PBPK modeling of the metabolic interactions of carbon tetrachloride and tetrachloroethylene in B6C3F1 mice.

Potential exists for widespread human exposure to low levels of carbon tetrachloride (CT) and tetrachloroethylene (TET). These halocarbons are metabolized by the cytochrome P450 system. CT is known to inhibit its own metabolism (suicide inhibition) and to cause liver injury by generation of metabolically derived free radicals. The objective of this research was to use develop a physiologically based pharmacokinetic (PBPK) model to forcast the metabolic interactions between orally administered CT and TET in male B6C3F1 mice. Trichloroacetic acid (TCA), a stable metabolite of TET, was used as a biomarker to assess inhibition of the cytochrome P450 system by CT. Metabolic constants utilized for CT were 1.0mg/kg/h for Vmaxc_CT and 0.3 for Km_CT (mg/l). Values for TET (based in TCA production), were 6.0mg/kg/h for Vmaxc_TET was 3.0mg/l for Km_TET. The rate of loss of metabolic capacity for CT (suicide inhibition) was describe as: Vmaxloss ( mg / h )=- Kd ( RAM × RAM ) , where Kd (h/kg) is a second-order rate constant, and RAM (mg/h) is the Michaelis-Menten description of the rate of metabolism of CT. For model simplicity, CT was assumed to damage the primary enzymes responsible for metabolism of CT (CYP2E1) and TET (CYP2B2) in an equal fashion. Thus, the calculated fractional loss of TET metabolic capacity was assumed to be equivalent to the calculated loss in metabolic capacity of CT. Use of a Kd value of 400h/kg successfully described serum TCA levels in mice dosed orally with 5-100mg/kg of CT. We report, for the first time, suicide inhibition at a very low dose of CT (1mg/kg). The PBPK model under-predicted the degree of metabolic inhibition in mice administered 1mg/kg of CT. This PBPK model is one of only a few physiological models available to predict the metabolic interactions of chemical mixtures involving suicide inhibition. The success of this PBPK model demonstrates that PBPK models are useful tools for examining the nature of metabolic interactions of chemical mixtures, including suicide inhibition. Further research is required to compare the inhibitory effects of inhaled CT vapors with CT administered by oral bolus dosing and determine the interaction threshold for CT-induced metabolic inhibition.

In vivo kinetics of trichloroacetate in male Fischer 344 rats.

Trichloroacetate (TCA) is a toxicologically important metabolite of the industrial solvents trichloroethylene and tetrachloroethylene, and a by-product of the chlorination of drinking water. Tissue disposition and elimination of 14C-TCA were investigated in male Fischer 344 rats injected iv with 6.1, 61, or 306 micromol TCA/kg body weight. Blood and tissues were collected at various time points up to 24 h. No metabolites were observed in plasma, urine, or tissue extracts. Overall TCA kinetics in tissues were similar at all doses. Based on similar terminal elimination rate constants, tissues could be divided into three classes: plasma, RBC, muscle, and fat; kidney and skin; and liver, small intestine, and large intestine. Nonextractable radiolabel, assumed to be biologically incorporated metabolites in both liver and plasma, increased with time, peaking at 6-9 h postinjection. The fraction of the initial dose excreted in the urine at 24 h increased from 67% to 84% as the dose increased, whereas fecal excretion decreased from 7% to 4%. The cumulative elimination of TCA as CO2 at 24 h decreased from 12% to 8% of the total dose. Two important kinetic processes were identified: a) hepatic intracellular concentrations of TCA were significantly greater than free plasma concentrations, indicating concentrative transport at the hepatic sinusoidal plasma membrane, and b) TCA appears to be reabsorbed from urine postfiltration at the glomerulus, either in the renal tubules or in the bladder. These processes have an impact on the effective tissue dosimetry in liver and kidney and may play an important role in TCA toxicity.

A human physiologically based pharmacokinetic model for trichloroethylene and its metabolites, trichloroacetic acid and free trichloroethanol.

