TCDD Levels Research Articles

Modelling of 2,3,7,8-tetrachlorodibenzo-p-dioxin levels in a cohort of workers with exposure to phenoxy herbicides and chlorophenols

Several cohort studies of herbicide manufacturing workers have been conducted over the last years. Most of these studies used simple proxies of exposure in the analysis such as a crude grouping in exposure categories based on job titles, presence in certain production areas over a period of time or during an accident, and duration of exposure. Current serum 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) levels available for a subset of workers can be used to back-extrapolate TCDD levels at the end of exposure using first order kinetic models, and relate TCDD levels to job history using regression models. The regression model obtained can be used to estimate TCDD levels for all cohort members. In this paper, the effect of changes in model assumptions on estimated TCDD levels is explored. TCDD levels are back-extrapolated assuming different values for TCDD half-life. A range of regression models with different sets of exposure determinants is used to relate back-extrapolated TCDD levels to determinants of exposure. These models were used to predict TCDD levels in the epidemiological analysis of data from a Dutch cohort study. The results show that the predicted serum TCDD level is strongly dependent on the assumed half-life. However, the ranking of all individuals on the exposure axis (from low to high) is not affected by changes in the half-life. Predicted serum TCDD levels seem not sensitive to changes in assumption regarding TCDD half-life. Predicted TCDD levels were positively associated with increased (cause specific) mortality.

Differences in kinetics of induction and reversibility of TCDD-induced changes in cell proliferation and CYP1A1 expression in female Sprague-Dawley rat liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent tumor promoter in two-stage initiation-promotion models and induces cell proliferation and development of enzyme-altered hepatic foci. It is believed that increased cell proliferation is a necessary step in carcinogenesis. Therefore, the analysis of the effect of TCDD on cell proliferation in rat liver may aid in the understanding of the mechanism of hepatocarcinogenesis induced by TCDD. The aim of this study was to investigate the time course and reversibility of cell proliferation in non-initiated and diethylnitrosamine-initiated female rats exposed biweekly to a daily averaged dose of 125 ng TCDD/kg/day for up to 60 weeks. In addition we evaluated the suitability of different dose metrics for the evaluation of TCDD-induced changes in cell proliferation and CYP1A1 enzyme induction. Cell proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into hepatocytes undergoing replicative DNA synthesis. Mean BrdU labeling indices in TCDD-treated animals were not increased over controls after 14 weeks exposure, but were increased 8- and 2-fold after 30 and 60 weeks' treatment respectively, despite similar liver levels of TCDD at all these times (23-30 p.p.b.). In comparison, CYP1A1 activity, as measured by ethoxyresorufin deethylase activity, was significantly induced at all times points analyzed. Sixteen weeks following cessation of TCDD treatment, labeling indices were still significantly elevated over controls, but after 30 weeks of withdrawal, labeling indices were no different from controls, indicating that TCDD-induced changes in cell proliferation were reversible. Dosimetric analysis indicated that rat liver tissue burden was suitable for prediction of CYP1A1 expression but not cell proliferation and that the area under the curve was unsuitable for prediction of both TCDD-induced changes in CYP1A1 expression and cell proliferation.

Dioxins and dioxin‐like chemicals in blood and semen of American Vietnam veterans from the state of Michigan

This exposure assessment pilot study tested the hypothesis that elevated blood levels of the dioxin congener 2,3,7,8-TCDD (“TCDD”), due to Agent Orange exposure, in American Vietnam veterans could be demonstrated two to three decades after Vietnam service. A second objective was to determine if dioxins, including TCDD, are present in the semen of adult males. In the early 1990s, blood samples from 50 Vietnam veterans and three pooled semen samples from 17 of them were analyzed by high-resolution gas chromatography-mass spectroscopy for dioxins, dibenzofurans, and the dioxin-like PCBs. Fifty volunteers from the Michigan Vietnam veteran bonus list, which documented Vietnam service, were invited to participate based on their self-reported exposure to Agent Orange in Vietnam. Screening of military and medical records was performed by an epidemiologist and a physician to assure that Agent Orange exposure was possible based on job description, location of service in Vietnam, and military Agent Orange spray records. Elevated 2,3,7,8-TCDD levels, over 20 ppt on a lipid basis, could still be detected in six of the 50 veterans in this nonrandomly selected group. The dioxin and dibenzofuran congeners commonly found in the U.S. population, including TCDD, were also detected in the three pooled semen samples. Quantification and comparison on a lipid basis were not possible due to low lipid concentrations where levels were below the detection limit. Therefore, semen samples were measured and reported on a wet-weight basis. Elevated blood TCDD levels, probably related to Agent Orange exposure, can be detected between two and three decades after potential exposure in some American veterans. Original levels were estimated to be 35–1,500-fold greater than that of the general population (4 ppt, lipid) at the time of exposure. In addition, the detection of dioxins in semen suggests a possible mechanism for male-mediated adverse reproductive outcomes following Agent Orange or other dioxin exposure. © 1996 Wiley-Liss, Inc.

