J.F. Quast

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.

Evaluation of the Developmental and Reproductive Toxicity of Chlorpyrifos in the Rat

Chlorpyrifos (O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)-phosphorothioate), an organophosphate insecticide, was evaluated for its potential to produce developmental and reproductive toxicity in rats following oral exposure. Pregnant Fischer 344 rats were given doses of 0 (corn oil vehicle), 0.1, 3.0, or 15 mg chlorpyrifos/kg/day, by gavage, on Gestation Days 6 through 15. Maternal effects noted at the two higher dose levels included decreased cholinesterase levels at 3.0 mg/kg/day and cholinergic signs (excessive salivation and tremors), decreased cholinesterase levels, and decreased body weight gain at 15 mg/kg/day. No maternal effects were apparent at 0.1 mg/kg/day. Although maternal toxicity was observed at these two higher exposure levels, no developmental effects were noted at any dose. In a two-generation reproduction study, Sprague-Dawley rats were maintained on diets supplying 0, 0.1, 1.0, or 5.0 mg chlorpyrifos/kg/day. Parental effects included decreased plasma and erythrocyte cholinesterase at 1.0 mg/kg/day, and decreased plasma, erythrocyte, and brain cholinesterase and histopathologic alterations of the adrenal zona fasciculata at 5.0 mg/kg/day. The histopathologic alterations of the adrenal were characterized as very slight to slight vacuolation (consistent with fatty change) in males, and very slight vacuolation and/or altered tinctorial properties in females. No effects on the reproductive or fertility indices or on the histopathology of reproductive tissues were observed at any dose level, and no neonatal effects were observed at 0.1 or 1.0 mg/kg/day in the F1 or F2 litters. Parental toxicity at the high dose was accompanied by decreased pup body weight and increased pup mortality in the F1 litters only. These data show that oral administration of chlorpyrifos to rats at parentally toxic dose levels was not embryolethal, embryo/fetotoxic, or teratogenic and did not adversely affect fertility or the function or structure of the reproductive organs. Although effects on neonatal growth and survival were observed at a maternally toxic dose level in one generation, this effect was not observed in the subsequent generation and, therefore, may not have been related to treatment.

In vitro kinetics of styrene and styrene oxide metabolism in rat, mouse, and human

Styrene oxide (SO), a labile metabolite of styrene, is generally accepted as being responsible for any genotoxicity associated with styrene. To better define the hazard associated with styrene, the activity of the enzymes involved in the formation (monooxygenase) and destruction of SO (epoxide hydrolase and glutathione-S-transferase) were measured in the liver and lungs from naive and styrene-exposed male Sprague-Dawley rats and B6C3F1 mice (three daily 6-h inhalation exposures at up to 600 ppm styrene) and Fischer 344 rats (four daily 6-h inhalation exposures at up to 1000 ppm styrene), and in samples of human liver tissue. Additionally, the time course of styrene and SO in the blood was measured following oral administration of 500 mg styrene/kg body weight to naive Fischer rats and rats previously exposed to 1000 ppm styrene. The affinity of hepatic monooxygenase for styrene, as measured by the Michaelis constant (Km), was similar in the rat, mouse, and human. Based on theVmax for monooxygenase activity and the relative liver and body size, the mouse had the greatest capacity and humans the lowest capacity to form SO from styrene. In contrast, human epoxide hydrolase had a greater affinity (i. e., lowerKm) for SO than epoxide hydrolase from rats or mice while the apparent Vmax for epoxide hydrolase was similar in the rat, mouse, and human liver. However, the activity of epoxide hydrolase relative to monooxygenase activity was much greater in the human than in the rodent liver. Hepatic glutathione-S-transferase activity, as indicated by theVmax, was 6- to 33-fold higher than epoxide hydrolase activity. However, the significance of the high glutathione-S-transferase activity is unknown because hydrolysis, rather than conjugation, is the primary pathway for SO detoxification in vivo. Human hepatic glutathione-S-transferase activity was extremely variable between individual human livers and much lower than in rat or mouse liver. Prior exposure to styrene had no effect on monooxygenase activity or on blood styrene levels in rats given a large oral dose of styrene. In contrast, prior exposure to styrene increased hepatic epoxide hydrolase activity 1.6-fold and resulted in lower (0.1>P>0.05) blood SO levels in rats given a large oral dose of styrene. Qualitatively, these data indicate that the mouse has the greatest capacity and the human the lowest capacity to form SO. In addition, human liver should be more effective than rodent liver in hydrolyzing low levels of SO. Quantitative evaluation of the species differences in enzyme levels are being evaluated with the development of a physiologically based pharmacokinetic model for styrene that includes SO.

