Assessing the reliability of PBPK models using data from methyl chloride-exposed, non-conjugating human subjects.

Physiologically based pharmacokinetic (PBPK) models are often optimized by adjusting metabolic parameters so as to fit experimental toxicokinetic data. The estimates of the metabolic parameters are then conditional on the assumed values for all other parameters. Meanwhile, the reliability of other parameters, or the structural model, is usually not questioned. Inhalation exposures with human volunteers in our laboratory show that non-conjugators lack metabolic capacity for methyl chloride entirely, and that elimination in these subjects takes place via exhalation only. Therefore, data from these methyl chloride exposures provide an excellent opportunity to assess the general reliability of standard inhalation PBPK models for humans. A hierarchical population PBPK model for methyl chloride was developed. The model was fit to the experimental data in a Bayesian framework using Markov chain Monte Carlo (MCMC) simulation. In a Bayesian analysis, it is possible to merge a priori knowledge of the physiological, anatomical and physicochemical parameters with the information embedded in the experimental toxicokinetic data obtained in vivo. The resulting estimates are both statistically and physiologically plausible. Model deviations suggest that a pulmonary sub-compartment may be needed in order to describe the inhalation and exhalation of volatile adequately. The results also indicate that there may be significant intra-individual variability in the model parameters. To our knowledge, this is the first time that the toxicokinetics of a non-metabolized chemical is used to assess population PBPK parameters. This approach holds promise for more elaborate experiments in order to assess the reliability of PBPK models in general.

Comparative genotyping and phenotyping of glutathione S-transferase GSTT1.

Only limited information is available so far concerning the human glutathione S-transferase isoenzyme class theta encoded by the GSTT1 gene. The aim of the study was to characterize individuals in respect to a polymorphic deletion of the GSTT1 gene and to validate these results with the phenotypical determination of the "conjugator status" according to Hallier et al. (1993). Determination of the GSTT1 genotype was done in 40 healthy adults by using an assay based on internal standard controlled polymerase chain reaction. The GSTT1-1 phenotype was determined by measuring the erythrocyte conjugating activity towards methyl chloride using a gas chromatographic assay. Genotypically, 34 individuals out of 40 were classified as GSTT1 positive; the remainder were negative. These results could be confirmed by phenotyping in all but one case. In the present study the frequency of "nonconjugators" was 15%. Our study demonstrates the reliability of the suggested PCR assay for GSTT1 genotyping which is easier to perform than the phenotyping assay and is not affected by confounding factors.

Polymorphic distribution of glutathione transferase activity with methyl chloride in human blood.

Interindividual variation in the in vitro conjugation of methyl chloride with glutathione by erythrocyte glutathione transferase was investigated in 208 healthy males and females from the southern and central parts of Sweden. It was found that 11.1% of the individuals lacked this activity, whereas 46.2% had intermediate activity and 42.8% had high activity. This distribution of three phenotypes is compatible with the presence of one functional allele with a gene frequency of 0.659 and one defect allele with a gene frequency of 0.341. The proportion of non-conjugators in this Swedish material was considerably smaller than that previously found in Germany (Peter et al., Arch Toxicol 1989: 63, 351-355). The polymorphic distribution of another glutathione transferase, GST mu, was determined in the same individuals with a PCR method. No connection between the genotype for GST mu (GSTM1) and the glutatione conjugation with methyl chloride in erythrocytes was found.

Metabolism of methyl chloride by human erythrocytes.

Erythrocyte cytoplasm of rats, mice and humans was incubated in head space vials with methyl chloride and the decline in concentration of the substance monitored as a parameter of metabolism. The production of S-methylglutathione was controlled by tlc. Rats, mice, bovines, pigs, sheep and rhesus monkeys showed no conversion of methyl chloride in erythrocyte cytoplasm. About 60% of the human blood samples showed a significant metabolic elimination of the substance (conjugators), whereas about 40% did not (non-conjugators). The production of S-methylglutathione indicated enzymatic metabolism of the substance by glutathione S-transferases. In literature, a "major" and "minor" form of human erythrocyte glutathione S-transferase has been described. The results indicate that the "minor" form is probably responsible for the unique metabolism of methyl chloride in human erythrocytes.

A physiological model for the pharmacokinetics of methylene chloride in B6C3F1 mice following i.v. administrations.

A physiologic mathematical model was developed to describe the time course of 14C-methylene chloride (14CH2Cl2) distribution and elimination in mice following single i.v. administrations of 10 and 50 mg/kg. A whole-body model was used to simulate 14CH2Cl2 concentrations in blood and tissues, pulmonary clearance of unchanged 14CH2Cl2, and metabolic conversion to 14CO2 and 14CO as monitored by the appearances of these metabolites in expired breath. This diffusion-limited model was identified via a sequential optimization scheme using hybrid models for each compartment. Pulmonary elimination of unchanged 14CH2Cl2 was modeled as a linear process while hepatic metabolism of 14CH2Cl2 to the compounds 14CO2 and 14CO was described by a saturable metabolic rate term. The model adequately described the dose dependence in methylene chloride distribution and metabolism when simulations were compared to experimental data.

Pharmacokinetics and metabolism of inhaled methyl chloride in the rat and dog.

Methyl chloride (MeCl) metabolism and pharmacokinetics were studied in male Fischer 344 rats and male beagle dogs. Apparent steady-state blood MeCl concentrations were proportionate to exposure concentration in rats and dogs exposed to 50 and 1000 ppm. Furthermore, blood MeCl concentrations were similar in both species when they were exposed to the same concentration. A linear two-compartment open model described the blood MeCl data: alpha and beta phase elimination half-times corresponded to approximately 4 and 15 min, respectively, in rats, and 8 and 40 min in dogs. Rats exposed for 6 hr to 0, 50, 225, 600, or 1000 [14C]MeCl were evaluated for tissue nonprotein sulfhydryl (NPSH), total 14C activity, nonextractable tissue 14C activity, and urinary metabolites. MeCl-induced NPSH depletion was dose-related and was greatest in liver. Total 14C in liver and kidney was approximately proportionate to exposure concentrations. Relative concentrations of nonextractable 14C decreased at 600 to 1000 ppm MeCl suggesting a dose-dependent metabolic pathway for MeCl in the rat. Metabolites in urine included N-acetyl-S-methylcysteine, methylthioacetic acid sulfoxide, and N-(methylthioacetyl)glycine. These metabolites are likely to be products of a reaction between MeCl and glutathione. A nonradiometric analysis of a putative MeCl metabolite (S-methylcysteine) was performed in dogs exposed to MeCl; this method was not a sensitive indicator of MeCl exposure.