Polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, pesticides, and diabetes in the Anniston Community Health Survey follow-up (ACHS II): single exposure and mixture analysis approaches.

Dioxins and dioxin-like compounds measurements were added to polychlorinated biphenyls (PCBs) and organochlorine pesticides to expand the exposure profile in a follow-up to the Anniston Community Health Survey (ACHS II, 2014) and to study diabetes associations. Participants of ACHS I (2005-2007) still living within the study area were eligible to participate in ACHS II. Diabetes status (type-2) was determined by a doctor's diagnosis, fasting glucose ≥125 mg/dL, or being on any glycemic control medication. Incident diabetes cases were identified in ACHS II among those who did not have diabetes in ACHS I, using the same criteria. Thirty-five ortho-substituted PCBs, 6 pesticides, 7 polychlorinated dibenzo-p-dioxins (PCDD), 10 furans (PCDF), and 3 non-ortho PCBs were measured in 338 ACHS II participants. Dioxin toxic equivalents (TEQs) were calculated for all dioxin-like compounds. Main analyses used logistic regression models to calculate odds ratios (OR) and 95 % confidence intervals (CI). In models adjusted for age, race, sex, BMI, total lipids, family history of diabetes, and taking lipid lowering medication, the highest ORs for diabetes were observed for PCDD TEQ: 3.61 (95 % CI: 1.04, 12.46), dichloro-diphenyl dichloroethylene (p,p'-DDE): 2.07 (95 % CI 1.08, 3.97), and trans-Nonachlor: 2.55 (95 % CI 0.93, 7.02). The OR for sum 35 PCBs was 1.22 (95 % CI: 0.58-2.57). To complement the main analyses, we used BKMR and g-computation models to evaluate 12 mixture components including 4 TEQs, 2 PCB subsets and 6 pesticides; suggestive positive associations for the joint effect of the mixture analyses resulted in ORs of 1.40 (95% CI: -1.13, 3.93) for BKMR and 1.32 (95% CI: -1.12, 3.76) for g-computation. The mixture analyses provide further support to previously observed associations of trans-Nonachlor, p,p'- DDE, PCDD TEQ and some PCB groups with diabetes.

An assessment of dioxin exposure across gestation and lactation using a PBPK model and new data from Seveso.

On July 10, 1976, an explosion at a chemical plant in Seveso, Italy, released up to 30kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-the most potent dioxin congener. Twenty years later, the Seveso Women's Health Study (SWHS) initiated a follow-up assessment of a cohort of female Seveso residents. Researchers collected serial blood, measured for TCDD levels, and recorded information about the women's medical history after the explosion. The study's aims were to: 1) modify the human PBPK model for TCDD (Emond et al. 2004; Emond et al. 2005; NCEA-USEPA, 2010) to include repetitive gestation and lactation; 2) simulate TCDD blood concentrations during different life stages including pregnancy and lactation, under different exposure scenarios; and 3) use this PBPK model to compare the influence of gestation and lactation on elimination of TCDD. After optimization of the model, it was assessed using data from the SWHS cohort. The 23 women in Subcohort A, were 4-39years old and in Subcohort B, the 18 women were 3-17years old when the explosion occurred. The model accurately predicted the blood concentrations during the 20years post-exposure, including periods of pregnancy and lactation. The model was also used to analyze the contribution of gestation and lactation to the mother's elimination of TCDD. The results suggest that gestation and lactation do not significantly impact TCDD blood elimination. Future efforts will focus on using additional data to evaluate the PBPK model and improving the mathematical descriptions of lactation and multiple gestations.

5th International PCB Workshop--summary and implications.

A summation of new and novel findings presented at "The Fifth PCB Workshop: New Knowledge Gained from Old Pollutants" workshop is provided in this overview, along with discussion of data gaps and research needs in the future. Relative to the previous workshop, the scientific presentations had a decreased emphasis on toxicology; rather, more than half of the sessions dealt with environmental sources, fate and transport, or transformations. Approximately 100 presentations in the form of talks and posters were included in the workshop. The presentations were generally divided into: emissions and transport of PCBs in natural and urban settings; chiral aspects of PCB transport; metabolism and distribution; new aspects of environmental metabolism of PCBs--from microbes to plants to animals; reproduction, developmental and cardiovascular effects of PCBs; updates on Anniston--the most highly exposed PCB community in the U.S. to date; and new and novel approaches for evaluating PCB mixtures (e.g., PCB toxic equivalency factors, and TEFs)--and the implications of such for risk assessment. An overarching state-of-the-science view is important to the goal of preventing negative health consequences. Currently, there are still many roadblocks to evaluating risk associated with this large group of 209 congeners--all of which have different physiochemical properties, variable fate and transport mechanism in the environment, and a range of ability for persistence, bioaccumulation, and biological activity.