Rapid distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to embryonic tissues in C57BL/6N mice and correlation with palatal uptake in vitro

2,3,7,8-Tetracholoridbenzo-p-dioxin (TCDD) is a developmentally toxic environmental contaminant capable of inducing cleft palate and hydronephrosis in embryonic C57BL/6N mice. In this study, the disposition of TCDD was determined in pregnant C57BL/6N mice in the 24 hr immediately following oral administration on Gestation Day (GD) 12. TCDD was detected in maternal blood, liver, and fat and in the placenta, embryonic liver, and palate within 30 min after dosing on GD 12. The levels peaked in blood and placenta at 3 hr and in the other tissues at 8 hr. Levels of TCDD decreased slightly after 8 hr in embryonic liver and palate. In vitro systems were used to study the mechanisms of action of TCDD and in these models exposure is typically reported as concentration of TCDD in the culture medium. The present study is the first to allow a direct comparison of the level of TCDD in embryonic tissue after in vivo and in vitro exposures. Uptake of TCDD was determined in embryonic palatal organ culture and tissue levels were then expressed in comparable units for both in vivo and in vitro exposures. The data provide new information on distribution in the pregnant mouse and the embryo and also show that the palatal organ culture model provides a reasonable dosimetric representation of in utero exposure.

Rapid distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to embryonic tissues in C57BL/6N mice and correlation with palatal uptake in vitro.

2,3,7,8-Tetracholoridbenzo-p-dioxin (TCDD) is a developmentally toxic environmental contaminant capable of inducing cleft palate and hydronephrosis in embryonic C57BL/6N mice. In this study, the disposition of TCDD was determined in pregnant C57BL/6N mice in the 24 hr immediately following oral administration on Gestation Day (GD) 12. TCDD was detected in maternal blood, liver, and fat and in the placenta, embryonic liver, and palate within 30 min after dosing on GD 12. The levels peaked in blood and placenta at 3 hr and in the other tissues at 8 hr. Levels of TCDD decreased slightly after 8 hr in embryonic liver and palate. In vitro systems were used to study the mechanisms of action of TCDD and in these models exposure is typically reported as concentration of TCDD in the culture medium. The present study is the first to allow a direct comparison of the level of TCDD in embryonic tissue after in vivo and in vitro exposures. Uptake of TCDD was determined in embryonic palatal organ culture and tissue levels were then expressed in comparable units for both in vivo and in vitro exposures. The data provide new information on distribution in the pregnant mouse and the embryo and also show that the palatal organ culture model provides a reasonable dosimetric representation of in utero exposure.

In Ovo2,3,7,8-Tetrachlorodibenzo-p-dioxin Exposure in Three Avian Species: 1. Effects on Thyroid Hormones and Growth during the Perinatal Period

