John E. Sagartz

p53 +=¡ Hemizygous Knockout Mouse: Overview of Available Data

The performance of the p53 +/- transgenic (knockout) mouse model was evaluated through review of the data from 31 short-term carcinogenicity studies with 21 compounds tested as part of the International Life Sciences Institutes (ILSI) Alternatives to Carcinogenicity Testing (ACT) project, together with data from other studies which used comparable protocols. As expected based on the hypothesis for the model, a significant number (12/16 or 75%) of the genotoxic human and/or rodent carcinogens tested were positive and the positive control, p-cresidine, gave reproducible responses across laboratories (18/19 studies positive in bladder). An immunosuppressive human carcinogen, cyclosporin A, was positive for lymphomas but produced a similar response in wild type mice. Two hormones that are human tumorigens, diethylstilbestrol and 17β-estradiol, gave positive and equivocal results, respectively, in the pituitary with p53-defi cient mice showing a greater incidence of proliferative lesions than wild type. None of the 22 nongenotoxic rodent carcinogens that have been tested produced a positive response but 2 compounds in this category, chloroform and diethylhexylphthalate, were judged equivocal based on effects in liver and kidney respectively. Four genotoxic noncarcinogens and 6 nongenotoxic, noncarcinogens were also negative. In total (excluding compounds with equivocal results), 42 of 48 compounds or 88% gave results that were concordant with expectations. The technical lessons learned from the ILSI ACT-sponsored testing in the p53+/- model are discussed.

Early cellular abnormalities induced by RET/PTC1 oncogene in thyroid-targeted transgenic mice

The RET/PTC1 oncogene, a rearranged form of the RET proto-oncogene, has been reported to be associated with human papillary thyroid carcinomas. We have shown that targeted expression of RET/PTC1 in the thyroid gland leads to the development of thyroid carcinomas in transgenic mice with histologic and cytologic similarities to human papillary thyroid carcinoma. To further investigate how RET/PTC1 expression contributes to the pathogenesis of papillary thyroid tumor, the time of tumor onset and the early phenotypic consequences of RET/PTC1 expression in thyrocytes were determined. All high copy transgenic mice developed bilateral thyroid tumors as early as 4 days of age. At embryological days 16 – 18, increased proliferation rate, distorted thyroid follicle formation and reduced radioiodide concentrating activity were identified in transgenic embryos. The reduced radioiodide concentrating activity was attributed to decreased expression of the sodium-iodide symporter. Our study showed that RET/PTC1 not only increased proliferation of thyrocytes, it also altered morphogenesis and differentiation. These findings provide a model for the role of RET/PTC1 in the formation of abnormal follicles with reduced iodide uptake ability observed in human papillary thyroid carcinoma.

Thyroid Carcinomas in RET/PTC Transgenic Mice

The RET/PTC oncogene, a rearranged form of the RET proto-oncogene, has been found to be associated with human papillary thyroid carcinomas. To investigate whether RET/PTC causes papillary thyroid carcinoma, we generated a transgenic mouse model of papillary thyroid carcinoma with targeted expression of RET/PTC1 in the thyroid gland. Thyroid tumors in these RET/PTC1 transgenic mice are characterized by a slow growth rate, thyroid-stimulating hormone (TSH)-responsive tumor progression, and loss of radioiodide-concentrating activity despite continued expression of thyroglobulin (Tg). The time of tumor onset appears to be dependent on the expression level of RET/PTC1 in these transgenic mice. In high-copy RET/PTC1 transgenic mice, cellular abnormalities, including a slightly increased proliferation rate, aberrant follicle formation, and loss of radioiodide-concentrating activity, can be readily identified at embryological day 18. To identify which signaling pathway or pathways perturbed by RET/PTC1 are essential for RET/PTC1 to induce tumor development, we generated transgenic mice carrying a thyroid-targeted RET/PTC1 triple mutant, which contains tyrosine to phenylalanine mutations at tyrosine residues 294, 404, and 451. Initial characterization of the thyroid glands of these RET/PTC1 triple-mutant transgenic mice showed no change in follicular morphology or radioiodide-concentrating activity. This finding suggests that signaling pathways mediated by one or more of these three phosphotyrosine binding sites are essential for RET/PTC1 to induce thyroid tumor development. Finally, in order to investigate whether tumors induced by RET/PTC3 are more aggressive than those tumors induced by RET/PTC1, we also generated thyroid-targeted RET/PTC3 transgenic mice.

