Pharmacokinetic analysis of chloral hydrate and its metabolism in B6C3F1 mice.

Chloral hydrate (CH) and its metabolites, trichloroacetate (TCA) and dichloroacetate (DCA), have been shown to induce liver tumors in male B6C3F1 mice. The pharmacokinetics of CH and its metabolites play an important role in its toxicity. This study was designed to characterize the kinetics of CH metabolism, and the formation and elimination of TCA, DCA, trichloroethanol (TCOH), and trichloroethanol glucuronide (TCOG) in male B6C3F1 mice. Mice were dosed with 67.8, 678, and 2034 micromol/kg of CH through the tail vein. At selected time points, mice were killed, and blood and liver samples were collected. Samples were assayed by GC for CH, TCOH, TCOG, TCA, and DCA concentrations. After intravenous administration, CH rapidly disappeared from blood with a terminal half-life ranging from 5 to 24 min. Systemic clearance decreased from 36.0 to 7.6 liters/kg-hr with increasing CH dose, demonstrating dose-dependent pharmacokinetics. TCOH, TCOG, TCA, and DCA were detected over the study period. Formation and metabolism of CH metabolites seemed to be dose-dependent. The terminal half-lives of TCOH and TCOG were similar, ranging from 0.2 to 0.7 hr. TCA and DCA were formed rapidly from the metabolism of CH and cleared slowly from systemic circulation. The area under the blood concentration-time curve for DCA was 10-20% of that for TCA. Both TCA and DCA were slowly eliminated from systemic circulation. The concentration-time profile of DCA seemed to be driven by the blood concentration of TCA, suggesting the possibility of DCA formation from TCA metabolism.

Chloroethylene mixtures: pharmacokinetic modeling and in vitro metabolism of vinyl chloride, trichloroethylene, and trans-1,2-dichloroethylene in rat.

Environmental and occupational exposures are typically to mixtures of chemicals, although most toxicity information is for individual compounds. Interactions between chemicals may involve pharmacokinetic and/or pharmacodynamic effects resulting in modulation of toxicity. Therefore, physiologically based pharmacokinetic modeling has been used to analyze data describing the metabolism of vinyl chloride (VC) and trichloroethylene (TCE) mixtures in rats. A single saturable pathway was modeled, representing cytochrome P450 2E1. This was partially validated using preexposure to trans-1,2-dichloroethylene (tDCE) which virtually eliminated in vivo metabolism of both VC and TCE at low concentrations. Microsomes from tDCE-exposed animals showed inhibition of metabolism of P450 2E1 substrates (chlorzoxazone, p-nitrophenol, and TCE) and no effect on 7-ethoxycoumarin deethylation. Studies with liver microsomes from VC-exposed animals found that neither suicide inhibition nor induction occurred during 6-hr exposures to high concentrations. Therefore, these effects were not modeled. Modeling of mixtures of VC and TCE was successful only using competitive inhibition, as might be predicted for cytochrome P450 2E1 substrates, and not uncompetitive or noncompetitive inhibition. These results were further confirmed by determining the depletion of glutathione due to VC metabolism. The validation of a detailed model for the inhibition kinetics of metabolism of these two compounds permits better understanding of the implications of coexposures for toxicity. It is notable that competitive inhibition only becomes significant at relatively high concentrations (tens of ppm), while at typical low environmental concentrations (ppb), absorption is perfusion limited and enzyme is in excess so that the chemicals will be metabolized independently.

Variability of physiologically based pharmacokinetic (PBPK) model parameters and their effects on PBPK model predictions in a risk assessment for perchloroethylene (PCE).

