Laboratory Manager: David Yu, Ph.D.
Cancer is the result of abnormal genetic control of cell growth that arises from structural changes in DNA or mutations. Many mutations occur in somatic cells during the lifetime of an individual and some may result in cancer. There is evidence that dietary factors can impede this process. For example, epidemiological studies reveal that the quarter of the population with the lowest dietary intake of fruit and vegetables has roughly twice the cancer risk for most types of cancer compared with the quarter with the highest intake. In recent years the possible chemoprevention of cancer by specific chemical constituents of fruit and vegetables has attracted much attention.
Established in April 2002, the Cancer Prevention and Intervention (CPI) Core Laboratory at the Linus Pauling Institute primarily provides genetic toxicology testing service to our researchers. The mission of this laboratory is to help investigators learn whether the dietary compound of interest is a potential chemoprotective agent and to elucidate the possible inhibitory mechanism(s). We employ several techniques, including the Salmonella mutagenicity assay (or Ames test), single cell gel electrophoresis assay (SCG or Comet assay), and micronucleus (MN) assay.
These assays enable investigators to assess the mutagenic, DNA-damaging and clastogenic effects of chemicals. Using compounds identified as mutagens or clastogens as "positive controls," we are able to study the potential beneficial activities of dietary compounds or any chemical entity of interest against those DNA-damaging agents.
Individuals or organizations outside of the OSU campus may also order these testing services. Please contact David Yu (541-737-5082 or David.Yu@oregonstate.edu) for further information.
The Salmonella Mutagenicity Assay
The Salmonella mutagenicity test, or bacterial reverse mutation assay, is also commonly known as the Ames test. It was developed by Dr. Bruce Ames, the recipient of 2001 LPI Prize for Health Research, and his colleagues in UC Berkeley in the 70s. The principle of the test is to expose histidine-dependent Salmonella typhimurium strains (the tester strains, which have artificially induced point mutations) to a compound to be examined in a histidine (His) deficient medium. His-independent bacterial colonies may arise from spontaneous reversions (backward mutations) or chemically induced reversions. The mutagenicity of a chemical can be assessed by comparing the control with the treated bacterial culture. Conversely, the antimutagenicity of a compound to a selected positive mutagen can be investigated when the two chemicals are co-administered to the bacteria.
National Toxicology Program overview for Ames test
FDA Redbook on bacterial reverse mutation test
Significance of the bacterial reverse mutation test as predictor of rodent and human carcinogenicity
The Comet Assay
The comet assay, or single cell gel electrophoresis assay, is used to detect DNA strand breaks as well as alkaline labile lesions. For many labs, it has become the tool of choice for detecting DNA damage in eukaryotic cells due to its versatility. Exposed and control cells are embedded in an agarose gel sandwich on a microscope slide. The cells are then subjected to electrophoresis. When the electrophoresis is performed in a neutral pH condition, DNA double strand breaks are revealed. In strong alkaline solution, all DNA strand breaks and alkaline labile lesions are revealed. The migration of DNA is visualized under a fluorescence microscope after the DNA is stained with a fluorescent dye. The shape of DNA distribution is very similar to a celestial comet. Measurement is usually carried out on a computerized image analysis system, like the one shown in the picture at right from the CPI Core Lab. In principle, all types of nucleated cells may be used in this assay.
Comet Assay Interest Group
NIH comet Listserv
The Micronucleus Assay
The micronucleus (MN) assay can be used to detect the clastogenic and aneugenic effects of test chemicals both in vitro and in vivo. It is much more cost effective than the metaphase chromosome aberration assay. The in vitro micronucleus assay is considered as the replacement for conventional metaphase analysis as the screening test of choice for clastogenicity. The in vivo assay, usually conducted in mice, is especially important since no in vitro alternative test has been validated to replace the MN test. The cells evaluated in this assay are typically erythrocyte populations in either the peripheral blood or bone marrow compartment. Clastogens or spindle poisons that cause chromosomal damage in stem cells result in the formation of readily detectable micronuclei in the anucleated immature erythrocytes.
FDA Redbook on Mammalian Erythrocyte MN
Histone Deacetylase (HDAC) Inhibition Assay
Epigenetic changes are thought to play an important role in carcinogenesis; for example, the methylation of DNA can affect the interaction of proteins (e.g. transcription factors) with DNA, thus affecting gene transcription. Histone acetylation is another type of epigenetic modification that is able to regulate gene expression. In general, inhibition of cellular histone deacetylase activity may lead to loosened histone-DNA binding, resulting in more permissive chromatin DNA to transcription factors. HDAC modulation can lead to transcriptional activation or silencing of a subset of genes controlling cell-cycle arrest, apoptosis, and differentiation. This makes HDAC an interesting and novel target for cancer chemoprotection. The CPI Core Lab provides access to HDAC inhibition assays, for evaluating the chemoprotective potentials of dietary components.
Guava PCA-based Assays
Studies of cell cycle kinetics and expression of cell surface/intracellular markers are important in cancer research. The CPI Core Lab acquired a Guava Personal Cell Analyzer (PCA), a compact and accessible platform to several cell analysis assays. The PCA provides forward scatter (cell size) and two fluorescent channels (580 and 675 nm) with a minimal sample requirement. The PCA can be used for:
- cell count and viability determination
- apoptosis (TUNEL, nexin, multi-caspases, etc.)
- cell cycle
- quantitation of cell surface/intracellular markers.