The Tg.AC (v-Ha-ras) transgenic mouse: nature of the model.

The Tg.AC (v-Ha-ras) transgenic mouse model provides a reporter phenotype of skin papillomas in response to either genotoxic or nongenotoxic carcinogens. In common with the conventional bioassay, the Tg.AC model responds to known human carcinogens and does not respond to noncarcinogens. It also does not respond to most chemicals that are positive in conventional bioassays principally at sites of high spontaneous tumor incidence. The mechanism of response of the Tg.AC model is related to the structure and genomic position of the transgene and the induction of transgene expression through specific mediated interactions between the chemicals and target cells in the skin.

Tg.AC genetically altered mouse: assay working group overview of available data.

In a Government/Industry/Academic partnership to evaluate alternative approaches to carcinogenicity testing, 21 pharmaceutical agents representing a variety of chemical and pharmacological classes and possessing known human and or rodent carcinogenic potential were selected for study in several rodent models. The studies from this partnership project, coordinated by the International Life Sciences Institute, provide additional data to better understand the models' limitations and sensitivity in identifying carcinogens. The results of these alternative model studies were reviewed by members of Assay Working Groups (AWG) composed of scientists from government and industry with expertise in toxicology, genetics, statistics, and pathology. The Tg.AC genetically manipulated mouse was one of the models selected for this project based on previous studies indicating that Tg.AC mice seem to respond to topical application of either mutagenic or nonmutagenic carcinogens with papilloma formation at the site of application. This communication describes the results and AWG interpretations of studies conducted on 14 chemicals administered by the topical and oral (gavage and/or diet) routes to Tg.AC genetically manipulated mice. Cyclosporin A, an immunosuppresant human carcinogen, ethinyl estradiol and diethylstilbestrol (human hormone carcinogens) and clofibrate, an hepatocarcinogenic peroxisome proliferator in rodents, were considered clearly positive in the topical studies. In the oral studies, ethinyl estradiol and diethylstilbestrol were negative, cyclosporin was considered equivocal, and results were not available for the clofibrate study. Of the 3 genotoxic human carcinogens (phenacetin, melphalan, and cyclophosphamide), phenacetin was negative by both the topical and oral routes. Melphalan and cyclophosphamide are, respectively, direct and indirect DNA alkylating agents and topical administration of both caused equivocal responses. With the exception of clofibrate, Tg.AC mice did not exhibit tumor responses to the rodent carcinogens that were putative human noncarcinogens, (di(2-ethylhexyl) phthalate, methapyraline HCl, phenobarbital Na, reserpine, sulfamethoxazole or WY-14643, or the nongenotoxic, noncarcinogen, sulfisoxazole) regardless of route of administration. Based on the observed responses in these studies, it was concluded by the AWG that the Tg.AC model was not overly sensitive and possesses utility as an adjunct to the battery of toxicity studies used to establish human carcinogenic risk.

The National Toxicology Program evaluation of genetically altered mice as predictive models for identifying carcinogens.

National Institute of Environmental Health Sciences researchers are exploring the utility of genetically altered mice to study mechanisms of carcinogenesis. Two of these mouse models, the Tg.AC (carrier of an activated mouse H-ras oncogene) and the p53+/- (heterozygous for the wild-type tumor suppressor gene Trp53), have genetic alterations that appear to hasten their expression of chemically induced tumors. These 2 models have been proposed as a basis for new strategies for identifying chemical carcinogens and for assessing risk. The National Toxicology Program (NTP) is conducting a series of studies with these 2 genetically altered strains to further examine their strengths and weaknesses for identification of documented rodent and human carcinogens. In this first evaluation, candidates for study were drawn from the NTP historical database of 2-yr rodent carcinogenicity studies and the open literature (primarily for drugs). Results with this first set of 11 chemicals tested in genetically altered mice, compared with previous findings in the traditional 2-yr rodent assays and literature on human tumor findings, appear to support the premise advanced by Tennant et al that these models have the potential to serve as more rapid and less expensive test systems to identify carcinogens.

Spontaneous and chemically induced proliferative lesions in Tg.AC transgenic and p53-heterozygous mice.

Recently, the use of selected genetically altered mouse models in the detection of carcinogens after short-term chemical exposures has been evaluated. Studies of several chemicals conducted by the National Toxicology Program in Tg.AC transgenic and heterozygous p53-deficient mice have been completed recently and represent a major contribution to this effort, as well as the largest accumulation to date of toxicologic pathology data in these 2 lines of mice. The purpose of this report is to describe the proliferative target organ effects observed in this set of studies, as well as to present the tumor profile in the control groups of this data set. These findings provide a comprehensive toxicologic assessment of these 2 genetically altered mouse strains, which are of emerging importance in toxicologic pathology.

