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

Severe 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) intoxication: insights into the measurement of hepatic cytochrome P450 1A2 induction.

The correct in vivo quantification of aryl hydrocarbon receptor-mediated induction of cytochrome P450 1A2 (CYP1A2) in humans is a long-standing question. We compared the performance of several modifications of the caffeine test for measurement of CYP1A2 activity in subjects with exceptionally high, low, or absent enzyme induction. CYP1A2 activity was measured in 2 women highly exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in 1 man moderately exposed, and in 50 control subjects (30 nonsmokers and 20 heavy smokers). After the application of a test dose, caffeine demethylation was detected with the carbon 13 breath test, the total clearance, and several serum and urinary metabolite ratios. In the highly TCDD-exposed persons, results of the breath test (cumulative 15-minute dose), the total caffeine clearance, the serum metabolic ratio paraxanthine/caffeine (30 and 120 minutes after application), and the urinary metabolic ratio sum of 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methyluric acid (1U), and 1-methylxanthine (1X) over 1,7-dimethyluric acid (17U) showed a CYP1A2 activity 8 to 10 times higher than the mean of nonsmokers. In contrast, two caffeine urinary metabolic ratios with the parent substance in the denominator did not reflect the CYP1A2 enzyme induction. These ratios strongly depended on urine flow. For the breath test, only results evaluated for a short sampling period (eg, 15 minutes after application) revealed the high induction. Compared with nonsmokers, higher mean values (maximally 1.8 times) were observed in smokers with all tests. After high TCDD exposure, hepatic CYP1A2 activity is inducible at least 10 times in humans. Moderate TCDD exposure (up to 1000 ppt in blood fat) does not cause a CYP1A2 induction that can be measured to differentiate from background exposure individually. Therefore direct quantification of such toxins is more specific and sensitive.

Promotion of Altered Hepatic Foci by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 17beta-estradiol in Male Sprague-Dawley Rats

The determination of differences in hormonal regulation of tumor promotion-related response to 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) between males and females may identify factors contributing to the female-specific hepatocarcinogenicity of TCDD in rats. In the current study, diethylnitrosamine-initiated male Sprague-Dawley rats were exposed to TCDD or corn oil vehicle in the presence and absence of 17beta-estradiol (E2), and cell proliferation and development of preneoplastic altered hepatic foci (AHF) were determined. After 30 weeks of exposure, gamma-glutamyltranspeptidase (GGT)-positive AHF and the number of placental glutathione-s-transferase (PGST)-positive AHF were significantly higher in TCDD-treated rats than in control rats. Both the number and volume fraction of GGT-positive AHF were significantly lower in rats cotreated with E2 regardless of TCDD exposure compared with corresponding non-E2-treated groups and were unaffected by TCDD. In contrast, the number of PGST-positive AHF was significantly higher in E2-treated rats in the absence of TCDD treatment. In addition, whereas E2 had no effect on the volume fraction of PGST-positive foci, the levels in rats cotreated with both E2 and TCDD were significantly higher than in controls. No differences were observed in cell proliferation between TCDD-treated and control rats, although cell proliferation was lower in rats exposed to E2 compared with placebo controls. The weaker potency of tumor promotion and lack of induction of cell replication and DNA damage in male rats likely explain the female-specific hepatocarcinogenicity of TCDD in chronic bioassays.

Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure during gestation has revealed reproductive anomalies in rat offspring, including inconclusive reports of stunted mammary development in females (Brown et al., 1998, Carcinogenesis 19, 1623-1629; Lewis et al., 2001, TOXICOL: Sci. 62, 46-53). The current studies were designed to examine mammary-gland development in female offspring exposed in utero and lactationally to TCDD, and to determine a critical exposure period and cellular source of these effects. Long-Evans rats were exposed to 1 microg TCDD/kg body weight (bw) or vehicle on gestation day (GD) 15. TCDD-exposed females sacrificed on postnatal days (PND) 4, 25, 33, 37, 45, and 68 weighed significantly less than control litter mates, and peripubertal animals exhibited delayed vaginal opening and persistent vaginal threads, yet did not display altered estrous cyclicity. Mammary glands taken from TCDD-exposed animals on PND 4 demonstrated reduced primary branches, decreased epithelial elongation, and significantly fewer alveolar buds and lateral branches. This phenomenon persisted through PND 68 when, unlike fully developed glands of controls, TCDD-exposed rats retained undifferentiated terminal structures. Glands of offspring exposed to TCDD or oil on gestation days 15 and 20 or lactation days 1, 3, 5, and 10 were examined on PND 4 or 25 to discern that GD 15 was a critical period for consistent inhibition of epithelial development. Experiments using mammary epithelial transplantation between control and TCDD-exposed females suggested that the stroma plays a major role in the retarded development of the mammary gland following TCDD exposure. Our data suggest that exposure to TCDD prior to migration of the mammary bud into the fat pad permanently alters mammary epithelial development in female rat offspring.