Thyroid hormones are important in the perinatal growth and development of avian species, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds have been shown to cause alterations in these hormones in laboratory animals. Since the decreased reproductive success in certain fish-eating bird populations exposed to TCDD and related compounds is characterized by high embryo and hatchling mortality, we examined the effects ofin ovoTCDD exposure on plasma thyroid hormone concentrations (total T3, total T4) and body and skeletal growth during the perinatal period in the domestic chicken (Gallus gallus), domestic pigeon (Columba livia), and great blue heron (Ardea herodias). Hepatic ethoxyresorufin O-deethylase (EROD) activity was also determined as an enzymatic marker of cytochrome P450IA induction by TCDD. [3H]TCDD was injected into the air cell of chicken eggs (21-day incubation period) on Embryonic Day 4.5 (0.1 μg/kg egg), pigeon eggs (18-day incubation period) on Embryonic Day 3.5 (1 μg/kg egg) and Embryonic Day 14 (3 μg/kg egg), and heron eggs (28-day incubation period) at approximately the midpoint of incubation (2 μg/kg egg). Chickens were euthanized on Embryonic Days 17 and 19, day of hatch (Embryonic Day 21), and Days 2 and 4 after hatch. Pigeons and herons were euthanized either at hatch (Embryonic Days 18 and 28, respectively), or fed an uncontaminated diet for 7 days prior to sacrifice. Although hepatic EROD activity was induced 13- to 43-fold above controls in chickens, there was no effect of TCDD exposure on hatchability, body growth, subcutaneous edema, or plasma thyroid hormone levels. In pigeons exposed to TCDD on Embryonic Day 3.5, EROD was induced 6- to 15-fold, hatchability was decreased, liver to body weight ratio was elevated, and body and skeletal growth were decreased (p< 0.01); however, there was no effect of TCDD exposure on plasma thyroid hormone levels. Similarly, in pigeons exposed to TCDD on Embryonic Day 14, EROD was induced 10- to 14-fold, liver to body weight ratio was elevated, and body and skeletal growth were decreased (p< 0.01), but there was no effect of TCDD treatment on plasma thyroid hormone levels. In herons, hepatic EROD activity was induced 2- to 3-fold above control birds, similar to EROD activities measured in heron hatchlings exposed to environmental levels of TCDD and related chemicals in the Strait of Georgia, British Columbia. However, this level of TCDD exposure had no effect on plasma thyroid hormone levels or body growth in herons. Collectively, these results suggest that perinatal plasma thyroid hormone levels cannot be used as relatively noninvasive biomarkers of TCDD exposure during embryonic development in chickens, pigeons, and great blue herons.

Levels of organochlorine compounds, including PCDDS and PCDFS, in the blubber of cetaceans from the west coast of North America

Levels of organochlorine compounds (PCDD, PCDF, PCB and organochlorine pesticides) were determined in cetaceans collected from the west coast of North America between 1986 and 1989. The samples included gray whale ( Eschrichtius robustus), killer whale ( Orcinus orca), false killer whale ( Pseudorca crassidens), Risso's dolphin ( Grampus griseus) and Dall's porpoise ( Phocoenoides dalli) collected in British Columbia, and harbour porpoises ( Phocoena phocoena) collected in British Columbia and central California. TCDD and TCDF levels ranged from 1 to 8 ng kg −1 and 2.0 to 109 ng kg −1, respectively. The highest levels of PCDDs were found in the harbour porpoises; the levels of 1,2,3,6,7,8-HxCDD in the samples from Victoria, Campbell River and Qualicum River were 128, 128 and 62 ng kg −1, respectively. Five other 2,3,7,8-substituted dioxins and dibenzofurans were detected in the cetaceans at levels ranging from 1 to 10 ng kg −1. In addition to the 2,3,7,8-substituted congeners, several non 2,3,7,8-substituted congeners were detected. The patterns of the PCDDs and PCDFs in the British Columbia porpoises were consistent with implication of chlorophenols as the source of the PCDDs and PCDFs, which were either present in wood chips used in bleached kraft paper mills, or came from direct contamination by chlorophenols. No PCDDs or PCDFs were detected in the California samples. One false killer whale sample had exceptionally high levels of DDT compounds (1700 mg kg −1 DDE, 120 mg kg −1 DDT and 40 mg kg −1 o,p′-DDT) and Toxaphene (89 mg kg −1). PCB levels in the cetaceans were highest in the false and killer whales (22 to 46 mg kg −1 GM), and lowest in the Risso's dolphin (1.7 mg kg −1). Levels of DDE in the British Columbia harbour porpoises were 6.0 mg kg −1, and probably reflect the accumulation of global background levels of DDE.

The need for cellular, biochemical, and mechanistic studies

The results of diverse in vitro neurotoxicity studies demonstrate that there are variations in cellular responsiveness between different types of neural cells. In contrast to experimental systems that have reported cellular responses to relatively high concentrations of various PCB congeners, our studies with rat hippocampal neural cells indicate that neurons and astroglia are responsive to relatively low levels of TCDD. However, these responses are probably not mediated through the classical Ah receptor pathway, which involves nuclear Ah receptor-mediated modulation of gene expression. It has recently been reported that TCDD-induced phosphorylation and other responses can be observed in some cell lines within minutes after treatment (20), and that cell membrane or cytosolic receptors may also play a role in mediating these effects. Future studies are required to determine both Ah receptor-dependent and -independent pathways associated with the neurotoxicity of PCBs, TCDD, and related compounds. The report that low-level dietary or background exposure to HAHs (32) results in neurobehavioral deficits is still a perplexing problem also requiring additional research and consideration of other dietary factors that may contribute to these effects. For example, we have recently been comparing the toxic effects and relative potencies of TCDD (exodioxins) and other “natural occurring” compounds (endodioxins) such as indole-3-carbinol (vegetables) and polynuclear aromatic hydrocarbons (PAHs, cooked foods), which also bind to the Ah receptor. Dr. Clynn Wilker has shown that in utero exposure of rats to indole-3-carbinol and chrysene (a PAH) cause demasculinization of the adult offspring as previously reported for TCDD (19). Thus, the neurotoxicity of low-level dietary exposure to HAHs should at least consider other possible confounding factors, including dietary endodioxins.

