P.G. Watanabe

Resolution of dose-response toxicity data for chemicals requiring metabolic activation: Example—Vinyl chloride☆

Abstract The toxicity of many chemicals results from biotransformation products formed from the chemical rather than from the chemical per se. In such cases, the incremental response may become diminishingly smaller with increasing dose or exposure because activation of the chemical to the toxic form follows apparent Michaelis—Menten rather than apparent first-order kinetics. To illustrate this concept, rats were exposed to concentrations ranging from 1.4 to 4600 ppm of vinyl chloride for 6 hr, and the total amount metabolized was determined. The amount metabolized followed apparent Michaelis—Menten kinetics. For rats, the logarithmic probability incidence of angiosarcoma versus the amount of vinyl chloride metabolized rather than the exposure concentration of vinyl chloride is linear. Assuming no threshold in spite of evidence to the contrary, extrapolation of the data below the range of doses causing experimentally observable responses predicted an incidence of 0.01% hepatic angiosarcoma in rats exposed to 4.6 ppm of vinyl chloride. Theoretical extension of the extrapolation to humans after adjusting for metabolic and body mass differences was undertaken. The theoretical extrapolation for man exposed daily for 8 hr to 1 ppm suggests an incidence of 1.5 per 100,000,000. This theoretical incidence, although a likely overestimate because of a less than predicted incidence in men exposed to 200 ppm and greater, as well as evidence for a threshold in rats, is less than that expected to occur spontaneously. The concepts evolved from this analysis reveals why pharmacokinetics must be considered in designing toxicology experiments as well as in interpretation of the resulting data.

In vitro activation of 1,2-dichloroethane by microsomal and cytosolic enzymes.

[1,2-14C]1,2-Dichloroethane was metabolized by rat liver enzyme systems to nonvolatile products and to products irreversibly bound to protein and calf thymus DNA. Cytosolic metabolism to all three types of metabolites was dependent upon the presence of reduced glutathione (GSH), suggesting the role of GSH transferases. Microsomal metabolism to all three types of products occurred via mixed function oxidation; microsomal GSH transferase(s) catalyzed the formation of metabolites irreversibly bound to DNA. GSH blocked microsomal mixed function oxidase (MFO)-catalyzed binding to protein but stimulated binding to DNA in a synergistic manner. 2-Chloroacetaldehyde, S-(2-chloroethyl)-GSH, and 1-chloroso-2-chloroethane are proposed as major species involved in irreversible binding but vinyl chloride, 2-chloroethanol, and chloroethyl radicals are not. The microsomal MFO, synergistic microsomal MFO-GSH transferase, and cytosolic GSH transferase systems differed in their preferences for irreversible binding of label from dichloroethane to various homopolyribonucleotides and only the latter system produced metabolites mutagenic to Salmonella typhimurium TA 1535. We postulate that several 1,2-dichloroethane activation pathways are operative which produce different adducts; the relative contribution of each pathway to total nonvolatile metabolites, mutagenic metabolites, and DNA and protein adducts under these in vitro assay conditions was estimated.

PRELIMINARY STUDIES OF THE FATE OF INHALED VINYL CHLORIDE MONOMER IN RATS

Rats were exposed to vinyl chloride monomer gas (VCM) in a closed recirculating system. The rate at which VCM was removed from the system via metabolism was determined for rats exposed to initial concentrations of VCM ranging from 50 to 1167 ppm. Upon exposure to initial concentrations of 50 to 105 ppm, the rate of metabolism was 8.04 ? 3.40 X 10-8 min-1. Upon exposure to initial concentrations ranging from 220 to 1167 ppm, the rate constants were less; the mean value being 2.65 ? 1.35 X 10-8 min-1. Regardless of concentration, the disappearance followed apparent first order kinetics.

The pharmacokinetics and macromolecular interactions of perchloroethylene in mice and rats as related to oncogenicity

Abstract The pharmacokinetic and macromolecular interactions of perchloroethylene were evaluated in B 6 C 3 F 1 mice and Sprague-Dawley rats in an attempt to explain, mechanistically, the sensitivity of the mouse and the resistance of the rat to perchloroethylene-induced hepatocellular carcinoma. When compared to rats, mice were found to metabolize 8.5 and 1.6 times more perchloroethylene per kilogram of body weight following inhalation of 10 ppm or a single oral dose of 500 mg/kg perchloro[ 14 C]ethylene, respectively. Since the initial metabolism of perchloroethylene is an activation process, the increased extent of metabolism in the mouse resulted in a greater extent of irreversible binding of radioactivity in hepatic macromolecules of the mouse compared to that in the rat after inhalation of 10 or 600 ppm or a single oral dose of 500 mg/kg perchloro[ 14 C]ethylene. Repeated oral administration of perchloroethylene for 11 days resulted in histopathological changes in the liver of mice at doses as low as 100 mg/kg/day, while minimal treatment-related effects were observed in the liver of rats only at the 1000 mg/kg/day level. Approximately a twofold increase in hepatic DNA synthesis, indicative of hepatic regeneration, was observed in mice but not in rats after repeated oral administration of perchloroethylene at dose levels which are tumorigenic to mice in lifetime studies. The absence of any pronouced direct interaction of perchloroethylene with hepatic DNA in mice at times of peak hepatic macromolecular binding suggests that hepatic tumors are induced in B 6 C 3 F 1 mice by recurrent cytotoxicity which enhances the spontaneous incidence of liver tumors in this highly susceptible strain of mouse. The implication of these results for hazard assessment is that recurrent tissue damage is necessary for tumors to be induced. Thus, levels of perchloroethylene which do not induce organ toxicity are not likely to pose a carcinogenic risk to man.

