2, or 1.0 microg/kg) on day 13 of pregnancy. On postnatal day 14, the body weight of male offspring was reduced after exposure to 1.0 microg/kg TCDD (from 33.9 +/- 1.66 g to 31.6 +/- 2.67 g). Relative testis weight, plasma testosterone, luteinizing hormone and follicle-stimulating hormone levels remained unaltered in all exposure groups. Moreover, in ex vivo incubations, testosterone and cAMP production was not affected. StAR protein level in the freshly isolated testes was increased in the 0.2 microg/kg group, and seminiferous cord diameter in the 0.04 microg/kg group. The present study confirms our earlier findings in in utero TCDD-exposed foetal testis indicating that maternal TCDD exposure does not negatively influence the developmental testosterone production of foetal type Leydig cells in rats.

Increased mortality associated with TCDD exposure in mice infected with influenza A virus is not due to severity of lung injury or alterations in Clara cell protein content

Most studies examining the cause of increased mortality in mice infected with a normally non-lethal dose of influenza A virus after exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) have focused on defects in the immune system. This study examined other possible consequences of TCDD exposure, which could alter pulmonary inflammation during infection. We measured bronchoalveolar lavage (BAL) fluid lactate dehydrogenase (LDH) and protein concentrations and lung wet to dry weight ratios to assess lung damage and edema formation. Immunohistochemistry for Cyp1A1 was used to evaluate the responsiveness of the lung to TCDD. Additionally, we characterized the effects of TCDD on Clara cell secretory protein (CCSP), which plays a regulatory role in pulmonary inflammation. There were no differences in BAL fluid LDH and protein levels, lung wet to dry weight ratios, or the amount of CCSP in the lungs from mice treated with TCDD or vehicle control. The amount of Cyp1A1 in endothelial cells, Clara cells, and Type II pneumocytes was greatly induced after TCDD exposure. Although lung tissue was clearly responsive to TCDD as shown by Cyp1A1 induction, the increased mortality in infected mice exposed to TCDD did not correlate with increased damage to the lung or decreased CCSP concentrations.

Heart Malformation Is an Early Response to TCDD in Embryonic Zebrafish

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which toxic compounds perturb normal development. When exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization, zebrafish embryos exhibit pericardial edema and reduced blood flow by 72 h post fertilization (hpf). To better understand the progression of dioxin toxicity in zebrafish, we have examined the effects of TCDD on heart development. At 72 hpf, TCDD-treated embryos exhibited altered looping, with the atria positioned distinctly posterior to the ventricles, contrary to the looping of control hearts, where the two chambers lied side by side. Moreover, the ventricles in dioxin-exposed hearts became more compact, and the atria elongated in comparison to controls. These defects are not secondary to pericardial edema because they were observed when edema formation was suppressed with osmotic support. In addition to morphological changes, TCDD produced functional deficits in the developing hearts, including blood regurgitation and a striking ventricular standstill that became prevalent by 120 hpf. We also assessed the effect of TCDD on the heart size using stereological measurements, which demonstrated significant reduction in heart tissue volume at 72 hpf. Perhaps our most significant finding was a decrease in the total number of cardiomyocytes in TCDD-exposed embryos by 48 hpf, one day prior to observable effects on peripheral blood flow. We conclude that the developing heart is an important target for TCDD in zebrafish.