A Mechanistic Model of Effects of Dioxin on Thyroid Hormones in the Rat

A physiological dosimetric model of the disposition of TCDD in the rat (Kohnet al., Toxicol. Appl. Pharmacol.120,138–154, 1993) was extended to include effects of dioxin on serum concentrations of thyroid hormones in the rat. The extended model included distribution of blood among major vessels and tissue capillary beds and resorption of TCDD released into the gut lumen from the liver by cell lysis consequent to cytotoxicity. TCDD metabolism was represented by Hill kinetics. Parameter values were estimated by fitting time-course data for a single subcutaneous injection of TCDD and dose–response data for biweekly oral dosing. The extended model included new compartments for the thyroid and thyroxine-sensitive tissues (e.g., pituitary, kidney, and brown fat), secretion and tissue uptake of thyroid hormones, binding of 3,5,3′-triiodothyronine (T3) and 3,5,3′,5′-tetraiodothyronine (thyroxine, T4) to proteins in blood and tissues, deiodination of iodothyronines, and glucuronidation of T4by the hepatic UDP-glucuronosyltransferase (UGT) activity induced by TCDD. Secretion of thyroid hormones was modeled as regulated by thyrotropin (TSH), whose secretion was modeled as regulated by the hypothalamic factors thyrotropin releasing hormone and somatostatin. Release of the hypothalamic factors was modeled as under feedback control by the blood T4level. Induction of UGT was modeled as stimulated by the Ah receptor–TCDD complex. The extended model fit the observed dose–response of P450 isozymes and Ah and estrogen receptors following repeated oral doses with comparable accuracy as the earlier model. The fit to liver and fat TCDD levels following single and repeated oral and subcutaneous doses was improved over the earlier model. The revised model's predicted liver TCDD concentrations at very low doses were verified experimentally. The model reproduced the responses observed for blood T3, T4, and TSH after 31 weeks of biweekly oral dosing of rats with TCDD. The model also predicted responses ofUGTmRNA and UGT enzymatic activity comparable to those observed in TCDD-treated rats in experiments whose data were not used in constructing the model. Calculated increases in blood TSH levels are consistent with prolonged stimulation of the thyroid and may represent an early stage in the induction of thyroid tumors identified in previous two-year bioassays. Thus, increases in UGT activity may be useful as a biomarker for tumorigenic changes in hormone levels subsequent to TCDD exposure.

Morphometric abnormalities in brains of great blue heron hatchlings exposed in the wild to PCDDs.

Great blue heron hatchlings from colonies in the Strait of Georgia, British Columbia, Canada are being monitored for environmental contaminant exposure and effects by the Canadian Wildlife Service. The contaminants of concern are polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), primarily derived from kraft pulp mill effluent. The levels of PCDDs and PCDFs in eggs from the most contaminated colonies peaked in 1988 and 1989 and dropped dramatically through 1990 to 1992. Brains of heron hatchlings (taken as eggs from the wild and hatched in the laboratory) were analyzed for gross morphological abnormalities. Brains from highly contaminated colonies (Crofton, British Columbia and University of British Columbia Endowment Lands) in 1988 exhibited a high frequency of intercerebral asymmetry. The frequency of this abnormality decreased in subsequent years as the levels of TCDD and TCDD-TEQs (toxic equivalence factors) decreased. The asymmetry was significantly correlated with the level of TCDD and TCDD-TEQs in eggs taken from the same nest. Yolk-free body weight negatively correlated and the brain somatic index positively correlated with the TCDD level in such pair-matched eggs. These results indicate that gross brain morphology, and specifically intercerebral asymmetry, may be useful as a biomarker for the developmental neurotoxic effects of PCDDs and related chemicals.

