Janet J. Diliberto

Effects of Perinatal PBDE Exposure on Hepatic Phase I, Phase II, Phase III, and Deiodinase 1 Gene Expression Involved in Thyroid Hormone Metabolism in Male Rat Pups

Previous studies demonstrated that perinatal exposure to polybrominated diphenyl ethers (PBDEs), a major class of brominated flame retardants, may affect thyroid hormone (TH) concentrations by inducing hepatic uridinediphosphate-glucoronosyltransferases (UGTs). This study further examines effects of the commercial penta mixture, DE-71, on genes related to TH metabolism at different developmental time points in male rats. DE-71 is predominately composed of PBDE congeners 47, 99, 100, 153, 154 with low levels of brominated dioxin and dibenzofuran contaminants. Pregnant Long-Evans rats were orally administered 1.7 (low), 10.2 (mid), or 30.6 (high) mg/kg/day of DE-71 in corn oil from gestational day (GD) 6 to postnatal day (PND) 21. Serum and liver were collected from male pups at PND 4, 21, and 60. Total serum thyroxine (T(4)) decreased to 57% (mid) and 51% (high) on PND 4, and 46% (mid) dose and 25% (high) on PND 21. Cyp1a1, Cyp2b1/2, and Cyp3a1 enzyme and mRNA expression, regulated by aryl hydrocarbon receptor, constitutive androstane receptor, and pregnane xenobiotic receptor, respectively, increased in a dose-dependent manner. UGT-T(4) enzymatic activity significantly increased, whereas age and dose-dependent effects were observed for Ugt1a6, 1a7, and 2b mRNA. Sult1b1 mRNA expression increased, whereas that of transthyretin (Ttr) decreased as did both the deiodinase I (D1) enzyme activity and mRNA expression. Hepatic efflux transporters Mdr1 (multidrug resistance), Mrp2 (multidrug resistance-associated protein), and Mrp3 and influx transporter Oatp1a4 mRNA expression increased. In this study the most sensitive responses to PBDEs following DE-71 exposure were CYP2B and D1 activities and Cyb2b1/2, d1, Mdr1, Mrp2, and Mrp3 gene expression. All responses were reversible by PND 60. In conclusion, deiodination, active transport, and sulfation, in addition to glucuronidation, may be involved in disruption of TH homeostasis due to perinatal exposure to DE-71 in male rat offspring.

The Acquisition and Application of Absorption, Distribution, Metabolism, and Excretion (ADME) Data in Agricultural Chemical Safety Assessments

A proposal has been developed by the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) for an improved approach to assessing the safety of crop protection chemicals. The goal is to ensure that studies are scientifically appropriate and necessary without being redundant, and that tests emphasize toxicological endpoints and exposure durations that are relevant for risk assessment. Incorporation of pharmacokinetic studies describing absorption, distribution, metabolism, and excretion is an essential tool for improving the design and interpretation of toxicity studies and their application for safety assessment. A tiered approach is described in which basic pharmacokinetic studies, similar to those for pharmaceuticals, are conducted for regulatory submission. Subsequent tiers provide additional information in an iterative manner, depending on pharmacokinetic properties, toxicity study results, and the intended uses of the compound.

Disposition of 2,3,7,8-tetrabromodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat: Biliary excretion and induction of cytochromes CYP1A1 and CYP1A2

