Xpa and Xpa/p53+/- knockout mice: overview of available data. (9/352)

DNA repair deficient Xpa-/- and Xpa-/-/p53+/- knock-out mice in a C57BL/6 genetic background, referred to as respectively the XPA and XPA/p53 model, were investigated in the international collaborative research program coordinated by International Life Sciences Institute (ILSI)/Health and Environmental Science Institute. From the selected list of 21 ILSI compounds, 13 were tested in the XPA model, and 10 in the XPA/p53 model. With one exception, all studies had a duration of 9 months (39 weeks). The observed spontaneous tumor incidence for the XPA model after 9 months was comparable to that of wild-type mice (total 6%). For the XPA/p53 model, this was somewhat higher (9%/13% for males/females). The 3 positive control compounds used, B[a]P, p-cresidine, and 2-AAF, gave positive and consistent tumor responses in both the XPA and XPA/p53 model, but no or lower responses in wild-type mice. From the 13 ILSI compounds tested, the single genotoxic carcinogen (phenacetin) was negative in both the XPA and XPA/p53 model. Positive tumor responses were observed for 4 compounds, the immunosuppressant cyclosporin A, the hormone carcinogens DES and estradiol, and the peroxisome proliferator WY-14,643. Negative results were obtained with 5 other nongenotoxic rodent carcinogens, and 2 noncarcinogens tested. As expected, both DNA repair deficient models respond to genotoxic carcinogens. Combined with previous results, 6 out of 7 (86%) of the genotoxic human and/or rodent carcinogens tested are positive in the XPA model. The positive results obtained with the 4 mentioned nongenotoxic ILSI compounds may point to other carcinogenic mechanisms involved, or may raise some doubts about their true nongenotoxic nature. In general. the XPA/p53 model appears to be more sensitive to carcinogens than the XPA model.  (+info)

Neonatal mouse model: review of methods and results. (10/352)

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 specificity 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 microL) 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 microL) 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-genotoxic rodent carcinogens were clearly negative (chlorpromazine, sulfisoxazole, sulfamethoxazole, clofibrate, 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 I 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.  (+info)

Background and framework for ILSI's collaborative evaluation program on alternative models for carcinogenicity assessment. International Life Sciences Institute. (11/352)

The willingness of the agencies involved in the regulation of pharmaceuticals to accept data from newly proposed models for carcinogenicity testing (eg, transgenic animals, neonatal rodent models, initiation-promotion models) has stimulated international interest in gaining experience and a greater understanding of the strengths and limitations of the specific models. Over a 4-year period, the International Life Sciences Institute (ILSI) Health and Environmental Science Institute (HESI) has coordinated a large-scale collaborative research program to help to better characterize the responsiveness of several models proposed for use in carcinogenicity assessment. The overall objective of this partnership among industry, government, and academic scientists was to evaluate the ability of these new models to provide useful information for human cancer risk assessment. This research program reflected a commitment of nearly US$35 million by over 50 industrial, govemment, and academic laboratories from the United States, Europe, and Japan. Evaluation of the models required the development of standardized protocols to allow reproducibility and comparability of data obtained across multiple laboratories. Test compounds were selected on the basis of mechanistically meaningful carcinogenic activity or noncarcinogenicity in the rodent bioassay as well as humans. Criteria were established for dose selection, pathology review, quality control, and for evaluation of study outcome. The database from these studies represents an important contribution to the future application of new models for human cancer risk assessment. Beyond the data, the collaborative process by which the models were evaluated may also represent a prototype for assessing new methods in the future.  (+info)

The syrian hamster embryo (SHE) cell transformation assay: review of the methods and results. (12/352)

