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Toxicity of the Protein Kinase C Inhibitor Safingol Administered Alone and in Combination with Chemotherapeutic Agents. Kedderis, L. B., Bozigian, H. P., Kleeman, J. M., Hall, R. L., Palmer, T. E., Harrison, S. D., Jr., and Susick, R. L., Jr. (1995). Fundam. Appl. Toxicol. 25, 201-217. Safingol [(2S,3S)-2-amino-1,3-octadecanediol] potentiates the toxicity of doxorubicin (DOX) and cisplatin (CIS) against tumor cells in vitro and in vivo . The present studies were conducted in rats and dogs to evaluate safingol toxicity when administered iv as a single agent and to evaluate safingol's ability to potentiate the toxicity of established chemotherapeutic agents to normal tissues in vivo . In an escalating dose study, dogs were administered safingol iv at 5, 10, 20, 30, 40, and 75 mg/kg on Days 1 through 6. Necropsies were performed on Day 7. Red urine was observed at 10 mg/kg and higher. Icterus was observed following 40 mg/kg with additional signs of hypoactivity and anorexia occurring after 75 mg/kg. Clinical and microscopic pathology revealed marked hepatotoxicity, venous degeneration and necrosis at injection sites, and evidence of intravascular hemolysis. Doses of 5, 20, or 40 mg safingol/kg were utilized in single iv dose rat and dog studies. No evidence of adverse systemic toxicity was seen up to 20 mg/kg in either species [for rats: C max = 12,600 (males) or 17,133 (females) ng/ml, AUC = 3853 (males) or 4365 (females) ng × hr/ml; for dogs: C max = 2533 ng/ml, AUC = 2851 ng × hr/ml (no sex differences)]. Local effects of venous irritation or intravascular hemolysis were observed at all doses in rats and at 20 and 40 mg/kg in dogs. A dose of 40 mg/kg [for rats: C max = 31,233 (males) or 91,300 (females) ng/ml, AUC = 11,519 (males) or 18,620 (females) ng × hr/ml; for dogs: C max = 9033 ng/ml, AUC = 11,094 ng × hr/ml (combined sex)] was associated with clinical pathologic and renal histomorphologic changes considered consequent to intravascular hemolysis in both species, lethality and testicular toxicity in rats, and clinical biochemical changes indicative of hepatobiliary injury in dogs. Studies indicated that hemolysis occurred during infusion, was not caused by circulating levels of safingol, and was a function of dose concentration and vein of delivery. Safingol at 10 or 20 mg/kg was administered iv to rats 30-60 rain prior to myelosuppressive iv doses of DOX, CIS, or cyclophosphamide (CYP). Hematology, plus renal function and morphology for CIS-treated animals, was assessed 4 and 14 days later. Safingol did not potentiate DOX-, CIS-, or CYP-mediated leukopenia/thrombocytopenia. A minimal enhancement of CIS-mediated decrease in GFR and increase in creatinine was observed at 20 mg safingol/kg. Dogs were administered 20 mg safingol/kg iv followed 60 min later by 0.5 or 1.25 mg DOX/kg or 0.75 or 2.0 mg CIS/kg. A complete toxicologic assessment 4 and 29 days postdose failed to show potentiation of DOX toxicity by safingol or vice versa. A renal lesion was inferred in dogs administered 20 mg/kg safingol and 2 mg/kg CIS based on minimal to slight renal tubular regeneration observed 4 weeks post-treatment. There were no effects of safingol on the pharmacokinetic profiles of DOX or CIS or vice versa.

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.

Effect of Dose, Time, and Pretreatment on the Biliary Excretion and Tissue Distribution of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Rat. Kedderis, L. B., Andersen, M. E., and Birnbaum, L. S. (1993). Fundam. Appl. Toxicol. 21, 405-411. Previous studies of the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) pretreatment on the biliary excretion and hepatic disposition indicated that TCDD did not induce its own metabolic elimination. Pretreatment with TCDD did enhance its hepatic uptake. The present work was designed to further examine the effects of dose, time, and pretreatment on the tissue distribution and biliary elimination of [3H]TCDD. Adult male F-344 rats were administered 0 or 100 nmol [14C]TCDD or [3H]TCDD/kg body weight po 3 days prior to bile duct cannulation and iv injection of 0 or 1 nmol [3H]TCDD or 1, 10, or 100 nmol [14C]TCDD/kg. Bile was collected for up to 8 hr while rats were maintained under pentobarbital anesthesia. Biliary TCDD and TCDD metabolites were quantified by liquid scintillation spectrometry. In naive animals which received no pretreatment, similar rates of excretion (% dose) were observed following iv administration of 1 nmol [3H]TCDD/kg or 10 or 100 nmol [14C]TCDD/kg. Metabolic elimination of highly purified [3H]TCDD (>99%) appeared to be linear with respect to time with ∼0.8% of the dose being excreted in the bile over a 5- to 8-hr collection period 0 or 24 hr after iv dosing (1, 10, or 100 nmol/kg) and 72 hr after oral dosing (100 nmol/kg). In all groups, higher concentrations of TCDD were found in liver versus fat, and perirenal fat concentrations were elevated relative to epididymal fat concentrations, probably reflective of the enhanced blood perfusion of the former tissue. Pretreatment enhanced hepatic concentrations and decreased fat concentrations of the challenge dose. The time dependence of factors involved in the dose-related hepatic accumulation of TCDD disposition was illustrated by the elevated liver:fat concentration ratios observed at the 100 versus 10 or 1 nmol/kg dose at 30 hr, but not by 5-6 hr after dosing. In studies designed to evaluate the role of CYP1A2 in the hepatic disposition of TCDD, pretreatment with isosafrole, a selective inhibitor of CYP1A2, diminished hepatic concentrations of [3H]TCDD. In conclusion, TCDD was metabolically cleared at a fairly constant rate over an 80-hr period, and the rate of elimination was proportional with regard to dose. Studies with isosafrole suggest that TCDD is bound to CYP1A2 in the liver.