The biologic activity and pharmacokinetic properties of 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) are similar to those of the chlorinated congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Metabolism of both compounds appears to be rate-limiting for excretion, which is primarily via the feces. Therefore, the biliary elimination of TBDD and TCDD was examined as an indirect assessment of metabolism. Male F344 rats were anesthetized with pentobarbital, and 1 nmol/kg [3H]TBDD or [3H]TCDD was administered iv. Bile was collected for up to 8 hr while rats were maintained under anesthesia. The rate of biliary excretion of radioactivity was slightly greater for TCDD than TBDD (10% vs 7% in 5 hr). All biliary radioactivity was attributable to metabolites. High pressure liquid chromatographic (HPLC) profiles of biliary radioactivity were similar for [3H]TBDD and [3H]TCDD. To determine if pretreatment altered elimination kinetics, a single dose of 100 nmol/kg TBDD or TCDD was administered to rats by oral gavage 3 days prior to iv injection of 1 nmol/kg [3H]TBDD or [3H]TCDD, respectively. Biliary excretion of the radiolabeled dose was quantitatively and qualitatively unaffected by pretreatment despite a twofold increase in hepatic levels of radiolabel in the pretreated animals. Therefore, under these experimental conditions, autoinduction of TCDD and TBDD metabolism did not occur in the rat in vivo at doses which elicited enhanced hepatic uptake. In a second set of studies, the dose-response profiles for induction of cytochromes CYP1A1 and CYP1A2 by TBDD were characterized. The ED50 value for CYP1A1 induction (measured by ethoxyresorufin O-deethylase activity and radioimmunoassay (RIA) was estimated to be 0.8-1.0 nmol/kg, similar to what has been reported for TCDD. Induction of CYP1A2 (RIA) by TBDD appeared to be a more sensitive response over the dose range studied. Finally, comparison of hepatic CYP1A2 induction vs hepatic concentrations of TBDD 3 days following treatment with 10 vs 1 nmol/kg TBDD suggested that induction of CYP1A2 alone may not account for nonlinearities in dioxin disposition exemplified by dose-related increases in the ratio of dioxin concentrations in liver and adipose tissue.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters embryonic palatal medial epithelial cell differentiation in vitro

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, inducing cleft palate and hydronephrosis. After exposure in vivo, TCDD specifically alters differentiation of embryonic palatal medial epithelial cells. In this study, the palatal epithelial cell response to TCDD is determined in vitro. C57BL/6N palatal shelves were placed in organ culture on gestation day (GD) 12 in Richters improved modified Eagles medium:Hams F12 medium (1:1) with 1% fetal bovine serum for 3 or 4 days. Medium contained 0.1% dimethylsulfoxide and TCDD at 0, 10(-13), 10(-12), 10(-11), 10(-10), and 10(-9) M, with some doses at 5 x 10(-11), 7.5 x 10(-11), and 5 x 10(-12) M. Epithelial cell responses to TCDD occurred over a narrow range of concentrations, with maximal response at 5 x 10(-11) M. Cytotoxicity was detected at 1 x 10(-10) M. At a stage when control medial cells ceased proliferation and EGF receptors were not detected immunohistochemically. TCDD-exposed medial cells incorporated [3H]thymidine and high levels of epidermal growth factor receptors were detected. TCDD prevented programmed cell death of medial peridermal cells, and induced a shift in the differentiation of medial cells toward an oral-like phenotype. The responses to TCDD observed after exposure in vitro were indistinguishable from previously reported effects observed after exposure in vivo. In the present study, the distribution of TCDD in the fetus after exposure in vivo was examined. The levels of exposure to TCDD are similar for in vitro and in vivo exposure routes. The levels of TCDD in 1 x 10(-11) to 1 x 10(-10) M solutions (3 to 32 pg/ml) were comparable to levels observed in fetal tissues after in vivo exposure on GD 11 to 30 microns/kg [3H]TCDD, where the palatal shelf contained 1.4 to 3.5. pg TCDD, representing 0.0003% of the total dose. In vivo, TCDD was detected in the GD 11 embryo 3 hr postexposure and the TCDD was equally distributed between the embryonic head and body. At 72 hr postexposure, 0.035% of the total dose was in fetal tissues, and 1% of the TCDD in the fetus was found in the palatal shelf. The present study shows that the palatal epithelium responds to TCDD in vitro in a manner comparable to that observed after in vivo exposure, and that the response occurs at a concentration comparable to in vivo levels in the fetus. The availability of an in vitro system will facilitate studies of TCDD toxicity that are difficult or impossible to perform in vivo, such as comparisons of TCDD effects between species, including human tissues.

The effect of dose on 2,3,7,8-TCDD tissue distribution, metabolism and elimination in CYP1A2 (-/-) knockout and C57BL/6N parental strains of mice.

