Tony R. Fox

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.

[14C]Methyl chloroform (1,1,1-trichloroethane): pharmacokinetics in rats and mice following inhalation exposure.

Studies on the pharmacokinetics of [14C]methyl chloroform (1,1,1-trichloroethane) in male Fischer 344 rats and B6C3F1 mice were undertaken to characterize the disposition of the inhaled chemical over a wide range of exposure concentrations. The animals were exposed to 150 or 1500 ppm of [14C]methyl chloroform vapor for 6 hr and the elimination of 14C activity was followed for 72 hr. Following exposure to either concentration of methyl chloroform, both species excreted >96% of the total recovered radioactivity during the first 24 hr. The major route of elimination of methyl chloroform was via exhalation of unchanged chemical in the expired air which constituted approximately 94–98% of the total recovered radioactivity in rats and 87–97% in mice at 150 and 1500 ppm, respectively. Mice were found to eliminate methyl chloroform in the expired air more rapidly than did rats. The remaining radioactivity (approximately, 2–13%) was detected as metabolized methyl chloroform in the expired air (14CO2) and as nonvolatile radioactivity in the urine, feces, carcass, and cage wash. Although mice were found to metabolize two to three times more methyl chloroform on a body weight basis, the biotransformation of methyl chloroform was shown to be a saturable, dose-dependent process in both species. Since the biotransformation of methyl chloroform occurred to such a limited extent, saturation of its metabolism did not impact markedly on the distribution or elimination of the parent chemical. The body burden, end-exposure blood level, and tissue concentration of methyl chloroform were found overall to increase in direct proportion with the exposure concentration. [14C]Methyl chloroform was more concentrated in the fat of both species than in the liver or kidneys immediately after exposure. However it was rapidly cleared from the fat so that by 24 hr <2% of the initial radioactivity remained. Thus, methyl chloroform shows little potential for significant bioaccumulation in rodents.

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.

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.

Activation of a Cellular Proto-Oncogene in Spontaneous Liver Tumor Tissue of the B6C3F1 Mouse

The controversy surrounding the interpretation of observed increases in the high spontaneous liver tumor incidence of the B6C3F1 mouse after administration of certain chemical agents necessitates a mechanistic understanding into the nature of tumor development in this particular strain of mouse. Recently, cancer genes (oncogenes) have been detected in the DNA from a variety of human tumors and tumor cell lines. These genes have been implicated to play a role in the transformation of normal cells into cancerous ones. To investigate the role that cellular oncogenes might play in the development of spontaneous liver tumors in the B6C3F1 mouse, DNA was isolated from spontaneously occurring liver tumors and transfected into NIH 3T3 fibroblasts. DNA from this tumor tissue was capable of transforming NIH 3T3 cells from 82% of the animals examined strongly suggesting the presence of an active cellular oncogene. In contrast, DNA isolated from surrounding non-tumorous liver tissue and liver tissue from non-tumor bearing mice did not cause any transformation in the NIH 3T3 assay. These data demonstrate that the active cellular oncogene is not present in the hepatic tissue via a germ-line transmission but is activated only in those cells of the tumor tissue. Experiments using Southern blot hybridization analysis have identified this active cellular oncogene to be a member of the ras oncogene family. Identification of this cellular oncogene will now allow the evaluation of factors which might modify its expression. These future studies will lead to an increased understanding of potential mechanisms by which hepatic tumors are enhanced and should provide more informed estimates of risk for man based on bioassay data generated in this strain of mouse.

The Role of Pharmacokinetics in Risk Assessment

Pharmacokinetics can aid in the formulation of risk estimations in several ways: (a) by selection of doses for toxicity studies, (b) by distinguishing between “internal dose or toxifor concentration” and “applied dose”, (c) by providing a physiological basis for extrapolating between species, and (d) by helping us to visualize the toxicological consequences of processes which we cannot (yet) quantify.