Olfactory epithelial metaplasia and hyperplasia in female Harlan Sprague-Dawley rats following chronic treatment with polychlorinated biphenyls

The National Toxicology Program recently completed a series of studies to evaluate the relative potency for toxicity and carcinogenicity of several polyhalogenated aromatic hydrocarbons including dioxin-like compounds (DLCs) and polychlorinated biphenyls. Female Sprague-Dawley rats were administered by gavage for up to 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); a tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118); control animals received corn oil-acetone vehicle (99:1) alone. Nasal epithelial changes were observed only in animals exposed for 2 years to the higher doses of the binary mixtures of PCB126 + PCB153 (1000 ng/kg and 1000 microg/kg) and PCB126 + PCB118 (216 and 360 ng TCDD equivalents/kg). In both studies, the changes were of the same nonneoplastic nature, localized to nasal sections II and III located, respectively, at the level of the incisive papilla anterior to the first palatial ridge (section II) and through the middle of the second molar teeth (section III). The changes consisted of hyperplasia of the respiratory epithelium (level II) and metaplasia of olfactory epithelium to respiratory epithelium with further hyperplasia of the metaplastic respiratory epithelium (levels II and III). Variable amounts of acute inflammatory exudate appeared within the lumen of the nasal cavity, overlying the affected epithelium. Occasionally, the inflammation eroded through the skull and into the adjacent olfactory bulbs.     

Thyroid follicular lesions induced by oral treatment for 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin and dioxin-like compounds in female Harlan Sprague-Dawley rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally-similar dioxin-like compounds affect thyroid function and morphology and thyroid hormone metabolism in animals and humans. The National Toxicology Program conducted eight 2-year gavage studies in female Harlan Sprague-Dawley rats to determine the relative potency of chronic toxicity and carcinogenicity of TCDD, 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 2,3',4,4',5-pentachlorobiphenyl (PCB118), 2,2',4,4',5,5'-hexachloro-biphenyl (PCB153), a tertiary mixture of TCDD/PCB126/PeCDF, and two binary mixtures (PCB126/PCB153 and PCB126/PCB118). Administration of these compounds was associated with increased incidences of thyroid follicular cell hypertrophy, variably observed in the 14-, 31-, and 53-week interim and 2-year sacrifice groups. In all studies, the incidences of follicular cell adenoma and carcinoma were not increased. Decreased levels of serum thyroxine were primarily noted in the 14-or-later -week interim groups of all chemicals. Serum triiodothyronine (T3) levels were increased in the TCDD, PCB126, PeCDF, TCDD/PCB126/PeCDF, and PCB126/PCB153 studies, while decreased levels were noted in the PCB153 and PCB126/PCB118 studies. TCDD, PCB126, PCB126/PCB153, and PCB126/PCB118 increased levels of serum thyroid-stimulating hormone almost in a dose-dependent manner in the 14-week groups. These data suggest that although dioxin-like compounds alter thyroid hormones and increase follicular cell hyperplasia, there is not an increase in thyroid adenoma or carcinoma in female Sprague-Dawley rats.     

Molecular basis of dioxin actions: evidence supporting chemoprotection

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin), a highly publicized environmental contaminant, was shown to be chemoprotective against breast cancer in both rats and mice in bioassays conducted in the late 1970s. This finding went largely unnoticed as investigators focused on animal tumors that were increased by dioxin. The position that dioxin causes human tumors remains a subject for debate; however, recent epidemiological studies of a population highly exposed to dioxin in 1976 as a result of an industrial accident suggest that women with higher dioxin body burdens may have a lower incidence of breast cancer. With the growth of new knowledge about the molecular basis of dioxin actions in humans and animals, it is clear that most of the responses produced by this agent are initiated by a specific recognition protein (designated the Ah receptor) found in almost all animal and human tissues and organs. The recognition event between the Ah receptor and environmental agents like dioxin is due to the formation of a complex. We have observed that in the presence of dioxin, the Ah receptor turns off proliferation in tumor cells and suppresses the ability of these cells to invade normal tissue. We believe that these findings provide a molecular and biochemical basis for understanding the chemoprotective mechanisms suggested by the findings of rodent bioassays and could lead to the development of novel therapeutic agents targeting the Ah receptor.     