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

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

Impact of repeated exposure on the toxicokinetics of BDE 47 in mice.

2,2',4,4'-Tetrabromodiphenyl ether (BDE 47) is the major polybrominated diphenyl ether (PBDE) found in environmental samples and human tissue despite its small contribution to global production and usage. Currently, three toxicokinetic studies are available investigating single-dose exposures; this is the first study to investigate toxicokinetic parameters following repeated exposure to BDE 47. The disposition and excretion of BDE 47 was monitored in adult female C57BL/6 mice for 5 days following ten consecutive 1.0-mg/kg oral doses and compared with results from our previous study. Results of the present study suggest greater retention of BDE 47 and nonlinear disposition patterns following repeated exposure to this dose in mice. No target tissues of sequestration or potential toxicity were determined; however, some tissues, such as the liver, demonstrated patterns of interest following repeated exposure that were not previously observed in acute toxicokinetic studies. Repeated exposure to BDE 47 results in higher concentrations remaining in adipose tissue, which demonstrates its potential for bioaccumulation. The data also suggest that excretion of BDE 47 may be decreased following repeated exposure. These results, in combination with evidence of its persistence and toxicity, underlie the need to further understand BDE 47 toxicokinetics across species at steady-state conditions.

Inhibition of human and rat CYP1A2 by TCDD and dioxin-like chemicals.

Dioxins have been shown to bind and induce rodent CYP1A2, producing a dose-dependent hepatic sequestration in vivo. The induction of CYP1A2 activity has been used as a noninvasive biomarker for human exposure to dioxins; while there is a consistent relationship between exposure and hepatic CYP1A2 induction in rodents, this relationship has only been observed in some of the highest exposed human populations. This may be explained by inhibition of CYP1A2 activity by dioxins as some rodent studies demonstrate that rodent CYP1A2 activity can in fact be inhibited by dioxins in vitro. CYP1A2 activity was examined using a series of dioxins to inhibit human and rat CYP1A2 activity in species-specific CYP1A2 SUPERSOMES using three common CYP1A2 substrates. Methoxyresorufin was a more efficient substrate than acetanalide or caffeine in this in vitro system. Rat and human CYP1A2 enzymatic activity is inhibited by TCDD, PCDD, TCDF, 4-PeCDF, and PCBs 126, 169, 105, 118, and 156 in a concentration-dependent manner. These data demonstrate that the in vitro metabolism of prototype substrates is similar between the rat and human CYP1A2 SUPERSOME preparations and that dioxins inhibit CYP1A2 activity in both species. Because of the potential for inhibition of CYP1A2 activity by TCDD and other dioxins, studies examining CYP1A2 induction in dioxin-exposed populations using these substrates should be viewed cautiously.

Toxicokinetics of BDE 47 in female mice: effect of dose, route of exposure, and time.

2,2',4,4'-Tetrabromodiphenyl ether (BDE 47) is present in commercial mixtures of polybrominated diphenyl ethers (PBDEs), which are used as flame retardants in a wide variety of consumer products. Despite its small contribution to PBDE global production and usage, BDE 47 is the major congener found in environmental samples and human tissue. No human data are currently available regarding the toxicokinetics of BDE 47 either as an individual congener or in the commercial mixture. Because previous studies have suggested potential toxicokinetic differences between rodent species, this study was conducted in an effort to fully characterize absorption, distribution, and excretion parameters following a single dose with respect to dose, time, and route of exposure in female C57BL/6 mice. Over 80% of the administered dose was absorbed after oral or intratracheal administration, whereas approximately 62% was absorbed when the dose was applied dermally. Disposition was dictated by lipophilicity as adipose and skin were major depot tissues. BDE 47 was rapidly excreted in the urine and feces. Of particular interest was the amount of parent compound found in the urine, which was a major factor in determining an initial whole-body half life of 1.5 days after a single oral exposure. Elimination, both whole-body and from individual tissues, was biphasic. Initial half-lives were 1-3 days, whereas terminal half-lives were much longer, suggesting the potential for bioaccumulation. This toxicokinetic behavior has important implications for extrapolation of toxicological studies to the assessment of health risk in humans.

