| Abstract |
 Studies in the laboratory of James Klaunig concentrate on understanding the mechanism by which chemicals induce cancer and the means to prevent or retard this process. Recent investigations have revolved around the role of oxidative stress and oxidative damage in the induction of cancer by nongenotoxic carcinogens. Hepatocarcinogenic compounds including selective chlorinated hydrocarbons appear to induce oxidative stress in the liver. This oxidative stress and oxidative damage in turn may be responsible for the tumor-promoting activity of these compounds. Reduction of oxidative damage by antioxidants or dietary-restrictions results in an ablation of the induction of selective cell growth by these agents.
Several lines of evidence support a role for oxidative stress in the prevention of colon cancer. A lower risk of colorectal cancer has been noted following high anti-oxidant intake. In addition, anti-oxidant enzyme activities were decreased and the extent of oxidative damage was increased in colon cancer tissue when compared with normal tissue. Reactive oxygen species were shown to cause DNA damage in genes that are commonly mutated in colorectal tumors. Interestingly, oxidative stress induction and vitamin D status appear to be associated, as it was shown that oxidative DNA damage levels and cell proliferation were increased in colon tissue from mice lacking the vitamin D receptor. These results suggest that chemoprevention strategies involving both antioxidant and vitamin D supplementation may be useful for preventing colon carcinogenesis.
It is well established that polymorphic gene expression may alter individual susceptibility to chronic diseases such as cancer. A search of the SNP500Cancer Database shows that a number of SNPs exist for genes involved in oxidant production, remediation of oxidative damage and/or reactive oxygen species, as well as vitamin D. Thus, identifying SNPs in these genes would provide information regarding potential risk factors for the development and etiology of colorectal cancer.
The pharmacogenomic analysis encompasses a comprehensive approach to understanding the relationship between known polymorphisms in critical genes associated with regulation of oxidative stress and damage, as well as the potential interaction of vitamin D status (including polymorphisms in vitamin D related genes), or other factors that may influence the development of colon carcinogenesis. This analysis will contribute information for the statistical modeling that will identify molecular signatures that define potential susceptibility factors for colorectal cancer development such that individuals at increased risk can be identified. This information may ultimately result in reduced risk through the establishment of targeted intervention strategies aimed at preventing or delaying disease onset.
Instruments
 The aliquots for the oxidative stress analysis are initially processed at IU Simon Cancer Center using this
blood sample processing methodology. The aliquots are then transferred to the Klaunig Laboratory in Indiana University, Bloomington Indiana for additional preparation.
Oxidative DNA damage is evaluated using the Comet assay. To quantitate the Comet assay, prepared samples are examined under a Nikon fluorescence microscope. Fifty to one hundred comets per slide are visually scored according to the amount of DNA present in the tail. The Olive tail moment = [(fluorescence intensity of the tail)/(fluorescence intensity of the head) x tail length], is used as a measure of DNA damage. See Procedure for Alkaline and Fpg Comet Assay and Instrument Setup for detailed descriptions of the sample processing methodologies.
 The Tecan Infinite M200 Microplate Reader is used to determine the amount of anti-oxidant in the sample. Magellan6 software converts instrument-generated WSP files to Excel spreadsheet format. Values are compared to standard curves. See Procedure for TEAC Assay and Instrument Setup for detailed descriptions of the sample processing methodologies.
Analysis Workflow
The following analyses will be performed on DNA extracted from white blood cells isolated from whole blood collected from colonoscopy patients at the IU Simon Cancer Center:
- TEAC. Reactive oxygen species (ROS) are produced by both endogenous and exogenous processes. Eukaryotic organisms have developed a complex antioxidant network to counteract ROS to reduce the deleterious actions of ROS. The Total Antioxidant Capacity (TAC), a parameter that provides information on the overall status of antioxidants within a biological sample, has proven to be a useful indicator for determining the ability of an organism to mitigate the potential damage produced by ROS.
