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Scientific and Technological Advancements for
Improving Current Genetic Toxicology Testing

Genetic Toxicology Association Fall Meeting
Thursday, October 19, 2006
Clayton Hall Conference Center, University of Delaware

New Insights from an Old Assay: Salmonella Revisited

David M. DeMarini (US EPA, RTP)

Although the Salmonella (Ames) mutagenicity assay is 36 years old, it is still the primary assay for routine mutagenicity testing and uniquely suited for some basic research questions. Using genomic hybridization, we showed that the Ames strains are missing 15-119 genes due to the ΔuvrB mutation. We now show that the ΔuvrB mutation does not cause the Ames assay to miss any mutagens (other than some base analogues such as hydroxyaminopurines) compared to strains that have just a point mutation in the uvrB gene. In fact, the ΔuvrB mutation actually makes the Ames strains quantitatively more sensitive to most mutagens compared to strains that are mutated in only the uvrB gene. Thus, most mutagens appear as more potent in the Ames strains than in those with only a point mutation in the uvrB gene. The basis for this appears to be the deletion of genes that inactivate mutagenic intermediates, such as hydroxyl radicals, and genes that are involved in DNA repair. The deletion or inactivation of homologous genes in humans may make humans carrying these SNPs or polymorphisms more sensitive to mutagenesis. In other studies using specially designed microarray chips, we found that the drinking-water mutagen MX altered expression of many genes, including those involved in porphyrin metabolism. This may be due to the structural similarity of MX, which is a furanone, to pyrrole. In addition, we have used the operon structure of bacteria to provide another level of bioinformatic analysis such that all genes on an operon are assumed to be coordinately expressed—permitting inclusion of all genes on an operon in the analysis even if not all of the genes show a reproducible change in gene expression by chip analysis. These studies show the continued value of the Ames assay for basic and applied research. [Abstract does not necessarily reflect the views of the US EPA.]

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Modernization of GLP Cytogenetic Studies With Flow Cytometry

Paula Muehlbauer (Pfizer Global Research and Development)

Comprehensive characterization of chemically-induced effects (i.e. cytotoxicity) on in vitro test systems is vital to assure the accurate interpretation of the data. Historically, the conduct of cytogenetic studies has shown a particular sensitivity to extreme culture conditions that oftentimes causes artifacts in the test system (e.g. non-genotoxic chromosome breakage and increased levels of polyploidy) hence making data interpretation difficult. Throughout the past several decades, cytogenetic studies have relied heavily on the mitotic index as a key cytotoxic indicator, particularly studies conducted in human lymphocyte cultures. Unfortunately during this time; the capability to consistently provide comprehensive characterization of mitotic frequencies was limited due to dependencies on methods established in the late 70s (i.e. slide making procedures with light microscopy). Recent advancements in the field using flow-based technology to assess mitotic index and polyploid frequencies have emerged, thus providing the opportunity for the cytogenetics laboratory at Pfizer to modernize portions of routine GLP cytogenetic studies. Consequently, process improvements were realized in several areas of the study conduct; e.g. slide making requirements were eliminated in the preliminary range-finder test and minimized in definitive tests to only the test concentrations identified for aberration scoring. More importantly, comprehensive characterization of mitotic indexes and polyploid frequencies across entire treatment regimens were achieved consistently in each test. As a result, the relationship between mitotic suppression and occurrence of polyploidy induction in the test system could be explored and non-genotoxic effects (i.e. chromosome breakage) could effectively be minimized in studies. Details of flow technology integration into routine GLP cytogenetic studies, with examples of comprehensive cytotoxicity characterizations and the relationship to polyploidy and non-genotoxic effects will be discussed.