Hepatic Microsomal Ethoxyresorufin O-Deethylase-Inducing Potency in Ovo and Cytosolic Ah Receptor Binding Affinity of 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Comparison of 4 Avian Species

The hepatic microsomal ethoxyresorufin O-deethylase (EROD)-inducing potency in ovo of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was determined in the domestic chicken (Gallus gallus), domestic pigeon (Columba livia), great blue heron, and double-crested cormorant. Dose-response curves were produced by injecting various doses of [3H]TCDD into the air sac of developing eggs during the latter third part of incubation. Hepatic EROD activities were measured in day-old hatchlings. Liver, yolk, and whole blood were analyzed for [3H]TCDD; no distributional differences among species were found. The ED50 for EROD induction was between one and two orders of magnitude lower in the chick (0.1 microgram/kg egg) than in the heron and cormorant (3-10 micrograms/kg egg). Consistent with this, the apparent affinity of TCDD for the hepatic cytosolic Ah receptor was about 15 times higher in the domestic chick (Kd = 0.75-1.6) than in the other avian species (pigeon, Kd = 11-14; heron, Kd = 10-20; cormorant, Kd = 12-16). Receptor binding affinities in the pigeon, heron, and cormorant were of the same order of magnitude as that reported for human placenta (D.K. Manchester, S.K. Gordon, C.L. Golas, E.A. Roberts, and A.B. Okey, 1987, Cancer Res. 47, 4861-4868). Subcutaneous edema was observed in TCDD-treated hatchlings of the chick, heron, and cormorant, but not of the pigeon, within the dose range examined. The laboratory dose-response relationships demonstrated that the heron and cormorant hatchlings that were exposed to TCDD and related chemicals in the Strait of Georgia (J.T. Sanderson, R.J. Norstrom, J.E. Elliott, L.E. Hart, K.M. Cheng, and G.D. Bellward (1994b) J. Toxicol. Environ. Health 41, 245-263; and J.T. Sanderson, J.E. Elliott, R.J. Norstrom, P.E. Whitehead, L.E. Hart, K.M. Cheng, and G.D. Bellward (1994a) J. Toxicol. Environ. Health 41, 435-450) had hepatic EROD activities at the lower end of the linear part of their respective dose-response curves. A further increase in levels of TCDD and related compounds in the environment would lead to a large increase in EROD activity and further increases in TCDD-induced toxicities, such as body weight loss and subcutaneous edema.

Induction of Cytochrome P450 Isoenzymes after Toxicokinetic Interactions between 2,3,7,8-Tetrachlorodibenzo- p-dioxin and 2,2′,4,4′,5,5′-Hexachlorobiphenyl in the Liver of the Mouse

Induction of Cytochrome P450 Isoeuzymes after Toxicokinetic Interactions between 2,3,7,8-Tetrachlorodibenzo- p-dioxin anti 2,2′,4,4′,5,5′-Hexachlorobiphenyl in the Liver of the Mouse. De Jongh, J., Devito, M., Nieboer, R., Birnbaum, L., and Van den Berg, M. (1995). Fundam. Appl. Toxicol. 25, 264-270. One group of male C57BL/6J mice received a single oral dose of 1 nmol 2,3.7,8-tetrachlorodibenzo- p-dioxin (TCDD)/kg. Six other groups received single oral doses of 100, 300, or 1000 μamol 2,2′,4,4′,5,5′-hexachlorobipheoyl (HxCB)/kg, alone or in combination with 1 nmol/kg TCDD. Liver deposition of both compounds was studied at Day 3 after dosage. Hepatic CYP1A1 and CYP1A2 protein levels and related 7-ethoxyresorufin-O-deethylation (EROD) and acetanilide 4-hydroxylation (ACOH) activities were also studied. A significant increase in the hepatic deposition of TCDD was observed in all three mixed dose groups but TCDD did not influence hepatic HxCB deposition. TCDD did increase both CYP1A1 and CYP1A2 protein levels. In the HxCB-treated groups, CYP1A2 levels were also increased in a dose-dependent way but CYP1A1 levels were not increased. CYP1A2 activities (ACOH), but not protein levels, in the TCDD groups cotreared with HxCB were higher than those in the group treated will TCDD alone. CYP1A1-dependent EROD activity and CYP1A2-dependent ACOH activity were induced in all treated dose groups. It is concluded that the present results do not confirm a direct role of CYP1A2 induction in the increase of hepatic TCDD levels by HxCB cotreatment in the mixed HxCB/TCDD dose groups. However, in this aspect, the discrepancy between CYP1A2 activities and protein levels remains to be explained.