TCDD and Puberty in Girls

We would like to comment on the article by Warner et al. (2004), in which the authors reported no significant associations between age at menarche and exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an extremely potent antiestrogenic xenobiotic. The exposure of girls to TCDD at Seveso, Italy, resulted in very high serum TCDD levels (> 100 pg/g lipid), 10–100 times levels usually seen today. Warner et al. noted that the literature is mixed regarding the agonist/ antagonist effects in humans of persistent exposures of this type. First, polybrominated biphenyl exposures have been associated with earlier menarche in girls, whereas experimental models show delayed puberty, a discordance that may be due to timing of exposure (Blanck et al. 2000). Second, as Warner et al. (2004) noted, the experimental data show that TCDD and other estrogen antagonists delay vaginal opening (VO) and disrupt cyclicity in rodents treated pre-natally (Gray et al. 1997; Levy et al. 1995). However, hormonal activity depends on both timing and level of dose, such that phytoestrogens, for example, may be estrogenic—hastening VO—at high doses given after birth (Lamartiniere et al. 1995; Whitten et al. 1995). Epidemiologic data regarding hormonally dependent female cancer are equivocal, such that there have been suggestions of a protective (i.e., antiestrogenic) effect of TCDD for breast and uterine cancer in TCDD-exposed women from Seveso (Bertazzi et al. 2001), whereas a carcinogenic effect has been observed in cohorts exposed for longer times (Manz et al. 1991; Warner et al. 2002). The findings of Warner et al. (2004), albeit not statistically significant, suggest earlier menarche with higher TCDD level among women who were younger than 8 years of age at the time of exposure [hazard ratio, 1.08 for 10-fold increase in TCDD levels; 95% confidence interval (CI), 0.89–1.30) but not among all women regardless of age. The study population appears to have the usual patterns of risk for menarche as indicated by associations that occur in the expected directions [e.g., for Seveso zone, body mass index (BMI), physical activity, alcohol intake]. Also, TCDD levels were higher among younger girls (median, 205 ppt) than in all girls (median, 140 ppt), an effect that may reflect lower BMI among younger girls and dilution of body burden by greater body size in older girls, but also a significantly higher target-organ dose. Warner et al. (2004) examined associations in premenarcheal girls who were a younger subset (0–8 years of age) during the exposure window in 1976. This age stratum should capture any strong underlying associations among girls exposed early in life. However, it is known that pubertal transition occurs around 5–7 years of age and that age at menarche is strongly correlated with age at first signs of development (de Ridder et al. 1992; Nicolson and Hanley 1953). Therefore, hormonal exposures before 5 years of age might alter the milestones of female development, including menarche, either more potently or in a different direction than peripubertal exposures. Therefore, the youngest girls in this population (Warner et al. 2004) may have been more susceptible to hormonal effects of environmental toxicants. Recognizing the limitation of small numbers available for further age stratification, it would be interesting to know whether risk of earlier (or later) puberty was raised among girls exposed at earlier ages, such as 0–4 years.

Altered thyroxin and retinoid metabolic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin in aryl hydrocarbon receptor-null mice

To determine whether the disruption of thyroid hormone and retinoid homeostasis that occurs after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mediated by the arylhydrocarbon receptor (AhR), pregnant AhR-heterozygous (AhR+/-) mice were administered a single oral dose of 10 microg kg(-1) TCDD at gestation day 12.5. Serum and liver were collected on postnatal day 21 from vehicle-treated control or TCDD-treated AhR+/- and AhR-null (AhR-/-) mouse pups. Whereas TCDD exposure resulted in a marked reduction of total thyroxin (TT4) and free T4 (FT4) levels in the serum of AhR+/- mice, TCDD had no effects on AhR-/- mice. Gene expression of UDP-glucuronosyltransferase (UGT)1A6, cytochrome P450 (CYP)1A1, and CYP1A2 in the liver was induced markedly by TCDD in AhR+/- but not AhR-/- mice. Induction of CYP1A1 in response to TCDD was confirmed by immunohistochemical evidence in that CYP1A1 protein was conspicuously localized in the cytoplasm of hepatocytes in the centrilobular region. Levels of retinyl palmitate were greatly reduced in the liver of TCDD-exposed AhR+/- mice, but not in vehicle-treated AhR+/- mice. No effects of TCDD on retinoid levels in the liver were found in AhR-/- mice. We conclude that disruption of thyroid hormone and retinoid homeostasis is mediated entirely via AhR. Induction of UGT1A6 is thought to be responsible at least partly for reduced serum thyroid hormone levels in TCDD-exposed mice.