Numerous metabolism studies have demonstrated that the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver-to-fat concentration ratios. This study was initiated to determine whether TCDD was inherently poorly metabolized or unavailable for metabolism because of sequestration to CYP1A2. [(3)H]TCDD was administered as a single, oral dose (0.1 and 10 microg/kg) to 12 male C57BL/6N mice or 12 CYP1A2 (-/-) mice. At 96 h, less than 5% of the dose was eliminated in the urine of all groups, and TCDD detected in urine was bound to mouse major urinary protein (mMUP). Feces were the major elimination pathway (24-31% of dose), and fecal extracts and non-extractables were quantitated by HPLC for metabolites. No great differences in urinary or fecal elimination (% dose) were observed between the high and low dose treatments. TCDD concentrations were the highest in adipose tissue for CYP1A2 knockout mice but in liver for C57BL/6N mice supporting the role of hepatic CYP1A2 in the sequestration of TCDD. Overall metabolism between parental and knockout strains showed no statistical differences at either the high or low doses. The data suggested that metabolism of TCDD is inherently slow, due principally to CYP1A1, and that hepatic CYP1A2 is not an active participant in the metabolism of TCDD in male mice. Rather, CYP1A2 governs the pharmacokinetics of TCDD by making it unavailable for hepatic CYP1A1 through sequestration and attenuating extrahepatic tissue disposition.

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.

Disposition and excretion of intravenous 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) in rats

Polybrominated dibenzodioxins and dibenzofurans are of toxicologic interest due to potential occupational and environmental exposure and because of their structural similarity to the highly toxic chlorinated analogues. The excretion and terminal tissue distribution of [3H]TBDD was studied in male F344 rats for 56 days following single iv doses of .001 or 0.1 mumol/kg. The major tissue depots of radioactivity were liver, adipose tissue, and skin, and tissue distribution was dose-dependent. At 56 days, liver concentrations in the high dose group were disproportionately increased compared to those of the low dose group. Liver:adipose tissue concentration ratios were 0.2 and 2.6 at the low and high doses, respectively. Elimination of radioactivity in the feces, the major route of excretion, and urine was also nonlinear with respect to dose. By Day 56, feces accounted for approximately 50% of the administered dose at the low dose versus 70% at the high dose. Based on fecal excretion, the apparent terminal whole body half-life was estimated to be 18 days for both dose groups. The time-dependent pattern of tissue disposition was characterized at the low dose over a 56-day period. Blood levels of radioactivity declined rapidly with 2% remaining in the blood by 24 hr. Radioactivity levels in the liver peaked by 7 hr and then gradually declined concomitant with a slow accumulation in adipose tissue. The terminal excretion half-life of radioactivity in adipose tissue was estimated to be 60 days. Liver:adipose tissue concentration ratios declined with time. Thus, the overall disposition of TBDD appears similar to that observed for the chlorinated analogue, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results of these studies are consistent with the hypothesis that TBDD, like TCDD, induces a binding species in the liver which accounts for higher liver:adipose tissue concentration ratios at the high dose. The dose-dependent tissue disposition and excretion kinetics of these compounds suggest important considerations for extrapolations from high to low doses.

Chemical characterization and disposition studies with 1,2,7,8‐tetrabromodibenzofuran in the rat

Polybrominated dibenzo-p-dioxins and dibenzofurans have been identified as potential environmental contaminants. The present studies were designed to characterize the chemical disposition of a tetrabrominated dibenzofuran. The isomer-specific pattern of 1,2,7,8-tetrabromodibenzofuran (TBDF) was chemically characterized using high-pressure liquid chromatography, gas chromatography/mass spectrometry, infrared absorption, and proton nuclear magnetic resonance techniques. The absorption, distribution, and elimination of 1,2,7,8-[4,6-3H]-TBDF were examined in the rat following a single oral, dermal, or intravenous dose of 1 nmol/kg. The 1,2,7,8-TBDF was rapidly excreted in the bile (approximately 50% of the dose in 8 h). Likewise, over half of the administered dose was found in the feces and intestine contents 24 h after iv administration and in feces 72 h after oral administration. Thus, the half-life of 1,2,7,8-TBDF is approximately 1 d. Major tissue depots included the liver, adipose tissue, and skin. The decline in hepatic concentrations observed in the iv and bile studies occurred in conjunction with metabolic elimination as well as a slight accumulation in adipose tissue. Dermal absorption of 1,2,7,8-TBDF, quantified as the amount contained in tissues (excluding the skin site) and excreta at 72 h, was estimated to be 29% of the administered dose. Thus, the general disposition profile of 1,2,7,8-TBDF in the rat is similar to that of other polyhalogenated aromatic hydrocarbons. Due to its rapid elimination, which is consistent with its predicted susceptibility to metabolic elimination, acute exposure to 1,2,7,8-TBDF would not be expected to result in the degree of toxicity associated with other more persistent congeners.