Richard H. Reitz

Physiologically based pharmacokinetics and the risk assessment process for methylene chloride

Methylene chloride (dichloromethane, DCM) is metabolized by two pathways: one dependent on oxidation by mixed function oxidases (MFO) and the other dependent on glutathione S-transferases (GST). A physiologically based pharmacokinetic (PB-PK) model based on knowledge of these pathways was used to describe the metabolism of DCM in four mammalian species (mouse, rat, hamster, and humans). Kinetic constants for the model were derived from in vivo experiments or the literature. The model was constructed to distinguish contributions from the two pathways of metabolism in lung and liver tissue, and to permit extrapolation from rodents to humans. Model validation was conducted by comparing predicted blood concentration time-course data in rats, mice, and humans with experimental data from these species. The tumor incidence in two chronic studies of DCM toxicity in mice was correlated with various measures of target tissue dose calculated with the PB-PK model. Tumor incidence correlated well with tissue AUC (area under the concentration/time curve) and amount of DCM metabolized by the GST pathway. However, tumor incidence did not correlate with the amount of DCM metabolized by the MFO pathway. Because of its low chemical reactivity, DCM is unlikely to be directly involved in carcinogenesis. Consequently, metabolism of DCM by GST appears to be important in carcinogenesis. The PB-PK model was used to estimate target doses of presumed toxic chemical species in humans exposed to DCM by inhalation or by drinking water. Target tissue doses in humans exposed to low concentrations of DCM are 140- to 170-fold lower (inhalation) or 50- to 210-fold lower (drinking water) than would be expected from the linear extrapolation and body surface area factors which have been used in conventional risk assessment methods (D. V. Singh, H. L. Spitzer, and P. D. White (1985). Addendum to the Health Assessment Document for Dichloromethane (Methylene Chloride). EPA/600/8-82/004F). The PB-BK analysis thus suggests that conventional risk analyses greatly overestimate the risk in humans exposed to low concentrations of DCM. PB-PK considerations provide a scientific basis for risk assessment, improve experimental design in chronic studies, and structure collection of quantitative metabolic constants required for risk assessment.

In vitro metabolism of methylene chloride in human and animal tissues: Use in physiologically based pharmacokinetic models

Physiologically based pharmacokinetic (PB-PK) models describe the dynamic behavior of chemicals and their metabolites in individual tissues of living animals. Because PB-PK models contain specific parameters related to the physiological and biochemical properties of different species as well as the physical chemical characteristics of individual chemicals, they are useful tools for performing high dose/low dose, dose route, and interspecies extrapolations in hazard evaluations. An example of such extrapolation has been presented by M. E. Andersen, H. J. Clewell III, M. L. Gargas, F. A. Smith, and R. H. Reitz (Toxicol. Appl. Pharmacol. 87, 185-205, 1987), who employed a PB-PK model for methylene chloride (CH2Cl2) to estimate the chronic toxicity of this material. However, one limitation of this PB-PK model was that the metabolic rate constants for the glutathione-S-transferase (GST) pathway in humans were estimated by allometric scaling rather than from experimental data. In this paper we report studies designed to estimate the in vivo rates of metabolism of CH2Cl2 from in vitro incubations of lung and liver tissues from B6C3F1 mice, F344 rats, Syrian Golden hamsters, and humans. A procedure for calculating in vivo metabolic rate constants from the in vitro studies is presented. This procedure was validated by making extrapolations with mixed function oxidase enzymes (MFO) acting on CH2Cl2, where both in vitro and in vivo rates of metabolism are known. The in vitro rate constants for the two enzyme systems are consistent with the hypothesis presented by Andersen et al. that metabolism of CH2Cl2 occurs in vivo by two competing pathways: a high-affinity saturable pathway (identified as MFO) and a low-affinity first-order pathway (identified as GST). The metabolic rate constants for GST obtained from these studies are also consistent with the hypothesis of Andersen et al. that production of large quantities of glutathione/CH2Cl2 conjugates in vivo may increase the frequency with which lung and liver tumors develop in some species of animals (e.g., B6C3F1 mouse). When in vivo studies in humans are unavailable, in vitro enzyme assays provide a reasonable method for estimating metabolic rate constants.