CYP1A2 is not required for 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced immunosuppression.

One of the most sensitive and reproducible immunotoxic endpoints of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure is suppression of the antibody response to sheep red blood cells (SRBCs) in mice. Immunosuppression occurs in concert with hepatomegaly and associated induction of several hepatic cytochrome P450 enzymes, including CYP1A2 which is responsible for the hepatic sequestration of TCDD. In this study, TCDD-induced immunosuppression was evaluated in C57BL/6N CYP1A2 (+/+) wild-type and compared with that of age-matched CYP1A2 (-/-) knockout and CYP1A2 (+/-) heterozygous female mice. Groups of mice were given a single gavage dose of 0, 0.03, 0.1, 0.3, 1.0, 3.0 or 10.0microg TCDD/kg, followed 7 days later by immunization with SRBCs. Serum was obtained 5 days after immunization and body, spleen, thymus and liver weights were measured. sheep red blood cell (SRBC) antibody titers were determined by an enzyme-linked immunosorbent assay (ELISA). Anti-SRBC titers were suppressed at 1.0, 1.0 and 0.3microg TCDD/kg for CYP1A2 (+/+), CYP1A2 (+/-), and CYP1A2 (-/-) mice, respectively, which indicated a three-fold increase in TCDD-induced immunosuppression for the CYP1A2 (-/-) mice. This increase in TCDD-induced immunosuppression may be due to the inability of CYP1A2 (-/-) mice to sequester TCDD in the liver leading to a higher dose to the immune system. In CYP1A2 (+/+) mice, a dose of 3.0microg TCDD/kg was sufficient to increase the liver weight, while in CYP1A2 (-/-) mice no increase in liver weight was observed. Application of analysis of variance and dose-response modeling approaches indicate that there is little evidence that the immunosuppression dose-response curves, for the three strains, differ in the lower part of the dose-response range. Thus, CYP1A2 is not required for TCDD-induced immunosuppression in the mouse.

A mixture of dioxins, furans, and non-ortho PCBs based upon consensus toxic equivalency factors produces dioxin-like reproductive effects.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and related polyhalogenated aromatic hydrocarbons (PHAHs) alter the reproductive development of laboratory animals. Therefore, we exposed animals to a mixture of dioxins, furans, and polychlorinated biphenyls (PCBs) that included TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), octachlorodibenzofuran (OCDF), 3,3',4,4'-tetrachlorobiphenyl (PCB77), 3,3',4,4',5-pentachlorobiphenyl (PCB126), and 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169). The mixture composition approximated the relative abundance of these compounds in foodstuff (L. S. Birnbaum and M. J. DeVito, 1995, Toxicology Vol. 105, pp. 391-401). Following the work of Gray et al. with TCDD (1997, Toxicology and Applied Pharmacology Vol. 146, pp. 11-20), we exposed time-pregnant dams on gestation day (GD) 15 at doses up to 1.0 microgram TCDD toxic equivalency (TEQ)/kg and the development of offspring was monitored. This mixture significantly increased the time to puberty in both male and female offspring. At postnatal day (PND) 32 seminal vesicle weights were decreased; however, only ventral prostate weight was affected at PND 49 and no effects were seen at PND 63. In female offspring, the mixture caused dose-dependent increases in the incidence of vaginal thread. Ethoxyresorufin-O-deethylase (EROD) activity was higher than with TCDD the comparable TEQ exposure. Based on the slightly lowered responsiveness to the mixture, we used 2.0 microgram TEQ/kg to examine reproductive effects. This dose elicited the responses observed with 1.0 microgram TCDD/kg. Results indicate that the mixture causes a similar spectrum of effects seen with TCDD and the slightly lowered degree of response based on administered dose appears to be due to decreased transfer of mixture components to the offspring. Thus, the use of the WHO consensus TEFs (M. Van den Berg et al., 1998, Environ. Health Perspec. 106, 775-792) reasonably predicts the developmental toxicity of this mixture of dioxin-like PHAHs.