Nine male and eight female healthy volunteers were exposed to 50 or 100 ppm trichloroethylene vapors for 4 h. Blood, urine, and exhaled breath samples were collected for development of a physiologically based pharmacokinetic (PBPK) model for trichloroethylene and its two major P450-mediated metabolites, trichloroacetic acid and free trichloroethanol. Blood and urine were analyzed for trichloroethylene, chloral hydrate, free trichloroethanol and trichloroethanol glucuronide, and trichloroacetic acid. Plasma was analyzed for dichloroacetic acid. Trichloroethylene was also measured in exhaled breath samples. Trichloroethylene, free trichloroethanol, and trichloroacetic acid were found in blood samples of all volunteers and only trace amounts of dichloroacetic acid (4-12 ppb) were found in plasma samples from a few volunteers. Trichloroethanol glucuronide and trichloroacetic acid were found in urine of all volunteers. No chloral hydrate was detected in the volunteers. Gender-specific PBPK models were developed with fitted urinary rate constant values for each individual trichloroethylene exposure to describe urinary excretion of trichloroethanol glucuronide and trichloroacetic acid. Individual urinary excretion rate constants were necessary to account for the variability in the measured cumulative amount of metabolites excreted in the urine. However, the average amount of trichloroacetic acid and trichloroethanol glucuronide excreted in urine for each gender was predicted using mean urinary excretion rate constant values for each sex. A four-compartment physiological flow model was used for the metabolites (lung, liver, kidney, and body) and a six-compartment physiological flow model was used for trichloroethylene (lung, liver, kidney, fat, and slowly and rapidly perfused tissues). Metabolic capacity (Vmaxc) for oxidation of trichloroethylene was estimated to be 4 mg/kg/h in males and 5 mg/kg/h in females. Metabolized trichloroethylene was assumed to be converted to either free trichloroethanol (90%) or trichloroacetic acid (10%). Free trichloroethanol was glucuronidated forming trichloroethanol glucuronide or converted to trichloroacetic acid via back conversion of trichloroethanol to chloral (trichloroacetaldehyde). Trichloroethanol glucuronide and trichloroacetic acid were then excreted in urine. Gender-related pharmacokinetic differences in the uptake and metabolism of trichloroethylene were minor, but apparent. In general, the PBPK models for the male and female volunteers provided adequate predictions of the uptake of trichloroethylene and distribution of trichloroethylene and its metabolites, trichloroacetic acid and free trichloroethanol. The PBPK models for males and females consistently overpredicted exhaled breath concentrations of trichloroethylene immediately following the TCE exposure for a 2- to 4-h period. Further research is needed to better understand the biological determinants responsible for the observed variability in urinary excretion of trichloroethanol glucuronide and trichloroacetic acid and the metabolic pathway resulting in formation of dichloroacetic acid.

Lactational transfer of volatile chemicals in breast milk.

Lactational transfer of chemicals to nursing infants is a concern for occupational physicians when women who are breast-feeding return to the workplace. Some work environments, such as paint shops, have atmospheric contamination from volatile organic chemicals (VOCs). Very little is known about the extent of exposure a nursing infant may receive from the mother's occupational exposure. A physiologically based pharmacokinetic model was developed for a lactating woman to estimate the amount of chemical that a nursing infant ingests for a given nursing schedule and maternal occupational exposure. Human blood/air and milk/air partition coefficients (PCs) were determined for 19 VOCs. Milk/blood PC values were above 3 for carbon tetrachloride, methylchloroform, perchloroethylene, and 1,4-dioxane, while the remaining 16 chemicals had milk/blood PC values of less than 3. Other model parameters, such as solid tissue PC values, metabolic rate constants, blood flow rates, and tissue volumes were taken from the literature and incorporated into the lactation model. In a simulated exposure of a lactating woman to a threshold limit value concentration of an individual chemical, only perchloroethylene, bromochloroethane, and 1,4-dioxane exceeded the U.S. Environmental Protection Agency non-cancer drinking water ingestion rates for children. Very little data exists on the pharmacokinetics of lactational transfer of volatile organics. More data are needed before the significance of the nursing exposure pathway can be adequately ascertained. Physiologically based pharmacokinetic models can play an important role in assessing lactational transfer of chemicals.

A species comparison of chloral hydrate metabolism in blood and liver.

Chloral hydrate (CH), [302-17-0], is a human sedative useful in premature infants. No current epidemiological study supports increased cancer risk. CH is also a rodent toxicant and a P450-derived metabolite of trichloroethylene (TRI). P450 induction increases TRI toxicity in rodents. CH is very rapidly metabolized to trichloroacetic acid (TCA) and trichloroethanol (TCOH). Because TCA mediates some responses following TRI exposure, we assessed the metabolism of CH to TCA and TCOH by liver and blood of the rat, mouse, and human. Both TCA and TCOH are formed in blood and liver. The constants for hepatic TCA and TCOH formation are presented. The K(m) for hepatic TCOH formation is at least ten-fold lower than for TCA formation in these species. Clearance values for TCOH are higher than for TCA. These data support TCOH as the first major metabolite of TRI and CH in vivo.