Various methods have been developed to assess TAC. The Trolox Equivalent Antioxidant Capacity (TEAC) assay, a method based on the scavenging of the 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical (ABTS•+), has been one of the most widely used methods. The original method used the methmyoglobulin radical to generate the ABTS•+ substrate, however use of this method has been limited due to the length of time needed to perform this assay, lack of reproducibility, and expense. Recently, an improved technique of TEAC assay was developed using pre-formed ABTS•+. In our lab, we scaled this assay to a 96-well, high throughput format. The ABTS•+ is prepared by the reaction of ABTS with potassium persulfate 12-16 hours before use. Samples or Trolox standards are reacted with ABTS•+, and the absorbance is measured at 734 nm on a Tecan M-200 plate reader after 5 minutes for the determination of TEAC.
This method has been used to evaluate the total antioxidant capacity of tissue homogenate, plasma/serum and cell lysates. It is also useful in the examination of antioxidant potential of chemicals. The results are presented as nmol per unit of sample, such ml or mg protein, and represent the quantity of ability total amount antioxidants equivalent to Trolox in the sample.
The Tecan Microplate Reader instrument-generated dataset workflow is shown here.
- Comet assay. The single cell gel electrophoresis or Comet Assay is a technique for quantitating DNA damage from in vivo and in vitro samples of eukaryotic cells. This technique is rapid, non-invasive, sensitive, visual and inexpensive compared to conventional techniques and is a powerful tool to study factors modifying mutagenicity and carcinogenicity. It is the only technique that directly measures DNA damage in individual cells and as a result has rapidly gained importance in the fields of genetic toxicology and human biomonitoring.
Comet Assay measures double strand breaks (DSBs), single strand breaks (SSBs), alkali labile sites, and DNA repair. Formamidopyrimidine glycosylase (Fpg) is a DNA repair enzyme. Fpg catalyzes the excision of damaged DNA bases including 8-OHdG sites, and this property has been successfully incorporated into Comet assay to detect the oxidative DNA damage, mainly the 8-OHdG formation. After slides are prepared, cells are evaluated under a Nikon fluorescent microscope and scored using Komet 5.5 imaging analysis software. In our lab, Olive tail moment is the parameter used to assess DNA damage. Olive tail moment is composite parameter without unit. Bigger Olive tail moment represents more DNA damage.
The Nikon Flourescent Microscope instrument-generated dataset workflow is shown here.
In addition, lifestyle and dietary records of colonoscopy patients will be collected during blood sample collection. The lifestyle and diet data will be analyzed and correlated with the SNP and biological analyses. Associations between oxidative stress, DNA damage, and antioxidants with colorectal cancer development will be determined through the statistical modeling approaches, as well as through independent bioinformatic approaches.
See The Role of Oxidative Stress In Chemical Carcenogenesis, James E. Klaunig, et al. in Environmental Health Perspectives, 1998 February; 106(Suppl 1): 289-295. PMCID: PMC1533298
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| references |
Klaunig, J.E. and Kamendulis, L.M. The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol, 2004. 44: p. 239-67.
Kallay, E., et al., Vitamin D receptor activity and prevention of colonic hyperproliferation and oxidative stress. Food Chem Toxicol, 2002. 40(8): p. 1191-6.
Park, Y.J., et al., Genetic changes of hOGG1 and the activity of oh8Gua glycosylase in colon cancer. Eur J Cancer, 2001. 37(3): p. 340-6.
Block, G., Patterson, B., and Subar, A. Fruit, vegetables and cancer prevention: a review of the epidemiological evidence. Nutr Cancer, 1992. 18(1): pp. 1-29.
Studzinski, G.P., McLane, J.A. and Uskokovic, M.R. Signaling pathways for vitamin D-induced differentiation: implications for therapy of proliferative and neoplastic diseases. Crit Rev Eukaryot Gene Expr, 1993. 3(4): p. 279-312.
Tong, W.M., et al., Establishment of primary cultures from human colonic tissue during tumor progression: vitamin-D responses and vitamin-D-receptor expression. Int J Cancer, 1998. 75(3): p. 467-72.
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| Cite this work |
Researchers should cite this work as follows:
Klaunig, J.E. and Kamendulis, L.M. The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol, 2004. 44: pp. 239-67.
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James E. Klaunig, Ph.D.; Xinzhu Pu (2009), "Klaunig Oxidative Stress Analysis Laboratory," http://ccehub.org/resources/201.
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