National Toxicology Program

A large number of chemicals (>80,000) are currently in use. The NTP continually faces the task of determining how to acquire the scientific information about a substance(s) being evaluated that will best address identification of any related hazard from exposure and strengthen the science base. Implementing new strategies, which provide additional or more accurate information, can strengthen the science base on which regulatory decisions are based. Through the NTP, efforts are focused on the development and validation of new alternative test systems (sensitive, specific, rapid) for toxicological research that will reduce, replace, or refine animal use.

Model systems under development include non-mammalian species, transgenic species, genetically engineered in vitro cell systems, microchip array technology, and computer-based predictive toxicology models. In addition, through the NTP Center for the Validation of Alternative Toxicological Methods, a concerted and coordinated Federal effort is being made to identify, validate, and promote regulatory acceptance of alternative test systems. University-based researchers are also involved in this alternative methods development and validation through the NIEHS/NIH extramural grants program.

TRANSGENIC MODELS

The conventional rodent bioassay has been used for over three decades and is accorded credibility in identifying carcinogens thought to pose risks for human health. An going goal of the NTP is to seek other model systems for toxiciology and carcinogenesis studies especially those that can provide mechanistic information relative to understanding an agent's mode of action. The use of transgenic models holds promise for improving both the accuracy and efficacy of experimental assessment of the carcinogenic potential of chemicals. Genetically altered or "transgenic" mouse models carry activated oncogenes or inactivated tumor suppressor genes known to be involved in neoplastic processes both in humans and rodents. This trait may allow them to respond to carcinogens more quickly than conventional rodent strains. In addition, the neoplastic effects of agents can be observed in transgenic models within a time frame in which few, if any, spontaneous tumors would arise. The high incidences of spontaneous or background tumors, which occur most often late in the two-year rodent cancer studies, are among the most confounding factors for interpreting the findings of chemical carcinogenesis and their implications for human health. The use of target or reporter genes also allows for direct molecular and cellular analysis of a chemical's effects in these models and can provide additional mechanistic information about mode of action.

Over the past few years, the NIEHS/NIH and NTP have been actively evaluating transgenic strains in toxicological testing strategies. Based on current evaluations, the models with greatest potential usefulness at this time are the p53def (p53+/-heterozygous) and Tg.AC (v-Ha-ras transgene). The Tg.AC mice carry a v-Ha-ras oncogene that represents a class of oncogenes that plays a key role in signal transduction pathways, which regulate cell proliferation, and are detectable in the early stages of tumor induction. The heterozygous p53def mouse lacks a member of a class of suppressor genes that has an important role in cell cycle control; loss of function is associated with progression of tumors to malignancy. These strains show specificity for being able to identify genotoxic agents (p53def) and both genotoxic and nongenotoxic agents (Tg.AC). Evaluation of the specificity and usefulness of these two transgenic models is continuing (French NIEHS/NIH; Spalding NIEHS/NIH; Tennant, NIEHS/NIH).

The NIEHS/NIH is also evaluating the usefulness of other transgenic models including

• TRAMP (Transgenic Adenocarcinoma Mouse Prostate) mice (Maronpot, NIEHS/NIH),
• p53 null mutants on an FVB/N background (Stasiewicz, NIEHS/NIH),
• p16 transgenic mice (determine role of p16 gene in ENU-induced brain tumors after transplacental exposure) (Stasiewicz, NIEHS/NIH),
• Tg.NK mouse transgenics (contains c-neu, the human breast cancer oncogene homologue of erbB2 and develops mammary tumors early in life) (Rao, NIEHS/NIH),
• COX 1 amd COX2 knockout mice (study physiological functions of cyclooxygenase isoforms) (Langenbach, NIEHS/NIH), and
• Tg.APC [contains a mutation in the mouse adenomatous polyposis coli (APC) gene, the mutation site in the majority of human colon cancers] (Stasiewicz, NIEHS/NIH).
• ERKO used to study estrogen receptor and reproductive function
• CB6F1-TgHras caries human CHras gene
• Inherited defects in the BRCA1 and BRCA2 genes account for most hereditary-linked human breast and ovarian cancers. Efforts at the NIEHS/NIH are focusing on development of experimental mouse models for Brca alterations. These mice should be useful to define functions of these genes and to provide important insights for understanding these cancers (Wiseman, NIEHS/NIH).

As part of the evaluation process, the NTP is participating in an industry/government consortium of Federal agencies and 25 pharmaceutical companies to study the utility of a number of alternative assays designed to augment or replace the two-year rodent bioassay. The International Life Sciences Institute/Health and Environmental Sciences Institute (ILSI/HESI), a non-profit foundation, is coordinating this effort. A set of about a dozen chemicals is being evaluated in five, short-term model systems including three genetically altered mouse models; the newborn mouse assay; and the Syrian hamster embryo cell transformation assay. The NTP is testing six chemicals in the Tg.AC model by two routes of administration. An ILSI/HESI organized meeting is scheduled for November 2000 in the Washington, DC area to discuss and evaluate the effort's findings. The results will contribute to an NTP evaluation of genetically engineered mouse models by the Interagency Coordinating Committee on the Validation of Alternative Methods that is scheduled for 2001 (Bucher, NIEHS/NIH).

Efforts are underway to determine the usefulness of transgenic fish as an alternate model for mice and cultured cells. Transgenic technology has been applied to the study of induced somatic mutation directly at the DNA level using PhiX174 bacteriophage as an identical marker in rodents, fish (Fundulus heterosclitus), and cultured cells to compare dose, adduction, and DNA repair and mutation. Initial studies comparing mice and fish exposed to the potent carcinogen, 7,12-bis-hydroxymethylbenz[a]anthracene, are encouraging. Future studies include expanding the target sequence in the transgenic vector, investigating mutagenicity in mice exposed to certain environmental mixtures, and combining this approach with developmental and endocrine disruptor endpoints (Burkhart, NIEHS/NIH).

The NIEHS/NIH is developing primary rat and mouse cell strains from the transgenic BlueÆ mouse (B6C3F1) and rat (Fischer 344) to allow for comparative analysis of cytogenetic and mutational endpoints in vivo and in vitro. Such studies can provide mechanistic insights into the processes by which xenobiotic agents induce genetic damage allowing for a more accurate estimate of genetic risk (Tindall, NIEHS/NIH).