Exfoliated buccal mucosa cells as a source of DNA to study oxidative stress.
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.
Borthakur
Gayatri
G
Department of Kinesiology and Nutrition, University of Illinois at Chicago M/C 517, 1919 West Taylor Street, Chicago, IL 60612, USA.
Butryee
Chaniphun
C
Stacewicz-Sapuntzakis
Maria
M
Bowen
Phyllis E
PE
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000293:Adolescent; D000328:Adult; D000368:Aged; D015415:Biomarkers; D004247:DNA / Q000235:genetics*; D018736:DNA Adducts / Q000235:genetics; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D005240:Feasibility Studies; D005260:Female; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009061:Mouth Mucosa / Q000166:cytology* / Q000601:surgery; D018384:Oxidative Stress / Q000187:drug effects*
D015415:Biomarkers; D018736:DNA Adducts; D000080242:8-Hydroxy-2'-Deoxyguanosine; D006493:Heparin; D004247:DNA; D003849:Deoxyguanosine