The potential role of exposure to environmental toxicants in the pathophysiology of endometriosis.

Humans and animals are exposed daily to a complex mixture of polyhalogenated aromatic hydrocarbons (PHAHs). Previous work has shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with a dose-dependent increase in the incidence and severity of endometriosis in the rhesus monkey. Dioxin-like chemicals can also exert effects in combination with TCDD via the aryl hydrocarbon receptor. Using a rhesus model of chronic TCDD exposure and endometriosis, serum concentrations of TCDD and 19 dioxin-like PHAHs were quantified 13 years after termination of exposure to TCDD. In additional studies, the immune status of TCDD-exposed monkeys was evaluated. For TCDD-exposed and unexposed animals, TCDD exposure correlated with an increased serum TCDD concentration. Furthermore, TCDD exposure and an elevated serum TCDD concentration were associated with increased serum levels of triglycerides, 1,2,3,6,7,8-hexachlorodibenzofuran (HxCDF), PCB77, and PCB126. Importantly, the animals with elevated serum levels of PCB77 and PCB126 and increased total serum TCDD equivalents (TEQs) had a high prevalence of endometriosis, and the severity of disease correlated with the serum concentration of PCB77. In immune studies, TCDD exposure correlated with increased tumor necrosis factor alpha (TNFalpha) production by peripheral blood mononuclear cells (PBMC) in response to stimulation by T cell mitogen and decreased NK cytolytic activity. Elevated serum concentrations of TCDD, 1,2,3,6,7,8-HxCDF, and PCB126 correlated with increased numbers of CD3+/CD25- and CD3-/CD25+ leukocytes and enhanced secretion of TNFalpha by mitogen-stimulated PBMC. This evidence suggests that TCDD exposure and endometriosis in the rhesus monkey may be associated with increased serum concentrations of specific coplanar PCB compounds and long-term alterations in systemic immunity. Furthermore, the data suggest a potential involvement of an increased body burden of PCB compounds in the etiology of endometriosis in the rhesus. Recent advances in the detection and assay of individual PHAH congeners in biological samples have made it possible to assess total PHAH body burden in humans and animals. Future studies are expected to exploit this advance to assess the health impact of PHAH body burdens in both exposed individuals and the general population. Serum PHAH concentrations and TEQs in TCDD-exposed monkeys with endometriosis are similar to or lower than blood levels in the general human population; thus, it is important to consider the implications of these findings for human health and the prevalence of endometriosis in humans. Additional studies are warranted, particularly in human subjects, to explore the potential implications of these data.

Effect of a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin on immune function in male NC/Nga mice.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immunosuppression in humans and animals. However, the effect of TCDD on Th2-type immune responses such as allergic reactions has been unclear. Using NC/Nga mice that developed atopic dermatitis-like skin lesions with marked elevation in plasma of total IgE when bred under conventional conditions, we investigated the effects of a single oral dose of TCDD on immune responses. NC/Nga mice received a single oral dose (0 or 20 microg/kg body weight) of TCDD. On day 7, treatment with TCDD alone decreased the cellularity of thymus. However, treatment with TCDD modified the cellularity of spleens and mesenteric lymph nodes (MLNs) but not of the thymus on day 28. When NC/Nga mice received ip immunization with OVA and alum on the same day as the TCDD treatment (0, 5, or 20 microg/kg body weight), TCDD markedly suppressed the concentrations of Th2-type cytokines (e.g., IL-4 and IL-5) in culture supernatants of spleen cells, whereas IFN-gamma production significantly increased. TCDD exposure reduced anti-OVA and total IgE antibody titers in plasma and did not induce the development of atopic dermatitis-like lesions in the pinnae or dorsal skin of NC/Nga mice. These results suggest that in NC/Nga mice, exposure to TCDD may impair the induction of Th2-type immune responses.