Agent Orange and the Vietnamese: the persistence of elevated dioxin levels in human tissues.

The largest known dioxin contamination occurred between 1962 and 1970, when 12 million gallons of Agent Orange, a defoliant mixture contaminated with a form of the most toxic dioxin, were sprayed over southern and central Vietnam. Studies were performed to determine if elevated dioxin levels persist in Vietnamese living in the south of Vietnam. With gas chromatography and mass spectroscopy, human milk, adipose tissue, and blood from Vietnamese living in sprayed and unsprayed areas were analyzed, some individually and some pooled, for dioxins and the closely related dibenzofurans. One hundred sixty dioxin analyses of tissue from 3243 persons were performed. Elevated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) levels as high as 1832 ppt were found in milk lipid collected from southern Vietnam in 1970, and levels up to 103 ppt were found in adipose tissue in the 1980s. Pooled blood collected from southern Vietnam in 1991/92 also showed elevated TCDD up to 33 ppt, whereas tissue from northern Vietnam (where Agent Orange was not used) revealed TCDD levels at or below 2.9 ppt. Although most Agent Orange studies have focused on American veterans, many Vietnamese had greater exposure. Because health consequences of dioxin contamination are more likely to be found in Vietnamese living in Vietnam than in any other populations, Vietnam provides a unique setting for dioxin studies.

Induction of Cytochrome P450 Isoenzymes after Toxicokinetic Interactions between 2,3,7,8-Tetrachlorodibenzo-p-dioxin and 2,2′,4,4′,5,5′-Hexachlorobiphenyl in the Liver of the Mouse

One group of male C57BL/6J mice received a single oral dose of 1 nmol 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg. Six other groups received single oral doses of 100, 300, or 1000µmol 2,2′,4,4′,5,5′-hexachlorobiphenyl (HxCB)/kg, alone or in combination with 1 nmol/kg TCDD. Liver deposition of both compounds was studied at Day 3 after dosage. Hepatic CYP1A1 and CYP1A2 protein levels and related 7-ethoxyres-orufin-O-deethylation (EROD) and acetanilide 4-hydroxylation (ACOH) activities were also studied. A significant increase in the hepatic deposition of TCDD was observed in all three mixed dose groups but TCDD did not influence hepatic HxCB deposition. TCDD did increase both CYP1A1 and CYP1A2 protein levels. In the HxCB-treated groups, CYP1A2 levels were also increased in a dose-dependent way but CYP1A1 levels were not increased. CYP1A2 activities (ACOH), but not protein levels, in the TCDD groups cotreated with HxCB were higher than those in the group treated with TCDD alone. CYP1A1-dependent EROD activity and CYPlA2-dependent ACOH activity were induced in all treated dose groups. It is concluded that the present results do not confirm a direct role of CYP1A2 induction in the increase of hepatic TCDD levels by HxCB cotreatment in the mixed HxCB/TCDD dose groups. However, in this aspect, the discrepancy between CYP1A2 activities and protein levels remains to be explained.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induced alterations of pyruvate carboxylase levels and lactate dehydrogenase isozyme shifts in C57BL/6J male mice.

A dose-dependent reduction of hepatic pyruvate carboxylase levels and activity occurs in C57BL/6J male mice given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) i.p. in a corn oil carrier. The dose range was from 1 to 75 micrograms/kg body weight and the analysis was done 8 days postinjection. At the maximum TCDD level investigated, we found a 10-fold reduction in pyruvate carboxylase activity. Furthermore, TCDD at a dose of 1 microgram/kg body weight blocks corn oil induction of an increase in the amount of pyruvate carboxylase in liver protein extracts. At doses beyond those required to initiate a reduction in pyruvate carboxylase, lactate dehydrogenase isozyme patterns shift. This is accompanied by an increase in blood lactic acid levels. We propose that TCDD-mediated reduction in pyruvate carboxylase and lactate dehydrogenase isozyme shifts may represent a major component in TCDD toxicity.

Lymphodepletion of the thymus cortex in rats after single oral intubation of 2,3,7,8-tetrachlorodibenzo- p-dioxin

Atrophy of the thymus and thymic lymphodepletion occurs after exposure to sublethal doses of TCDD. Previous studies using high doses of TCDD (150 μg/kg) have indicated a preferential lymphodepletion of the thymus cortex on day 4 after a single oral intubation. Here we describe the effects of a single oral intubation of lower dose levels of TCDD (1, 5, and 25 μg/kg) on thymic weights and thymocyte subpopulations in Wistar rats on day 4 after exposure. A single administration of 1 μg/kg TCDD induced a significant reduction in the number of immature CD4 +CD8 + double-positive (DP) thymocytes. At this time point the numbers of mature CD3 high medullary thymocytes were not affected at any dose level tested. We conclude that also lower dose levels of TCDD induce thymic atrophy via a preferential lymphodepletion of the thymus cortex.

