Dataset Information

Direct LC-MS/MS Detection of Guanine Oxidations in Exon 7 of the p53 Tumor Suppressor Gene.

ABSTRACT: Oxidation of DNA by reactive oxygen species (ROS) yields 8-oxo-7,8-dihydroguanosine (8-oxodG) as primary oxidation product, which can lead to downstream G to T transversion mutations. DNA mutations are nonrandom, and mutations at specific codons are associated with specific cancers, as widely documented for the p53 tumor suppressor gene. Here, we present the first direct LC-MS/MS study (without isotopic labeling or hydrolysis) of primary oxidation sites of p53 exon 7. We oxidized a 32 base pair (bp) double-stranded (ds) oligonucleotide representing exon 7 of the p53 gene. Oxidized oligonucleotides were cut by a restriction endonuclease to provide small strands and enable positions and amounts of 8-oxodG to be determined directly by LC-MS/MS. Oxidation sites on the oligonucleotide generated by two oxidants, catechol/Cu2+/NADPH and Fenton's reagent, were located and compared. Guanines in codons 243, 244, 245, and 248 were most frequently oxidized by catechol/Cu2+/NADPH with relative oxidation of 5.6, 7.2, 2.6, and 10.7%, respectively. Fenton's reagent oxidations were more specific for guanines in codons 243 (20.3%) and 248 (10.4%). Modeling of docking of oxidizing species on the ds-oligonucleotide were consistent with the experimental codon oxidation sites. Significantly, codons 244 and 248 are mutational "hotspots" in nonsmall cell and small cell lung cancers, supporting a possible role of oxidation in p53 mutations leading to lung cancer.

SUBMITTER: Jiang D

PROVIDER: S-EPMC5777150 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Direct LC-MS/MS Detection of Guanine Oxidations in Exon 7 of the p53 Tumor Suppressor Gene.

Jiang Di D Malla Spundana S Fu You-Jun YJ Choudhary Dharamainder D Rusling James F JF

Analytical chemistry 20171122 23

Oxidation of DNA by reactive oxygen species (ROS) yields 8-oxo-7,8-dihydroguanosine (8-oxodG) as primary oxidation product, which can lead to downstream G to T transversion mutations. DNA mutations are nonrandom, and mutations at specific codons are associated with specific cancers, as widely documented for the p53 tumor suppressor gene. Here, we present the first direct LC-MS/MS study (without isotopic labeling or hydrolysis) of primary oxidation sites of p53 exon 7. We oxidized a 32 base pair ...[more]

PMID: 29116749

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Project description:A reliable and fast liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of three oxidized nucleic acid damage products in urine, 8-oxoguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo). We applied this method to assess the effect of various urine workup procedures on the urinary concentrations of the oxidized nucleic acid products. Our results showed that frozen urine samples must be warmed (i.e., to 37 °C) to re-dissolve any precipitates prior to analysis. We showed that common workup procedures, such as thawing at room temperature or dilution with deionized water, are not capable of releasing fully the oxidized nucleic acid products from the precipitates, and result in significant underestimation (up to ~ 100% for 8-oxoGua, ~ 86% for both 8-oxodGuo and 8-oxoGuo). With this method, we further assessed and compared the ability of the three oxidized nucleic acid products, as well as malondialdehyde (MDA, a product of lipid peroxidation), to biomonitor oxidative stress in vivo. We measured a total of 315 urine samples from subjects with burdens of oxidative stress from low to high, including healthy subjects, patients with chronic obstructive pulmonary disease (COPD), and patients on mechanical ventilation (MV). The results showed that both the MV and COPD patients had significantly higher urinary levels of 8-oxoGua, 8-oxodGuo, and 8-oxoGuo (P < 0.001), but lower MDA levels, compared to healthy controls. Receiver operating characteristic curve analysis revealed that urinary 8-oxoGuo is the most sensitive biomarker for oxidative stress with area under the curve (AUC) of 0.91, followed by 8-oxodGuo (AUC: 0.80) and 8-oxoGua (AUC: 0.76). Interestingly, MDA with AUC of 0.34 failed to discriminate the patients from healthy controls. Emerging evidence suggests a potential clinical utility for the measurement of urinary 8-oxoGuo, and to a lesser extent 8-oxodGuo, which is strongly supported by our findings.

Project description:BackgroundBisphenol A (BPA) is typically measured in urine using an indirect method that involves enzymatic deconjugation and extraction. In contrast, the direct method measures free and conjugated BPA concurrently and sums them to estimate urinary BPA concentrations. Statistical comparison of total BPA results using the direct and indirect methods is necessary to accurately interpret biomonitoring data for risk assessments.ObjectivesTo compare urinary BPA concentrations estimated from the indirect and direct methods in duplicate first trimester urine samples collected from 1879 pregnant women from the MIREC Study.MethodsFor the indirect method, we measured urinary BPA concentrations using GC-MS/MS. For the direct method, we summed free and conjugated BPA concentrations measured using LC-MS/MS. We evaluated deviation between the two methods using the Bland-Altman analysis in the total sample and stratified (1) by specific gravity and (2) at the limit of quantification (LOQ).ResultsMedian urinary BPA concentrations for the direct and indirect methods were 0.89 µg BPA equivalents/L and 0.81 µg/L respectively. Concentrations from the direct method were, on average, 8.6% (95% CI: 6.7%, 10.5%) higher than the indirect method in a Bland-Altman analysis. The percent differences between the two methods was 4.0% in urines with specific gravities < 1.02 (n = 1348, 72%) and 20.3% in urine with specific gravity ≥ 1.02. In values below the LOQ (n = 663, 35%), we observed smaller average percent deviation (4.8%) between the two methods but wider limits of agreement.DiscussionResults from this study, based on the largest statistically rigorous comparison of the direct and indirect methods of BPA measurement, contrast previous findings reporting that the indirect method underestimates total BPA exposure. The difference in urinary BPA concentrations we observed with the indirect and direct methods is unlikely to alter the interpretation of health outcome data.

Dataset Information

Direct LC-MS/MS Detection of Guanine Oxidations in Exon 7 of the p53 Tumor Suppressor Gene.

Publications

Direct LC-MS/MS Detection of Guanine Oxidations in Exon 7 of the p53 Tumor Suppressor Gene.

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