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Of Mice and Men: Micronucleated Reticulocyte Measurements as a Cross-Species Endpoint of Genotoxicity

Stephen D. Dertinger, Ph.D. (Litron Laboratories)

The in vivo rodent micronucleus test benefits from automated scoring, as this enhances the objectivity, reproducibility, and throughput capacity of the assay, while simultaneously reducing labor and time requirements. A flow cytometric method for scoring micronucleated reticulocytes in mouse or rat blood has been developed by this laboratory, and is based on anti-CD71 labeling (commercially known as MicroFlow®). More recently, this approach has been applied to other species of toxicological interest. Data to date suggest that this endpoint has the potential to serve a cross-species role, one that can bridge pre-clinical safety assessments to human exposure scenarios. Using this technique, the following human populations have been studied, and will be discussed here: healthy adults; patients undergoing cancer therapy; pediatric sickle cell disease patients (with and without hydroxyurea exposure); pregnant women and newborn infants treated with zidovudine. We conclude that this endpoint is likely to find widespread after more complete characterization of: 1) its ability to assess acute and chronic genotoxicant exposures, 2) its utility for studying clinically and environmentally relevant exposures, and 3) the degree that host factors influence micronucleated reticulocyte frequencies.

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Prevention of Spontaneous and X-Ray Induced Genomic Instability in Breast Cancer Cells by the Dietary Antimutagens Genistein and Lycopene

Audrey A. King, Joanna Leszczynska, and Catherine B. Klein (New York University School of Medicine)

Elevated spontaneous mutation rates are one consequence of dysregulation of genomic stability in cancer. Various dietary antimutagens have been shown by us and others to be effective at reducing elevated spontaneous mutation rates found in colon tumor HCT116 cells. In studies utilizing breast tumor cell lines, genistein (3.125 μM, 24 hrs) and lycopene (2 μM, 24 hrs) were similarly found to be effective antimutagens. Specifically, genistein or lycopene reduced the spontaneous HPRT mutant fraction in MCF-7 (ERα+) cells by an average of 49% and 39%, respectively. In MDA-MB-468 (ERα−) breast cancer cells, the spontaneous HPRT mutant fraction was reduced by genistein or lycopene by an average of 31% and 35%, respectively. In the BT-474 (ERα+/−) breast tumor cells, genistein or lycopene reduced the spontaneous HPRT mutant fraction by an average of 41% and 37%, respectively. Chromosomal and microsatellite instability (MSI) are also other endpoints of genomic instability that have been examined. MCF-7, MDA-MB-468, and BT-474 breast tumor cells treated with genistein (3.125 μM, 24 hrs) or lycopene (2 μM, 24 hrs) exhibited reduced frequencies of micronuclei. In non-tumor MCF10A breast cells pre-treated with genistein (12.5 μM, 24 hrs) or lycopene (20 μM, 24 hrs), X-ray induced aneuploidy was also reduced to varying degrees. In MSI studies, protective differences were noted in lycopene (2 μM, 1 week) treated MCF-7 single cell clones, but not in BT-474 single cell clones. Further, treatment of MCF10A cells with genistein (3.125 μM, 24 hrs) or lycopene (2 μM, 24 hrs) prior to X-ray exposure protected against X-ray induced MSI. To begin to elucidate possible mechanisms by which genistein and lycopene prevent genomic instability, modulation of gene-specific methylation patterns by these antimutagens were studied. Lycopene reversed the hypermethylation of GSTP1 in MDA-MB-486 breast cancer cells and RARβ2 and HIN-1 in MCF10A breast cells. The genome stabilizing effects of genistein or lycopene could have protective roles in normal breast or breast cancer cells at the chromosomal and mutational level, either to protect normal cells against cancer development, or possibly to prevent further tumor progression of breast tumor cells. This research was supported in part by the Susan G. Komen Breast Cancer Foundation BTCR0402531.

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Toxicogenomic Analysis of Cellular Stress Responses and its Potential for Differentiating Genotoxic and Carcinogenic Mechanisms.

Jiri Aubrecht (Pfizer, Safety Sciences)

The standard in vitro genetic toxicity testing provides relatively simple and accurate hazard detection. However, interpretation of positive findings in terms of relevant risk, oncogenic potential in animals and humans, is difficult due to the limited insight into underlying genotoxic/carcinogenic mechanisms. Thus development of broad mechanism-based approaches capable of differentiating a wide range of genotoxic and oncogenic mechanisms is expected to significantly improve risk assessment. The goal of this presentation is to summarize current developments in toxicogenomic analysis of cellular stress response in vitro and in vivo and provide a perspective on application of genomic approaches in risk assessment.

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ICCVAM: What It Is and What It Does

Rodger Curren (Institute for In Vitro Sciences)

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