Relative Sensitivities of 2,3,7,8-Tetrachlorodibenzo- p-dioxin-Induced Cyp1a-1 and Cyp1a-2 Gene Expression and Immunotoxicity in Female B6C3F1 Mice

Relative Sensitivities of 2,3,7,8 Tetracholorodibenzo- p-dioxin-Induced Cypla-1 and Cypla-2 Gene Expression and Immunotoxicity in Female B6C3F1 Mice. Narasimhan, T. R., Craig, A., Arellano, L., Harper, N., Howie, L., Menache, M., Birnbaum, L., and Safe, S. (1994). Fundam. Appl. Toxicol. 23, 598-607. Improvements in risk assessment require better linkage of exposure to response by the determination or target tissue dose. The relative sensitivity of several responses in female B6C3F1 mice was compared on the basis of administered and target tissue dose spanning 3 orders of magnitude. Twenty-four hours after administration, [ 3H]TCDD was detected in the heart, spleen, kidney, uterus, thymus, lung, and liver and the highest concentrations were noted in the liver, uterus, and lung. At doses from 5 to 25 ng/kg, hepatic [ 3H]TCDD levels associated with the cytosolic and nuclear subcellular fractions increased from 12 to 62% of the total liver levels and then decreased at higher doses. At the two lowest doses used in the enzyme induction study, 5 and 10 ng/kg, the levels of specifically bound nuclear Ah receptor complex liganded with [ 3H]TCDD were 2.3 and 2.5 fmol/mg protein. Slightly higher levels of nuclear Ah receptor complex were observed at doses between 25 and 100 ng/kg (i.e., 3.6 to 4.2 fmol/mg protein) and a steep dose-dependent increase in nuclear Ah receptor levels was noted at doses of 500, 1000, and 5000 ng/kg (8.0, 39.3, and 92.8 fmol/mg protein, respectively). The dose-dependent effects of [ 3H]TCDD on hepatic Cyp1a-1 and Cyp1a-2 mRNA levels, ethoxyresorufin O-deethylase (EROD) activity, and the splenic antibody plaque-forming cell (PFC) response to sheep red blood cells were also determined; the latter response was determined 9 days after administrations of TCDD. Statistically significant induction of hepatic Cyp1a-1 was observed at lower doses (25 ng/kg) than any other marker, followed by induction of EROD and PFCs expressed per spleen or per 10 6 cells which was observed at 100 ng TCDD/kg and at higher doses. Cyp1a-2 was elevated significantly relative to control at doses ⩾ 1000 ng/kg. The ED50 value for PFCs/106; cells was the lowest of the variables analyzed and was not statistically significantly different from control (91 ± 92 ng/kg). A 500% increase in Cyp1a-2 and Cyp1a-1 mRNA levels was observed at doses of 736 ± 132 and 1630 ± 431 ng/kg, respectively. Due to variability in response in PFCs/spleen and the submaximal induction of EROD activity, ED50 values could not be calculated for these responses. The analyses indicate that the immunosuppressive response (when normalized for the number of spleen cells) may be depressed by administered doses as low as 90 ng TCDD/kg body weight. A 50% in crease in Cyp1a-1 or Cyp1a-2 was observed at higher administered doses (1630 or 736 ng/kg, respectively). This suggests that the immunosuppressive response is depressed at lower doses of TCDD than the other variables studied.

Elevated dioxin blood levels in Russian chemical workers and their children following maternal exposure