The U.S. National Toxicology Program evaluation of transgenic mice as predictive models for identifying carcinogens.

National Institute of Environmental Health Sciences researchers have invested considerable effort in exploring the utility of transgenic mice to detect carcinogens and study mechanisms of carcinogenesis. Work has assessed several mouse models genetically altered to enhance their expression of chemically induced tumors. Results with the p53def (hemizygous for the tumor-suppressor gene) and the Tg.AC (carrier of an activated H-ras oncogene) mice have been used as a basis for a proposed new strategy for identifying chemical carcinogens and assessing risk. The U.S. National Toxicology Program is conducting a series of studies with these two transgenic strains to further examine their strengths and weaknesses for identification of documented rodent and human carcinogens and to explore their ability to provide information concerning the effective dosimetry for target organ mutation.

Effects of D&C yellow no. 11 ingestion on F344/N rats and B6C3F1 mice.

D&C yellow no. 11 (CAS no. 8003-22-3) was administered in the feed at concentrations of 500-50,000 ppm to groups of F344/N rats and B6C3F1 mice of each sex for 13 wk to determine the toxicity. In addition, a perinatal study was conducted to determine the effects of feeding diets containing D&C yellow no. 11 to female rats during reproduction and to their offspring. Although the estimated intake (g/kg) of D&C yellow no. 11 of mice was more than twice that of rats, the results were generally similar for both rats and mice. In both species, D&C yellow no. 11 caused no mortality, but it did reduce body weight gain slightly in both sexes of rats exposed to 17,000 and 50,000 ppm. Absolute and relative liver weights were significantly increased in all groups of rats and mice administered D&C yellow no. 11 in the feed. There was minimal to mild degeneration of the periportal hepatocytes in rats at doses of 1700 ppm and higher and in mice at 5000 ppm and above. A dose-related yellow-brown pigment was observed in hepatocytes, Kupffer cells, and biliary epithelium of the liver of both sexes of both species and in the renal tubule epithelium in both sexes of rats. In male rats, all treated groups had increased number and size of hyaline droplets in the renal tubule epithelium of the cortex and outer medulla. To determine if these renal and hepatic lesions were reversible, male rats were administered 5000 ppm dietary D&C yellow no. 11 for 70 d and then examined at 3, 14, and 28 d after the chemical was removed from the diet. Pigment persisted in the kidney and liver for as long as 28 d following removal of D&C yellow no. 11 from the diet, but hepatocellular degeneration and cytoplasmic alteration in the kidney completely resolved by d 3 and 14, respectively. In the perinatal toxicity study, body weight gain in rat dams given diets containing as much as 50,000 ppm D&C yellow no. 11 for 4 wk before mating to untreated males was similar to that of controls at the time of mating but was lower at parturition and weaning. However, fertility, gestation length, litter size, and pup birth weights were unaffected by treatment. At weaning, there was a significant dose-related decrease in pup body weights from the 5000, 17,000, and 50,000 ppm groups. At 8 wk of age, pups fed the same dosed-feed concentrations as the dams had depressed body weights in the 17,000 and 50,000 ppm treated groups. Microscopic lesions in the liver and kidney of the pups in all dose groups were similar to those described in the 13-wk study. The results of these studies indicate that compound-related effects occurred at all dietary concentrations of D&C yellow no. 11. Liver weights were increased in dosed rats and mice, minimal to mild hepatocellular degeneration was seen in rats receiving dietary concentrations of 1700 ppm and above and in mice at 5000 ppm and above, and there was an increase in the number and size of hyaline droplets in all dosed groups of male rats. Similar compound-related effects were also seen in all dosed rats in the perinatal toxicity study. With the exception of pigment accumulation, the treatment-related kidney and liver lesions in male rats were reversible by 14 d after chemical was withdrawn from the diet.

Evidence for a common genetic pathway controlling susceptibility to mouse skin tumor promotion by diverse classes of promoting agents.