Fate of [14C]vinyl chloride after single oral administration in rats☆

Male rats were given single oral doses of 0.05, 1, and 100 mg/kg of [14C]vinyl chloride (VC), and the routes and rates of elimination of 14C activity followed for 72 hr. Following 0.05 and 1 mg/kg, excretion in the urine as nonvolatile metabolites and as 14CO2 in expired air accounted for 59–68% and 9–13%, respectively of the administered dose. Only 1–2% of the dose was expired by the lungs as VC. Conversely, after 100 mg/kg, 67% of the dose was eliminated by the lungs as VC, while urinary nonvolatile metabolites and 14CO2 comprised 11 and 3%, respectively. Pulmonary elimination after 100 mg/kg showed an apparent biphasic clearance with haif-times (t12) of 14.4 and 40.8 min for the respective fast and slow phases. Following 0.05 and 1 mg/kg the pulmonary clearance of VC was monophasic with t12 of 53.3 and 57.8 min. The percentage of the dose remaining in the carcass after 72 hr was 10, 11, and 2% for the 0.05-, 1- and 100-mg/kg doses, respectively. The urinary radio-activity was separated by high pressure liquid chromatography into three major metabolites. Two of the three major urinary metabolites have been identified as N-acetyl-S-(2-hydroxyethyl)-cysteine and thiodiglycolic acid by gas chromatography-mass spectrometry. The proportions of the urinary metabolites were not influenced by the dose. The fate of VC following an oral dose between 1 and 100 mg/kg was clearly dose-dependent. Consistent with our previous studies on the fate of VC following inhalation exposure in rats, the metabolism of VC appears to be a saturable process.

Fate of [14C]Vinyl Chloride following inhalation exposure in rats

Inhalation exposure to vinyl chloride (VC) has been shown to be carcinogenic in rats and man. It is important in assessing the toxicological potential of inhaled VC to understand the disposition of VC in the body. Therefore, the objective of the present study was to determine the fate of inhaled [ 14 C]VC at different exposure concentrations in rats. Male rats were exposed to 10 or 1000 ppm [ 14 C]VC for 6 hr and the routes and rates of elimination of 14 C activity were followed for 72 hr after termination of exposure. Following exposure to 10 ppm of VC, urinary 14 C activity and expired VC comprised 68 and 2%, respectively, of the recovered radioactivity. After exposure to 1000 ppm of VC, the proportion of the radioactivity in the urine decreased while that expired as VC increased representing 56 and 12%, respectively. The pattern of pulmonary elimination of VC per se was described by similar apparent first-order kinetics following 10 or 1000 ppm with respective half-lives of 20.4 and 22.4 min. The elimination of 14 C activity in the urine occurred in accordance with a two-exponential equation; the half-lives for the initial phase of excretion were 4.6 and 4.1 hr following 10 and 1000 ppm, respectively. The percent of the recovered 14 C activity remaining in the carcass after 72 hr was 14 and 15% at the respective low and high exposure level. VC per se was not found in tissues. The urinary 14 C activity was separated by high pressure liquid chromatography into three major metabolites corresponding to N -acetyl- S -(2-hydroxyethyl)cysteine, thiodiglycolic acid, and a third unidentified metabolite. The proportions of the urinary metabolites were not markedly influenced by the exposure magnitude. The fate of inhaled [ 14 C]VC was shown to be dose-dependent; this is consistent with previous studies on the fate of VC following ingestion as well as inhalation.

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.

Disposition of tetrachloro(14C)ethylene following oral and inhalation exposure in rats

Abstract Tetrachloro[ 14 C]ethylene (Perc) was administered to adult, male Sprague-Dawley rats by gavage (1 or 500 mg/kg) or by inhalation (10 or 600 ppm, 6 hr duration). Within 72 hr following oral administration of 1 mg/kg or inhalation of 10 ppm [ 14 C]Perc, approximately 70% of the body burden of radioactivity was excreted in expired air as Perc, 26% as 14 CO 2 and nonvolatile metabolites in urine and feces, and 3 to 4% remained in the carcass. After oral administration of 500 mg/kg or inhalation of 600 ppm [ 14 C]Perc, 89% of the radioactivity was recovered in expired air as Perc, 9% as 14 CO 2 and urinary and fecal metabolites, and 1 to 2% remained in the carcass. The major urinary metabolite of Perc was identified as oxalic acid. Pulmonary elimination of Perc was monophasic with a half life ( t 1 2 ) of approximately 7 hr independent of dose or route of administration. Radioactivity remaining in the carcass 72 hr after exposure by either route was primarily distributed within liver, kidney and fat tissue. In liver, 85 to 90% of the total radioactivity was cleared within 72 hr following inhalation exposure to 10 or 600 ppm. Nonextractable radioactivity, either bound or incorporated into hepatic macromolecular material, was cleared at a slower rate. The tissue concentration of nonextractable radioactivity was dependent upon body burden and metabolic capacity but apparently not upon route of administration. Thus, the data indicate that disposition of Perc is a saturable, primarily dose-dependent process in rats.

Vinyl chloride-induced depression of hepatic non-protein sulfhydryl conten and effects on bromosulphalen (BSP) clearance in rats

Rats were exposed to atmospheres of 2000, 1000, 250, 150, 50 and 10 ppm vinyl chloride (VC) for 1-7 h to determine the effect of VC on the hepatic non-protein sulhydryl content, Exposure to 2000, 1000, 250 and 150 ppm VC caused a progressive depression of the hepatic non-protein sulfhydryl content. Following exposure to 50 ppm VC for 7 h the depression was inconsistent, and no depression was observed after 10 ppm VC for 7 h. Also, exposure to 1000 ppm VC did not alter the serum clearance of bromosulphalein (BSP).