Deborah E. Devor

Emerging Issues in Mouse Liver Carcinogenesis

The mouse liver is the primary target site for carcinogenesis of more than 200 chemicals (including pesticides. food additives, pharmaceuticals, and industrial intermediates) tested in long-term toxicity safety assessment assays. Mouse liver tumors develop through defined morphological stages (similar to those found in other species) whether their origin is of undetermined etiology (spontaneous) or induced by chemicals. The morphologic type of hepatocytes in the various stages of hepatocarcinogenesis is sometimes associated with the specific inducing agent. Liver tumors developing in toxic livers often have more benign appearances and may progress to carcinomas at a slower rate than tumors developing in histologically normal livers. Specific tumors, dependent on the inducing chemical, may regress under defined protocols. Genotoxic and nongenotoxic mouse hepatocarcinogens each may induce tumors of either high malignant or low malignant potential. Liver tumors with specific H-ras oncogene mutations may appear morphologically and biologically similar to those without proven ras mutations. Thus, distinguishing mechanism of carcinogenesis by liver tumor morphology and mutation spectra may be difficult. Additionally, the presence of liver tumors with a morphology and a ras oncogene mutation spectrum characteristic of spontaneous tumors in histologically normal livers of mice exposed to a nongenotoxic test chemical may indicate promotion of spontaneous hepatocarcinogenesis by one of several potential mechanisms. Further research into the mechanisms responsible for the increased incidences of liver tumors in mice exposed to test chemicals could enhance human cancer risk assessments.

Acute effects of 4-ipomeanol on experimental lung tumors with bronchiolar or alveolar cell features in Syrian hamsters or C3H/HeNCr mice

Abstract4-Ipomenaol (IPO) has been shown to induceP-450-mediated necrosis of Clara cells in experimental animals, and clinical trials were initiated to treat people with bronchioloalveolar cancers with this novel drug. We therefore performed experiments to examine two different animal lung tumor models for acute IPO cytotoxicity: hamster Clara-cell-derived adenocarcinomas and mouse alveolar type II cell tumors. Clara cells serve as stem cells for airway cell renewal and, therefore, tumors derived from Clara cells may likewise differentiate into various bronchiolar cell types, or undergo squamous cell metaplasia. Bronchiolar cell tumors were induced in Syrian hamsters by a single weekly gavage with 6.8 mgN-nitrosomethyl-n-heptylamine (NMHA)/animal for 35 weeks. NMHA-induced bronchiolar tumors were classified as well-differentiated lepidic bronchioloalveolar carcinomas, acinar adenocarcinoma, adenosquamous carcinoma, and squamous-cell carcinoma. After 35 and 46 experimental weeks, control and carcinogen-treated hamsters were injected once with doses of 40–110 mg IPO/kg i.p. and necropsied 15–48 h later. Solid and papillary tumors with alveolar cell features were induced transplacentally in C3H/HeNCr mice, by treating pregnant animals on gestation day 16 with 0.5 mmolN-nitrosoethylurea/kg, i.p. Offspring of control and carcinogen-treated mice were injected at 2–3 months of age with 35 mg or 50 mg IPO/kg i.p. and necropsied either 24–48 h or 5 and 12 days after injection. Light microscopic studies were carried out to assess cytotoxic effects in various tissues in both hamsters and mice; in hamsters, additional ultrastructural studies were performed. When administered to hamsters, IPO induced moderate to severe cytotoxicity in normal and dysplastic bronchiolar lining cells, in most lepidic bronchioloalveolar carcinomas, and in some glandular areas of adenosquamous cell carcinomas. Susceptible cells included normal, anaplastic, and neoplastic nonciliated and some ciliated bronchiolar cells. Undifferentiated and squamous tumor cells were resistant to IPO, as were resident normal alveolar type II cells. However, some adenocarcinomas composed primarily of ciliated and mucous cells also showed no IPO-induced necrosis, indicating a deficiency in appropriate activating enzymes. In the mice, IPO induced bronchiolar cell necrosis and, at the high dose, also severe pulmonary edema. No cytotoxicity was observed in normal or hyperplastic alveolar epithelium, nor in either solid or papillary growth forms of mouse alveolar cell tumors. In conclusion, these experiments show, in original tumor settings of the lung, that it is possible to achieve cell-specific cytotoxic effects based on cellular composition and functional maturity, i.e., toxicity in carcinomas of predominantly nonciliated bronchiolar cells but not in tumors of alveolar type II cell lineage.