2,3,7,8-Tetrachlorodibenzo- p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos

Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC 50 and LD 50 of TCDD for Fundulus embryos were ∼19.7±9.5 pg TCDD/μl (water bath) and 0.25±0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction.

Increased tumor necrosis factor-alpha production by peripheral blood leukocytes from TCDD-exposed rhesus monkeys.

Previous work has shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with a dose-dependent increase in the incidence and severity of endometriosis in the rhesus monkey. Studies also suggest that immune mechanisms participate in TCDD-mediated toxicity and the pathogenesis of endometriosis. Thirteen years after TCDD treatment was terminated, we characterized the phenotypic distribution of peripheral blood mononuclear cells (PBMC) from TCDD-exposed and -unexposed rhesus monkeys and determined the ability of these cells to produce cytokines and exert cytolytic activity against NK and T-cell-sensitive cell lines. We also determined whether elevated serum levels of TCDD, dioxin-like PHAH congeners, and triglycerides correlated with changes in PBMC phenotype or function. For all animals, TCDD exposure correlated with increased PBMC tumor necrosis factor-alpha (TNF-alpha) secretion in response to stimulation by T-cell mitogen and decreased cytolytic activity against NK-sensitive target cells. Furthermore, increased production of this cytokine by PHA-stimulated leukocytes was associated with elevated serum triglyceride levels. Leukocyte TNF-alpha secretion in response to viral antigen and PBMC production of interferon gamma (IFNgamma), IL-6, and IL-10 following exposure to mitogen or antigen were unaffected by previous TCDD treatment. Although TCDD exposure was not associated with changes in PBMC surface antigen expression, elevated serum concentrations of TCDD, 1,2,3,6,7,8-hexachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl correlated with increased numbers of CD3+/CD25- and CD3-/CD25+ leukocytes and enhanced secretion of TNF-alpha by mitogen-stimulated PBMC. These findings indicate that TCDD-exposed rhesus monkeys with endometriosis exhibit long-term alterations in systemic immunity associated with elevated serum levels of specific PHAH congeners.

Serum levels of TCDD and dioxin-like chemicals in Rhesus monkeys chronically exposed to dioxin: correlation of increased serum PCB levels with endometriosis.

Humans and animals are exposed daily to a complex mixture of polyhalogenated aromatic hydrocarbons (PHAHs). Previous work has shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with a dose-dependent increase in the incidence and severity of endometriosis in the rhesus monkey. Dioxin-like chemicals can also exert effects in combination with TCDD via the aryl hydrocarbon receptor. This study demonstrates that the serum levels of TCDD and specific dioxin-like PHAH congeners were increased in TCDD-treated animals with endometriosis 13 years after the TCDD exposure. Nine TCDD-exposed and 6 unexposed female rhesus monkeys were evaluated for serum content of relevant compounds and for endometriosis by surgical laparoscopy. Additional studies were done on 4 animals that died 7 to 11 years after exposure to TCDD and 4 lead-treated animals with no history of PHAH treatment. For TCDD-exposed and unexposed animals, TCDD exposure correlated with an increased serum TCDD concentration. Furthermore, TCDD exposure and an elevated serum TCDD concentration were associated with increased serum levels of triglycerides, 1,2,3,6,7,8-hexachlorodibenzofuran, 3,3',4,4'-tetrachlorobiphenyl (TCB) and 3,3'4,4',5-pentachlorobiphenyl (PnCB). Importantly, the animals with elevated serum levels of 3,3',4,4'-TCB, 3,3',4,4',5-PnCB and an increased total serum TEQ had a high prevalence of endometriosis, and the severity of disease correlated with the serum concentration of 3,3,',4,4'-TCB. Increased serum concentrations of coplanar PCBs were also present in lead-treated animals. Implications of these findings for human health and the prevalence of endometriosis in humans will be discussed.

The transcriptional signature of dioxin in human hepatoma HepG2 cells

We have used a high density microarray hybridization approach to characterize the transcriptional response of human hepatoma HepG2 cells to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). We find that exposure to 10 nM TCDD for 8 hr alters by at least a factor of 2.1 the expression of 310 known genes and of an equivalent number of expressed sequence tags. Treatment with TCDD in the presence of 20 μg/mL of cycloheximide blocked the effect on 202 of these genes, allowing us to distinguish between primary effects of TCDD exposure, which take place whether cycloheximide is present or not, and secondary effects, which are blocked by inhibition of protein synthesis. Of the 310 known genes affected by TCDD, 30 are up-regulated and 78 are down-regulated regardless of cycloheximide treatment, and 84 are up-regulated and 118 are down-regulated only when protein synthesis is not inhibited. Functional clustering of genes regulated by TCDD reveals many potential physiological interactions that might shed light on the multiple biological effects of this compound. Our results, however, suggest that arriving at a sound understanding of the molecular mechanisms governing the biological outcome of TCDD exposure promises to be orders of magnitude more complicated than might have been previously imagined.

Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental defects in the rat vagina.

At puberty, female rats exposed in utero to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exhibit a persistent thread of mesenchymal tissue surrounded by keratinized epithelium that partially occludes the vaginal opening. Our objective was to determine the earliest time during fetal development that morphological signs of this vaginal canal malformation could be detected and to obtain greater insight into mechanisms involved in this effect. Pregnant rats were administered a single dose of vehicle (control) or TCDD (1.0 microg/kg, po) on gestation day (GD) 15 and were sacrificed on GD 18, 19, 20, and 21 for histological evaluation of female. Gestational exposure to TCDD affected vaginal morphogenesis as early as GD 19, 4 days after exposure of pregnant dams. In exposed fetuses, the thickness of mesenchymal tissue between the caudal Mullerian ducts was increased, which resulted in a failure of the Mullerian ducts to fuse, a process normally completed prior to parturition. In addition, TCDD exposure appeared to inhibit the regression of Wolffian ducts. Thus, TCDD interferes with vaginal development by impairing regression of the Wolffian ducts, by increasing the size of interductal mesenchyme, and by preventing fusion of the Mullerian ducts. Taken together, these effects appear to cause the persistent vaginal thread defect observed in rats following in utero and lactational TCDD exposure.

Expression of Dioxin-Responsive Genes in Human Endometrial Cells in Culture

To investigate expression of dioxin-responsive genes in human endometrial cells with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), human endometrial stromal cells immortalized with temperature-sensitive SV40 T antigen were used for the experiments. Cells were treated with 0.1% DMSO or 0.1, 1, 10, or 100 nM TCDD for 24 h. Induction of interleukin-1β (IL-1β) and plasminogen activator inhibitor-2 (PAI-2) mRNAs was analyzed by reverse-transcription polymerase chain reaction. Expression of IL-1β or PAI-2 mRNA in response to TCDD was increased in a dose-dependent fashion. The maximum increases of PAI-2 and IL-1β mRNAs were observed at 100 and 10 nM TCDD, respectively. While cycloheximide treatment did not show a significant difference of PAI-2 mRNA levels between control and TCDD-treated cells, mRNA stability assay using actinomycin D showed that PAI-2 mRNA in TCDD-treated cells was about twofold more stable than the control cells. While expression of CYP1A1 mRNA was not detected and levels of ARNT mRNA were not altered by TCDD exposure, the amount of AhR mRNA was decreased dose dependently. The present study represents an initial attempt to determine the responses of dioxin-responsive genes in human endometrial cells following TCDD exposure. The results demonstrated that IL-1β and PAI-2 genes are induced dose dependently in human endometrial cells with exposure to TCDD and expression of PAI-2 mRNA is controlled at the posttranscriptional level.

A malignant transformation of human cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibits altered expressions of growth regulatory factors.

The neoplastic transformation of human cells in culture with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has recently been reported. In this study, expressions of growth regulatory factors were analyzed to examine their possible roles in TCDD-induced malignant transformation of human cells. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analysis were performed to detect altered expressions of genes associated with dioxin responses. The RT-PCR analysis showed that expressions of the growth regulatory factors, such as transforming growth factor-beta1 (TGF-beta1), plasminogen activator inhibitor-2 (PAI-2) and tumor necrosis factor-alpha (TNF-alpha), were significantly changed in the transformed cells as compared with the parental cells. Whereas parental cells showed a dose-dependent increase of PAI-2 mRNA levels following TCDD exposure, the transformed cells did not show any significant induction. In addition, constitutive levels of PAI-2 mRNA were 25 times lower in the transformed cells than in the parental cells. The mRNA stability assay suggests that downregulation of PAI-2 mRNA in the transformed cells may be associated with the posttranscriptional control. Expression of TGF-beta1 mRNA in the transformed cells was also four times lower than the parental cells. However, levels of TNF-alpha mRNA in the transformed cells were increased 3-fold. These results suggest that dysregulation of growth regulatory factors may be involved in TCDD-induced cellular transformation. Whereas plenty of studies demonstrated a number of immediate toxic effects by TCDD, this study revealed an initial evidence that altered expression of growth regulatory genes, such as PAI-2, TGF-beta1 or TNF-alpha, are some of the genetic events fixed in the genome following the successive cell divisions of TCDD-damaged cells. It is suggested that these changes may be associated with TCDD-induced malignant transformation of human cells.

