The alkaline comet assay is a sensitive and relatively inexpensive technique used for the detection of DNA damage. We have developed, and implemented modifications to the alkaline comet assay which may increase ability of the assay to detect subtle differences in DRC as well as baseline and induced levels of DNA damage measured as strand breaks and oxidative damage to DNA. Currently, there is no universally accepted panel of useful markers of oxidative stress in humans even though many lines of evidence suggest that oxidative DNA damage and other forms of oxidative stress influence the development of age-associated disease.
The aim of this work is to determine whether demographic factors, such as age, sex and race affect the baseline level of oxidative DNA damage and to examine whether there were consistent relationships between frequently studied and novel measures of oxidative stress in diverse population. We are examining the level of single strand breaks and oxidative base lesions detected by endonuclease III and Fpg in cryopreserved PBMCs. In addition, we want to examine whether there is a correlation between the level of oxidative DNA damage in lymphocytes and other oxidative stress-related measures including: red blood cell glutathione (RBC GSH), heme degradation products, serum alpha-tocopherol, protein carbonyls in plasma and high-sensitivity C-reactive protein (hs-CRP). This study examines standard and unique markers of oxidative DNA damage and repair, oxidative stress and inflammation. We are studying four age-matched male and female whites and African-Americans aged 30-64 years. Our preliminary findings suggest that females have higher single strand break (SSB) levels than males (p=0.013). We observe a significant negative correlation between SSB repair capacity and SSB level (p=0.041). Thus far plasma carbonyl levels have shown no significant correlation with other markers. Our preliminary results suggest a complex relationship between measures of oxidative stress and clinical parameters used frequently and believed to reflect inflammation or oxidative stress. Our studies highlight the need for further examination to clarify the relationship between commonly used oxidative stress measures and DNA damage measures in diverse population cohorts so that we can understand how to accurately assess the potential clinical applications of these measures or to identify other more relevant and correlative markers. The lack of a strong overall relationship between hs-CRP, a marker of systemic inflammation, and markers of oxidative DNA damage may suggest that each of these markers represents a different pathway through which inflammation and oxidative stress occur and are monitored and/or modulated. Alternatively, it could also be difficult to correlate hs-CRP levels in serum with DNA damage in PMBCs. Therefore, we have begun to evaluate another marker of oxidant stress and oxidative DNA damage that can be detected in serum, the common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) and its correlation with inflammation, aging and other clinical parameters. We have assessed the relationship of 8-oxo-G levels with selected clinical and laboratory measures in a select part of our cohort. Our preliminary analyses revealed that the relationship between both systolic blood pressure and pulse pressure were significant. The relationship between 8-oxo-G and poverty status with race were not significant. 8-oxo-G levels increase with age and though we found no relationship between hs-CRP with SSB we have found that 8-oxo-G levels increase with increasing hs-CRP levels, suggesting a potential important relationship between a marker of inflammation and a marker of oxidative stress. We have also begun to pursue other biomarkers of age and disease. Human aging is a highly complex process that is characterized by an increase in age-associated diseases. Important in the study of aging is the discovery of new biomarkers that serve as indicators of tissue age and development and that also can aid in the diagnosis of age-related diseases. Studies in model systems suggest that longevity can be modulated by changes in gene and protein expression. However, little is known about how these small RNAs contribute to human aging. We have profiled the expression of over 800 miRNAs in peripheral blood mononuclear cells from young and old individuals by real-time RT-PCR analysis. This genome-wide assessment of miRNA expression revealed that the majority of miRNAs studied decreased in abundance with age. We identified nine miRNAs (miR-103, miR-107, miR-128, miR-130a, miR-155, miR-24, miR-221, miR-496, miR-1538) that were significantly lower in older individuals. Among them, five have been implicated in cancer pathogenesis. Predicted targets of several of these miRNAs, including PI3 kinase (PI3K), c-Kit and H2AX, were found to be elevated with advancing age, supporting a possible role for them in the aging process. Furthermore, we found that decreasing the levels of miR-221 was sufficient to cause a corresponding increase in the expression of the predicted target, PI3K. Taken together, these findings demonstrate that changes in miRNA expression occur with human aging and suggest that miRNAs and their predicted targets have the potential to be diagnostic indicators of age or age-related diseases.
| Kim, Yoonseo; Noren Hooten, Nicole; Evans, Michele K (2018) CRP Stimulates GDF15 Expression in Endothelial Cells through p53. Mediators Inflamm 2018:8278039 |
| DeMarino, Catherine; Pleet, Michelle L; Cowen, Maria et al. (2018) Antiretroviral Drugs Alter the Content of Extracellular Vesicles from HIV-1-Infected Cells. Sci Rep 8:7653 |
| Eitan, Erez; Green, Jamal; Bodogai, Monica et al. (2017) Age-Related Changes in Plasma Extracellular Vesicle Characteristics and Internalization by Leukocytes. Sci Rep 7:1342 |
| Noren Hooten, Nicole; Evans, Michele K (2017) Techniques to Induce and Quantify Cellular Senescence. J Vis Exp : |
| Dluzen, Douglas F; Noren Hooten, Nicole; Evans, Michele K (2017) Extracellular RNA in aging. Wiley Interdiscip Rev RNA 8: |
| Dluzen, Douglas F; Kim, Yoonseo; Bastian, Paul et al. (2017) MicroRNAs Modulate Oxidative Stress in Hypertension through PARP-1 Regulation. Oxid Med Cell Longev 2017:3984280 |
| Eicher, John D; Chami, Nathalie; Kacprowski, Tim et al. (2016) Platelet-Related Variants Identified by Exomechip Meta-analysis in 157,293 Individuals. Am J Hum Genet 99:40-55 |
| Plank-Bazinet, Jennifer L; Kornstein, Susan G; Clayton, Janine Austin et al. (2016) A Report of the Women's Health Congress Workshop on The Health of Women of Color: A Critical Intersection at the Corner of Sex/Gender and Race/Ethnicity. J Womens Health (Larchmt) 25:4-10 |
| Chami, Nathalie; Chen, Ming-Huei; Slater, Andrew J et al. (2016) Exome Genotyping Identifies Pleiotropic Variants Associated with Red Blood Cell Traits. Am J Hum Genet 99:8-21 |
| Dluzen, Douglas F; Noren Hooten, Nicole; Zhang, Yongqing et al. (2016) Racial differences in microRNA and gene expression in hypertensive women. Sci Rep 6:35815 |
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