Neonatal Mouse Model: Review of Methods and Results

The neonatal mouse model, in various forms, has been used experimentally since 1959 and a large number of chemicals have been tested. The neonatal model is known to be very sensitive for the detection of carcinogens that operate via a genotoxic mode of action. In contrast, it is known not to respond to chemicals that act via epigenetic mechanisms, commonly observed in the two-year carcinogenicity studies. As such, the model has a high sensitivity and specifi city in its response. Dose selection for the neonatal model is based on the maximum tolerated or feasible dose. Traditionally, compounds have been tested via the IP route of administration in this model. In some cases, this has limited the amount of material that can be administered because of the low dosing volumes (10 to 20 μL) that can be administered IP. For the ILSI project, the neonatal model was adapted for oral administration, which has the advantages of being the same route for which most pharmaceuticals are administered. In addition, a 10-fold increase in the volume of administration (100 to 200 μL) and the ability to dose drugs in suspension, permits much higher doses to be used as compared to the IP route of administration. The spontaneous tumors in the neonatal model occurred mainly in the liver of male mice and lung of male and female mice with a few tumors observed in the Harderian gland. The positive control, DEN produced a robust, uniform, and reproducible tumor response with the target organs essentially limited to liver and lung. A total of 13 compounds out of the 21 ILSI ACT compounds were evaluated in the neonatal model involving 18 studies with duplicate studies for some compounds. The genotoxic carcinogens including those used as positive controls were clearly positive (cyclophosphamide, diethylnitrosamine, 6-nitrochrysene). The non-genotoxicrodent carcinogens were clearly negative (chlorpromazine, sulfi soxazole, sulfamethoxazole, clofi brate, DEHP, haloperidol, metaproteranol, and phenobarbital). The non-genotoxic human carcinogen (cyclosporin) was clearly negative. The two other human carcinogens phenacetin and DES were negative and interestingly estradiol was negative in one of the two oral studies, but was clearly positive in the other. Considering the mode of action for three of the human carcinogens (DES, cyclosporin and phenacetin), which were negative in this model, the mode of action in humans is likely to be epigenetic. Overall, for the 3 clearly genotoxic chemicals, all were positive. For the 9 clearly non-genotoxic chemicals, all 9 were negative. The two human carcinogens for which genotoxicity may or may not play a role (DES and phenacetin) were negative and estradiol was positive in 1 of the two oral studies. Overall, the extensive database for compounds tested in the neonatal mouse model would support its use as an alternative model for the assessment of the carcinogenic potential of a chemical. The model responds to chemicals that act via a genotoxic mode of action that represent a greater concern for human cancer risk.

NSAID-Induced Acute Phase Response is Due to Increased Intestinal Permeability and Characterized by Early and Consistent Alterations in Hepatic Gene Expression

Toxicogenomics using a reference database can provide a better understanding and prediction of toxicity, largely by creating biomarkers that tie gene expression to actual pathology events. During the course of building a toxicogenomic database, an observation was made that a number of non-steroidal anti-inflammatory compounds (NSAIDs) at supra-pharmacologic doses induced an acute phase response (APR) and displayed hepatic gene expression patterns similar to that of intravenous lipopolysaccharide (LPS). Since NSAIDs are known to cause injury along the gastrointestinal tract, it has been suggested that NSAIDs increase intestinal permeability, allowing LPS and/or bacteria into the systemic circulation and stimulating an APR detectable in the liver. A short term study was subsequently conducted examining the effects of aspirin, indomethacin, ibuprofen, and rofecoxib to rats and a variety of endpoints were examined that included serum levels of inflammatory cytokines, histologic evaluation, and hepatic gene expression. Both indomethacin and ibuprofen injured the gastrointestinal tract, induced an APR, and increased serum levels of LPS, while rofecoxib and aspirin did not affect the GI tract or induce an APR. In treatments that eventually showed a systemic inflammatory response, hepatic expression of many inflammatory genes was noted as early as 6 hours after treatment well before alterations in traditional clinical pathology markers were detected. This finding led to the creation of a hepatic gene expression biomarker of APR that was effectively shown to be an early identifier of imminent inflammatory injury. In terms of the relative gastrointestinal safety and the NSAIDs studied, an important safety distinction can be made between the presumptive efficacious dose and the APR-inducing dose for indomethacin (1—2-fold), ibuprofen (5-fold), and rofecoxib (~250-fold). Our data support the notion that NSAID-induced intestinal injury results in leakage of commensural bacteria and/or LPS into the circulation, provoking a systemic inflammatory response and that hepatic gene expression-based biomarkers can be used as early and sensitive biomarkers of APR onset. [The table referenced in this paper is not printed in this issue of Toxicologic Pathology. It is available as a downloadable text file in the online edition of Toxicologic Pathology, 34(2). In order to access the full article online, you must have either an individual subscription or a member subscription accessed through www.toxpath.org.]