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.

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.

Results of Two-Year Toxicity and Reproduction Studies on Pentachlorophenol in Rats

Male and female Sprague-Dawley rats were maintained on diets containing pentachlorophenol (PCP), characterized by a low content of nonphenolic impurities, for up to 24 months. Pentachlorophenol (PCP) was not found to be carcinogenic when administered to rats in their diet on a chronic basis at dose levels sufficiently high to cause mild signs of toxicity (1, 3, 10, or 30 mg/kg/day). Effects at the high dose level included decreased body weight gain (females), increased serum glutamic pyruvic transaminase activity (males and females), and increased urine specific gravity (females). An accumulation of pigment was observed in the livers and kidneys of females at 30 and 10 mg PCP/kg/day and males at 30 mg/kg/day. Ingestion of 3 mg PCP/kg/day or less by females and 10 mg/kg/day or less by males was not associated with significant toxicologic effects. To evaluate the effects on reproduction, rats were fed 3 or 30 mg PCP/kg/day for 62 days prior to mating, during 15 days of mating, and subsequently throughout gestation and lactation. A reduction in the mean body weight was observed among the adult rats at the highest dose level. Except for a significant decrease in neonatal survival and growth among litters of females ingesting 30 mg PCP/kg/day, measures of reproductive capacity were unaffected at both dose levels of pentachlorophenol. Ingestion of 3 mg PCP/kg/day had no effect on reproduction or neonatal growth, survival, or development.

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.

The chronic toxicity and oncogenicity of inhaled technical-grade 1,3-dichloropropene in rats and mice

Male and female Fischer 344 rats and B6C3F1 mice were exposed by inhalation to target concentrations of 0, 5, 20, or 60 ppm (0, 22.7, 90.8, or 272 mg/m3) technical-grade 1,3-dichloropropene (DCPT) 6 hr/day, 5 days/week, for up to 2 years. Ancillary groups of rats and mice were exposed for 6- and 12-month periods. Significant treatment-related nonneoplastic changes following exposure for 2 years were morphological alterations in the nasal tissues of rats exposed to 60 ppm and mice exposed to 20 or 60 ppm DCPT. In addition, mice exposed to 20 or 60 ppm had hyperplasia of the transitional epithelium lining the urinary bladder. Survival of male and female rats and mice exposed to DCPT was similar to that of the corresponding controls. No statistically increased tumor incidence was observed in treated rats. The only neoplastic response observed in mice was an increased incidence of benign lung tumors (bronchioloalveolar adenomas) in male mice exposed to 60 ppm DCPT (22/50 versus 9/50 in controls).

Two-year toxicity and oncogenicity study with acrylonitrile incorporated in the drinking water of rats.

Sprague-Dawley rats (80 per sex per control and 48 per sex in each treatment group) were given drinking water formulated to contain 0, 35, 100, or 300 ppm acrylonitrile (AN) for up to 2-years. An additional ten rats per sex per group were added for a 1-year interim necropsy. The equivalent doses of AN consumed were 0, 3.4, 8.5, and 21.3 mg/kg per day for males and 0, 4.4, 10.8, and 25.0 for females. Rats were closely monitored clinically with body weight, feed and water consumption measured at numerous intervals. Hematology, clinical chemistry, and urinalysis were evaluated six times. All rats were necropsied when moribund, found dead, or at scheduled termination, with extensive histopathology of all rats. Numerous adverse toxic and oncogenic effects were observed in both sexes of all AN treatment groups. Decreased water consumption, feed consumption, and concomitant body weight suppression occurred within days of study initiation and persisted throughout the study in all treatment groups. An early onset of Zymbal gland tumors in high dose male and female rats, and in the mammary gland of all treated groups of females, was detected in-life. Hematology, clinical chemistry, and urinalysis, repeatedly evaluated, were without significant biological effects, except for an increased urine specific gravity due to the rats lower water intake. Organ weights at study termination were not significantly affected. Mortality was high in all female treated groups, with no surviving male or female 300 ppm rats during the last 2 months of the study. The most significant findings in this study were detected following gross and microscopic examination of an extensive list of tissues from all rats in the study. Nontumorous and tumorous lesions were found at an increased and/or decreased rate in a number of tissues of both sexes at all treatment levels. The primary nontumorous histopathologic effects of AN exposure occurred in the forestomach and the central nervous system of rats of both sexes and involved all treatment groups. A statistically significant increased incidence of tumors in one or more dose levels of either sex occurred in the central nervous system, Zymbal gland, forestomach, tongue, small instestine, and mammary gland. A no-observed-effect level (NOEL) was not identified in this study for toxicity or oncogenicity in either sex.