Measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine in peripheral blood mononuclear cells: optimisation and application to samples from a case-control study on cancers of the oesophagus and cardia.
8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker to evaluate the level of oxidative stress. This study describes in its first part the optimisation of our analytical procedure (HPLC/electrochemical detection). Particular care was exercised to avoid artefactual oxidation and in the precision of measurement, which was evaluated with blood bags from hemochromatosis patients. The best results were obtained with a DNA extraction step using the "chaotropic method" recommended by the European Standards Committee on Oxidative DNA Damage (ESCODD). Other approaches such as anion exchange columns gave ten times as much 8-oxodG as this method. Moreover, a complete DNA hydrolysis using five different enzymes allowed improved precision. The optimised protocol was applied to peripheral blood mononuclear cells (PBMC) sampled during a case-control study on cancers of the oesophagus and cardia. With 7.2 +/- 2.6 8-oxodG/10(6) 2'-deoxyguanosines (2'-dG) (mean +/- SD), patients (n = 17) showed higher levels of 8-oxodG than controls (4.9 +/- 1.9 8-oxodG/10(6) 2'-dG, n = 43, Student's t-test: p < 0.001). This difference remained significant after technical (storage, sampling period, 2'-dG levels) and individual (age, sex, smoking, alcohol) confounding factors were taken into account (p < 0.0001, Generalised Linear regression Model). To our knowledge, this is the first report to demonstrate an increase of 8-oxodG in PBMCs of patients suffering from a cancer of the upper digestive tract. This elevated level of DNA damage in patients can raise interesting issues: is oxidative stress the cause or the result of the pathology? Could this biomarker be used to evaluate chemoprevention trials concerning digestive tract cancers?
Breton
Jean
J
University of Leeds, Faculty of Medicine and Health, Leeds Institute for Health and Therapeutics, Molecular Epidemiology Unit, Leeds LS2 9JT, UK. j.breton@leeds.ac.uk
Sichel
François
F
Pottier
Didier
D
Prevost
Virginie
V
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood*; D004563:Electrochemistry; D004938:Esophageal Neoplasms / Q000097:blood* / Q000517:prevention & control; D005260:Female; D006801:Humans; D006868:Hydrolysis; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D008956:Models, Chemical; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D013274:Stomach Neoplasms / Q000097:blood* / Q000517:prevention & control; D013997:Time Factors
D014408:Biomarkers, Tumor; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine; D010100:Oxygen