An evaluation of benchmark dose methodology for non-cancer continuous-data health effects in animals due to exposures to dioxin (TCDD)

The U.S. Environmental Protection Agency (EPA) has conducted extensive reviews and analyses of health effects associated with exposures to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and related compounds. Because the carcinogenicity of TCDD has received considerable attention from EPA and others, this paper focuses on animal data for non-cancer health effects that sometimes appear to be almost as sensitive as cancer to TCDD exposures. Benchmark dose (BMD) methodology can be used to identify point-of-departure (POD) estimates for use in derivation of reference doses or evaluation of margins of exposure. However, selection of an appropriate BMD methodology for assessment of non-cancer data, which are usually continuous (non-quantal), needs to be considered. One option available for a benchmark dose is to use a small percentage change in the mean response relative to the estimated maximum effect of TCDD at large doses. The benchmark based on a change estimated to equal 1% of the estimated maximum change from background to the asymptotic response at large doses (denoted as the relative ED 01) was used by EPA in a reassessment of TCDD health risks. A lower confidence limit (LED 01) could serve as a point of departure for setting a reference dose (RfD). This is a somewhat arbitrary effect level, generally within the background range of variation among unexposed animals, with an unknown risk. An alternative approach is recommended in which the risk of abnormal levels can be estimated. For continuous-data effects, a low and/or high percentile (e.g., 1st and/or 99th) in unexposed control animals can be used to define abnormal (not necessarily adverse) levels. From a dose–response curve and the standard deviation, it is possible to estimate the excess risk (proportion) of animals with abnormal levels as a function of dose for normally distributed levels. With this approach, the risk-based benchmark dose (BMD 01) represents the dose with an estimated excess risk of 1% of the animals in the abnormal range rather than an arbitrary change in the value of the measured endpoint. Values for the relative and risk-based benchmark doses are computed from published data for a variety of non-cancer health effects associated with exposure to TCDD. For the 30 cases investigated, the BMD 01 tended to vary around the lowest experimental dose tested, whereas the relative ED 01 tended to be about a factor of three below the lowest dose, and the BMD 01 was more precisely estimated than the ED 01 as reflected by narrower confidence intervals. The BMDL 01 values were on average more than fivefold higher than the corresponding LED 01 values. However, these values still provide a conservative assessment for POD assessment, because the BMDL 01 tends to be about an order of magnitude lower (more conservative) than the no-observed-adverse-effect level. This analysis demonstrates the potential impact of alternative choices in benchmark dose methodology. In combination with selection of appropriate adverse health effect endpoint(s) and studies, use of the risk-based BMD results in identification of more valid and meaningful POD estimates for non-cancer effects compared to the use of the relative ED approach.

An evaluation of benchmark dose methodology for non-cancer continuous-data health effects in animals due to exposures to dioxin (TCDD)

The U.S. Environmental Protection Agency (EPA) has conducted extensive reviews and analyses of health effects associated with exposures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Because the carcinogenicity of TCDD has received considerable attention from EPA and others, this paper focuses on animal data for non-cancer health effects that sometimes appear to be almost as sensitive as cancer to TCDD exposures. Benchmark dose (BMD) methodology can be used to identify point-of-departure (POD) estimates for use in derivation of reference doses or evaluation of margins of exposure. However, selection of an appropriate BMD methodology for assessment of non-cancer data, which are usually continuous (non-quantal), needs to be considered. One option available for a benchmark dose is to use a small percentage change in the mean response relative to the estimated maximum effect of TCDD at large doses. The benchmark based on a change estimated to equal 1% of the estimated maximum change from background to the asymptotic response at large doses (denoted as the relative ED01) was used by EPA in a reassessment of TCDD health risks. A lower confidence limit (LED01) could serve as a point of departure for setting a reference dose (RfD). This is a somewhat arbitrary effect level, generally within the background range of variation among unexposed animals, with an unknown risk. An alternative approach is recommended in which the risk of abnormal levels can be estimated. For continuous-data effects, a low and/or high percentile (e.g., 1st and/or 99th) in unexposed control animals can be used to define abnormal (not necessarily adverse) levels. From a dose–response curve and the standard deviation, it is possible to estimate the excess risk (proportion) of animals with abnormal levels as a function of dose for normally distributed levels. With this approach, the risk-based benchmark dose (BMD01) represents the dose with an estimated excess risk of 1% of the animals in the abnormal range rather than an arbitrary change in the value of the measured endpoint. Values for the relative and risk-based benchmark doses are computed from published data for a variety of non-cancer health effects associated with exposure to TCDD. For the 30 cases investigated, the BMD01 tended to vary around the lowest experimental dose tested, whereas the relative ED01 tended to be about a factor of three below the lowest dose, and the BMD01 was more precisely estimated than the ED01 as reflected by narrower confidence intervals. The BMDL01 values were on average more than fivefold higher than the corresponding LED01 values. However, these values still provide a conservative assessment for POD assessment, because the BMDL01 tends to be about an order of magnitude lower (more conservative) than the no-observed-adverse-effect level. This analysis demonstrates the potential impact of alternative choices in benchmark dose methodology. In combination with selection of appropriate adverse health effect endpoint(s) and studies, use of the risk-based BMD results in identification of more valid and meaningful POD estimates for non-cancer effects compared to the use of the relative ED approach.

