Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers

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Abstract

Background: 8-hydroxydeoxyguanosine (8-OHdG) is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Urinary 8-OHdG is now considered as a biomarker of generalized, cellular oxidative stress and is linked to degenerative diseases including cancer. Methods: We developed a competitive enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG by coating BSA conjugated 8-hydroxyguanine (8-OHG) on a microplate. Urine specimens containing 8-OHdG and monoclonal anti-8-OHdG antibody were incubated together in the microwell. Final quantification of bound anti-8-OHdG antibody was estimated by the addition of HRP-conjugated sheep–anti-mouse antibody. Results: The concentration range of the calibration curve was 0–60 ng/ml. The sensitivity of the assay was 0.5 ng/ml. The within-day precision and day-to-day precision were <10%. The ELISA correlated well with a commercial kit (r=0.9). Our assay measured not only 8-OHdG but also 8-OHG and 8-hyroxyguanine in urine. Increased urinary concentration of 8-OHdG and its analogs were detected in both patients with bladder cancer (70.5±38.2 ng/mg creatinine) and prostate cancer (58.8±43.4 ng/mg creatinine) as compared to the healthy control (36.1±24.5 ng/mg creatinine). Conclusion: Our preliminary data suggest that the competitive ELISA for 8-OHdG and its analogs appears to be a simple method for quantifying the extent of oxidative stress and may have potential for identifying cancer risk.

Introduction

It is well known that reactive oxygen species (ROS) of cellular metabolism can directly cause oxidative damage of DNA. ROS hydroxylates predominantly the base in DNA. Among all bases in the nucleic acid, guanine is most prone to oxidative damage. Following oxidative damage, cells have evolved mechanisms capable of removing the oxidative DNA lesion (repair). Mismatch repair appears to be one pathway associated with the repair of 8-hydroxylated guanine lesions [1], [2].

8-hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Therefore, the amount of urinary 8-OHdG has been considered not only as indicator of DNA repair capacity but also a biomarker of generalized, cellular oxidative stress [3], [4].

In DNA, the hydroxylation of guanine by ROS at the 8-position, the 8-hydroxylation of guanine, leads to the lack of specific base pairing and misreading of the modified base and adjacent residues. When this occurs, extensive and specific repair is performed by the cell for survival and to maintain genomic integrity. Within DNA, hot-spots of oxidative modification and subsequent mutation have been described [5]. Oxidative damage to cellular DNA has been proposed to play an important role in a number of pathological conditions, including carcinogenesis.

Recently, increasing evidences have shown that malignant cells contain high concentrations of oxidized DNA lesion [6]. Tumor also tends to generate large amounts of hydrogen peroxide [7]. Increased 8-OHdG also has been detected in various tumors. In uterine myomas, the concentration of 8-OHdG was found to increase with the size of tumor [8]. In breast cancer, higher concentrations of 8-OHdG and 5-hydroxymethyl-2′-deoxyuridine in DNA were detected in both cancer tissues and blood cells [9], [10]. In addition, higher 8-OHdG/creatinine ratios were found in urine of patients with small cell lung cancer [11]. It appears that urinary 8-OHdG may be used to indicate cancer risk.

8-OHdG is most frequently measured with high-performance liquid chromatography (HPLC) coupled with an electrochemical (EC) detector [12]. Although HPLC-EC method is sensitive and accurate, it is not a convenient procedure for analyzing 8-OHdG contents in a clinical laboratory because of its cost, technical involvement and low throughput. An alternative way to measure 8-OHdG is by enzyme-linked immunosorbent assay (ELISA) [13]. ELISAs are sensitive and specific and do not require sophisticated equipments. Measuring 8-OHdG in urine is also noninvasive, requiring less purification of the specimen. Because ELISA kit for 8-OHdG is not widely available, we decided to establish a competitive ELISA for 8-OHdG.

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Materials

Pure 8-OHdG and 8-hydroyguanosine (8-OHG) were from Cayman Chemical (Ann Arbor, MI). Sodium meta-periodate was from Pierce (Rockford, IL). Bovine serum albumin (BSA, fraction V) and other chemicals were from Sigma (St. Louis, MO). Mouse monoclonal anti-8-OHdG antibody (clone 1F7) was from Trevigen (Gaithersburg, MD). The antibody has similar affinity for 8-OHdG and 8-OHG. The antibody cross-reacts with guanine and guanosine but with 20,000-fold lower affinity. The antibody will also cross-react

Establishment of the competitive ELISA

Fig. 1 shows the structures of the normal guanine base, 8-hydroxyguanine (8-OHGua) and its nucleoside derivatives, 8-OHdG and 8-OHG. According to the manufacturer, the monoclonal anti-8-OHdG antibody used in the in-house assay has the same affinity for 8-OHdG, 8-OHG and 8-OHGua. Therefore, our ELISA measures not only 8-OHdG but also its analogs in the urine, all products of oxidative damage.

Fig. 2 shows a typical calibration curve using pure 8-OHdG as calibrator. Since in this assay, coated

Discussion

The antibody used in our assay, i.e., anti-8-hydroxyguanine monoclonal antibody from Trevigen, was raised against 8-OHG coupled to keyhole limpet hemocyanine. The manufacturer claims that the monoclonal antibody has similar binding affinity for the oxidized free base, 8-OHGua, and the oxidized nucleosides, 8-OHdG and 8-OHG. The antibody has much less affinity for unmodified free base and nucleoside. It appears that our in-house assay using this antibody will then detect all oxidation modified

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