TCDD-mediated oxidative stress in male rat pups following perinatal exposure.

2,3,7,8-tetrachlorododibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known. Exposure to TCDD has been shown to cause oxidative stress in a variety of animal models. In this study, pregnant Long Evans rats were dosed with 1 microg TCDD/kg on gestational day (GD) 15 so as to investigate oxidative stress in the liver of male pups following gestational exposure to TCDD. Lipid peroxidation (TBARS), production of reactive oxygen species (ROS), and total glutathione (GSH) were assayed to identify changes in oxidative stress parameters in the pup liver at GD 21 and postnatal days (PND) 4, 25, 32, 49, and 63. Mean ROS levels in pups were elevated at all time points tested with a significant elevation at PND 4 and PND 25. However, pup hepatic lipid peroxidation was unchanged throughout the time course. In addition, hepatic total GSH levels were not significantly changed although the means for the TCDD-treated groups were less than those of the controls at all time points except PND 49. The results indicate that although the levels of ROS are increased following gestational/lactational exposure, this increase does not translate to direct oxidative damage or significant changes to endogenous antioxidant defense mechanisms. Further investigation into the effect of gestational/lactational exposure in pups should include additional endpoints for further characterization of the time course of the response, the effect upon extrahepatic tissues, and investigation of differences between male and female offspring.

Differential effects of two lots of aroclor 1254: congener-specific analysis and neurochemical end points.

Aroclor 1254 is a widely studied commercial polychlorinated biphenyl (PCB) mixture which, by definition, contains 54% chlorine by weight. Recent reports indicate substantial differences in the congener composition among Aroclor lots and hence their biologic effects. We designed the current study to compare the effects of two lots of Aroclor 1254 (lots 6024 and 124-191). We analyzed these two lots for PCB congeners, polychlorinated dibenzofurans (PCDFs), polychlorinated naphthalenes (PCNs), and polychlorinated dibenzodioxins (PCDDs). We used previously established techniques for analyzing intracellular Ca(2+) buffering and protein kinase C (PKC) translocation to test their biologic activity in neuronal preparations. PCB congener-specific analysis indicated that ortho and non-ortho congeners in these two lots varied in their percent contribution. Among all congeners, the percentages of non-ortho congeners (PCBs 77, 81, 126, and 169) were higher in lot 6024 (2.9% of total) than in lot 124-191 (0.02% of total). We detected no dioxins in these two lots (< 2 ppb). Although there are some differences in the congener composition, total PCNs were similar in both lots: 171 ppm in lot 6024 and 155 ppm in lot 124-191. However, total PCDFs were higher in lot 6024 (38.7 ppm) than in lot 124-191 (11.3 ppm). When we tested these two Aroclors on Ca(2+) buffering and PKC translocation in brain preparations, the effects were significantly different. Although lot 124-191 was more potent on PKC translocation than lot 6024, lot 6024 was slightly more active on Ca(2+) buffering than lot 124-191. These effects could not be attributed to the differences in the percentage of non-ortho congeners or PCDFs because they were inactive on these two parameters. The effects could not be attributed to PCNs because the levels were almost similar. The effects seen with two lots of Aroclor 1254 in neuronal cells were also not predicted based on the TCDD toxic equivalents (TEQs), although TEQs predicted the effects on ethoxyresorufin-O-deethylase (EROD) or methoxyresorufin-O-deethylase (MROD) activities. It is possible that the differential effects seen in neuronal cells could be caused by differences in the composition of ortho-congeners in these two mixtures, because PCBs with ortho-lateral substitutions can exhibit different activities on the selected neurochemical end points. Because of these differential effects with different lot numbers, the composition of Aroclor mixtures used in investigations should be disclosed.

Differential effects of two lots of aroclor 1254 on enzyme induction, thyroid hormones, and oxidative stress.