Development of an Animal Model for Testing Human Breast Implantation Materials

Although breast implant materials have been tested in laboratory species since the early 1950s, a standardized evaluation system does not currently exist in which human-made polymers are exposed directly to the mammary milieu of female animals. The present study evaluated such a model as the basis for future experiments on long-term tissue effects. Polyesterurethane disks, 6 mm in diameter × 3 mm thick, were inserted bilaterally beneath the axillobrachial and inguinal mammary/fat pads of 50 9-wk-old female B6D2F1 mice (4 implants each). Implant sites were examined histologically at time points 24 hr to 47 wk after surgery. An acute inflammatory reaction at the implant edges began within 24 hr, and macrophages were found lining the smooth polyurethane fiber surfaces at the periphery by day 2. Multinucleated foreign body giant cells formed by day 4, and by week 4 giant cells contained polyurethane fragments within the cytoplasm, implying degradation of the material. Implant sites showed declining subacute inflammatory responses and increasing fibrosis by week 5. By 13 wk, the polyurethane disks appeared to be integrated into the growing adipose and mammary tissues. Although not apparent on gross inspection, microscopic examination showed that polyurethane fibers moved progressively into adjacent tissues and were always associated with chronic granulomatous inflammation. Histologic findings in the present study are strikingly similar to the human response to polyurethane-coated breast implants. These results suggest the applicability of this model to appropriately test mammaplasty materials in mammary tissues.

Activation of neu by missense point mutation in the transmembrane domain in schwannomas induced in C3H/HeNCr mice by transplacental exposure to N -nitrosoethylurea

Abstract Transplacentally initiated schwannomas in mice and rats arise preferentially in the Gasserian ganglion of the trigeminal nerve and spinal root ganglia, while those of the Syrian golden hamster most commonly occur subcutaneously. Rat and hamster schwannomas almost invariably contain a mutationally activated neu oncogene. In rat schwannomas, the mutant allele predominates, while the relative abundance of mutant alleles is very low in hamster nerve tumors. We investigated whether neu is mutated in mouse schwannomas and whether the pattern and allelic ratio of the mutation resemble those for the hamster or the rat. Pregnant C3H/HeNCr mice received 0.4 μmol N-nitrosoethylurea/g body weight on day 19 of gestation. Ten trigeminal and one peripheral nerve schwannomas developed in 11 of the 201 offspring. Missense T → A transversion mutations were detected in the neu transmembrane domain in eight of ten schwannomas analyzed, as determined by MnlI digestion of polymerase chain reaction products. The mutant allele was predominantly detected in two tumors and was abundant in six others. Transfection of eight out of ten mouse tumor DNAs into hamster cells yielded transformed foci; seven out of eight contained mutant mouse neu. Mouse schwannomas closely resembled those of rats both in the preferred anatomical site and in the mutant/wild-type neu allele ratios.