Acute Lymphoid and Gastrointestinal Toxicity Induced by Selective p38α Map Kinase and Map Kinase–Activated Protein Kinase-2 (MK2) Inhibitors in the Dog

Exposure to moderately selective p38α mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38α MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38α MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38α MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Phenobarbital Does Not Promote Hepatic Tumorigenesis in a Twenty-Six-Week Bioassay in p53 Heterozygous Mice

The tumorigenic potential of phenobarbital was examined in a 26-wk carcinogenesis bioassay using p53 heterozygous mice and wild-type controls. Fifteen mice/sex/genotype were exposed to either 500 or 1,000 ppm phenobarbital in the diet. Dietary administration of 3,750 ppm p-cresidine, a transspecies mutagenic carcinogen, to both heterozygous and wild-type mice served as a positive control. Phenobarbital treatment caused increases in liver: body weight ratios and histologic evidence of centrilobular hepatocellular hypertrophy. No tumors were observed in any phenobarbital-treated mice. Mice given p-cresidine exhibited a moderate reduction in body weight gain over the couise of the study. Heterozygous mice treated with p-cresidine exhibited a high incidence of urinary bladder tumors. Similar tumors were also present in a small number of p-cresidine-treated wild-type mice. Our results demonstrate the lack of a hepatic tumor response to phenobarbital, a compound that is a potent and prototypic hepatic microsomal enzyme inducer, a nongenotoxic rodent carcinogen, and a human noncarcinogen. This finding supports the continued utility of this model as an alternative to the mouse bioassay for human carcinogenic safety assessment of potentially genotoxic carcinogens because it did not produce a false-positive response to this potent nongenotoxic agent.

The Pathologist and Toxicologist in Pharmaceutical Product Discovery

Significant change is occurring in the drug discovery paradigm; many companies are utilizing dedicated groups from the toxicology/ pathology disciplines to support early stage activities. The goal is to improve the efficiency of the discovery process for selecting a successful clinical candidate. Toxicity can be predicted by leveraging molecular techniques via rapid high-throughput, low-resource in vitro and in vivo test systems. Several important activities help create a platform to support rapid development of a new molecular entity. The proceedings of this symposium provide excellent examples of these applied concepts in pharmaceutical research and development. Leading biopharmaceutical companies recognize that a competitive advantage can be maintained via rapid characterization of animal models, the cellular identification of therapeutic targets, and improved sensitivity of efficacy assessment. The participation of the molecular pathologist in this quest is evolving rapidly, as evidenced by the growing number of pathologists that interact with drug discovery organizations.

Phagocytosis of Fluorescent Beads by Rat Thyroid Follicular Cells (FRTL-5): Comparison with Iodide Trapping as an Index of Functional Activity of Thyrocytes In Vitro

The ability of FRTL-5 rat thyroid follicular cells to engulf latex beads by phagocytosis was evaluated using flow cytometry and compared to iodide trapping in response to selected growth factors, second messengers, and chemicals. Cell suspensions were analyzed to determine the percentage of fluorescence-positive cells as well as the fluorescence intensity of positive cells. Phagocytosis was stimulated by forskolin, cholera toxin, 8-Br-cAMP, calcitriol, and transforming growth factor-β. In contrast, phagocytosis was inhibited by insulin, calcium, and aminotriazole, but not by sodium iodide. The results of this study showed that phagocytosis of latex beads was regulated in a manner similar to iodide trapping and could be altered by the addition of numerous compounds. Phagocytic activity was stimulated by both cAMP-dependent and cAMP-independent pathways. Flow cytometric evaluation of phagocytosis of fluorescent latex beads represents a simple, rapid, nonradioactive index of thyroid function in vitro.