Perinatal TCDD exposure and the adult onset of autoimmune disease

Modulation of the developing immune system can occur following perinatal exposure to a number of immunotoxic compounds, including polyhalogenated hydrocarbons like 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD; dioxin), the most toxic of the congeners. Studies in rodents have shown immunologic effects from perinatal TCDD exposure are more severe and persistent than following exposure in the adult, and include what appears to be life-long immunosuppression. Whether prenatal TCDD exposure may predispose an individual to postnatal autoimmune disease remains largely unknown. TCDD crosses the placenta and alters normal prenatal thymocyte maturation, T-cell receptor expression and expression of thymic major histocompata-bility complex Class II molecules. During the juvenile stage, mice exposed to TCDD prenatally show increased peripheral T-cells possessing “autoreactive” variable-β receptors. These data suggest that gestational exposure to TCDD may interfere with normal development of central tolerance in the thymus. In possible support of this theory, when autoimmune disease-prone mice were treated with TCDD during gestation, postnatal autoimmunity had an accelerated onset and was exacerbated. This review provides an overview of the currently available information, which appears to support a hypothesis for increased risk of postnatal autoimmune responses as a result of TCDD exposure during the sensitive time of immune system establishment.     

Pulmonary lesions in female Harlan Sprague-Dawley rats following two-year oral treatment with dioxin-like compounds

Dioxin and dioxin-related compounds have been associated with high incidences of pulmonary dysfunctions and/or cancers in humans. To evaluate the relative potencies of effects of these compounds, the National Toxicology Program completed a series of two-year bioassays which were conducted using female Harlan Sprague-Dawley rats. The rats were treated orally for up to 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and a ternary mixture of TCDD, PCB126 and PeCDF. In addition to treatment-related effects reported in other organs, a variety of pulmonary lesions were observed that were related to exposure. Pulmonary CYP1A1-associated 7-ethoxyresorufin-O-deethylase (EROD) activity was increased in all dosed groups. The most common non-neoplastic lesions, which occurred in all studies, were bronchiolar metaplasia and squamous metaplasia of the alveolar epithelium. Cystic keratinizing epithelioma was the most commonly observed neoplasm which occurred in all studies. A low incidence of squamous cell carcinoma was associated only with PCB126 treatment. Potential mechanisms leading to altered differentiation and/or proliferation of bronchiolar and alveolar epithelia may be through CYP1A1 induction or disruption of retinoid metabolism.     

Whole genome sequencing and mutation rate analysis of trios with paternal dioxin exposure

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD) or dioxin, is commonly considered the most toxic man‐made substance. Dioxin exposure impacts human health and diseases, birth defects and teratogenesis were frequently observed in children of persons who have been exposed to dioxin. However, the impact of dioxin on human mutation rate in trios has not yet been elucidated at the whole genome level. To identify and characterize the genetic alterations in the individuals exposed to dioxin, we performed whole genome sequencing (WGS) of nine Vietnamese trios whose fathers were exposed to dioxin. In total, 846 de novo point mutations, 26 de novo insertions and deletions, 4 de novo structural variations, and 1 de novo copy number variation were identified. The number of point mutations and dioxin concentrations were positively correlated (P‐value < 0.05). Considering the substitution pattern, the number of A > T/T > A mutation and the dioxin concentration was positively correlated (P‐value < 0.05). Our analysis also identified one possible disease‐related mutation in LAMA5 in one trio. These findings suggested that dioxin exposure might affect father genomes of trios leading to de novo mutations in their children. Further analysis with larger sample sizes would be required to better clarify mutation rates and substitution patterns in trios caused by dioxin.     

Effects of environmental toxins on lymphocyte function: studies in rhesus and man

The immune system is a potential target of environmental toxins. Impairment of immune function could have a disastrous effect upon the affected individual. We had the unique opportunity to study the results of a prolonged exposure to TCDD (2,3,7,8-tetrachlorodibenzo-P-dioxin) in rhesus monkeys and their offspring. Subsequently, we performed similar studies on humans exposed to the nematode pesticide, Aldicarb. This report summarizes those previous studies. In the monkeys, no major deficits of the immune system were found and the animals did not have excessive numbers of infections. However, at higher doses of dietary TCDD (25 ppt), only 22% of the offspring survived to 1 year of age. Thus, the failure to demonstrate effects on the young may simply relate to the essential equivalence of the lethal to an immunosuppressive dose. In humans, exposure to the acetylcholinesterase inhibitor, Aldicarb, was received through contaminated well water. The known exposure was for at least 1 year and could have been as long as 5 years. Various tests of the immune system, including lymphocyte subset counts, proliferative responses, total immunoglobulin levels and specific antibody responses did not reveal immunodeficiency. Increases in the numbers of CD8 positive T lymphocytes was observed. There was no evidence of any increase in clinical illness in the exposed compared with the control group.     