Aryl hydrocarbon receptors in urogenital sinus mesenchyme mediate the inhibition of prostatic epithelial bud formation by 2,3,7,8-tetrachlorodibenzo- p-dioxin

In utero exposure of male C57BL/6 mice to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) prevents prostatic epithelial buds from forming in the ventral region of the urogenital sinus (UGS) and reduces the number of buds that form in the dorsolateral region. This inhibition of budding is aryl hydrocarbon receptor (AHR) dependent and appears to be the primary cause of lobe-specific prostate abnormalities in TCDD-exposed mice. TCDD can inhibit prostatic epithelial bud formation by acting directly on the UGS in vitro, but whether it does so via AHR in UGS mesenchyme, epithelium, or both was unknown. To address this issue, UGS mesenchyme and epithelium from gestation day (GD) 15 wild-type C57BL/6J male mice were isolated, recombined, and cultured in vitro for 5 days with 10 −8 M 5α-dihydrotestosterone (DHT) and either 10 −9 M TCDD or vehicle. Prostatic epithelial buds were viewed by light microscopy after removal of mesenchyme. Effects depended greatly on which portions of the mesenchyme were used: TCDD had little if any effect when whole UGS epithelium (UGE) was recombined with ventral plus dorsolateral mesenchyme, tended to reduce bud numbers in recombinants made with UGE and dorsolateral mesenchyme, and severely reduced bud numbers in recombinants made with UGE and ventral mesenchyme (VM). [VM + UGE] recombinants prepared from wild-type and AHR knockout ( Ahr −/−) mice were then cultured with DHT to determine the site of action of TCDD. AHR null mutation alone had no effect on budding. TCDD severely inhibited prostatic epithelial bud formation in recombinants that contained mesenchymal AHR, whereas bud formation was not inhibited by TCDD in recombinants lacking mesenchymal AHR, regardless of epithelial AHR status. These results demonstrate that UGS mesenchyme and not UGS epithelium is the site of action of TCDD. Therefore, the initial events responsible for abnormal UGS (and ultimately prostate) development occur within the UGS mesenchyme, and changes in gene expression in the UGS epithelium responsible for inhibited prostatic budding are secondary to the direct effects of TCDD on UGS mesenchyme.

T cell receptor transgenic mice provide novel insights into understanding cellular targets of TCDD: suppression of antibody production, but not the response of CD8 + T cells, during infection with influenza virus

Although exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) clearly impairs T cell-dependent immune responses, the mechanisms underlying TCDD-induced T cell dysfunction are unclear. With the goal of determining precisely how exposure to TCDD impairs the activation of CD8 + T cells in vivo, we used a well-defined T cell receptor (TCR) transgenic system. Greater than 95% of the CD8 + T cells in F5 transgenic mice possess TCR specific for a peptide from influenza A virus expressed in the context of H-2D b. Unexpectedly, we discovered that exposure to TCDD did not alter CD8 + T cell function in the transgenic mice. Specifically, treatment of F5 mice with TCDD did not affect the recruitment of virus-specific CD8 + T cells to the lung, nor did it impair the ability of CD8 + T cells in the lymph node to produce cytokines, or to clonally expand or differentiate. This is in direct contrast to the suppressive effects of TCDD on the response of CD8 + T cells in wild-type mice. Exposure of F5 mice to TCDD induced CYP1A1 and suppressed the production of virus-specific antibodies. Likewise, upon adoptive transfer into wild-type mice, TCDD suppressed the expansion and differentiation of F5-derived CD8 + T cells. This indicates that the F5 mice and lymphocytes derived from them are not inherently resistant to the immunosuppressive effects of TCDD. Rather, our data suggest that in the context of a supraphysiological number of antigen-specific CD8 + T cells, the function of these cells was not affected by exposure to TCDD. Given that antibody production in the F5 mice was sensitive to suppression by TCDD, while the CD8 response was resistant, our data provide a new perspective on the ways in which exposure to TCDD adversely affects B and T lymphocyte function.

Influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the antigen-presenting activity of dendritic cells.

We have previously shown that exposure of mice to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induces activation-like changes in splenic dendritic cells (DC) in the absence of antigen challenge. Since activation of DC reduces their ability to phagocytize antigen, we examined the effects of TCDD on the ability of DC to process and present antigen to antigen-specific T cells and to internalize latex beads. Additionally, the expression of costimulatory and adhesion molecules was examined on DC from TCDD-treated mice injected with allogeneic tumor cells. The ability of DC from C57Bl/6 mice to induce proliferation of keyhole limpet hemocyanin (KLH)-specific 10.5.17 T cells and production of IL-4 was not significantly altered by TCDD exposure, either when KLH was added in vitro or when the mice were injected with KLH prior to DC isolation. In contrast, ovalbumin (OVA) presentation by DC from TCDD-treated Balb/c mice induced enhanced proliferation of OVA-specific D011.10 T cells, although the production of IL-2 and IFN-gamma was not affected. Enhanced in vivo proliferation of adoptively transferred, CFSE-labeled DO11.10 T cells was also observed in TCDD-treated Balb/c mice that were challenged with OVA. TCDD treatment modulated the expression of major histocompatibility complex (MHC) class II, CD24, ICAM-1, CD40, and LFA-1 on splenic DC from C57Bl/6 mice injected with allogeneic tumor cells; however, the effects of TCDD were identical to changes seen previously in nonimmune mice, suggesting that these effects were not antigen-dependent. Finally, TCDD treatment did not affect the ability of splenic DC to internalize latex beads administered in vivo. Taken together, these results suggest that the activation-like changes induced in DC by TCDD do not suppress the ability of DC to process and present antigen, but may enhance their ability to provide activation signals to T cells. This, in turn, may alter the survival of the T cells, the DC, or both, and might lead to dysregulation of the immune response.

2,3,7,8-tetrachlorodibenzo-p-dioxin interacts with endogenous estradiol to disrupt prostate gland morphogenesis in male rat fetuses.

Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interferes with normal development of the male reproductive system in rats and mice. We examined the effects of TCDD on the initial development of the urogenital system (urethra, prostate, and seminal vesicles) in male rat fetuses on gestation day (GD) 20. The number of prostatic buds and size of prostate glands as well as seminal vesicle size was determined by computer-assisted 3D reconstruction. Pregnant Holtzman rats received a single oral dose of TCDD (1 microg/kg) on GD 15. The intrauterine position (IUP) of male fetuses was identified based on the sex of adjacent fetuses: 2F males were located between 2 females and 2M males were located between 2 males. Control 2F males had elevated serum estradiol and larger prostates than control 2M males, which had elevated serum testosterone and larger seminal vesicles, confirming prior findings. There was no effect of TCDD on serum testosterone. TCDD significantly decreased the number of buds in the dorsocranial and dorsolateral regions of the urogenital sinus and overall prostate size, and was associated with a significant decrease in serum estradiol only in 2F males. In contrast, in 2M males both serum estradiol and the number and size of prostatic buds in these same regions of the prostate were unaffected by TCDD, although seminal vesicle size was reduced. These findings show that individual differences in gonadal steroid levels influence the response of the developing prostate to TCDD in male fetuses. In addition, these TCDD effects may be mediated in part by a decrease in serum estradiol levels.