Aroclor 1254 is a commercial mixture of polychlorinated biphenyls (PCBs), which is defined as being 54% chlorine by weight. However, the congener composition varies from lot to lot. Two lots which have been used in toxicity studies, 124-191 and 6024 (AccuStandard), were analyzed for their congener composition. Lot 6024 has approximately 10 times the dioxin toxic equivalents (TEQ) of lot 124-191. The purpose of this study was to determine if the difference in the TEQ of the two lots explains the different in vivo responses seen on a weight basis. Male Long-Evans rats (70 days old) were treated orally with a single dose of 0-1,000 mg/kg of each lot. Hepatic ethoxy-, methoxy-, and pentoxyresorufin O-deethylase (EROD, MROD, and PROD, respectively) activities as well as serum thyroxine (T(4)) concentrations and measures of oxidative stress were determined 4 days after treatment. Results, on a weight basis, indicate that lot 6024 led to a greater induction of EROD, MROD, and PROD but not total T(4) reduction. The differences in TEQ between the lots explained the differential induction of EROD and MROD but did not account for the induction of PROD nor decreases in T(4). PROD induction is not due to dioxin-like congeners, whereas the decrease in serum T(4) levels may involve multiple mechanisms. Effects on the antioxidants ascorbic acid and uric acid were seen only at the highest mass dose for both lots and were not explained by the difference in TEQ. These results illustrate that the differences in the TEQ explain the differences in the strict dioxin-like effects (EROD, MROD induction), but the non-dioxin-like congeners cause other effects that are not associated with the aryl hydrocarbon receptor (e.g., PROD). In addition, supra-additive effects also occur in the mixture (T(4), oxidative stress). Thus, current results demonstrate that overall toxicity cannot be predicted on the basis of the TEQ values. It is also critical that the lot number is reported in studies conducted with Aroclor 1254 because the congener composition and therefore the effects observed can be very different.

Disposition of polychlorinated dibenzo-p-dioxins, dibenzofurans, and non-ortho polychlorinated biphenyls in pregnant long evans rats and the transfer to offspring.

Pharmacokinetic properties of polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDFs), and non-ortho biphenyls (PCBs) play a critical role in their relative toxicity. The present study examined the transfer of these chemicals to offspring and placenta. Pregnant Long Evans rats received 0.0 (control), 0.05, 0.2, 0.8, and 1.0 microg/kg of dioxin toxic equivalence (TEQ) by oral gavage on the 15th gestational day (GD 15), using a dosing mixture that contained 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), octachlorodibenzofuran (OCDF), 3,3',4,4'-tetrachlorobiphenyl (PCB 77), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169) in ratios approximating that in food. Rats were euthanized on GD 16, GD 21, and postnatal day 4 (PND 4). The chemical concentrations in fetus, pup, placenta, and maternal liver, serum, and adipose tissue were determined using gas chromatography/high-resolution mass spectrometry. A dose-dependent increase in hepatic sequestration was seen with TCDD, PeCDD, 4-PeCDF, OCDF, PCB 126, and PCB 169, and the transfer to offspring was reduced at higher doses. 4-PeCDF, PeCDD and PCB 126 showed higher liver affinity than TCDD. TCDF, 1-PeCDF, and PCB 77 were metabolized rapidly. On GD 16, TCDD and the three PCBs reached equilibration between the fetus and placenta, but this did not occur with PeCDD and 4-PeCDF until GD 21, according to the lipid-based concentrations. Offspring compartments received more of the dosed compounds lactationally than transplacentally (7-28% versus 0.5-3%). The behavior of each congener was dose-dependent; therefore, extrapolation of high-dose experimental data should be used with caution.

Subchronic Exposure of [3H]- 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in female B6C3F1 mice: relationship of steady-state levels to disposition and metabolism.

The present study of subchronic low exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at or near steady-state levels tries to emulate the most probable mode for human exposure, dietary consumption. This study is the first and most intensive pharmacokinetic study to be reported with repeated dosing, multiple times, and multiple doses examining disposition of TCDD-derived radioactivity and CYP1A activities in mice. For time-course relationships, animals were dosed (daily, Monday-Friday) with 0, 1.5, or 150 ng [3H]TCDD/kg for 4, 8, 13, or 17 weeks and also for 13 weeks followed by 4 weeks with no dosing. For dose-response relationships, animals were dosed for 13 weeks (daily, Monday-Friday) with 0, 0.15, 0.45, 1.5, 4.5, 15, 45, 150, or 450 ng [3H]TCDD/kg. Additional animals dosed for 13 weeks (daily, Monday-Friday) with 1.5 or 150 ng [(3)H]TCDD/kg were housed in metabolism cages. Time- and dose-dependencies of TCDD were confirmed in all measured tissues. Liver/fat (L/F) concentration ratios ranged from 0.2-3.4 (low to high dose). Hepatic CYP1A1 enzymatic activity increased (p < 0.05) starting at 0.15 ng/kg/day with L/F of 0.2 and body burden of 2.8 ng TCDD/kg body weight. By examining TCDD exposures at or near steady state, this study reports for the first time and provides direct evidence of low-dose effects on a measured reversible response at body burdens that are within background levels of the general human population. In addition, this study emphasizes cumulative effects of daily dosing and suggests the importance of tissue dosimetry or body burden for a persistent chemical such as TCDD.

