An observation: role of the M.D., Ph.D. in science.
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Animals↗
Biomedical subjects
P G Watanabe
Publications and source records attributed to P G Watanabe.
Toxicologists: have we been honest with the public?
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Communications Media↗
Mutational analysis of the H-ras oncogene in spontaneous C57BL/6 x C3H/He mouse liver tumors and tumors induced with genotoxic and nongenotoxic hepatocarcinogens.
The frequency and mutational profile of H-ras gene activation were determined in spontaneous liver tumors of male C57BL/6 x C3H/He mice and in tumors induced with the genotoxic hepatocarcinogen benzidine.2 HCl or the nongenotoxic hepatocarcinogens phenobarbital, chloroform, and ciprofibrate. DNA sequence analysis of the H-ras gene from representative tumors revealed that 32 of 50 (64%) spontaneous tumors and 13 of 22 (59%) benzidine.2 HCl-induced tumors contained a point mutation in codon 61. Tumors induced with the nongenotoxic agents had a much lower frequency of codon 61 mutations, i.e., phenobarbital, 1 of 15 (7%); chloroform, 5 of 24 (21%), and ciprofibrate, 8 of 39 (21%). No mutations were observed at codons 12, 13, and 117 in tumors from any of the groups. Only three base pair substitutions within codon 61 were found. The one most frequently detected in all of the groups was a C.G to A.T transversion at the first nucleotide position, occurring at a 59%, 85%, 100%, 80%, and 88% frequency in the spontaneous tumors and in the tumors induced with benzidine 2.Hcl, phenobarbital, chloroform, and ciprofibrate, respectively. In these same groups an A.T to G.C transition or an A.T to T.A transversion at the second nucleotide position occurred at a frequency of 34%, 8%, 0%, 0%, and 12%, and 6%, 8%, 0%, 20%, and 0%, respectively. The number of tumors carrying an activated H-ras gene in the nongenotoxic treatment groups is within the range that would be expected if those animals had not received any treatment. This indicates that the activation of the H-ras gene in those tumors is probably the result of a spontaneous event. The data suggest that these toxicologically and pharmacologically diverse nongenotoxic hepatocarcinogens increase the frequency of liver tumors but do not induce mutations in the H-ras gene. Instead these agents appear to interact with a population of cells that do not contain an activated H-ras gene. This suggests that the mechanisms of tumor development by these nongenotoxic carcinogens differ at least partially from the mechanisms responsible for the development of spontaneous tumors or those induced by a typical genotoxic agent.
Animals↗
Metabolism and disposition of ethylene carbonate in male Fischer 344 rats.
Ethylene carbonate (EC) has a toxicity profile which resembles that of ethylene glycol (EG). To determine whether the toxicity of EC could be explained on the basis of its metabolism to EG, male Fischer 344 rats were given 200 mg/kg of uniformly labeled [14C]EC in water by gavage and the disposition of the radiolabel was then followed for 72 hr. EC was rapidly metabolized, with approximately 57 and 27% of the administered dose eliminated in the expired air as 14CO2 and in the urine, respectively; the remainder was found in the carcass. Separation of the urinary metabolites using liquid chromatography revealed a single radioactive peak. This metabolite was unequivocally identified as ethylene glycol via gas chromatography-mass spectrometry with the aid of 13C enrichment of the EC dose. Measurement of whole blood levels of EC and EG in rats given 200 mg/kg of EC by gavage revealed blood levels of EG approximately 100-fold higher than the levels of EC in these same animals, with a half-life of EG in blood of 2 hr, indicating rapid conversion of EC to EG. In a separate group of animals administered an equimolar dose of [14C]EG (141 mg/kg), approximately 37% of the dose was expired as 14CO2 and 42% was excreted in the urine as parent compound. When expressed on the basis of the ethanediol moiety, the disposition of EC was identical to that of EG. In view of the rapid and extensive biotransformation of EC to EG and the similarity of the existing (though limited) toxicity data base of EC compared to EG, utilization of the extensive EG systemic toxicity data base for assessing the safety of EC appears justified.
Administration, Oral↗
Assessment of cell cycle duration on the incidence of sister chromatid exchanges in somatic and spermatogonial cells of the rat.
In order to examine the influence of the length of cell cycle on the incidence of sister chromatid exchanges (SCEs), average generation times and SCEs were studied in the presence of bromodeoxyuridine from spontaneously dividing rat lymph node, bone marrow, spleen, and spermatogonial cells. Average generation time differences among the three somatic cell types (lymph node, 7.6 h; bone marrow, 12.0 h; spleen, 14.9 h) were statistically significant as were the differences between the germinal cell (37.4 h) and each of the somatic cells. The SCE per cell frequencies were significantly higher in the somatic cells (lymph node, 6.8; bone marrow, 5.8; spleen, 6.1) as compared to the spermatogonial cells (1.6). However, no difference in SCE incidence was detected among the cells from the different somatic tissues. It was concluded that there was no simple relationship between cell cycle duration time and SCE formation.
