A study on UHPLC-MS/MS analyses of DNA and RNA oxidative damage metabolites in patients with cervical carcinoma: 8-oxoG in urine as a potential biomarker of cervical carcinoma.

Objective: The purpose of this study was to compare the levels of 8-oxoG and 8-oxodG in urine of patients with cervical carcinoma and healthy women to evaluate their influences on cervical carcinoma. Methods: In this study, urine samples were collected from 70 patients with cervical carcinoma, 24 patients with one-year follow-up, and 100 healthy women. The contents of 8-oxodG and 8-oxoG in urine were assayed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Results: The levels of 8-oxoG and 8-oxodG were higher in patients with cervical carcinoma (P < 0.000), while AUC of 8-oxoG and 8-oxodG was higher than 0.7. Specifically, the high-risk HPV (HR-HPV) positive group had higher 8-oxoG levels (P < 0.000), but there was no difference in 8-oxodG levels. Yet, 8-oxoG level was associated with lymphatic metastasis, lymph-vascular space infiltration (LVSI) and stromal infiltration, while 8-oxodG level was affected by the differentiation degree and stromal infiltration. According to statistics, the distinct cut-off index of lymphatic metastasis was 7.282 nmol/mmol creatinine. After operation, the concentrations of 8-oxoG and 8-oxodG dropped significantly (8-oxoG P < 0.000, 8-oxodG P = 0.004). Except for chemotherapy group, the urinary 8-oxoG dose of all treatment groups and 8-oxodG dose of chemo-radiotherapy group declined obviously. Conclusions: 8-oxoG may be a potential biomarker for cervical carcinoma.

Oxidative DNA and RNA damage and their prognostic values during Salmonella enteritidis-induced intestinal infection in rats.

Emerging evidence suggests that microbial pathogens may induce oxidative stress in infected hosts. The aim of the present study was to investigate the relationship between changes in oxidative stress and intestinal infection with and without antibiotic treatment in animal models. Sprague-Dawley (SD) rats were divided into three groups: rats infected with Salmonella enterica serovar Enteritidis (S. enteritidis), rats infected with S. enteritidis followed by norfloxacin treatment, and the control group. To evaluate oxidative stress changes, levels of 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn), which represented oxidative damage to RNA and DNA, respectively, were analysed in urine and tissue samples. In urine, the level of 8-oxo-Gsn increased significantly after oral exposure to S. enteritidis (p ≤ 0.001) and returned to baseline after recovery. Notably, norfloxacin treatment decreased the level of 8-oxo-Gsn in urine significantly (p = 0.001). Changes of 8-oxo-Gsn measured in tissues from the small intestine, colon, liver and spleen were consistent with 8-oxo-Gsn measured in urine. Our study suggested that 8-oxo-Gsn in urine may serve as a highly sensitive biomarker for evaluating the severity of S. enteritidis infection and the effectiveness of antibiotic treatment against infection.

The Ratio of Plasma and Urinary 8-oxo-Gsn Could Be a Novel Evaluation Index for Patients with Chronic Kidney Disease.

Nucleic acid oxidation plays an important role in the pathophysiology progress of a variety of diseases. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn), which originate from DNA and RNA oxidation, were the most widely used indicators for oxidative stress. The study investigated the relation between 8-oxo-dGsn, 8-oxo-Gsn, and CKD. 146 patients with CKD were divided into five disease stages, and their fasting blood and morning urine were collected. The levels of 8-oxo-dGsn and 8-oxo-Gsn in plasma and urine were quantified by LC-MS/MS. The ratio of urinary 8-oxo-Gsn to creatinine increased from stages 1 to 4 corresponding to the increased severity of CKD, but it decreased in stage 5. And plasma 8-oxo-Gsn gradually increased with the decline of renal function. In particular, the increased ratio of plasma and urine 8-oxo-Gsn in stage 5 exceeded the concentration of creatinine. This trend was similar to the estimated glomerular filtration rate (eGFR), which indicates that 8-oxo-Gsn could be an appropriate indicator for renal function. Our finding indicates that as the disease progresses, RNA oxidation is increased. The significant increase in the ratio of plasma and urinary 8-oxo-Gsn is a novel evaluation index of end-stage renal disease.

Levels of 8-oxo-dGsn and 8-oxo-Gsn in random urine are consistent with 24 h urine in healthy subjects and patients with renal disease.

Oxidatively generated damage to nucleic acids may play an important role in the pathophysiological processes of a variety of diseases. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) are oxidatively generated products of DNA and RNA, respectively. Our previous studies have suggested that the amounts of 8-oxo-dGsn and 8-oxo-Gsn in urine were considerably higher than other body fluid or tissue. The aim of this study was to investigate whether 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples are consistent with those in 24 h urine samples in healthy subjects and patients with renal disease. A total of 16 healthy subjects and 104 renal disease patients were enrolled in this study, and their random and 24 h urine samples were collected. The levels of urinary 8-oxo-dGsn and 8-oxo-Gsn were quantified by LC-MS/MS and corrected by creatinine. Regardless of healthy subjects or renal disease patients, the levels of oxidised nucleosides in random urine samples were consistent with 24 h urine samples. Regardless of the age bracket, there is no significant difference between random samples and 24 h urine samples. In conclusion, 8-oxo-dGsn and 8-oxo-Gsn levels in random urine samples could replace those in 24 h urine samples, and were considered as the representative of the level of systemic oxidative stress for the whole day.

Effect of CYP2D6 variants on venlafaxine metabolism in vitro.

1. CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme superfamily, we recently identified 22 CYP2D6 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of venlafaxine in vitro. 2. The wild-type and 24 CYP2D6 variants were expressed in insect cells, and each variant was characterized using venlafaxine as the substrate. Reactions were performed at 37 °C with 5-500 µM substrate (three variants was adjusted to 1000 µM) for 50 min. By using high-performance liquid chromatography to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of O-desmethylvenlafaxine were determined. 3. Among the 22 CYP2D6 variants, the intrinsic clearance (Vmax/Km) values of all variants were significantly decreased (from 0.2% to 84.5%) compared with wild-type CYP2D6*1. In addition, the kinetic parameters of two CYP2D6 variants could not be detected because they have no detectable enzyme activity. 4. The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards venlafaxine in vivo.

The role of CYP2C9 genetic polymorphism in carvedilol O-desmethylation in vitro.

We aimed at investigating the role of CYP2C9 in carvedilol O-desmethylation and identifying the effect of 35 CYP2C9 allelic variants we found in Chinese Han population on the in vitro metabolism of carvedilol. Recombinant CYP2C9 and CYP2D6 microsomes of the wild type were used to test and verify the enzymes involved in carvedilol O-desmethylation. Recombinant CYP2C9 microsomes of distinguished genotypes were used to characterize the corresponding enzyme activity toward carvedilol. 2-100 µM carvedilol was incubated for 30 min at 37 °C. The products were detected using high-performance liquid chromatography. CYP2C9 plays a certain role in carvedilol metabolism. Compared with wild-type CYP2C9*1, the intrinsic clearance (V max/K m) values of all variants toward carvedilol O-desmethylation were significantly altered. The variants exhibited significantly decreased values (from 30 to 99.8 %) due to increased K m and/or decreased V max values. We conclude that recombinant system could be used to investigate the enzymes involved in drug metabolism and these findings complement the database where CYP2C9 polymorphism interacts with biotransformation of exogenous substances like drugs and toxins.