Joel F. Mahler

Spontaneous Lesions in Aging FVB/N Mice

The FVB/N mouse strain was created in the early 1970s and has since been used extensively in transgenic research because of its well-defined inbred background, superior reproductive performance, and prominent pronuclei of fertilized zygotes, which facilitate microinjection of DNA. Little is known, however, about the survivability and spontaneous disease of nontransgenic FVB/N mice. Therefore, the purpose of this study was to determine survival to 24 mo of age and the incidence of neoplastic and nonneoplastic disease at 14 and 24 mo of age. At 14 mo of age, the incidence of tumor-bearing mice was 13% in males (n = 45) and 26% in females (n = 98). All tumors in males and most in females at this time were alveolar-bronchiolar (AB) neoplasms of the lung. Survival to 24 mo of age was approximately 60% in both sexes (29/50 males, 71/116 females), and the incidence of mice with tumors at this time was 55% in males and 66% in females. In decreasing order of frequency, the following neoplasms were observed in >5% of subjects: in males, lung AB tumors, liver hepatocellular tumors, subcutis neural crest tumors, and Harderian gland adenomas; in females, lung AB tumors, pituitary gland adenomas, ovarian tumors (combined types), lymphomas, histiocytic sarcomas, Harderian gland adenomas, and pheochromocytomas. Compared with other mouse strains, the observed incidences of tumors in FVB/N mice suggest a higher than usual rate of lung tumors and a lower than usual incidence of liver tumors and lymphomas. This tumor profile should be considered in the interpretation of neoplastic phenotypes in FVB/N-derived transgenic lines.

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.

Spontaneous lesions in control B6C3F1 mice and recommended sectioning of male accessory sex organs.

Because sampling of the paired lobes (ventral, dorsal, lateral, and anterior) of the mouse prostate has often been inconsistent, comparisons among different investigations have lacked validity. The absence of site identifi cation for prostatic lesions has made reported incidences relatively nonspecific. We present here the lobe-specifi cincidences and degree of severity of spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands in 612 control B6C3F1 mice from 12 recent National Toxicology Program 2-year carcinogenicity and toxicity studies conducted in 1 of 4 different laboratories. Lymphocytic infi ltration, infl ammation, epithelial hyperplasia, mucinous cyst, and mucinous metaplasia were observed in the dorsolateral lobes. Lymphocytic infi ltration, infl ammation, epithelial hyperplasia, and edema were present in the ventral lobes. Lymphocytic infi ltration, acinar dilatation, infl ammation, epithelial hyperplasia, and atrophy occurred in the coagulating glands. No neoplastic lesions were observed in the prostate or coagulating gland. Lymphocytic infi ltration, acinar dilatation, inflammation, atrophy, adenoma, adenocarcinoma, and a granular cell tumor were observed in the seminal vesicles. Lymphocytic infi ltration was also present in the ampullary glands. The results of our survey indicate that the amounts of glandular tissues were not present consistently in slides from the different laboratories. Landmarks for uniform tissue trimming are needed. We therefore suggest an optimal trimming and embedding method for mouse prostate and seminal vesicles to ensure adequate, consistent sampling.

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 fromgovernmentand 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 clofi brate, an hepatocarcinogenicperoxisome 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 clofi brate, 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.

A Retrospective Analysis of Background Lesions and Tissue Accountability for Male Accessory Sex Organs in Fischer-344 Rats

Because the paired lobes (ventral, dorsal, lateral, and anterior) of the rat prostate have not been consistently sampled in many carcinogenicity and toxicity studies, comparison among different investigations has been compromised. The lack of specifi c site identifi cation for prostatic lesions further lessens the value of incidences reported. We present here the lobe-specific incidences and degree of severity of background prostatic, seminal vesicular, and ampullary glandular lesions in 1768 control Fischer-344 rats from 35 recent National Toxicology Program 2-year carcinogenicity and toxicity studies conducted in 4 laboratories. The dorsal and lateral lobes were combined and considered the dorsolateral lobe where inflammation, epithelial degeneration, mucinous cysts, and edema were observed. Infl ammation in the dorsolateral lobes was signifi cantly associated with pituitary gland adenoma whose prolactin was suggested to play an important role in pathogenesi s of prostatic inflammation. Epithelial degeneration, epithelial hyperplasia, infl ammation, edema, and adenoma were conspicuous in the ventral lobes. Infl ammation and edema occurred in the anterior lobes (coagulating glands). Inflammation, dilatation, epithelial hyperplasia, edema, and adenoma were observed in the seminal vesicles. Infl ammation was also present in the ampullary glands. We suggest an optimal embedment and trimming method in rat prostate and seminal vesicle to ensure adequate, consistent sampling.