The Syrian hamster embryo (SHE) cell-transformation assay represents a short-term in vitro assay capable of predicting rodent carcinogenicity of chemicals with a high degree of concordance (LeBoeuf et al [1996]. Mutat Res 356: 85-127). The SHE assay models the earliest identifiable stage in carcinogenicity, morphological cell transformation. In contrast to other short-term in vitro assays, both genotoxic and epigenetic carcinogens are detected. The SHE assay, originally developed by Berwald and Sachs (J Natl Cancer Inst 35: 641-661) and modified as described by LeBoeuf and Kerckaert (Carcinogenesis 7: 1431-1440), was included in the International Life Sciences Institute, Health and Environmental Sciences Institute (ILSI/HESI). Alternative Carcinogenicity Testing (ACT) collaboration to provide additional information on the use of short-term in vitro tests in predicting carcinogenic potential. A total of 19 ILSI compounds have been tested in the SHE assay: 15 were tested for this project, whereas clofibrate, methapyrilene, reserpine, and Di(2-ethylhexyl)phalate (DEHP) were tested previously. Of the 3 noncarcinogenic compounds tested, 2 were negative in the SHE assay, whereas ampicillin was tested positive. The remaining 16 compounds tested were either known rodent carcinogens and/or human carcinogens. From this group, 15 tested positive in the SHE assay whereas phenacetin, a genotoxic carcinogen, was tested negative. Therefore, overall concordance between the SHE assay and rodent bioassay was 89% (17/19), whereas concordance with known or predicted human carcinogens was 37% (7/19). Based on these data, it is concluded that the SHE cell-transformation assay has utility for predicting the results of the rodent carcinogenesis bioassay but lacks the selectivity to distinguish between rodent and human carcinogens.  (+info)

Is p53 haploinsufficient for tumor suppression? Implications for the p53+/- mouse model in carcinogenicity testing. (13/352)

The p53 tumor suppressor gene has been shown to be critical in preventing cancer in humans and mice. We have generated and extensively characterized p53-deficient mice lacking one (p53+/-) or both (p53-/-) p53 alleles. The p53-deficient mice are much more susceptible to an array of different tumor types than their wild-type (p53+/+) littermates. The enhanced tumor susceptibility of the p53+/- mice has made them one of several transgenic mouse models that are being considered as substitutes for standard 2-year rodent carcinogenicity assays. In order to fully exploit this model, it will be important to understand some of the basic biological and molecular mechanisms that underlie its enhanced tumor susceptibility. With this in mind, we have explored the fate of the remaining wild-type p53 allele in spontaneously arising p53+/- tumors and have shown that over half of these tumors retain an intact, functional wild-type p53 allele. This suggests that p53 is haploinsufficient for tumor suppression and that mere reduction in p53 dosage is sufficient to promote cancer formation. To support the idea that p53 is indeed a haploinsufficient tumor suppressor, we show here that normal p53+/- cells exhibit reduced parameters of growth control and stress response compared to their p53+/- counterparts. We hypothesize that the reduced p53 dosage in the p53+/- cells provides an environment more conducive to the development of further oncogenic lesions and the initiation of a tumor. Finally, we have assessed p53 loss of heterozygosity (LOH) in carcinogen-induced p53+/- tumors and have found that some agents induce tumors that almost invariably exhibit p53 LOH, whereas other agents induce tumors that often retain the wild-type p53 allele. Our preliminary data suggest that LOH is dependent on both the mechanism of genotoxicity of the agent utilized and the tissue type targeted.  (+info)

Mechanisms of hormonal carcinogenesis in the p53+/- hemizygous knockout mouse: studies with diethylstilbestrol. (14/352)

The 2-year rodent bioassay has long had a central role in determining whether a compound is carcinogenic. It has recently been suggested that the use of 6-month studies in transgenic mice could reduce costs and animal numbers, without impairing the validity of cancer risk assessment. The p53+/- hemizygous knockout mouse model is phenotypically stable and develops tumors during the 6-month study period only in response to chemical and physical stimuli, showing a high concordance with genotoxic rodent carcinogens. We treated p53+/- mice and wild-type parent strain (C57BL/6J) animals with diethylstilbestrol (DES). 500 micromol/kg i.p. for 4 days. Following sacrifice, DNA was extracted from various tissues and adducts measured by a modified monophosphate version of the 32P-postlabelling assay. Major DES adducts were detected in the liver DNA of DES-treated wild-type mice at a level of 118.7+/-17.0 (mean +/- SD relative adduct level [RAL]/10(10) nucleotides) compared with 207.7+/-36.4 in p53+/- mice. No such adducts were detected in vehicle-treated animals. Total adduct levels, including endogenous I-compound adducts, in wild-type mice were 192.4+/-17.5 and 311.5+/-58.6 in p53+/- animals. These data support the hypothesis that deficient p53-dependent global genomic repair of DES adducts in p53+/- mice may result in the persistence of exogenous and endogenous DNA adducts that could contribute to earlier carcinogenicity in this model. We also prepared hepatic microsomes from male and female p53+/- and wild-type mice exposed to DES or vehicle. Western blot analysis demonstrated modestly higher basal levels of various cytochrome P450 (CYP) enzymes in the untreated p53+/- mice compared to the wild-type mice. Furthermore, P450 levels were higher in female DES-treated p53+/- mice compared to treated wild-type mice. For the p53+/- knockout mice to be used with contidence in drug safety studies, a further understanding of the metabolic capacity of these animals is needed.  (+info)