Responsiveness of the Adult Male Rat Reproductive Tract to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exposure: Ah Receptor and ARNT Expression, CYP1A1 Induction, and Ah Receptor Down-Regulation

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) either in adulthood or during late fetal and early postnatal development causes a variety of adverse effects on the male rat reproductive system. It was therefore of interest to identify male rat reproductive organs and cell types within these organs that might be direct targets of TCDD exposure. Because TCDD toxicity could possibly be the result of alterations in gene transcription mediated by the TCDD/aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) complex, the presence of the AhR and ARNT in the various organs of the adult male reproductive tract was examined using Western blotting. Both proteins were detectable in all organs examined (testis, epididymis, vas deferens, ventral prostate, dorsolateral [combined dorsal and lateral] prostate, and seminal vesicle). Although technical difficulties precluded the immunohistochemical evaluation of AhR distribution in these organs, ARNT was localized in all organs in a variety of cell types, including germ cells, epithelial cells, fibroblasts, smooth muscle cells, and endothelial cells. Subcellular localization varied across organs and across cell types within these organs. In order to determine whether TCDD exposure could alter gene expression in these organs, animals were dosed with TCDD (25 μg/kg po) or vehicle and euthanized at 24 h, and cytochrome P4501A1 (CYP1A1) expression was evaluated. By Western blotting, only the ventral and dorsolateral prostates exhibited significant induction of CYP1A1. Immunohistochemistry confirmed this induction and localized CYP1A1 expression to epithelial cells of the ventral and lateral lobes of the prostate. Immunohistochemistry also revealed CYP1A1 induction in select epithelial cells in the epididymis and seminal vesicle, as well as endothelial cells in the vas deferens and seminal vesicle. No induction was observed in the testis. Finally, AhR and ARNT expression in TCDD-exposed and control animals was evaluated by Western blotting. Results revealed no effect of TCDD exposure on ARNT protein expression, while AhR expression was decreased to 5–51% of control in all organs examined. In summary, both AhR and ARNT were expressed in all organs of the adult male rat reproductive tract examined, and epithelial and/or endothelial cells within each of these organs (with the exception of the testis) were responsive to TCDD exposure in terms of CYP1A1 induction. In addition, all tissues exhibited marked reductions in AhR protein content after TCDD exposure that did not correlate with the magnitude of the CYP1A1 response.

Evaluation of cardiovascular outcomes among U.S. workers exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Some animal studies and some human studies suggest that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may be associated with adverse effects on the cardiovascular system. As part of a cross-sectional medical study comparing workers employed 15 years earlier in the manufacture of 2,4,5-trichlorophenol or one of its derivatives at two U.S. chemical plants with an unexposed comparison group, we examined the association between TCDD exposure and various cardiovascular outcomes. A total of 281 workers and 260 unexposed referents participated. The workers had substantial exposure to TCDD, as demonstrated by significantly elevated mean serum TCDD concentration of 220 pg/g of lipid, compared with 7 pg/g of lipid among the referents. No significant association was found between TCDD exposure and any of the cardiovascular outcomes including myocardial infarction, angina, cardiac arrhythmias, hypertension, and abnormal peripheral arterial flow. Although our study had sufficient statistical power to detect an elevated risk for cardiac arrhythmias, hypertension, and abnormal peripheral arterial flow, it had low power (approximately 50%) to detect an elevated risk for myocardial infarction and angina. Our review of the literature suggests that our negative findings are consistent with those from other cross-sectional medical studies. Although several mortality studies of TCDD-exposed cohorts found significantly increased risks for cardiovascular disease mortality, similar increased risks were not observed in other mortality studies. The data available do not provide definitive conclusions but indicate that further examination of the association between TCDD exposure and cardiovascular disease should be pursued.

Induction and localization of cytochrome P450 1B1 (CYP1B1) protein in the livers of TCDD-treated rats: detection using polyclonal antibodies raised to histidine-tagged fusion proteins produced and purified from bacteria.