Characterization of bronchiolar metaplasia of the alveolar epithelium in female Sprague-Dawley rats exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB126)

To test the dioxin toxic equivalency factor methodology, the National Toxicology Program conducted a series of 2-year rat bioassays of dioxin-like compounds. Following gavage exposure of female Harlan Sprague-Dawley rats to 2,3',4,4',5-pentachlorobiphenyl (PCB126), pulmonary alveolar epithelium at the junction of terminal bronchioles and along alveolar ducts was replaced by cuboidal to columnar ciliated cells. Scattered among these were cells exhibiting characteristics consistent with those of Clara cells; they lacked cilia and had a smooth apical surface that protruded into the alveolar space. This lesion was not typical of alveolar epithelial hyperplasia seen in rodent lungs; therefore, studies were done to characterize the lesion. Results of periodic acid-Schiff (PAS) staining, alcian blue (AB) staining, and GSTPi immunohistochemical staining of the lesions seen in treated rats were more similar to normal bronchiolar epithelium than normal alveolar epithelium or alveolar epithelial hyperplasia. These findings, along with the morphology of the cells, provide evidence that this lesion is closer in character to bronchiolar epithelium than alveolar type I or alveolar type II epithelium, and as a result, was called bronchiolar metaplasia.     

Identification of mammary carcinogens in rodent bioassays

Results from chemical carcinogenesis studies in rodents are useful to identify substances in our environment that may contribute to cancer development. The National Toxicology Program (NTP) was established in 1978 to coordinate research and testing of potential human carcinogens and to publish the Report on Carcinogens, which lists human carcinogens. The results for over 500 chemicals tested in the NTP 2-year bioassays have been published in Technical Reports and include data for chemical, agent, or complex mixture exposures. The bioassays have identified 42 chemicals that induce tumors in the rodent mammary gland. The physical and chemical characteristics of the carcinogens vary, but epoxides (including chemicals metabolized to epoxides) and nitro-containing compounds are well represented. The 9th Report on Carcinogens, issued in 2000, lists 21 of the 42 chemicals as human carcinogens including benzene, ethylene oxide, 1,3-butadiene, isoprene, chloroprene, C.I. basic red 9, and C.I. acid red 114. Ethylene oxide was associated with increased breast cancer risk in an epidemiologic study, whereas other listed chemicals, for which human data are available, display different target organ specificity. Bioassays other than those conducted by the NTP also provide information about rodent mammary gland carcinogens. Several carcinogen exposures are associated with breast tumor induction in both humans and rodents including radiation, diethylstilbestrol, and estrogens. These studies demonstrate that route, timing and frequency of exposure, and genetic factors contribute to the overall susceptibility to breast cancer development. More information is needed on the effects of chemicals to which humans are exposed and the manner by which they influence breast cancer risks.     

The effect of maternal exposure to dioxin on fetal steroidogenesis in the steroidogenic organs

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposed to pregnant or lactational mother impairs the reproduction and development of the pups. The defect is a serious problem, because it is caused by TCDD at much lower doses than that needed for acute toxicity in the mother. However, the toxic mechanism underlying the defect remains to be obscure. We have previously revealed that maternal exposure to TCDD (1 microg/kg) causes a reduction in luteinizing hormone in the fetal pituitary, leading to the reduced expression of testicular steroidogenic proteins such as steroidogenic acute-regulatory protein (StAR) and cytochrome P450 (CYP) 17. In addition, we have provided evidence that such a reduction imprints defects in sexual behaviors at adulthood. In this study, we investigated TCDD effect on fetal steroidogenesis in the extra-gonadal tissues. Even when pregnant Wistar rats at gestational day (GD) 15 were orally treated with TCDD (0.25, 1 or 3 microg/kg), neither expression of StAR nor CYP17 mRNA was affected in the adrenal gland, placenta and hypothalamus of male fetuses (GD20). However, TCDD induced placental StAR (3 microg/kg) and adrenal CYP17 mRNAs (0.25 microg/kg) in female fetuses. Therefore, our study suggests that while TCDD gives damage to male fetal steroidogenesis in a testis-specific manner, the dioxin enhances the steroidogenesis of the fetal adrenal gland and placenta in females. Thus, the mechanism whereby TCDD exerts its endocrine-disrupting properties is considered to differ, at least partially, between male and female fetuses.     