Promotion of hepatocellular foci and adenomas by di(2-ethylheyl) phthalate and phenobarbital in C3H/HeNCr mice following exposure to N-nitrosodiethylamine at 15 days of age

Previous studies have shown that phenobarbital (PB), as well as another known liver tumor promoter, alpha-hexachlorocyclohexane (HCH), inhibits hepatic tumor formation in infant N-nitrosodiethylamine (NDEA)-initiated C57BL/6 x C3H/He (B6C3F1) male mice. These inconsistencies in detecting PB and HCH as tumor promoters have raised important questions on the mechanism of tumor promotion in mice, as well as the reliability of the infant B6C3F1 mouse as an initiation model in two-stage carcinogenesis experiments. Therefore, in an effort to avoid the inconsistencies associated with the B6C3F1 mouse, the present study evaluated the ability of two known hepatic liver tumor promoters, di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, and phenobarbital (PB), a barbiturate, to promote hepatocellular tumorigenesis in mice of the C3H/HeNCr strain initiated during infancy. At 15 days of age, male and female C3H/HeNCr mice received either a single ip injection of NDEA (5 micrograms/g body weight) or saline. At weaning (4 weeks of age), mice were divided into 3 groups and treated with either DEHP in the diet (12,000 ppm), PB in the drinking water (500 ppm), or control drinking water and diet for 24 weeks. All mice were killed at 28 weeks of age and the number and size of hepatic foci and adenomas were evaluated. Mice exposed to NDEA+DEHP or NDEA+PB showed significant increases in the number and size of hepatic tumors compared to those receiving NDEA alone. DEHP treatment in males yielded larger adenomas than those seen in PB-treated males.(ABSTRACT TRUNCATED AT 250 WORDS)

neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N-nitrosoethylurea: high incidence but low allelic representation.

Abstract Peripheral nerve tumors (PNT) and melanomas induced transplacentally on day 14 of gestation in Syrian golden hamsters by N-nitrosoethylurea were analyzed for activated oncogenes by the NIH 3T3 transfection assay, and for mutations in the neu oncogene by direct sequencing, allele-specific oligonucleotide hybridization, MnlI restriction-fragment-length polymorphism, single-strand conformation polymorphism, and mismatch amplification mutation assays. All (67/67) of the PNT, but none of the melanomas, contained a somatic missense T → A transversion within the neu oncogene transmembrane domain at a site corresponding to that which also occurs in rat schwannomas transplacentally induced by N-nitrosoethylurea. In only 2 of the 67 individual hamster PNT did the majority of tumor cells appear to carry the mutant neu allele, in contrast to comparable rat schwannomas in which it overwhelmingly predominates. The low fraction of hamster tumor cells carrying the mutation was stable through multiple transplantation passages. In the hamster, as in the rat, specific point-mutational activation of the neu oncogene thus constitutes the major pathway for induction of PNT by transplacental exposure to an alkylating agent, but the low allelic representation of mutant neu in hamster PNT suggests a significant difference in mechanism by which the mutant oncogene acts in this species.

Mast Cell Neoplasms, Mouse

Mast cell neoplasms of the mouse appear grossly as soft, white, opaque nodules originating in the subcutis or in other organs such as the lymph nodes, omentum, spleen, and stomach where mast cells are normally found (Deringer and Dunn 1947; Furth et al. 1957). Occasionally, hemorrhagic foci or a canary-yellow margin can accompany the neoplastic growth, and the liver, spleen, or lymph nodes may be swollen. Some cases, however, are detectable only by light microscopic examination (Deringer and Dunn 1947; Dunn and Potter 1957; Frith et al. 1976).

Squamous Cell Carcinoma Arising in Induced Papilloma, Skin, Mouse

Most frequently, squamous cell carcinomas are studied in skin-painting studies (complete carcinogenesis or initiation-promotion regimens) and are identified as arising from induced papillomas, keratoacanthomas, or epidermal inclusion cysts (Shubik et al. 1953; Foulds 1954). Squamous cell carcinomas are also seen developing within the epidermis without preceding neoplastic lesions or in association with ulcers (Bogovski 1979; Klein-Szanto 1984; Knutsen et al. 1986). Characteristic clinical features of squamous cell carcinomas developing from papillomas are loss of the papillomatous stalk and wartlike appearance, followed by ulceration of the papillomas and crater formation. The borders are elevated and poorly demarcated (Bogovski 1979; Knutsen et al. 1986). The neoplasms grow rapidly and may bleed severely.