Unique renal tubule changes induced in rats and mice by the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D) and WY-14643.

Peroxisome proliferators are non-mutagenic carcinogens in the liver of rodents, acting both as initiators and promoters. The National Toxicology Program (NTP) conducted a study of several peroxisome proliferators (PPs), including Wyeth (WY)-14643 as a prototypical PP and 2,4-dichlorophenoxyacetic acid (2,4-D) as a weak PP, in Sprague-Dawley rats, B6C3F1 mice, and Syrian hamsters. In the kidney, an unusual change was observed in the outer stripe of the outer medulla, especially in rats treated with 2,4-D or WY-14643. This change was characterized by foci of tubules that were partially or completely lined by basophilic epithelial cells with decreased cytoplasm and high nuclear density. Changes typical of chronic nephropathy such as interstitial fibrosis or basement membrane thickening were not associated with these foci. Results of immunohistochem- ical staining for catalase and cytochrome P-450 4A in the kidney indicated increased staining intensity in renal tubular epithelial cells primarily in the region where the affected tubules were observed; however, the altered cells were negative for both immunohistochemica l markers. Ultrastructurally, affected cells had long brush borders typical of the P3 tubule segment. The most distinguishing ultrastructural change was a decreased amount of electronlucent cytoplasm that contained few differentiated organelles and, in particular, a prominent reduced volume and number of mitochondria; changes in peroxisome s were not apparent. In addition to the lesion in rats, mice treated with the highest dose of 2,4-D, but not WY-14643, manifested similar renal tubular changes as seen by light microscopy. Neither chemical induced renal tubular lesions in hamsters. Hepatocellular changes characteristic of PPs were present in all 3 species treated with WY-14643, but not 2,4-D. These results indicate that the rat is the species most sensitive to the nephrotoxic effects of PPs and there is a site specificity to this toxicity related to areas of PP-related enzyme induction. Although 2,4-D is considered a weak PP for the liver, it was the most effective at inducing renal lesions, indicating that the toxic potency of various PPs will depend on the target organ.

Disruption of the Mouse Cyclooxygenase 1 Gene

We recently reported the characteristics of mice deficient in cyclooxygenase (COX) 1 (Langenbach et al., 1995) and COX-2 (Morham et al., 1995). This chapter will summarize the known characteristics of COX-1 deficient mice and describe some future studies utilizing these mice which will lead to a better understanding of the physiological functions of the COX’s and their roles in the therapeutic and toxic effects of NSAIDs (non-steroidal anti-inflammatory drugs).

Glycine Modulates the Toxicity of Benzyl Acetate in F344 Rats

The influence of supplemental glycine on benzyl acetate (BA; a compound metabolized via the hippurate pathway)-induced toxicity was investigated. Groups of male F344 rats were fed NIH-07 diet containing 0, 20,000, 35,000, or 50,000 ppm BA for up to 28 days. Two additional groups were fed NIH-07 diet with 50,000 ppm BA and 27,000 ppm glycine or 50,000 ppm BA and 32,000 ppm L-alanine; supplemental glycine and L-alanine were equimolar. The L-alanine group served as an amino nitrogen control. A third group was fed NIH-07 diet with 32,000 ppm L-alanine and served as an untreated isonitrogenous control. BA caused increase in mortality, body weight loss, the incidence of abnormal neurobehavioral signs such as ataxia and convulsions, along with astrocyte hypertrophy and neuronal necrosis in the cerebellum, hippocampus, and pyriform cortex of the brain. These effects were reduced significantly by supplementation with glycine but not with L-alanine. These results suggest that the neurodegeneration induced by BA is mediated by a depletion of the glycine pool and the subsequent excitotoxicity.

Have you seen this ? Inflammatory lesions in the lungs of rats

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