Human health effects of dioxins: cancer, reproductive and endocrine system effects

Polychlorinated dioxins, furans and polychlorinated benzene constitute a family of toxic persistent environmental pollutants. In Europe, environmental concentrations increased slowly throughout this century until the late 1980s. Dioxins have been shown to be carcinogenic in animals and humans. In humans, excess risks were observed for all cancers, without any specific cancer predominating. In specific cohorts, excess risks were observed for reproductive cancers (breast female, endometrium, breast male, testis) but, overall, the pattern is inconsistent. In animals, endocrine, reproductive and developmental effects are among the most sensitive to dioxin exposure. Decreased sperm counts in rats and endometriosis in rhesus monkeys occur at concentrations 10 times higher than current human exposure. In humans, results are inconsistent regarding changes in concentrations of reproductive hormones. A modification of the sex ratio at birth was described in Seveso. There exist no data on effects such as endometriosis or time-to-pregnancy. Small alterations in thyroid function have occasionally been found. Increased risk for diabetes was seen in Seveso and a herbicide applicators cohort but, overall, results were inconsistent. Experimental data indicate that endocrine and reproductive effects should be among the most sensitive effects in both animals and humans. Epidemiological studies have evaluated only a few of these effects.     

Disruption of erythropoiesis by dioxin in the zebrafish.

2,3,7,8-Tetrachlorodibenzo-p- dioxin (TCDD, or dioxin) causes early life stage mortality in a variety of fish species. We have used the zebrafish (Danio rerio) to study the cardiovascular effects of TCDD treatment over the time course of zebrafish development. Early TCDD exposure (6 ng/ml) starting at 4 hr postfertilization (hpf) produced reductions in blood flow and in the number of circulating erythrocytes. These defects were consistently observable by 72 hpf. However, these responses were not observed when TCDD exposure was delayed until 96 hpf or later. These results suggest a model in which TCDD interferes with cardiovascular and erythropoietic developmental processes that are normally completed by 96 hpf. This model is strengthened by the finding that TCDD exposure blocks the step in hematopoiesis in which developing zebrafish switch from the primitive phase to the definitive phase of erythropoiesis. We observed no effect of TCDD on the levels of circulating primitive erythrocytes before 72 hpf and the expression of markers for early hematopoiesis, GATA-1 and GATA-2. However, early TCDD exposure prevented the appearance of definitive phase erythrocytes. TCDD produced a small delay in the migration of blood cells expressing SCL from the intermediate cell mass to the dorsal mesentery and dorsal aorta. Despite the decrease in blood flow produced by TCDD, confocal microscopy of the trunk vasculature by using a Tie2/green fluorescence protein endothelial marker at 48, 60, 72, and 96 hpf of TCDD-exposed (4 hpf) revealed no apparent defects in blood vessel structure.     

Up-regulation of methionine-enkephalin-like immunoreactivity by 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment in the forebrain of the Long-Evans rat

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is considered to be one of the most toxic environmental contaminants, named dioxin. Exposure to TCDD induces a plethora of intoxication symptoms, including anorexia and hypothermia, in several mammals and human. Enkephalin, an endogenous pentapeptide, is an important neuroregulator of autonomic functions, such as food intake and body temperature. In this study, we investigated the effects of TCDD gastric administration on methionine-enkephalin (MEK) immunoreactivity in the brain of the Long-Evans rat, the species strain considered to be the most TCDD-susceptible, using immunohistochemical staining. A single dose of TCDD (dissolved in olive oil, 50 microg/kg) or olive oil alone was administrated to the rats by gavage. Compared with the vehicle-treated rat, a marked increase in the density of MEK immunoreactive cell bodies, fibers and terminals was found 2 weeks after TCDD treatment in the forebrain of the TCDD-treated rat, i.e. the central amygdaloid nucleus, field CA3 of the hippocampus, paraventricular hypothalamic nucleus, medial preoptic nucleus, interstitial nucleus of the posterior limb of the anterior commissure, lateral globus pallidus, ventral pallidum and lateral division of the bed nucleus of the stria terminalis. These results demonstrated for the first time a site-specific increased enkephalinergic activity in certain brain regions of the Long-Evans rat. It is suggested that the increased MEK immunoreactivity may act as a compensatory adaptation for the pathophysiological alterations caused by TCDD exposure.     