Knowledge of the response of cytochrome P450 1B1 (CYP1B1) to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in both humans and rodents is limited. To improve the analysis of CYP1 proteins, specific CYP1B1 and CYP1A1 polypeptides were expressed as hexahistidine-tagged fusion proteins in Escherichia coli, purified to homogeneity and used to produce polyclonal antibodies in rabbits. Immunoblot analyses showed that these antibodies were specific and sensitive, detecting both the human and rat forms of the respective isozymes and exhibiting negligible cross-reactivity between the two known CYP1 subfamilies. We show that CYP1B1, CYP1A1 and CYP1A2 protein levels were induced in the livers of female Sprague-Dawley rats following either acute (single dose of 25 microg TCDD/kg) or chronic (125 ng TCDD/kg/day for 30 weeks) exposure to TCDD. CYP1B1 protein exhibited a dose-response to TCDD that was different from those of CYP1A1 and CYP1A2. CYP1B1 induction appeared to be less sensitive to TCDD exposure, with induction occurring at higher doses of TCDD than that required for induction of CYP1A1 or CYP1A2. Immunohistochemical analysis showed that in animals chronically exposed to TCDD (35 ng/kg/day for 30 weeks), CYP1B1 was induced only in centrilobular hepatocytes, a pattern of expression similar to that of CYP1A1 and CYP1A2. These observations of cellular co-localization of the CYP1 cytochromes in livers of TCDD-treated rats and apparent differences in both protein amounts and dose-response are indicative of both common and unique regulation of CYP1 induction.

Dioxin exposure and non-malignant health effects: a mortality study.

OBJECTIVE: To investigate, in a population heavily exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the possible unusual occurrence of diseases other than cancer. METHODS: Five year extension of the follow up of the...

Developmental neurotoxic effects of dioxin and dioxin‐like compounds on domestic and wild avian species

AbstractIn ovo exposure to dioxin and dioxin‐like compounds has been shown to be associated with the development of grossly asymmetric avian brains. This asymmetry is primarily seen in two major brain regions, the forebrain and the tectum, and is detectable in the intact (but isolated) brain or in cross sections, as in computer tomographic (CT) images of the brain. Brain asymmetry has been observed in several wildlife species (heron, cormorant, and eagle) exposed to a mixture of contaminants in the wild, as well as in a domestic species (chicken) exposed to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) in ovo under controlled conditions. Asymmetry appears with increasing frequency and severity in embryos and hatchlings exposed to increasing doses of TCDD beginning early in embryonic development. Since asymmetry is evident in early embryos (embryonic day 9) when the braincase is just a thin covering, it is unlikely that brain asymmetry is caused by effects on the braincase rather than on the brain itself. In addition, preliminary histological evaluation of heron hatchling brains indicates that the pyriform (striate) cortex has increased cell density in contaminated versus control brains, although it is not yet clear whether this change in cell density is also manifested asymmetrically. Immunohistochemical analysis of embryonic chicken spinal cord demonstrated that TCDD exposure in ovo also induces a dysmyelination.

Modulation of ovarian follicle maturation and effects on apoptotic cell death in holtzman rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) in utero and lactationally

Recent reports have described the reproduction-modulating and endocrine-disrupting effects following exposure to toxic substances such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Herein, we set out (1) to determine whether TCDD exposure exerts detrimental effects on follicle maturation in the Holtzman rat ovary and (2) to determine whether the effects of TCDD are mediated in part via apoptotic cell death. In certain species, dioxin exposure is correlated with reduced fecundity, reduced ovulatory rate, an increased incidence of endometriosis, and various reproductive cancers. Although some of the effects of TCDD are mediated via the hypothalamic-pituitary axis, direct effects on the ovary have also been observed. In the present study, an oral dose of l μg TCDD/kg maternal body weight was administered on Day 15 of gestation. Female pups were sacrificed on Postnatal Day 21 22 , and the ovaries were excised, fixed for histologic analysis, and analyzed in a double-blind paradigm. The analysis included a count and measurement and classification of preantral and antral follicles throughout the entire ovary. The contralateral ovary from each animal was analyzed for DNA fragmentation indicative of apoptotic cell death. The results indicate that TCDD treatment significantly reduced the number of antral follicles in the size classes 50,000 to 74,999 μm 2 and > 100,000 μm 2. We also observed a reduction in the number of preantral follicles less than 50,000 μm 2. No difference was observed in the degree of apoptotic cell death in antral (50,000 to > 100,000 μm 2) and preantral follicles (50,000 μm 2 to > 75,000 μm 2) between TCDD-treated and control-treated tissues. These data support the hypothesis that TCDD results in a diminution in the number of antral and preantral follicles of certain size classes in animals exposed during critical periods of development, but that apoptosis does not appear to be the underlying mechanism in these particular follicles. This does not preclude apoptosis occurring in pools of smaller precursor follicles.