Physiologically Based Pharmacokinetic Modeling with Dichloromethane, Its Metabolite, Carbon Monoxide, and Blood Carboxyhemoglobin in Rats and Humans'

Dichloromethane (methylene chloride, DCM) and other dihalomethanes are metabolized to carbon monoxide (CO) which reversibly binds hemoglobin and is eliminated by exhalation. We have developed a physiologically based pharmacokinetic (PB-PK) model which describes the kinetics of CO, carboxyhemoglobin (HbCO), and parent dihalomethane, and have applied this model to examine the inhalation kinetics of CO and of DCM in rats and humans. The portion of the model describing CO and HbCO kinetics was adapted from the Coburn-Forster-Kane equation, after modification to include production of CO by DCM oxidation. DCM kinetics and metabolism were described by a generic PB-PK model for volatile chemicals (RAMSEY AND ANDERSEN, Toxicol. Appl. Pharmacol. 73, 159-175, 1984). Physiological and biochemical constants for CO were first estimated by exposing rats to 200 ppm CO for 2 hr and examining the time course of HbCO after cessation of CO exposure. These CO inhalation studies provided estimates of CO diffusing capacity under free breathing and for the Haldane coefficient, the relative equilibrium distribution ratio for hemoglobin between CO and O2. The CO model was then coupled to a PB-PK model for DCM to predict HbCO time course behavior during and after DCM exposures in rats. By coupling the models it was possible to estimate the yield of CO from oxidation of DCM. In rats only about 0.7 mol of CO are produced from 1 mol of DCM during oxidation. The combined model adequately represented HbCO and DCM behavior following 4-hr exposures to 200 or 1000 ppm DCM, and HbCO behavior following 1/2-hr exposure to 5160 ppm DCM or 5000 ppm bromochloromethane. The rat PB-PK model was scaled to predict DCM, HbCO, and CO kinetics in humans exposed either to DCM or to CO. Three human data sets from the literature were examined: (1) inhalation of CO at 50, 100, 250, and 500 ppm; (2) seven 1/2-hr inhalation exposures to 50, 100, 250, and 500 ppm DCM; and (3) 2-hr inhalation exposures to 986 ppm DCM. An additional data set from human volunteers exposed to 100 or 350 ppm DCM for 6 hr is reported here for the first time. Endogenous CO production rates and the initial amount of CO in the blood compartment were varied in each study as necessary to give the baseline HbCO value, which varied from less than 0.5% to greater than 2% HbCO. The combined PB-PK model gave a good representation of the observed behavior in all four human studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Incorporation of in vitro enzyme data into the physiologically-based pharmacokinetic (PB-PK) model for methylene chloride: implications for risk assessment.

Physiologically-based pharmacokinetic (PB-PK) models provide a mechanism for reducing the uncertainty inherent in extrapolating the results of animal toxicity tests to man. This paper discusses a technique for incorporating data from in vitro studies of xenobiotic metabolism into in vivo PB-PK models. Methylene chloride is used as an example, and carcinogenic risk estimates incorporating PB-PK principles are presented.

Molecular mechanisms involved in the toxicity of orthophenylphenol and its sodium salt