1142292808Developmental exposure of mice to dioxin exhibit endometrial dysfunction which mimics endometrium from women with endometriosis

Problem: The environmental contaminant 2,3,7,8‐tetracholorodibenzo‐p‐dioxin (TCDD) has been suggested to play a role in the etiology of endometriosis, but whether women develop this disease due to a prior exposure to endocrine disruptors is difficult to ascertain. Several studies have failed to demonstrate a link between adult TCDD exposure and the development of endometriosis, but we suspect it is the very early exposures that may be most relevant to alterations in adult tissue function. To this end, we have recently developed a mouse model in which we can examine the impact of developmental TCDD exposure on the adult uterus. The endometrial phenotype of these mice is markedly similarly to the phenotype found in women with endometriosis. Method of Study: TCDD was administered to mice at multiple developmental stages (in utero, prepubertal and pubertal). Adult mice were ovariectomized and treated with either estradiol (E2) or with E2 and progesterone (P4). The uteri were collected and PR expression examined by immunohistochemistry. As an indicator of P4 responsiveness, tissues were further examined for expression of TGF‐β2. Human endometrial samples from women with and without endometriosis were similarly examined for PR and TGF‐β2 expression. Results: Minimal uterine expression of PR and TGF‐β2 was observed in TCDD exposed mice while control mice demonstrated abundant expression of both proteins. Analysis of normal human endometrium revealed intense staining for both PR and TGF‐β2 during the P4‐dominated secretory phase while these proteins were greatly diminished in tissues from women with endometriosis. Conclusions: A reduced responsiveness to P4 has recently been demonstrated in women with endometriosis. Although development of endometriosis is likely multifactorial, our studies are consistent with a role of in utero/developmental exposure to TCDD in the pathophysiology of this disease. Acknowledgement: This work was supported by NIEHS # R21ES12298 and The Endometriosis Association.     

Potential for genotoxic and reprotoxic effects of vanadium compounds due to occupational and environmental exposures: An article based on a presentation at the 8th International Symposium on Vanadium Chemistry,Biological Chemistry,and Toxicology,Washington DC,August 15–18, 2012

Abstract

Research on the biological effects of vanadium in humans has shown that acute poisoning in workers can manifest itself in a number of symptoms. There are no reports in humans about reproductive and developmental effects induced by vanadium compounds in humans; however, some studies with rats and mice indicate that vanadium can cross the placental barrier and accumulate in fetal membranes rather than the fetus itself. In this case, probably most consequences of administration of vanadium to pregnant females like reabsorptions, fetal death and reduction in size can be the result of maternal toxicity. Concerning genetic and related effects in humans exposed to different vanadium compounds, data are controversial. Data on genotoxic effects in workers exposed to vanadium indicate that they can have an increased risk to develop cancer, and DNA instability can give rise to an onset of genetic syndromes, fetal malformations, and cancer. This paper presents materials presented at the 8th International Symposium on Vanadium Chemistry, Biological Chemistry, and Toxicology in a session titled ‘Relationship between occupational and environmental exposure to vanadium compounds and the reprotoxic and genotoxic effects’.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits the activation of antigen-specific T-cells in mice.

There are conflicting data in the literature regarding target cells in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity. In the present study, adult male C57BL/6 mice were exposed to TCDD (50 micrograms/kg) 4 days prior to immunization with ovalbumin (OVA). The effect of TCDD on the specific immune response in vivo was determined by T-cell proliferation and IL-2 production in response to either OVA or anti-mouse-CD3 antibodies plus PMA in vitro. The antigen-specific T-cell proliferation and IL-2 production in response to OVA were significantly suppressed by TCDD, while the polyclonal response to anti-CD3 antibodies plus PMA was not affected. This indicates that even at a high dose of TCDD the intra T-cell signalling pathways in resting cells are not disturbed, but TCDD selectively impairs the antigen-specific activation of T-cells. Since activated T-cells are required in antibody responses to T-dependent antigens, the low number of such cells observed in the present study, may well explain the suppressive effects of TCDD on humoral immunity reported previously.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits cell proliferation through arylhydrocarbon receptor-mediated G1 arrest in SK-N-SH human neuronal cells

The neurotoxic mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has not been completely elucidated. In this study we investigated the possible role of cell cycle regulators and their dependence on arylhydrocarbon receptor (AhR) in the TCDD-mediated inhibition of cell proliferation using a human neuronal cell system. TCDD suppressed DNA synthesis of SK-N-SH human neuronal cells determined by [(3)H]thymidine incorporation which was significantly prevented either by pretreatment with alpha-naphthoflavone (alpha-NF), a partial AhR antagonist, or 8-methoxypsoralen (MOP), a binding inhibitor of activated AhR to dioxin response elements. Cell cycle analysis showed that TCDD induced a G(1) cell cycle arrest, which was also significantly prevented by pretreatment with alpha-NF and MOP. TCDD did not alter the expression of cyclin D, cyclin E, p21 and p53. However, TCDD induced an enhanced expression of p27 and a hypophosphorylation of pRb, which was prevented by alpha-NF and MOP. Combined, these results suggest that the TCDD-induced inhibition of neuronal cell proliferation may be due to the AhR-dependent G(1) arrest through an enhanced expression of p27 and a hypophosphorylation of pRB.