Dichloroacetic acid: metabolism in cytosol.

Dichloroacetic acid (DCA) arises from the chlorination of drinking water and the metabolism of trichloroethylene (TRI) and is used therapeutically. The toxicity of TRI exposure is dependent on metabolism, and DCA has been proposed to be one contributor to this toxicity. Beyond the identification of some metabolites of DCA and some pharmacokinetic studies, little is known about the tissue distribution and enzymology of DCA metabolism. We present data that indicate that DCA degradation occurs primarily in the cytosol. Low molecular weight components of cytosol are required for the reaction, including nicotinamide cofactor and glutathione (GSH). GSH plays a role in the removal of DCA from cytosol, although not through transferase-mediated conjugation. In rat cytosol, the KM is approximately 0.3 mM, and the apparent Vmax approximates 12 nmoles/min/mg cytosolic protein. These results set DCA apart from other chlorinated compounds that are metabolized by the cytochrome P450 enzyme family.

Gas uptake studies of deuterium isotope effects on dichloromethane metabolism in female B6C3F1 mice in vivo.

In common with a diverse group of low-molecular-weight volatile substrates, dichloromethane (DCM; methylene chloride) is a high-affinity, low-capacity substrate for oxidation by several cytochrome P450 isoenzymes in vivo. DCM oxidation, catalyzed primarily by the 2E1 and 2B1 cytochrome P450 isoforms, yields carbon monoxide (CO) and carbon dioxide. We have studied the characteristics of DCM oxidation in vivo by examining the metabolism of DCM and of both deuterated forms ([2H2]-DCM and [2H]DCM) in female B6C3F1 mice with gas uptake methods. Gas uptake and CO production curves were analyzed by physiologically based pharmacokinetic (PBPK) modeling techniques, permitting differentiation of isotope effects on specific metabolic parameters from those associated with blood flow or diffusion limitations in vivo. A marked isotope effect was observed on the moles of CO produced per mole of DCM oxidized (0.76 +/- 0.06, 0.33 +/- 0.006, and 0.31 +/- 0.07, with DCM, [2H]DCM, and [2H2]DCM, respectively). Based on these ratios, the calculated kH/kD ratio for the rate constant of disproportionation of the putative formyl chloride intermediate was about 7, indicating a significant role of C-H bond breaking in this reaction. Deuterium substitution altered the apparent Km for metabolism; there was 14-fold increase in the apparent Km between DCM and [2H2]DCM (6.5 +/- 0.69 to 97 +/- 3.5 microM) with little effect on Km with [2H]DCM (14.4 +/- 0.015 microM). Vmax was not greatly affected by deuteration (151 +/- 1.2, 116 +/- 0.82, and 149 +/- 2.3 mumol/hr/kg with DCM, [2H]DCM, and [2H2]DCM, respectively). Two kinetic mechanisms are discussed, both of which are consistent with these observations. One, a conventional cytochrome P450 mechanism has a rate-limiting product-release step after the isotopically sensitive step; a second, more like a peroxidase mechanism, has a flux-limiting oxygen activation step followed by a second-order reaction between an activated oxygen-enzyme complex and DCM. Regardless of the correct mechanism, the in vivo kinetic constants for oxidation of DCM are complex and represent more than simple rate-limiting bond-breaking (Vmax) and enzyme-substrate binding (Km). Current PBPK models for metabolism of these volatiles may have to be restructured to account for this unusual kinetic mechanism.

A partition coefficient determination method for nonvolatile chemicals in biological tissues.

Partition or distribution coefficients are critical elements in efforts designed to describe the uptake, distribution, biotransformation, and excretion of organic chemicals in biological systems. In order to estimate the partition coefficients needed to describe the biological distribution of low-volatility compounds, an experimental method was developed to measure partitioning of nonvolatile compounds into biological tissues. Blood, fat, muscle, liver, and skin were individually incubated in a saline solution containing the chemical of interest. Each sample was centrifuged and 2.0 ml of the supernatant was removed and placed into a prewashed, low binding 10,000 MW cutoff Millipore filter cell. Each cell was fitted with a magnetic stirrer and 32 psi nitrogen was applied to the closed cell. The filtrate was collected, extracted, and analyzed for the chemical of interest. The chemicals evaluated were parathion, lindane (hexachlorocyclohexane), paraoxon, perchloroethylene, trichloroacetic acid, and dichloroacetic acid. These chemicals were chosen to develop this method because their vapor pressures range from 9 x 10(6) to 14.2 mm Hg at 20 degrees C. For the one volatile chemical evaluated, perchloroethylene, the method provided partition coefficient results that were in good agreement with values obtained using the vial equilibration method. The nonvolatile partition coefficient method described in this paper demonstrates an approach for evaluation of chemicals with diverse chemical structure and solubility properties.