Comparison of the levels of enzymes involved in drug metabolism between transgenic or gene-knockout and the parental mice. (15/352)

Drug-metabolizing enzymes are involved in the metabolic activation or detoxification of carcinogens. To evaluate animals developed as models for alternative carcinogenicity testing, we investigated whether or not a gene manipulation including the transgene of ras and the knocking out of a tumor suppressor gene such as p53 or XPA could alter the expression of representative drug-metabolizing enzymes directly or indirectly. Expression of several isoforms of cytochrome P450 (CYP) in the liver of rasH2, p53 (+/-), Tg.AC, and XPA (-/-) mice with or without treatment of prototype inducer. phenobarbital or 3-methylcholanthrene, was analyzed by Western immunoblotting in comparison with their parental strains of mice. In addition, the activities of 3 major phase II enzymes, UDP-glucronosyltransferase, sulfotransferase, and glutathione S-transferase, were compared between the gene-manipulated and the corresponding parental strains of mice. Results demonstrate that XPA gene knockout appeared to increase constitutive expression of CYP2B and CYP3A isoforms. Overexpression of human c-Ha-ras gene or p53 gene knockout appeared to increase constitutive UGT activity toward 4-nitrophenol. The content or activities of almost all other enzymes examined in the present study do not appear to be affected by the gene manipulation.  (+info)

A perspective on current and future uses of alternative models for carcinogenicity testing. (16/352)

This perspective is based upon the data presented at the International Life Sciences Institute (ILSI), Health and Environmental Sciences Institute Workshop on the Evaluation of Alternative Methods for Carcinogenicity Testing (ILSI Workshop). It is important to understand that all models discussed at the Workshop have limitations and that they are not designed to be employed as stand-alone assays. Although they may have other, appropriate applications. I do not recommend use of the SHE cell assay and the Tg.AC model for the regulatory purposes of a safety assessment. In my view, the neonatal mouse, p53+/-, XPA-/-, XPA-/- and p53+/-, and the rasH2 models can, as a component of an overall assessment, provide information on potential carcinogenicity of a chemical that is appropriate for consideration in a regulatory context. Generally, these models exhibit the ability to detect genotoxic compounds. In most cases these compounds would be detected in a standard battery of genotoxicity tests and, therefore, quite often the use of an alternative is not necessary. Actually, I believe that a bioassay in rats will suffice most of the time, that is, in my view, a routine bioassay in mice is not necessary. Specific circumstances where data obtained from one of the "recommended" alternative models might be helpful are discussed. With regard to lessons for the future, there is a particular need for models that are responsive to chemicals that exhibit a nongenotoxic mode of action. Additionally, new models will continue to be developed and their half-life will likely be substantially shorter than the time required for traditional validation. The development of enhanced paradigms for validation should be a priority so that improved safety assessment decisions can be made more quickly. However, while evaluating and validating such models, it is important to consider the fundamental issues, for example, rational dose selection, evaluation of mode of action in the context of dose-response relationships including the existence of thresholds and secondary mechanisms, and species-to-species extrapolation. The alternatives to carcinogenicity testing project was a very major undertaking. In addition to the valuable information provided, it serves to illustrate the value of cooperation between academia, government, and industry. Furthermore, the involvement of the International Life Sciences Institute as the overall organizing, facilitating umbrella was crucial for the success of the project.  (+info)