Correlation of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Induced Apoptotic Cell Death in the Embryonic Vasculature with Embryotoxicity

Vertebrate embryos are particularly sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Identification of tissues that are susceptible to the adverse effects of TCDD is requisite for understanding the embryo toxic effects of TCDD. The objective of the present study was to quantitate the temporal appearance of and dose dependence of apoptosis in TCDD-exposed medaka embryos (Oryzias latipes). A fluorescent-based DNA end-labeling assay provided a sensitive method for detection of TCDD-induced apoptosis in tissue sections of medaka embryos. Apoptotic cells were readily apparent in the medial yolk vein at all observed embryonic stages in TCDD-exposed embryos. Slope-comparison analysis indicated that TCDD-induced programmed cell death in the embryonic medial yolk vein was mechanistically linked to embryo mortality. These data are consistent with the hypothesis that vascular damage contributes to the acute embryo toxic effects of TCDD. However, as sublethal concentrations of dioxin-like compounds are more typical of environmental exposures, tissue damage was also assessed in medaka fry that were exposed to low doses of TCDD during embryonic development. Cell death was detected in gill and digestive tissues in visibly healthy medaka fry that had been exposed to low doses of TCDD during embryonic development. Increased expression of cytochrome P450 1A is a major biochemical consequence of TCDD exposure and is often used as a biomarker for exposure to dioxin-like compounds. Therefore, we compared the tissue distribution of TCDD-induced P450 1A expression and TCDD-induced programmed cell death. TCDD-induced programmed cell death co-localized with TCDD-induced P450 1A expression in both embryos and in visibly healthy post-hatch fry. Our results suggest that aberrant programmed cell death may be a suitable marker for exposure of feral organisms to dioxin-like compounds.

2,3,7,8-Tetrachlorodibenzo-p-dioxin in Pregnant Long Evans Rats: Disposition to Maternal and Embryo/Fetal Tissues

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interferes with fetal development at doses lower than those causing overt toxicity in adult animals. In a multigeneration study (Murray et al., 1979), female rats that were administered 0.01 microgram TCDD/kg/day in their diet did not experience reduced fertility; however, reduced fertility was seen in the F1 and F2 generations. Exposure to TCDD during development produces alterations in the reproductive system of the developing pups, such as delayed puberty and reduced sperm counts in males (Mably et al., 1992a; Gray et al., 1995) and malformations in the external genitalia of females (Gray and Ostby, 1995). Therefore, the objectives of this study were to determine maternal and fetal tissue concentrations of TCDD that are associated with the adverse reproductive effects seen by Gray and co-workers. Pregnant Long Evans rats received a single oral dose of 1.15 micrograms [3H]TCDD/kg on Gestation Day (GD) 8 and maternal as well as fetal tissue concentrations of TCDD were measured on GD9, GD16, and GD21. On GD9, the highest level of TCDD localized in the maternal liver (25.1% dose). In addition, the amount reaching all the embryos on GD9 was 0.01% of the administered dose, which resulted in a concentration of 0.02% dose/g. The amount of TCDD reaching the fetal compartment (fetuses + placentas) increased to 0.12% dose/tissue on GD16 and 0.71% by GD21. The concentration of TCDD within the fetal compartment (0.01% dose/g) on GD16 was comparable to that found in the maternal blood and spleen. Concentrations of TCDD in a single embryo/fetus were 39.6, 18.1, and 22.1 pg/g on GD9, GD16, and GD21, respectively. Estimates of hepatic half-life of elimination in pregnant rats suggested that TCDD may be eliminated faster in pregnant LE rats. Therefore, measurements of biliary elimination were made in pregnant and nonpregnant LE rats to compare rates of metabolism; however, biliary elimination of TCDD is not affected by pregnancy. In conclusion, this dose administered during a critical period of organogenesis causes adverse effects on the developing reproductive system of rodents. This dose produced a body burden of 22.1 pg TCDD/g within a single fetus on GD21. This indicates that low-level TCDD exposure during the perinatal stage of life can produce adverse effects within the developing pups.