Hiraga and Fujii have recently reported that F344 rats consuming diets with high levels of sodium orthophenylphenate (SOPP) developed bladder tumors after 13–91 weeks (Fd. Cosmet. Toxicol., 19 (1981) 303). Several dose levels were tested and doses above 1.0% SOPP by weight appeared to cause an increase in both toxicity and bladder carcinogenicity. In order to put these studies into better perspective, the effects of feeding diets containing SOPP or orthophenylphenol (OPP) to F344 male rats for varying lengths of time were characterized. Hyperplasia of the bladder epithelium was noted in rats consuming diets containing 2% SOPP (equivalent to 1000–1500 mg/kg/day) after 1–2 weeks, with epithelial thickening increasing through 90 days. No bladder lesions were seen in the group consuming 2% OPP but focal kidney lesions were noted. In contrast to the results reported by Hiraga and Fujii, no tumors of the urinary tract were observed following 90 days of consumption of the 2% SOPP diet. The potential of these chemicals to induce genotoxic lesions was studied. No detectable increases in the reversion rates of Salmonella typhimurium (strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538) were seen at concentrations of SOPP up to 5.8 · 10−4 M. SOPP also failed to produce a detectable increase in unscheduled DNA synthesis in primary rat hepatocytes at concentrations up to 1 · 10−4 M. No covalently-bound radioactivity was observed in DNA purified from the bladders of rats gavaged with 500 mg/kg [14C]SOPP or [14C]OPP (detection limit < 1 alkylation/106 nucleotides). These results suggest little or no genotoxicity for OPP or SOPP. The metabolism of OPP and SOPP in male F344 rats was shown to be dose-dependent. After gavage with 50 mg/kg or less, most of the administered material was recovered in the urine as glucuronide or sulfate conjugates of the parent material. After gavage with 500 mg/kg a new metabolite, apparently produced by mixed function oxidases, was observed. This metabolite was characterized by gas chromatography/mass spectroscopy as a conjugate of dihydroxybiphenyl. It is postulated that the potentially reactive metabolites produced by this oxidative pathway may be associated with the toxicity induced by high concentrations of OPP or SOPP. Thus the bladder toxicity and carcinogenicity of SOPP and the renal toxicity of OPP appear to occur only following the administration of high doses which saturate the normal conjugation pathways. However, since no genotoxicity was detected even at saturating doses, it appears unlikely that exposure to subtoxic doses would cause any significant increase in carcinogenic risk.

Genetic and nongenetic events in neoplasia

It has become increasingly evident that all chemical carcinogens do not act via the same mechanism of tumorigenicity. Based upon the extent of a chemicals interaction with DNA, a general classification scheme of various mutational and nonmutational theories of chemical carcinogenesis is presented. Compounds that directly interact with DNA are classified as genotoxic whereas those that do not interact directly with DNA are classified as epigenetic carcinogens. Under each general heading, several mutational and nonmutational mechanisms of carcinogenesis are believed to be possible. Data are also presented to support the existence of one such mechanism, an epigenetic-mutational theory of chemical carcinogenesis based upon recurrent cytotoxicity. In this case, increased regenerative DNA synthesis in response to tissue injury is believed to result in an enhancement of the normal spontaneous mutation rate, conceivably leading to a cellular transformation. The carcinogenic risk posed by such epigenetic carcinogens appears to differ greatly from that posed by genotoxic carcinogens. Thus, consideration of data concerning the possible mechanism of carcinogenicity of a chemical, along with pharmacokinetic data, will allow a better understanding of bioassay results and a more accurate assessment of carcinogenic risk.

Effects of vinylidene chloride on DNA synthesis and DNA repair in the rat and mouse: A comparative study with dimethylnitrosamine

Abstract Exposure to vinylidene chloride (VDC) vapor has been reported to induce tumors in mice, but rats are apparently insensitive to this effect of VDC. This species difference has been correlated with the greater capacity of mice to activate VDC to a reactive electrophile which can react with macromolecules. To increase our understanding of the molecular events associated with this species difference, we have investigated the potential of VDC to cause DNA alkylation, DNA repair, and DNA replication in the liver and kidneys of rats and mice. For comparative purposes, the potent carcinogen dimethylnitrosamine (DMN) was also studied. Male Sprague-Dawley rats and CD-1 mice were exposed to 10 and 50 ppm VDC for 6 hr. DNA alkylation after 50 ppm [ 14 C]VDC was minimal in liver and kidney of both rats and mice (one or two orders of magnitude less than reported for DMN in rats). Similarly, DNA repair in the kidney of mice exposed to 50 ppm VDC was only 38% higher than control values, while DNA repair in the liver of mice injected with 20 mg/kg DMN was elevated 637%. However, tissue damage and increased DNA replication (25-fold) were seen in the kidneys of mice exposed to 50 and 10 ppm VDC. Comparable effects were not seen in the liver of mice exposed to VDC (50 or 10 ppm) or in the liver or kidneys of rats exposed to 10 ppm VDC. Thus an important distinction between DMN and VDC has been demonstrated. Tumorigenic doses of DMN produced relatively little tissue damage, but were associated with a high degree of DNA alkylation and DNA repair synthesis. In contrast, exposure to tumorigenic doses of VDC resulted in massive tissue damage but induced minimal DNA alkylation or DNA repair synthesis. This suggests that the tumors observed in mice exposed to VDC arise primarily through effects of the chemical on nongenetic components of the cells. Consequently protection of humans from levels of VDC sufficient to cause tissue damage should also serve to preclude any carcinogenic activity of VDC.