Tissue disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in maternal and developing long-evans rats following subchronic exposure.

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces alterations in the reproductive system of the developing pups. The objective of this study was to determine the disposition of TCDD in maternal and fetal Long-Evans (LE) rats following subchronic exposure, since the adverse reproductive and developmental effects have been extensively characterized in this strain of rat. LE rats were dosed by gavage with 1, 10, or 30 ng [(3)H]TCDD/kg in corn oil, 5 days/week for 13 weeks. At the end of 13 weeks, females were mated and dosing continued every day throughout gestation. Dams were sacrificed on gestation day (GD) 9, GD16, GD21, and post-natal day 4 and analyzed for [(3)H]TCDD-derived activity in maternal and fetal tissues. Maternal body burdens were equivalent at different time points, indicating that the dams were at steady state. Maternal body burdens were approximately 19, 120, and 300 ng TCDD/kg following doses of 1, 10, and 30 ng TCDD/kg, respectively. Individual embryo concentrations on GD9 were 1.6, 7, and 16 pg TCDD/g after maternal exposure of 1, 10, and 30 ng/kg/d, respectively. On GD 16, fetal liver, urogenital tract, head, and body concentrations were similar and averaged 1.4, 7.8, and 16.4 pg TCDD/g after administration of 1, 10, or 30 ng TCDD/kg/d, respectively, indicating no preferential sequestration within the different fetal tissues. These concentrations of TCDD within fetal tissues after subchronic exposure are comparable to those seen after a single dose of 50, 200, or 1000 ng TCDD/kg administered on GD15, a critical period of gestation.

Dose-response relationships for induction of CYP1A1 and CYP1A2 enzyme activity in liver, lung, and skin in female mice following subchronic exposure to polychlorinated biphenyls.

The Toxic Equivalency Factor (TEF) method is used to estimate potential health risks associated with exposure to dioxin-like chemicals. The TEF method is a relative potency (REP) scheme that assumes dose additivity, that the chemicals produce the same response through the same mechanism, and that the REP of a chemical is equivalent for all responses. The present study estimates the REP for five PCBs with dioxin-like activity. Mice were exposed to either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,3',4, 4'-pentachlorobiphenyl (105), 2,3',4,4',5-pentachlorobiphenyl (118), 3,3',4,4',5-pentachlorobiphenyl (126), or 2,3,3',4,4'-, 5-hexachlorobiphenyl (156), five days/week for 13 weeks by oral gavage in a corn oil vehicle. Three days after the last dose, animals were euthanized and the ethoxyresorufin-O-deethylase activity was determined in liver, lung, and skin. Acetanilide-4-hydroxylase activity was determined in liver. In addition, liver and skin disposition of the chemicals were determined. REPs were estimated using a statistical method previously described (DeVito et al., Toxicol. Appl. Pharmacol.147, 267-280, 1997). For any given compound, the REP generally varied by less than a factor of four across endpoints when calculated based on an administered dose. However, typically there was one response for every chemical in which the REP was different by an order of magnitude or more from the other responses. There was some evidence that the REPs may be dose-dependent. While, in general, these data support the use of a single point estimate of the REP, the issue of dose-dependency requires targeted investigation.

Non-carcinogenic effects of TCDD in animals.