The present study has compared different mouse stocks and strains with known sensitivity to phorbol ester skin tumor promotion for their sensitivities to skin tumor promotion by a prototypic organic peroxide (benzoyl peroxide, BzPo) and anthrone (chrysarobin, Chr) tumor promoter. Following initiation with either 7,12-dimethylbenz(a)anthracene and/or N-methyl-N'-nitro-N-nitrosoguanidine, groups of mice were promoted with several different doses of each promoting agent. Among mice selectively bred for sensitivity to phorbol ester promotion, the order of sensitivity to BzPo was inbred SENCAR (SSIn) greater than SENCAR greater than CD-1. With Chr as the promoter, the order of sensitivity was SENCAR greater than SSIn greater than CD-1. Concurrent tumor promotion experiments examined the responsiveness of two common inbred mouse strains, DBA/2 and C57BL/6. The phorbol ester-responsive mouse strain, DBA/2, was more sensitive to skin tumor promotion by Chr than was C57BL/6 at all doses tested but was clearly less sensitive than both SENCAR and SSIn mice. Finally, DBA/2 and C57BL/6 mice were similar in their responsiveness to BzPo promotion, but again both of these inbred strains were significantly less sensitive than were SSIn and SENCAR mice to this organic peroxide type of skin tumor promoter. Histological evaluations comparing SENCAR and C57BL/6 mice revealed that a major difference between these strains in response to multiple Chr and BzPo treatments was in the inflammatory response (measured by edema formation). Unlike 12-O-tetradecanoylphorbol-13-acetate, Chr and BzPo did not induce dramatic differences in the epidermal hyperplasia (as measured by epidermal thickness) in these two mouse lines. The results presented in this paper suggest that there is a common pathway controlling susceptibility to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate, BzPo, and chrysarobin. These results are discussed in terms of a possible genetic model(s) for skin tumor promotion in mice.

Intestinal absorption of two glucose analogues in rats of different ages.

Intestinal absorption of nutrients and xenobiotics has been suggested to change during aging. Transport processes were examined using nonmetabolizable glucose analogues as markers in an in situ single pass intestinal perfusion procedure. Male Fischer 344 rats of 2, 4, 10, 16, and 27 months of age were used in this study. At least 7 rats were used per age group. Net active and passive transport were estimated using 0.85% NaCl solutions containing [14C]-3-O-methylglucose (3OMG) and [3H]-2-deoxy-D-glucose (2DG) and phenol red as nonabsorbed volume marker. Rats were anesthetized and approximately 30 cm of jejunum was perfused for 30 min. Final glucose analogue concentrations were corrected for volume changes, and compared with initial concentrations. Changes in marker concentrations were normalized to the dry weight of the perfused intestinal segment. Concurrent exposure to both glucose analogues allowed simultaneous monitoring of active and passive components. Under these conditions, there was no significant change in the net transport of 2DG, a compound absorbed by nonspecific processes (range 17-33 mumoles/g/h). In the same animals, the rate of net 3OMG absorption, an actively transported compound, was generally an order of magnitude greater than 2DG. In addition, net 3OMG absorption was significantly (p less than .05) decreased in the 16- and 27-month-old rats (237 and 181 mumoles/g/h) compared with the 2-, 4- and 10-month-old animals (325, 364 and 398 mumoles/g/h, respectively). There appears to be a gradual decrease in net active transport of glucose with age. The mechanism responsible for this age-related change remains to be explained.

Disposition of 4,4'-thiobis(6-t-butyl-m-cresol) in rats.

The absorption, distribution, metabolism, and excretion of 14C-labeled 4,4'-thiobis(6-t-butyl-m-cresol) (TBBC) was studied in male rats. Oral treatment showed a dose-related decrease in the rate of absorption due to a dose-related increase in retention time in the stomach. TBBC was incompletely absorbed after oral treatment, although the rate of absorption was proportional to the dose once the compound reached the small intestine. TBBC was rapidly distributed throughout the body with the liver being the major tissue depot. Significant amounts of the compound were also present in blood, muscle, skin, and adipose tissue. TBBC was initially rapidly cleared from all tissues except adipose, although a small percentage of the total dose tended to persist in liver and skin. Over half of the compound was excreted the first day, primarily via the bile into the feces. Little TBBC-derived radioactivity appeared in the urine. Metabolites of TBBC were present in the tissues at early times after administration, but were rapidly excreted. The major metabolite(s) appeared to be glucuronide conjugates of the parent compound. Thus, TBBC, an important antioxidant in the rubber and plastic industries, would tend to accumulate in liver and lipid-rich tissues upon chronic exposure, which if by the oral route, could also result in direct damage to the gastrointestinal tract.

Effects of lindane, paraquat, toxaphene, and 2,4,5-trichlorophenoxyacetic acid on mallard embryo development.