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.

Structural determination of the carboxylic acid metabolites of polychlorotrifluoroethylene.

1. Polychlorotrifluoroethylene (PCTFE) is a perhalogenated hydrocarbon which consists mainly of C-6 and C-8 oligomers of chlorotrifluoroethylene (CTFE) end-capped with chlorine and referred to as trimer and tetramer, respectively. PCTFE is a hydraulic fluid considered for use in advanced weapon systems. 2. Inhalation studies have shown that PCTFE causes a dose-related hepatotoxicity in rats that is accompanied by proliferation of hepatic peroxisomes and increased liver weight. 3. Carboxylic acid metabolites of PCTFE have been isolated from rats exposed to PCTFE via inhalation. These metabolites, or their formation, may be involved in the toxicity of PCTFE. 4. Trimer carboxylic acids have been isolated from rat urine and identified, and tetramer carboxylic acids have been isolated from rat liver, and identified. 5. Our investigation of trimer and tetramer carboxylic acid metabolites of PCTFE has shown that the terminal carbon bearing two chlorine atoms is the exclusive site of oxidation. No evidence was found indicating oxidation of terminal carbon atoms having one chlorine.

Confirmation of a carboxylic acid metabolite of polychlorotrifluoroethylene and a method for its GC-ECD analysis in biological matrices.

3.1 Oil, referred to as polychlorotrifluoroethylene (pCTFE), is a polymeric mixture consisting primarily of trimers and tetramers of chlorotrifluoroethylene (CTFE) end-capped with chlorine. Inhalation studies have associated dose-related body weight loss, increased organ weights, and abnormal hepatic enzyme activities with exposure to pCTFE. The carboxylic acid metabolites of pCTFE have been shown to cause hepatotoxicity in rats, which is manifested by increased liver weights and the proliferation of hepatic peroxisomes. A method was developed to derivatize these carboxylic acid metabolites. Tissue homogenates and feces were extracted with methanol, and urinary metabolites were extracted on octadecylsilane (ODS) solid-phase extraction columns. Aliquots of the extracts and whole blood were methylated with 3N methanolic HCl to transesterify the carboxylic acid metabolites to volatile methyl esters. The pCTFE methyl esters were analyzed by gas chromatography (GC) with electron capture detection (ECD). The on-column limit of detection was 5 pg for each methyl ester. Solid-phase extraction of spiked urine gave extraction efficiencies of 90.4% for the trimer acid and 84.7% for the tetramer acid. This method was successfully applied to toxicity studies in rats and nonhuman primates. The identities of the derivatized metabolites were confirmed by GC/MS.

Effects of vacuum and pulsation ratio on udder health.

Effects of vacuum and pulsation ratio on udder health were studied for 36 first parity animals in a 60-day trial. Treatments consisted of vacuum at 33.3, 41.6, and 50 kPa at pulsation ratios of 50:50, 60:40, and 70:30 at 60 pulsations per min. All teats were exposed to a culture broth of Staphylococcus aureus after machine removal. Numbers of intramammary infections and somatic cells were determined. Analysis for number of intramammary infections indicated no significant differences among treatments for number of infected available quarters. Trend for intramammary infections was that as pulsation ratio widened, the number of infected available quarters increased, especially at the ratio 70:30. As vacuum increased, number of infected available quarters increased. Least squares means of Wisconsin Mastitis Test scores were 6.29, 5.57, and 6.68 for 33.3 kPa; 12.18, 3.82, and 7.86 for 41.6 kPa; and 9.11, 6.40, and 15.02 for 50 kPa at pulsation ratios 50:50, 60:40, and 70:30. There were significant differences among treatments for vacuum and pulsation ratio. Wisconsin Mastitis Test data indicated an interaction between vacuum and pulsation ratio. Optimum predicted vacuum and pulsation ratio based on Wisconsin Mastitis Test data were 27.2 kPa and 62:38.