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.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
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Special Emphasis Panel (ZRG1-IMST-D (16))
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Shaughnessy, Daniel
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Litron Laboratories, Ltd.
United States
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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
Bhalli, Javed A; Shaddock, Joseph G; Pearce, Mason G et al. (2013) Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens. Mutagenesis 28:447-55
Bhalli, Javed A; Ding, Wei; Shaddock, Joseph G et al. (2013) Evaluating the weak in vivo micronucleus response of a genotoxic carcinogen, aristolochic acids. Mutat Res 753:82-92
Phonethepswath, Souk; Avlasevich, Svetlana L; Torous, Dorothea K et al. (2013) Flow cytometric analysis of Pig-a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD-1 mice. Environ Mol Mutagen 54:294-8
Bemis, Jeffrey C; Hall, Nikki E; Dertinger, Stephen D (2013) Erythrocyte-based Pig-a gene mutation assay. Methods Mol Biol 1044:51-77
Lemieux, Christine L; Douglas, George R; Gingerich, John et al. (2011) Simultaneous measurement of benzo[a]pyrene-induced Pig-a and lacZ mutations, micronuclei and DNA adducts in Mutaýýý Mouse. Environ Mol Mutagen 52:756-65
Dertinger, Stephen D; Phonethepswath, Souk; Weller, Pamela et al. (2011) Interlaboratory Pig-a gene mutation assay trial: Studies of 1,3-propane sultone with immunomagnetic enrichment of mutant erythrocytes. Environ Mol Mutagen 52:748-55
Dertinger, Stephen D; Bryce, Steven M; Phonethepswath, Souk et al. (2011) When pigs fly: immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis. Mutat Res 721:163-70
Shi, Jing; Krsmanovic, Ljubica; Bruce, Shannon et al. (2011) Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies. Environ Mol Mutagen 52:711-20
Dertinger, Stephen D; Heflich, Robert H (2011) In vivo assessment of Pig-a gene mutation-recent developments and assay validation. Environ Mol Mutagen 52:681-4

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