The effects were determined of externally treating mallard (Anas platyrhynchos) eggs with two insecticides (lindane and toxaphene) and two herbicides (paraquat and 2,4,5-T) with formulations and concentrations similar to field applications. Paraquat was the most embryotoxic of the four compounds regardless of the type of vehicle. The LC50 for paraquat was 1.5 lb of active ingredient/acre in aqueous emulsion and 0.1 lb/acre in the oil vehicle. The other compounds had LC50's that were several orders of magnitude higher. Both paraquat and toxaphene caused some mortality at 1/2 of the field level of application. Paraquat impaired growth and was slightly teratogenic at 1/2 of the field level of application, but required higher concentrations (1.5 to 3 times the field level) to produce brain and visceral defects. Lindane was teratogenic, resulting in multiple defects but only at doses that were greater than five times the field level of application. Toxaphene resulted in defects of the joints at doses close to or exceeding the LC50. The herbicide 2,4,5-T resulted in few toxic effects and relatively few abnormal survivors with gross defects. The overall embryotoxicity with either vehicle was paraquat greater than lindane greater than toxaphene greater than 2,4,5-T on a lb per acre basis. However the potential hazard at exposures of up to five times the field level of application was paraquat greater than toxaphene; neither lindane nor 2,4,5-T constituted much of a hazard. Both paraquat and lindane were more toxic on lb-per-acre basis when administered in oil vehicle but only paraquat represented a potential hazard at five times the field level of application.

Organophosphate inhibition of avian salt gland Na, K-ATPase activity.

1. Adult black ducks (Anas rubripes) were given freshwater or saltwater (1.5% NaCl) for 11 days and half of each group was also given an organophosphate (17 p.p.m. fenthion) in the diet on days 6-11. 2. After 11 days, ducks drinking saltwater had lost more weight and had higher plasma Na and uric acid concentrations and osmolalities than birds drinking freshwater. 3. Saltwater treatment stimulated the salt gland to increased weight and Na, K-ATPase activity. 4. Fenthion generally reduced plasma and brain cholinesterase activity and depressed cholinesterase and Na, K-ATPase activities in salt glands of birds drinking saltwater.

Effects of crude oil ingestion on avian intestinal function.

Intestinal function in mallard ducklings (Anas platyrhynchos) on a freshwater regime was studied after a 7-day dietary ingestion of 0.25% and 2.5% Prudhoe Bay crude oil (PBCO) or a 2.5% paraffin mixture with an in vivo luminal perfusion technique. Dietary ingestion of 2.5% PBCO may have an effect on the integrity of the duckling intestine. There were no significant effects of PBCO on the absorption of Na, Cl, K, or H2O compared with control animals. The ducklings fed 2.5% paraffin had a significant depression in Na and H2O absorption compared with controls. However, this depression dose not seem to be related to an effect on intestinal mucosa Na-K-ATPase activity.

Effects of industrial effluents, heavy metals, and organic solvents on mallard embryo development.

Mallard eggs were externally exposed at 3 and 8 days of incubation to 7 different industrial effluents and to 7 different heavy metal, organic solvent, and petroleum solutions to screen for potential embryo-toxic effects. This route of exposure was chosen in order to simulate the transfer of pollutant from the plumage of aquatic birds to their eggs. Five of the effluents including mineral pigment, scouring effluent, sludge, and tannery effluent resulted in small but significant reductions in embryonic growth. Treatment with methyl mercury chloride solution of 50 ppm (Hg) impaired embryonic growth but much higher concentrations were required to affect survival and cause teratogenic effects. Oil used to suppress road dust was the most toxic of the pollutants tested and only 0.5 microliter/egg caused 60% mortality by 18 days of development. These findings, in combination with other studies suggest that petroleum pollutants, or effluents in combination with petroleum, may pose a hazard to birds' eggs when exposure is by this route.

Effects of dietary nickel on mallards.

Thirty breeding pairs of mallards (Anas platyrhynchos) were randomly assigned to one of five treatment groups and were fed breeder mash containing 0, 12.5, 50.0, 200.0, or 800.0 ppm Ni (as the sulfate) for 90 d. Ni ingestion had no effect on egg production, hatchability, or survival of ducklings. After 90 d birds were bled, sacrificed, and necropsied. There were no significant differences in hematocrit; concentrations of hemoglobin, plasma triglyceride, and cholesterol; of plasma activities of ornithine carbamoyltransferase and alanine aminotransferase. A black tarry feces was noted in the high Ni dose group at necropsy, but no gross or histopathologic lesions were observed. Although absolute concentrations of Ni in tissues were low, there were significant accumulations in kidneys of birds fed Ni at all dietary levels and in feathers, blood, and livers of birds fed high doses of Ni compared with controls.