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Sorcerer Colony Counter and cancer chemoprevention using the Ames test

“Operating from the core philosophy that optimum diet is the key to optimum health, researchers at The Linus Pauling Institute (LPI) at Oregon State University are actively involved in research on dietary components and their beneficial effects for human health. An NCCAM (National Center for Complementary and Alternative Medicine) Center of Excellence, the institute’s mission is to determine the function and role of micronutrients (vitamins and minerals) and phytochemicals (chemicals from plants) in promoting health and preventing and treating disease. Researchers are particularly interested in the role of oxidative and nitrative stress and antioxidants in human health and debilitating diseases.”

case_pauling_himselfCancer chemoprevention

Established in April 2002, the Cancer Chemoprotection Program (CCP) Core Laboratory at LPI primarily provides genetic toxicology testing services to the institute’s research scientists. The laboratory offers a range of assays to help investigators to determine whether various dietary compounds offer chemoprotection and also investigates the possible inhibitory mechanisms involved. Techniques employed to assess mutagenic and DNA-damaging effects include the Salmonella mutagenicity assay (or Ames test) and the single cell gel electrophoresis assay (SCG or Comet assay). Using known mutagenic compounds as ‘positive controls’, it is possible to study whether specific dietary components can reduce DNA damage. Recent research projects include studying the potentially chemoprotective properties of spearmint and assessing the effect of antioxidant vitamins on exercise induced DNA damage.

Anti-genotoxic effects of spearmint

A popular additive in foods and beverages such as herbal tea, spearmint (Mentha spicata) has long been considered beneficial to health. The Core Laboratory’s studies have specifically looked for possible anti-genotoxic effects, using the Ames test to detect in vitro anti-mutagenic activity. 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 in a histidine deficient medium. Histidine-independent bacterial colonies may arise from spontaneous reversions (backward mutations) or chemically induced reversions. The mutagenicity of a compound can be assessed by comparing the control with the treated bacterial culture. Conversely, the anti-mutagenicity of a compound to a selected positive mutagen can be investigated when the two chemicals are co-administered to the bacteria.

A plate incorporation version of the Ames test based on previously described methodologies (2) was used. Varying amounts of mutagen and spearmint were added to 0.5 ml of phosphate buffered saline and 2 ml of molten top agar (45ºC – 46ºC) in sterile disposable tubes, finally adding 0.1 ml of Salmonella typhimurium strain TA102 culture. After vortexing for a few seconds each mixture was poured onto a minimal glucose plate. Once the agar had set, the inverted plates were incubated at 37ºC in the dark for 48 hr. The histidine positive revertant colonies were then counted with a Sorcerer Colony Counter.

The colony counter combines image processing and analysis with an innovative Petri-viewer to provide fast, accurate counts of revertant colonies using dark-field illumination. Following on-screen prompts, the operator selects the Ames test from a range of pre-configured applications, enters the sample detail and places the dish in the Petri viewer. A single button press then initiates the measurement sequence – displaying a clear image of the detected colonies and computing the count. All of the experiments were repeated at least twice, and triplicate plates were used for each data-point.

The studies found that spearmint tea did not inhibit the mutagenicity of the dye 4-nitro-1, 2-phenylenediamine, but did inhibit the mutagenicity of the cooked-meat mutagen 2-amino-3-methylimidazo [4,5-f] quinoline (IQ), a food borne carcinogen formed in proteinaceous food cooked at high temperatures. This observation led the team to test spearmint tea in rats treated with IQ, where the mutagen stimulates the formation of aberrant crypt foci (ACF) in the colon – precancerous lesions that can develop into colon cancer. Rats given IQ plus spearmint tea developed less than half the number of ACF per colon compared to rats given IQ alone, demonstrating that spearmint possesses anticancer potential. Collectively, these findings suggest that spearmint tea protects against IQ and possibly other heterocyclic amines through inhibition of carcinogen activation and via direct effects on the activated metabolite(s).

case_pauling_systemExercise induced DNA damage and the effect of antioxidant vitamins

The Comet assay was used to determine whether 6 weeks of supplementation with vitamins C and E can alleviate exercise-induced DNA damage (3). The 21 runners involved in the study were randomly assigned to one of two groups and given either placebos (PL) or antioxidants (AO) (1000 mg vitamin C and 400 IU natural vitamin E) for 6 weeks prior to completing a 50 km ultra marathon. DNA damage in circulating leukocytes was assessed at selected time points: pre-, mid-, and 2 hours post race and daily for 6 days post race.

The comet assay is used to detect DNA strand breaks as well as alkaline labile lesions. For many laboratories it has become the tool of choice for detecting DNA damage in eukaryotic cells due to its versatility, and the name is derived from the shape of DNA distribution seen which bears resemblance to a celestial comet.

This study used the comet assay protocol based on that of Singh et al (4) with some modification. Exposed and control cells were embedded in an agarose gel ‘sandwich’ on a microscope slide. Following overnight immersion in cold lysing solution, the slides were rinsed and the cells subjected to electrophoresis. When the electrophoresis is performed in a neutral pH condition, DNA double strand breaks are revealed. In this case, a strong alkaline solution (pH > 13) was used to reveal all DNA strand breaks and alkaline-labile lesions. The slides were rinsed and neutralized before staining with 60 µl of 20µg/ml ethidium bromide to visualize DNA migration under a fluorescence microscope. 25 randomly chosen nuclei per each duplicate slide were analyzed using the Comet Assay image analysis system from Perceptive Instruments. Using a highly sensitive video camera attached to a microscope, the system immediately transfers a live video image to the computer screen to provide real-time cell scoring.

Each comet is brought into sharp focus and the operator initiates the measurement, which includes correction for background with a click of the mouse cursor. Measurements are saved to an Excel spreadsheet for statistical analysis. Each experiment was repeated at least twice in this study.

‘Percentage DNA in tail’ was used as the indicator of DNA damage in the published study (3). The results showed that DNA damage in leukocytes, induced by the extreme exercise of an ultra-marathon, increased transiently at mid race but returned to baseline by 2 hours post race, indicating that the exercise bout induced non-persistent DNA damage. One day post-race, women taking antioxidant vitamin supplements had 62% less DNA damage than women taking the placebo. In contrast, there were no statistically significant differences between the two treatment groups of men at any time point. Thus, endurance exercise resulted in DNA damage as shown by the Comet assay and antioxidants seemed to enhance recovery in women but not in men.

Update

This article has been published in the May 2007 edition of American Biotechnology Laboratory magazine. You can download a PDF copy of the article below:

Download article (PDF)

Useful links

Linus Pauling Institute
LPI – Cancer Chemoprotection Program Core Laboratory
Oregon State University

Contact

Professor Roderick Dashwood – rod.dashwood@oregonstate.edu

References

1. Tian-Wei Yu, Meirong Xu and Roderick H Dashwood, Antimutagenic Activity of Spearmint. Environ Mol Mutagenesis, 44:387-393, 2004.

2. Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res 113: 173-215, 1983.

3. Mastaloudis A, Yu T-W, O’Donnell RP, Frei B, and Dashwood R and Traber M, Endurance exercise results in DNA damage as detected by the comet assay. Free Radical Biol Med, Free Radical Biology and Medicine, 36(8): 966-975, 2004.

4. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175: 184-191, 1988.