Elevated dioxin levels in children of female workers with elevated dioxin body burden following workplace exposure has not been previously reported. We previously reported elevation of polychlorinated dibenzo-p-dioxins, particularly 2,3,7,8-TCDD, in Russian male and female chemical production workers' blood. Exposure is thought to have occurred in the early 1960s and blood was drawn in 1991. (1) Among those with elevated dioxin levels were women with grown children. Since dioxins can be transferred transplacentally (2) and by nursing, (3,4) it was decided to see if a relationship existed between blood dioxin levels in mothers and their children. Individual samples were obtained from factory office workers, production workers and their now adult offspring. A pooled blood sample (n=100) from adult Ufa residents was obtained for comparison. Samples were analyzed for polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (CoPCBs). Office workers, production workers and workers' children 2,3,7,8-TCDD (TCDD) blood levels ranged from 11 to 273 parts per trillion (ppt) on a lipid basis, while the pooled blood sample (n=100) from adult Ufa residents had a TCDD level of 12 ppt. Elevated TCDD was detected in all adult children of female production workers tested while two adult children of a male production worker had dioxin levels similar to the general population of Ufa. All children of workers had been nursed by their mothers for the first year of life and none have worked at a chemical plant. The data suggest that relatively large amounts of TCDD were transferred from mother to child approximately 18 to 28 years prior to blood collection. A sample of 2,4,-D amine salt produced at this factory was also analyzed to determine if its dioxin congeners might be contributing to dioxin contamination. 2,3,7,8-TCDD was not detected in the 2,4-D (detection limit of 0.02 ppb), although other dioxins were present, including some with 2,3,7,8- chlorine substitution.

A Pharmacodynamically Responsive Model of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Transfer Between Liver and Fat at Low and High Doses

We have constructed a pharmacokinetic model for TCDD in the rat, emphasizing its transfer between plasma, lipid, and cytoplasmic compartments of the liver, white adipose tissue (WAT), and brown adipose tissue (BAT). Volumes of the lipid pools are controlled by a submodel of triglyceride (TG) metabolism and transport that responds via a receptor for TCDD in WAT cytoplasm with a Kd of about 2 nM. This submodel, and one for cytochrome P450IA2 induction, allowed us to simulate binding of TCDD to the induced P450IA2 binding sites at low doses (1 ng/kg to 10 μg/kg) independently of the decreased feed intake and hyperlipidemia associated with higher doses (20 to 120 μg/kg). In low-dose simulations, the induction of cytochrome P450IA2 binding sites for TCDD dominated the redistribution of TCDD between WAT and liver. When simulations were performed using a partitioning model with constant gastrointestinal flows, increased WAT lipolysis driven by reduced feed intake (from 10 to 120 μg/kg) is predicted to result in a decreased WAT volume and a sharp drop in the mass of WAT-associated TCDD, while initially increasing the levels of TCDD in liver. However, the observed concentration of TCDD in WAT increased in rats treated with a high dose (72 μg/kg) of TCDD. The rise in tissue concentrations could not be explained without incorporating decreased intestinal flows into the gastrointestinal absorption process, which increased the resorption of TCDD. TCDD concentrations in tissue increased only when the relative tissue volumes decreased more rapidly than the wholebody TCDD elimination rate.

Toxic Potency of 2,3,3′,4,4′,5-Hexachlorobiphenyl Relative to and in Combination with 2,3,7,8-Tetrachlorodibenzo- p-dioxin in a Subchronic Feeding Study in the Rat

Interactive effects on toxicity and biochemical parameters were studied between 2,3,3′4,4′5-hexachlorobiphenyl (PCB 156) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in a 13-week feeding study of female Sprague-Dawley rats. The diets were supplemented with PCB 156 (1.2, 6, or 12 mg/kg), with TCDD (5 μg/kg), or with combinations of both compounds. Estimated daily intake of 365 μg/kg body wt/day (6 mg/kg diet group) of PCB 156 caused a decrease in body weight gain, thymic atrophy, liver enlargement, a loss in hepatic retinoids, induction of CYP2B activity, and a decrease in plasma thyroxine concentrations. At an estimated daily intake of 81 μg PCB 156/ kg body wt/day CYP1A1 and CYP1A2 activities were induced. Compared to a simultaneous subchronic feeding study with TCDD a toxic equivalency factor (TEF) between 0.00004 and 0.001 was estimated for PCB 156 with respect to the mentioned effects. Antagonistic effects were found between TCDD and PCB 156 for CYP2B activity and hepatic retinol levels. These effects concurred with a PCB 156 dose-dependent decrease in hepatic TCDD levels. Hepatic PCB 156 levels were found to be increased at the 1.2-mg PCB 156/kg dose group in coadministration with TCDD. In conclusion, at least part of the antagonistic effects between PCB 156 and TCDD observed have a toxicokinetic base. Furthermore, the magnitude of the antagonistic effects may be neglected in comparison with the uncertainty in the TEF value. Therefore, the interactive effects found between PCB 156 and TCDD may have no implications for the additivity of the TEF concept.