Pharmacokinetics and macromolecular interactions of ethylene dichloride in rats after inhalation or gavage

Ethylene dichloride (EDC) induces tumors in rats and mice when administered chronically by gavage. However, chronic inhalation of EDC vapor failed to induce any treatment-related tumors. To help understand the consequences of environmental exposure to EDC by either route, [14C]EDC was administered to male Osborne-Mendel rats by gavage (150 mg/kg in corn oil) or inhalation (150 ppm, 6 hr). EDC was extensively metabolized following either exposure. No significant differences were observed in the route of excretion of nonvolatile metabolites. In each case, ∼85% of the total metabolites appeared in the urine, with 7 to 8, 4, and 2% found in the CO2, carcass, and feces, respectively. The major urinary metabolites were thiodiacetic acid and thiodiacetic acid sulfoxide, suggesting a role for glutathione in biotransformation of EDC. Gross macromolecular binding (primarily protein binding) was studied after inhalation or gavage. No marked differences were noted between the two routes, or between “target” and “nontarget” tissues, after in vivo administration of EDC. Covalent alkylation of DNA by EDC was studied in Salmonella typhimurium and rats. DNA alkylation in S. typhimurium was directly related to the frequency of mutation in these bacteria. However, when DNA was purified from the organs of rats exposed in vivo to EDC, very little alkylation was observed after either gavage or inhalation (2 to 20 alkylations per million nucleotides). DNA alkylation after gavage was two to five times higher than after inhalation, but no marked differences were noted between target and nontarget organs. Pharmacokinetic studies indicated that peak blood levels of EDC were approximately five times higher after gavage than after inhalation. When pharmacokinetic data were modeled, it appeared that the elimination of EDC may become saturated when high blood levels are produced and that such saturation is more likely to occur when equivalent doses are administered by gavage versus inhalation. Since toxicity often occurs when the normal detoxification pathways are overwhelmed, this toxicity may represent the most reasonable explanation for the apparent differences between the two bioassays.

Pharmacokinetics, biochemical mechanism and mutation accumulation: a comprehensive model of chemical carcinogenesis

Chemical carcinogenesis is a process beginning with carcinogen absorption and ending with development of a malignant tumor. Individual elements of this process have been studied intensively but no comprehensive model has been developed. This report describes a comprehensive model which incorporates carcinogen pharmacokinetics, biochemical mechanism of action, and the resultant mutation of normal cells to malignancy. Model parameters correspond to specific physiological and biochemical structures and processes. The model was encoded in a simulation language and used to examined biochemical and cellular effects of exposure to an initiator and a promoter. With laboratory validation, the model should be useful for interpretation and design of studies on carcinogenic mechanisms and for risk assessment.

Biochemical factors involved in the effects of orthophenylphenol (OPP) and sodium orthophenylphenate (SOPP) on the urinary tract of male F344 rats.

Carbon-14 labeled sodium orthophenylphenate (SOPP) was incubated with purified microsomes isolated from rat liver. During this incubation, macromolecular binding of radioactivity (MMB) was observed. MMB was dependent upon the presence of both active microsomes and NADP. In vivo studies of MMB were also conducted. MMB was measured in the liver, kidney, and bladder of male F344 rats administered SOPP (0.19 to 1.88 mM/kg) or orthophenylphenol (OPP) (0.29 to 2.97 mM/kg). The levels of MMB were not linearly related to administered dose. Disproportionate increases in MMB were observed in each tissue after administration of 0.75 to 1.88 mM/kg of SOPP. Disproportionate increases in MMB in liver and bladder tissue were also observed with OPP at somewhat higher doses. These studies indicate that the intermediate(s) produced by the oxidative pathway for metabolism of SOPP and OPP are capable of binding to biological macromolecules. The disproportionate increases in MMB observed in vivo after high doses are probably associated with saturation of the primary (conjugative) metabolic pathway for SOPP and OPP metabolism.