Exfoliated Buccal Mucosa Cells as a Source of DNA to Study Oxidative Stress

Abstract

The extent of oxidative DNA damage is considered a biomarker of carcinogenic process and could be investigated in population studies using easily obtained cells. The oxidized DNA base adduct 8-hydroxy-2-deoxyguanosine (8-OHdG) released by enzymatic hydrolysis of DNA is commonly assayed by high performance liquid chromatography with electrochemical detection. It is expressed as a ratio of 8-OHdG to unoxidized deoxyguanosine. We modified and improved this method, determined the optimal time for harvesting buccal mucosa cells (BMC), assessed whether they mirror peripheral circulating blood cell DNA damage, and compared the anticoagulants, heparin, and EDTA for consistency in measurement of leukocyte 8-OHdG. Thirty-one healthy participants, randomized into two groups, donated BMC and blood samples. Samples were collected at baseline and either 3 or 7 days after baseline. Results showed no correlation between 8-OHdG/deoxyguanosine ratios in BMC and peripheral blood leukocytes at any time point regardless of harvest time. BMC had much higher oxidative DNA damage, but displayed a 25.6% reduction in the oxidized DNA adduct level (P < 0.04) at 3 days after baseline. Leukocytes collected in heparin and EDTA had similar 8OHdG/deoxyguanosine ratios; however, EDTA was preferred, as it produced a clean nuclear pellet without hemoglobin contamination, and the results were less variable. This improved assay shows within subject stability over time in both leukocyte and BMC DNA damage, increasing the probability that small intervention differences can be detected in healthy subjects. Buccal cells provide an accessible pool of epithelial cells that represents higher levels of DNA damage than circulating leukocytes. (Cancer Epidemiol Biomarkers Prev 2008;17(1):212 -9)

Altered cytological parameters in buccal cells from individuals with mild cognitive impairment and Alzheimer's disease

“…Furthermore micronuclei are a biomarker of chromosome malsegregation and tend to be elevated in lymphocytes, fibroblasts and buccal cells of AD cases (8,28,29). Other DNA damage markers such as cH2AX (DNA double strand breaks) and 8HOdG (oxidative DNA damage) were also found to be linked with AD (30)(31)(32) and previously detected in human buccal cells (33,34). Therefore, the genomic instability events observed previously may partly explain the results of increased DNA content observed in this study when measured quantitatively by LSC.…”

Oral Cell DNA Adducts as Potential Biomarkers for Lung Cancer Susceptibility in Cigarette Smokers

“…Oral mucosa cells are an excellent source of material for evaluating DNA adducts and molecular changes potentially related to cancer. 28-31 Collection of oral mucosa cells is relatively simple, in contrast to bronchial brushings and sputum, which can be difficult, expensive, and impractical to obtain. Multiple studies have demonstrated similarities in molecular changes between oral mucosa cells and bronchial cells obtained from the same individuals, particularly in smokers, consistent with the field carcinogenesis concept of lung and upper aerodigestive tract cancer.…”

Biomarkers of Alzheimer’s Disease Risk in Peripheral Tissues; Focus on Buccal Cells

“…Buccal cells have been used as a source of tissue in a variety of biochemical and molecular biology studies using an assortment of different techniques to collect the cells including; cotton swabs [92], cytobrushes [92-94], a “swish and spit” method [95-97], a modified Guthrie card [98] and a method of rubbing cheeks against teeth to exfoliate cells [94]. The results from those studies demonstrated that high quantities of buccal cells (more than a million per sampling) could be obtained and then subsequently used in a variety of assays; such as DNA analysis using PCR or other genotype tests [95, 96, 99-102], for isolation of mRNA for gene expression profiling, Western blots for detection of proteins and immunocytochemistry [103-105], high-performance liquid chromato-graphy (HPLC) [106] and ion transporter assays [107]. Ideally invasive procedures should be avoided in AD patients due to age and presenting medical issues, therefore buccal cells could offer an appropriate alternative as a relatively non-invasive and easily accessible source of tissue for analysis.…”

“…Additionally, DNA double strand breaks have been detected in human buccal cells using an immunofluorescent antibody against γH2AX [158], therefore confirming that MN and γH2AX are two important DNA damage biomarkers that can be detected and may be altered in buccal cells from patients with AD. Oxidative stress has also been studied in leukocytes and exfoliated BM using HPLC after DNA isolation [106] and because the association between accumulated oxidative DNA damage and ageing is well documented, it is possible that the BM may show changes in 8-OHdG levels from AD buccal samples; however this has yet to be tested.…”