The Effect of Pretreatment on the Biliary Excretion of 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 2,3,7,8-Tetrachlorodibenzofuran, and 3,3′,4,4′-Tetrachlorobiphenyl in the rat. McKinley, M. K., Kedderis, L. B., and Birnbaum, L. S. (1993). Fundam. Appl. Toxicol. 21, 425-432. The laterally halogenated chemicals 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 3,3′,4,4′-tetrachlorobiphenyl (TCB) exhibit the same spectrum of toxic effects as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototype and most toxic member of the halogenated aromatic hydrocarbon family. Metabolism of all three compounds appears to be the rate-limiting step for excretion, which is primarily via the bile into the feces. Therefore, the biliary elimination of TCDF, TCDD, and TCB was examined as an indirect measure of metabolism. Male F344 rats were anesthetized with pentobarbital, the bile duct was cannulated, and 0.1 μmol [3H]TCDD, [14C]TCDF, or [14C]TCB/kg body wt was administered iv. Bile was collected for 0-8 hr while the animals were kept under anesthesia. To determine if TCDF was able to induce its own metabolism in vivo, a single dose of 1.0 μmol TCDF/kg was administered to rats by oral garage 3 days prior to iv injection of 0.1 or 0.3 μmol [14C]TCDF/kg. Biliary excretion and hepatic concentrations of [14C]TCDF were significantly increased in the pretreated animals. These results suggest an autoinduction of TCDF metabolism. Essentially all biliary [14C]TCDF radioactivity was attributable to metabolites. High-pressure liquid chromatography profiles of biliary radioactivity from 0 to 4 hr were qualitatively different between naive and pretreated rats. To determine if pretreatment with TCDD altered the metabolism of TCDF and vice versa, a single dose of 1.0 μmol TCDF/kg or 0.1 μmol TCDD/kg was administered by oral gavage 3 days prior to iv injection of 0.1 μmol [3H]TCDD or [14C]TCDF/kg, respectively. TCDD pretreatment increased the metabolism and hepatic concentrations of [14C]TCDF in pretreated rats, while biliary elimination and hepatic concentrations of [3H]TCDD were unaffected by pretreatment with TCDF. In a fourth experiment, the ability of TCDD to alter the metabolism of TCB, a laterally substituted polychlorinated biphenyl (PCB), was examined. Pretreatment with TCDD increased the metabolism of [14C]TCB by approximately twofold, but no differences in hepatic concentrations were seen due to the rapid elimination of TCB. Rats pretreated with Aroclor 1254, a commercial mixture of PCBs, demonstrated significantly increased metabolism of [14C]TCB compared to the naive group. Therefore, under these experimental conditions, induction of TCDF metabolism occurred in the rat upon pretreatment with either TCDD or TCDF at doses which also elicited enhanced hepatic uptake. TCDD and Aroclor 1254 induced the metabolism of TCB. These findings suggest that repeated exposure to a chemical or to a mixture of these halogenated aromatic hydrocarbons can result in more rapid metabolism and elimination from the body than following a single, acute exposure.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD), a widespread polychlorinated aromatic hydrocarbon, caused tumors in the liver and other sites when administered chronically to rats at doses as low as 0.01 microgram/kg/day. It functions in combination with a cellular protein, the Ah receptor, to alter gene regulation, and this resulting modulation of gene expression is believed to be obligatory for both dioxin toxicity and carcinogenicity. The U.S. EPA is reevaluating its dioxin risk assessment and, as part of this process, will be developing risk assessment approaches for chemicals, such as dioxin, whose toxicity is receptor-mediated. This paper describes a receptor-mediated physiologically based pharmacokinetic (PB-PK) model for the tissue distribution and enzyme-inducing properties of dioxin and discusses the potential role of these models in a biologically motivated risk assessment. In this model, ternary interactions among the Ah receptor, dioxin, and DNA binding sites lead to enhanced production of specific hepatic proteins. The model was used to examine the tissue disposition of dioxin and the induction of both a dioxin-binding protein (presumably, cytochrome P4501A2), and cytochrome P4501A1. Tumor promotion correlated more closely with predicted induction of P4501A1 than with induction of hepatic binding proteins. Although increased induction of these proteins is not expected to be causally related to tumor formation, these physiological dosimetry and gene-induction response models will be important for biologically motivated dioxin risk assessments in determining both target tissue dose of dioxin and gene products and in examining the relationship between these gene products and the cellular events more directly involved in tumor promotion.