Exposure to TCDD and related chemicals leads to a plethora of effects in multiple species, tissues, and stages of development. Responses range from relatively simple biochemical alterations through overtly toxic responses, including lethality. The spectrum of effects shows some species variability, but many effects are seen in multiple wildlife, domestic, and laboratory species, ranging from fish through birds and mammals. The same responses can be generated regardless of the route of exposure, although the administered dose may vary. The body burden appears to be the most appropriate dosimetric. Many of the effects often attributed to TCDD are associated with relatively high doses: lethality, wasting, lymphoid and gonadal atrophy, chloracne, hepatotoxicity, adult neurotoxicity, and cardiotoxicity. Changes in multiple endocrine and growth factor systems have been reported in a manner which is tissue, sex, and age-dependent. The most sensitive adverse effects observed in multiple species appear to be developmental, including effects on the developing immune, nervous, and reproductive systems. Such effects have been observed at maternal body burdens in the range of 30-80 ng/kg in both non-human primates and rodents. Biochemical effects on cytokine expression and metabolizing enzymes occur at body burdens which are within a factor of ten of the clearly adverse developmental responses. Thus, effects on the immune system, learning, and the developing reproductive system of multiple animals occur at body burdens which are close to those present in the background human population.

Extrapolation of a PBPK model for dioxins across dosage regimen, gender, strain, and species.

A physiologically based pharmacodynamic (PBPK) model for 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was developed based on pharmacokinetic data from acute oral exposures of TCDD to female Sprague-Dawley rats (Wang et al., 1997, Toxicol Appl. Pharmacol 147, 151-168). In the present study, the utility of this model to predict the disposition of TCDD in male and female Sprague-Dawley and female Wistar rats exposed to TCDD through different dosage regimens was examined. The ability of the model to predict the disposition of 2-iodo-3,7,8-trichlorodibenzo-p-dioxin (ITrCDD) in mice (Leung, et al., 1990, Toxicol. Appl. Pharmacol. 103, 399-410) was also examined. The ability of the model to predict across routes of exposure was assessed with intravenous injection data (5.6 microg/kg bw) (Li et al., 1995, Fundam. Appl. Toxicol. 27, 70-76) in female rats. Analysis across gender extrapolations used data for male Sprague-Dawley rats exposed intravenously to 9.25 microg TCDD/kg bw (Weber et al., 1993, Fundam. Appl. Toxicol. 21, 523-534). The analysis of across-dosage regimen and stains of rats extrapolations were assessed using data from rats exposed to TCDD through a loading/maintenance dosage regimen (Krowke et al., 1989, Arch. Toxicol. 63, 356-360). The physiological differences between gender, strain, and species were taken into account when fitting the PBPK model to these data sets. The results demonstrate that the PBPK model for TCDD developed for female Sprague-Dawley rats exposed by acute oral dosing accurately predicts the disposition of TCDD, for different gender and strain of rats across varying dosage regimens, as well as in a strain of mice. Minimal changes in fitted parameters were required to provide accurate predictions of these data sets. This study provides further confirmation of the potential use of physiological modeling in understanding pharmacokinetics and pharmacodynamics.

Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known to man. Hassoun et al. (1998, Toxicol. Sci. 42, 23-27) reported an increase in the production of reactive oxygen species (ROS) in the brain of female B6C3F1 mice following subchronic exposure to TCDD at doses as low as 0.45 ng/kg/day. In the present study, oxidative stress was characterized in liver, spleen, lung, and kidney following subchronic (0.15-150 ng/kg; 5 days/week for 13 weeks, po) or acute exposure (0.001-100 microg/kg, po) to TCDD in order to investigate the interaction between tissue concentration and time for production of ROS. Seven days following acute administration of TCDD, mice were sacrificed; they demonstrated increases in liver superoxide anion production (SOAP) and thiobarbituric acid reactive substances (TBARS) at doses of 10 and 100 microg/kg, associated with hepatic TCDD concentrations of 55 and 321 ng/g, respectively. Liver obtained from mice following subchronic TCDD exposure demonstrated an increase in SOAP and TBARS above controls at doses of 150 ng/kg/day with liver TCDD concentration of only 12 ng/g. Interestingly, glutathione (GSH) levels in lung and kidney following sub-chronic TCDD exposure were decreased at the low dose of 0.15 ng/kg/day. This effect disappeared at higher TCDD doses. The data suggest that higher tissue TCDD concentrations are required to elicit oxidative stress following acute dosing than with subchronic TCDD exposure. Therefore, the mechanism of ROS production following TCDD exposure does not appear to be solely dependent upon the concentration of TCDD within the tissue. In addition, very low doses of TCDD that result in tissue concentrations similar to the background levels found in the human population produced an effect on an oxidative stress endogenous defense system. The role of this effect in TCDD-mediated toxicity is not known and warrants further investigation.