When used in the risk assessment process, the output from physiologically based pharmacokinetic (PBPK) models has usually been considered as an exact estimate of dose, ignoring uncertainties in the parameter values used in the model and their impact on model predictions. We have collected experimental data on the variability of key parameters in a PBPK model for tetrachloroethylene (PCE) and have used Monte Carlo analysis to estimate the resulting variability in the model predictions. Blood/air and tissue/blood partition coefficients and the interanimal variability of these data were determined for tetrachloroethylene (PCE). The mean values and variability for these and other published model parameters were incorporated into a PBPK model for PCE and a Monte Carlo analysis (n = 600) was performed to determine the effect on model predicted dose surrogates for a PCE risk assessment. For a typical dose surrogate, area under the blood time curve for metabolite in the liver (AUCLM), the coefficient of variation was 25% and the mean value for AUCLM was within a factor of two of the maximum and minimum values generated in the 600 simulations. These calculations demonstrate that parameter uncertainty is not a significant potential source of variability in the use of PBPK models in risk assessment. However, we did not in this study consider uncertainties as to metabolic pathways, mechanism of carcinogenicity, or appropriateness of dose surrogates.

In vivo metabolism of chloroform in B6C3F1 mice determined by the method of gas uptake: the effects of body temperature on tissue partition coefficients and metabolism.

Mice exposed to various chemicals have been shown to respond by decreasing their core body temperature. To examine what effect such a response might have on the determination of in vivo metabolism, core body temperatures of B6C3F1 mice were recorded with temperature telemetry devices during exposure to chloroform (CHCl3) in a closed, recirculating chamber (100 to 5500 ppm). Significant decreases in body temperature occurred in all mice exposed to greater than 100 ppm CHCl3, with the greatest decrease of 14 degrees C occurring at 5500 ppm. A starting CHCl3 concentration of 4000 ppm had no effect on the 7-ethoxycoumarin O-deethylase (ECOD) activity or P450 levels determined at the end of a 5-hr gas uptake exposure. A physiologically based pharmacokinetic (PB-PK) model was developed to describe the effects of decreased body temperature on the analysis of metabolic data. In vitro ECOD activity as a measure of in vivo P450 metabolism was determined for temperatures ranging from 24 to 40 degrees C. In vitro enzyme activity decreased linearly from a maximum at 37 degrees C to one-third of this activity at 24 degrees C. A linear equation describing this enzymatic activity-temperature correlation was incorporated into the PB-PK model structure to describe decreases in metabolic activity resulting from decreases in core body temperature. In vitro blood/air and tissue/air partition coefficients were determined for CHCl3 at temperatures ranging from 24 to 40 degrees C. All blood/air and tissue/air partitions increased with decreasing temperature, while the tissue/blood partition coefficients calculated from the tissue/air and blood/air partitions decreased with decreasing temperature. Adding these temperature corrections to the model greatly improved the overall fit of the gas uptake curves at all concentrations. Incorporation of a first-order metabolic rate constant was also required to provide an adequate representation of the data at high concentrations. The analysis of gas uptake data by the use of a PB-PK computer model is a very powerful technique for determining in vivo metabolism of many volatile compounds, but the incorporation of significant deviations from a generally used model structure (i.e., Ramsey-Andersen model) to account for shortcomings of the model's ability to adequately analyze a gas uptake data set should be based on data collection when possible.

Polychlorotrifluoroethylene (PCTFE) oligomer pharmacokinetics in Fischer 344 rats: development of a physiologically based model.

The hydraulic fluid oil polychlorotrifluoroethylene (PCTFE) is hepato- and nephrotoxic in the rat. Male Fischer 344 rats were exposed to PCTFE either for a single 6-hr exposure (0.5 or 0.25 mg/liter) or daily 5 days/week, 6 hr/day, for 13 weeks (0.5, 0.25, or 0.01 mg/liter). Blood, tissue, and urinary PCTFE concentrations measured postexposure were used to develop a physiologically based pharmacokinetic (PB-PK) model. The PCTFE hydraulic fluid used was a mixture of trimeric and tetrameric oligomers with minor amounts of other chain lengths. The PB-PK model was designed to describe the behavior, not of individual oligomers, but of total mass for the trimer and tetramer in each tissue. Partition coefficients were estimated using the model to optimize tissue/blood concentration ratios measured at the end of the 13-week exposure. First-order metabolic rate constants for both trimeric (2.0 hr-1) and tetrameric (1.0 hr-1) portions were estimated by optimization against urinary fluoride data assuming release of 0.77 mole fluoride per mole trimer and 0.844 mole fluoride per mole tetramer metabolized. To obtain accurate simulation of pharmacokinetic data it was necessary to hypothesize two fat compartments with diffusion-limited exchange of PCTFE oligomer with the blood. Relative concentrations of trimer and tetramer in venous blood, liver, and fat after a single 6-hr exposure were proportional to inhaled concentrations. Tetramer accumulated preferentially with multiple exposure. Components of PCTFE were metabolized to carboxylic acids with release of fluoride. Due to their persistence tetrameric oligomers appear to be more important than trimeric oligomers as causative agents of PCTFE hepato- and nephrotoxicity in the rat.

