Oxidative Stress and DNA Damage in Human Gastric Carcinoma: 8-Oxo-7'8-dihydro-2'-deoxyguanosine (8-oxo-dG) as a Possible Tumor Marker

Abstract

We characterized the oxidative stress (OS) status by the levels of reduced/oxidized glutathione (GSH/GSSG), malondialdehyde (MDA) and the mutagenic base 8-oxo-7'8-dihydro-2'-deoxyguanosine (8-oxo-dG) in human gastric carcinoma (HGC) samples and compared the results with normal tissue from the same patients. We also analyzed 8-oxo-dG in peripheral mononuclear cells (PMNC) and urine from healthy control subjects and in affected patients in the basal state and one, three, six, nine and twelve months after tumor resection. The levels of DNA repair enzyme mRNA expression (hOGG1, RAD51, MUYTH and MTH1) were determined in tumor specimens and compared with normal mucosa. Tumor specimens exhibited increased levels of MDA and 8-oxo-dG compared with normal gastric tissue. GSH levels were also increased, while GSSG levels remained stable. DNA repair enzyme mRNA expression was induced in the tumor tissues. Levels of 8-oxo-dG were significantly elevated in both urine and PMNC of gastric cancer patients compared with healthy controls. After gastrectomy, the levels of the damaged base in urine and PMNC decreased progressively to values close to those found in the healthy population. The high levels of 8-oxo-dG in urine may be related to the increased induction of DNA repair activity in tumor tissue, and the changes observed after tumor resection support its potential use as a tumor marker.

Figure 1

Oxidative stress by-products in gastric carcinoma vs. normal mucosa. Tissues were obtained as outlined in the Experimental Section and dealt with appropriately for metabolite assays. A total of 28 paired samples were used. Results are expressed as means ± SD. *** p < 0.001 in the comparison between tumor and normal mucosa.

Figure 2

mRNA expression of DNA repair enzymes in tumor tissue and normal mucosa. The figure presents relative mRNA expression levels for the indicated enzymes and their significance. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was analyzed to normalize gene expression for sample-to-sample differences in RNA input, RNA quality and reverse transcriptase efficiency. Each sample was analyzed in triplicate. Results are expressed as means ± SD of ten different tumors and mucosa from the same patient. ** p < 0.05; *** p < 0.001.

Figure 3

Time course effect of tumor resection on the levels of 8-oxo-dG in (a) urine and (b) peripheral mononuclear cells of gastric carcinoma patients. Control: healthy subjects; Pre: samples 24 h before surgical intervention; Post: 24 h and following months (1–12) after surgery. Results are expressed as means ± SD with the number of samples (n) indicated in the figure. ⁺⁺⁺p < 0.001 for the comparison of control subjects with cancer patients at the basal state; *** p < 0.001 for the comparison of post-surgical time periods with basal state in the patient group.

Figure 3

Time course effect of tumor resection on the levels of 8-oxo-dG in (a) urine and (b) peripheral mononuclear cells of gastric carcinoma patients. Control: healthy subjects; Pre: samples 24 h before surgical intervention; Post: 24 h and following months (1–12) after surgery. Results are expressed as means ± SD with the number of samples (n) indicated in the figure. ⁺⁺⁺p < 0.001 for the comparison of control subjects with cancer patients at the basal state; *** p < 0.001 for the comparison of post-surgical time periods with basal state in the patient group.

Figure 4

Schematic representation of the roles played by oxidative stress and oxygen free radicals in the induction of genetic instability during tumor progression. Increased oxidative stress and the production of hydroxyl radicals in tumor tissue interact and oxidize DNA, inducing the mutagenic base 8-oxo-dG, leading to increased gene instability in the tumor tissue. Different genetic alterations affecting oncogenes and tumor suppression genes take place during tumor growth and progression, contributing to the pathogenicity of the disease [19]. Induced expression of DNA repair enzymes results in the release of high levels of the damaged base in urine. The 8-oxo-dG levels in the urine of affected patients are reduced after tumor resection.