Biologically based models serve as valuable tools for integration of mechanistic pharmacokinetic data by their explicit definition of important determinants of chemical disposition. The objective of the present work was to develop a physiologically based pharmacokinetic model to describe the disposition and enzyme induction properties of 2,3,7,8-tetrabromodibenzo- p -dioxin (TBDD). The TBDD model, which was based on models previously developed for 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), incorporated: ternary interactions between TBDD, the Ah receptor, and specific DNA-binding sites; induction of a TBDD-binding protein specific to the liver; and diffusion-limited tissue uptake. In the model for TBDD, CYP1A2, which had been measured directly by radioimmunoassay, was assumed to be the hepatic binding protein. The model employed physiologic parameters based on recent data in unanesthetized rats, growing tissue compartments, transluminal excretion of parent TBDD via the gut into the feces, and a separate skin compartment. The model was developed using tissue distribution and excretion data following a single intravenous dose of 1 nmol [ 3 H]TBDD per kilogram. The TBDD model was then used unchanged to analyze several experimental data sets illustrating the time, dose, and route of exposure dependency of TBDD disposition. The model successfully described the dose-dependent tissue distribution of [ 3 H]TBDD following intravenous and oral administration and following a single dermal dose. These studies show that diffusional clearance from blood to tissue was slower for skin than for fat (PA sk ∼ 0.1 PA f ). When compared with TCDD: (i) TBDD had a higher fat partition coefficient (P f = 1000 vs 400) and a lower diffusional clearance into fat (PA f = 0.1 vs 0.2) than TCDD; (ii) the binding affinity of CYP1A2 for TBDD was slightly lower than that for TCDD (9.0 vs 6.5 nM); and (iii) TBDD exhibited a slightly greater rate of metabolic elimination (2.0 vs 1.65). Small differences were noted in DNA binding parameters derived for the induction of CYPlAl and CYP1A2 for TBDD versus TCDD. With minor modifications, the biologically based model for TCDD accurately described the behavior of the brominated congener. The present model, which relied on measured values ofCYP1A2 and specified CYP1A2 as the hepatic dioxin binding species, successfully describes the hepatic disposition of TBDD, providing further evidence that CYP1A2 is the primary hepatic binding species in the rat.

Polybrominated dibenzo-p-dioxins and dibenzofurans are of major concern because of potential occupational and environmental exposures and their structural similarity to the highly toxic chlorinated analogues. 2,3,7,8-Tetrabromodibenzo-p-dioxin (TBDD) is a closely related analogue in both structure and activity to the most toxic isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin. The objectives of this study were to characterize the effects of dose and routes on absorption, excretion, and terminal tissue distribution of [3H]TBDD in the rat 72 hr after dosing. Rats were treated orally by gavage with 1, 10, 100, or 500 nmol/kg, intratracheally with 1 nmol/kg, or dermally with 1 nmol/kg (200 pmol/1.8 cm2). TBDD exhibited nonlinear oral absorption kinetics with maximum absorption (approximately 80%) occurring at doseor = 10 nmol/kg, similar to the transpulmonary absorption. In contrast, dermal absorption of TBDD was low (approximately 12%). The major tissue depots of radioactivity were liver, adipose tissue, and skin. Tissue distribution of the oral dose was dose-dependent, with disproportionally greater hepatic concentrations occurring at absorbed doses of8 nmol/kg. Liver:adipose tissue (L:F) concentration ratios were 2.9 to 6.6 (lowest to highest oral dose, respectively). The lower L:F ratios observed for the dermal and intratracheal doses at 1 nmol/kg (1.5 and 2, respectively) were likely due to differences in absorbed dose and dose-related tissue distribution. Elimination of radioactivity in feces, the major route of excretion for all dose groups and routes, and urine was also nonlinear with respect to the oral dose. The results of the present study provide important considerations for high- to low-dose and route-to-route extrapolations with TBDD and other dioxins and furans in human risk assessments.

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.