Animals↗
Toxicokinetics in the evaluation of toxicity data.
Toxicokinetics provides a powerful tool, which is not used sufficiently in the conduct and interpretation of animal toxicity studies. In selecting doses for toxicity studies toxicokinetic data can be used effectively. The tools of toxicokinetics are limited only by the toxicologists' understanding of basic biologic mechanisms. More knowledge on mechanisms of action implicates the possibility of more detailed toxicokinetic models. Physiologically based pharmacokinetic models are valuable in the interpretation of animal toxicity studies. They provide a physiological basis for extrapolating between species and routes of administration, and the future use of these models in setting acceptable levels of exposure for non-cancer toxicity in humans deserves serious consideration.
Animals↗
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.
Animals↗
Research strategy in industrial toxicology.
While much of industrial toxicology is observational in character, pursuit of specific research is needed to facilitate the overall evaluation of potential toxicity for man. Two such areas are the application of physiologic pharmacokinetic models to inter-species extrapolation of toxic effects and an understanding of the role of cellular oncogenes in the process of spontaneous tumor formation in animals. A physiologic pharmacokinetic model was developed for methylene chloride (MeCl2) which describes the fate of MeCl2 and its metabolic products in numerous species including the mouse, rat, hamster and man. This model has been used to predict specific tissue concentrations of critical metabolic reaction products in target tissues between animals and man. If it is assumed that toxicity is related to target tissue concentrations such methodology provides a means of relating interspecies toxicity to absorbed dose. This methodology precludes the necessity of using arbitrary factors in relating animal toxicity data to man. A particular controversial issue in animal toxicology is the significance of the enhancement of animal tumors in tissues which already have a high spontaneous incidence. Without a better understanding of the basic process of spontaneous tumor formation it remains difficult to interpret results from chemical treatment. In particular spontaneous liver tumors in the B6C3F1 mouse have been shown to contain an activated cellular oncogene identified as H-RAS. The activated cellular oncogene is present in tumor tissue only and not in surrounding normal liver tissue. Of particular significance is the high frequency of activation in these mouse liver tumors (82%) compared to a 10-20% incidence of oncogenes present in a variety of human tumors. This suggests the ultra sensitivity of this mouse strain to liver tumor induction. Additional studies in progress are designed to determine whether genotoxic and nongenotoxic hepatocarcinogens show differences in oncogene activation.
Animals↗
The role of pharmacokinetics in risk assessment.
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Animals↗
Effect of food and water deprivation on the parameters of the mouse bone marrow micronucleus test.
This study reports the influence of acute starvation on spontaneous and cyclophosphamide (CP) induced micronucleus (MN) frequencies in the bone marrow polychromatic erythrocytes (PCE) of CD-1 mice. Groups of mice (5/sex) were deprived of either food alone or food and water for 0, 24, 48 and 72 h prior to sacrifice. Although there was no evidence of a significant increase in MN-PCE frequencies among the starved groups, a significant depressant effect of starvation on erythropoietic activity was observed. Peak levels of CP-induced (40 mg/kg b.w.) MN-PCE's appeared later in male mice deprived of food and water after treatment compared to mice given food and water ad libitum. The results indicate that starvation is detrimental to bone marrow erythropoiesis and that starvation may alter the response of mice to clastogens.
Animals↗
Detection of a cellular oncogene in spontaneous liver tumors of B6C3F1 mice.
An active cellular oncogene was demonstrated in hepatocellular neoplasms arising spontaneously in 24-month-old B6C3F1 mice. DNA isolated from the tumorous tissue and transfected into NIH 3T3 cells showed an 82 percent (9 of 11 animals) frequency of foci induction. In contrast, DNA isolated from the surrounding nontumorous hepatic tissue from the same animals and DNA from other 24-month-old B6C3F1 mice without tumors did not cause transformation in the NIH 3T3 cell assay. This strain of mouse is used extensively in carcinogen bioassays, and the observed high frequency of transformation (82 percent, compared to 10 to 20 percent in humans) supports the concept that the B6C3F1 mouse is hypersusceptible to liver tumor development. It also emphasizes the need to further understand the mechanisms of oncogene activation in animals used for long-term studies of toxicity and oncogenicity before evaluating potential human risk.
Animals↗
Measurement of solid feed consumption patterns in neonatal rats by 141Ce-radiolabeled microspheres.