Effects of short-term oral dosing of polychlorotrifluoroethylene (polyCTFE) on the rhesus monkey.

Polychlorotrifluoroethylene (polyCTFE--primarily oligomers with 3-4 monomer units), a non-flammable hydraulic fluid for aircraft, was given daily for 15 days by oral gavage to four Rhesus monkeys at a concentration of 0.725 g kg-1. The administered dose was at a level that had caused toxicity in rats. Steady-state blood and liver concentrations reached were the same in both species. In monkeys, polyCTFE did not cause the electrolyte, serum protein, liver enzyme and anemic disturbances previously seen in rats. Liver sections taken at 15 days, analyzed for palmitoyl Co-A beta-oxidation rates or by electron microscopy, showed no significant indication of peroxisomal proliferation. An increased blood urea nitrogen (BUN) at 15 days was the only clinical pathological abnormality seen in both monkeys and rats. Previously unobserved effects were increased triglycerides and glycogen depletion.

Absorption of glucose polymers from canine jejunum deprived of pancreatic amylase.

We evaluated the absorption of glucose polymers in canine jejunal Thiry-Vella fistulas proven to be free of pancreatic amylase. Medium-length oligomers with degrees of polymerization of 6 through 10 glucose units (DP 6-10) and long-chain material (DPavg23) were isolated from a cornstarch hydrolysate. We perfused 90, 180, and 360 mg/dl solutions of glucose, DP 6-10, and DPavg23 at 0.4, 1.9, and 3.4 ml/min. At all perfusion rates carbohydrate absorption decreased as the chain length of the oligomers increased, and these differences persisted even at the slowest perfusion rate employed. In two additional animals the fistulas were perfused at 3.4 ml/min with the three test carbohydrates at concentrations of 90, 180, 225, 270, 315, 360, 405, and 450 mg/dl. At this flow rate, the assimilative process of DP 6-10 and the long-chain fraction appeared to be saturated at carbohydrate concentrations above 360 mg/dl, whereas the absorption of glucose was linearly related to concentration throughout the range studied. With both groups of polymers, the fluid emerging from the fistula was virtually free of glucose, a finding that suggests that polymer digestion, not glucose absorption, is the rate-limiting step for polymer assimilation.

Large scale production of glucose oligomers and polymers for physiological studies in humans.

Methods for isolating relatively large quantities of glucose oligomer and polymer subfractions from a partial corn starch hydrolysate (PCSH) are described. To ensure that the products are suitable for physiological studies in humans, potentially toxic substances were excluded from the preparative processes. For long chain glucose polymer fractions with degrees of polymerization (DP) averaging 43 glucose units, we employed molecular filtration through Amicon YM5 membranes. For fractions containing glucose oligomers with DP's 3 through 8, we employed yeast fermentation followed by ethanol fractionation.

Large scale preparation of selected glucose oligomers and polymers by gel filtration chromatography.

A method for isolating relatively large quantities of glucose oligomer and polymer subfractions from a corn starch hydrolysate is described. Employing large columns of Bio-Gel P-2 (40-80 microns) at room temperature, we can prepare each day 0.5 to 1.2 grams of oligomeric fractions containing three to four adjacent homologues. The columns are homemade, require no flow adapters, and are operated by gravity elution with water as the solvent. The means for avoiding and overcoming potential difficulties, such as microbial contamination and declining flow rate, are described. With the use of the described method, we can operate a single column continuously for up to twelve months.