Mutation to DNA is a primary mechanism by which cancers arise. These events have also been implicated in diseases such as atherosclerosis, and processes such as aging. Therefore, there is an important need for sensitive analytical methods that facilitate the study of mutagenesis, as well as the identification of chemical or physical agents that can mutate DNA. Methods for measuring in vivo mutation currently exist, each with their own advantages and limitations. While some are based on colony formation and require tissue culture work, others rely on expensive, proprietary trangenic rodents. This laboratory has developed an in vivo mutation assay that is based on the Pig-a locus. The Pig-a gene product is essential for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Mutations giving rise to nonfunctional GPI anchors prevent certain proteins from being expressed on the cell surface, and this represents a phenotype that can be measured by flow cytometry. The work proposed herein extends this line of investigation through an extensive inter-laboratory validation effort whereby rats will be treated with known mutagens and non-mutagens using both a short-term as well as a 28-day repeat dosing schedule. This assay validation effort will focus on an erythrocyte-based assay, a target cell population that has been studied most intensely to date. Concurrently, other work will be directed at developing means to measure Pig-a mutation in other tissues. Society will benefit from the proposed work as pharmaceutical and chemical companies are provided improved methods for conducting safety assessment work.

Public Health Relevance

It is well known that DNA damage is a precursor to the development of cancer and other significant diseases. Therefore, it is in the interest of public health to reduce the occurrence of mutagenic chemicals in the environment, in our drugs, and from our workplaces. This research project will validate a powerful blood-based method that detects mutagenic agents, thereby enhancing the nation's ability to effectively reduce exposure to these toxic compounds. Additional work will be directed at developing mutant cell scoring methods that are compatible with other tissues.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
Project #
5R44ES018017-03
Application #
8197945
Study Section
Special Emphasis Panel (ZRG1-IMST-D (16))
Program Officer
Shaughnessy, Daniel
Project Start
2010-06-01
Project End
2012-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
3
Fiscal Year
2012
Total Cost
$688,086
Indirect Cost
Name
Litron Laboratories, Ltd.
Department
Type
DUNS #
085992055
City
Rochester
State
NY
Country
United States
Zip Code
14623
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Labash, Carson; Avlasevich, Svetlana L; Carlson, Kristine et al. (2016) Mouse Pig-a and micronucleus assays respond to N-ethyl-N-nitrosourea, benzo[a]pyrene, and ethyl carbamate, but not pyrene or methyl carbamate. Environ Mol Mutagen 57:28-40
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Stankowski Jr, Leon F; Aardema, Marilyn J; Lawlor, Timothy E et al. (2015) Integration of Pig-a, micronucleus, chromosome aberration and comet assay endpoints in a 28-day rodent toxicity study with urethane. Mutagenesis 30:335-42
Coffing, Stephanie L; Kenyon, Michelle O; Ackerman, Joel I et al. (2015) Evaluation of the in vivo mutagenicity of isopropyl methanesulfonate in acute and 28-day studies. Environ Mol Mutagen 56:322-32
Dertinger, Stephen D; Avlasevich, Svetlana L; Bemis, Jeffrey C et al. (2015) Human erythrocyte PIG-A assay: an easily monitored index of gene mutation requiring low volume blood samples. Environ Mol Mutagen 56:366-77
Graupner, Anne; Instanes, Christine; Andersen, Jill M et al. (2015) Genotoxic effects of two-generational selenium deficiency in mouse somatic and testicular cells. Mutagenesis 30:217-25
Graupner, Anne; Instanes, Christine; Dertinger, Stephen D et al. (2014) Single cell gel electrophoresis (SCGE) and Pig-a mutation assay in vivo-tools for genotoxicity testing from a regulatory perspective: a study of benzo[a]pyrene in Ogg1(-/-) mice. Mutat Res Genet Toxicol Environ Mutagen 772:34-41
Bemis, Jeffrey C; Hall, Nikki E; Dertinger, Stephen D (2013) Erythrocyte-based Pig-a gene mutation assay. Methods Mol Biol 1044:51-77

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