Dams nursing litters of Fischer 344 rats were allowed access to feed containing nonabsorbable 141Ce-labeled NEN-TRAC microspheres for 24-hr intervals during the later half of a 28-day nursing period. Neonatal solid feed consumption began during the third week of nursing and rose to peak amounts as high as 2.1 times normal adult levels on a gram consumed per kilogram body weight per day basis at 28 days of age, with an average daily feed consumption (g kg-1 day-1) of approximately 1.5 times that normally eaten by adults. Feed consumption in lactating females peaked at concentrations as high as 3.2 times and averaged approximately 2.5 times the amount normally consumed by nonpregnant adult females of comparable age. These results should be considered when interpreting the results observed in reproduction studies when exposure is via feed. Effects which could be attributed to "enhanced neonatal sensitivity" may, in fact, merely reflect the toxicity resulting from increased chemical exposure. It is thus clear that, if reproduction studies are to be conducted at or near the MTD, adjustment of dietary concentration of test agents during lactation is necessary to maintain target dose levels and to prevent overexposure.
Animals↗
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.
Animals↗
Tetrachloroethylene: balance and tissue distribution in male Sprague-Dawley rats by drinking-water administration.
While low levels of organic contaminants have been reported to occur in drinking water, often little information is available concerning the toxicological significance of ingestion by this route. For tetrachloroethylene, considerable animal toxicity data exist for gavage or inhalation exposure. Since extrapolation of toxicity between routes of administration implies similar pharmacokinetic disposition, the pharmacokinetics of tetrachloroethylene was examined in rats following administration in the drinking water. Tetrachloro[14C]ethylene was administered to adult, male Sprague-Dawley rats in saturated drinking water solutions (approximately 150 ppm) resulting in a mean dose of 8.1 +/- 3.1 mg/kg. Within 72 hr from termination of a 12-hr drinking exposure period, approximately 88% of the body burden was eliminated as unmetabolized parent compound in expired air. As much as 96% of the activity recovered in this form was excreted during the first 24 hr. The remaining 14C activity recovered was the result of metabolic conversion to 14CO2 (2.2%), nonvolatile metabolites in urine (7.2%) and feces (1.7%), and minor amounts (0.9%) in the carcass. Radioactivity in the carcass 72 hr following termination of the exposure period was distributed within liver, kidneys, fat, lungs, heart, and adrenals. Pulmonary elimination of unchanged tetrachloroethylene was monophasic with a calculated elimination half life of 7.1 hr. The elimination kinetics were consistent with results previously generated by both gavage and inhalation exposure. The fate of tetrachloroethylene following drinking-water administration was not substantially different from the disposition resulting from these two routes of administration.
Animals↗
Molecular mechanisms involved in the toxicity of orthophenylphenol and its sodium salt.
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Alkylation↗
Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies.
Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.
Animals↗
Evaluation of ELISA for detecting in vivo chemical immunomodulation.
Immunotoxicology is a relatively new field of investigation and is becoming recognized and used by toxicologists involved in drug and chemical testing. The purpose of this study was to evaluate the enzyme-linked immunosorbent assay (ELISA) as a means of quantitating humoral immune responses in rats exposed to immunomodulating chemicals. The ELISA proved to be highly sensitive and quantitative, simple to perform, and reliable. The assay is also economically feasible and automated so that large numbers of samples can be analyzed at one time. The antigen used in the study was bovine serum albumin, and the immunosuppressive chemicals used to validate the system were lead, polychlorinated biphenyl, cyclophosphamide, and dexamethasone. Practical application of the procedure to immunotoxicology testing is discussed.
Animals↗
Relevance of experimental studies to human risk.
Confidence in the extrapolation of animal toxicity data to humans can be enhanced by the application of pharmacokinetic concepts integrated with chronic toxicity data and knowledge of a chemical's mechanism(s) of toxicity. Basic pharmacokinetic concepts (including dose-dependent or Michaelis-Menten kinetics) and their relationship to the risk estimation process are discussed using vinyl chloride and styrene as specific examples. Species differences in metabolic rates must be considered in order to arrive at realistic estimates of human risk to vinyl chloride-induced liver angiosarcomas utilizing vinyl chloride toxicity data observed in rats. Because small animal species generally metabolize chemicals more rapidly than larger species on a body surface area basis, small animals should be more sensitive to chemicals (such as vinyl chloride) that exert their toxicities via the metabolic formation of toxic products. Inhaled styrene is a chemical whose clearance from the blood at low exposure levels in both rats and humans follows first-order kinetics. However, at higher exposure levels, the pharmacokinetic fate of styrene in rats is dose-dependent, suggesting a saturation of styrene metabolism. These data indicate that any extrapolation of observable toxicity at elevated exposure levels in rats to anticipated responses at lower levels in either rats or humans may be invalid. An integration of the foregoing concepts provides a sound scientific basis for the use of experimental animal data to predict the risk to humans from chemical exposure.
Animals↗