To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHalpha and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma, the gene-transfected cells HepG2/HBx which stably expressed HBx was established, and the effect of HBx on the cell cycle and proliferation of HepG2 was examined. By using the beta-actin as the interior control, real-time polymerase chain reaction (Real-time qPCR) was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHalpha in the HepG2/HBx, the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector (HepG2/pDNA3.1). The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1. Our results showed that the expression of DNA repair enzyme hMYHalpha in the HepG2/HBx (0.021+/-0.007) was significantly lower than that of HepG2 (0.099+/-0.041) (P<0.05) and HepG2/pDNA3.1 (0.121+/-0.005) (P<0.05). However, the no significant differences existed in the expression of DNA repair enzyme hOGG1 among the three cell strains (P>0.05). The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 (P<0.05). It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHalpha mRNA to impair the ability to repair the intracellular DNA oxidative damage, to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function, thus participate in the occurrence and development of hepatocellular carcinoma.
DNA Glycosylases/genetics ; DNA Glycosylases/*metabolism ; Deoxyguanosine/analogs & derivatives ; Deoxyguanosine/metabolism ; Hep G2 Cells ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Trans-Activators/*genetics

OBJECTIVE: To discuss seminal plasma oxidative stress and sperm DNA oxidative damage in patients with idiopathic asthenozoospermia. METHODS: Infertile couples were selected from the clinic outpatients of the Reproductive Center of Xiangya Hospital, Central-South University from December 2010 to March 2011. Fresh semen of 28 men with idiopathic asthenozoospermia was collected as an experiment group, and 24 fertile men with normal semen and normal reproductive history served as a control group. Level of reactive oxygen species (ROS) in the seminal plasma was assessed with luminer chemiluminescence method. Density of sperm DNA oxidation product 8-hydroxy-2'-deoxyguanosine (8-OHdG) was assessed with enzyme linked immunosorbent assay. RESULTS: 1) ROS level in the experiment group was higher than that in the control group (P<0.01). There was negative correlation between the percentage of progressive motility spermatozoa and the ROS level in the seminal plasma in the 2 groups (r=-0.72, P<0.01). 2) Density of sperm 8-OHdG in the experiment group was higher than that in the control group (P<0.01). There was negative correlation between the percentage of progressive motility spermatozoa and the density of sperm 8-OHdG (r=-0.73, P<0.01). 3) There was positive correlation between the ROS level in the seminal plasma and the density of sperm 8-OHdG (r=0.77, P<0.01). CONCLUSION There is sperm DNA oxidative damage in patients with idiopathic asthenozoospermia, which may be related with the oxidative stress. Excessive generation of reactive oxygen species may be a cause of low sperm motility in patients with idiopathic asthenozoospermia.
8-Hydroxy-2'-Deoxyguanosine ; Adult ; Asthenozoospermia ; etiology ; genetics ; metabolism ; Case-Control Studies ; DNA Damage ; Deoxyguanosine ; analogs & derivatives ; metabolism ; Humans ; Infertility, Male ; genetics ; Male ; Oxidative Stress ; physiology ; Spermatozoa ; metabolism ; Young Adult

Animals ; Antineoplastic Agents ; toxicity ; Brain ; metabolism ; Cisplatin ; toxicity ; DNA Damage ; drug effects ; Deoxyguanosine ; analogs & derivatives ; analysis ; Female ; Kidney ; metabolism ; Male ; Mice ; Mice, Inbred ICR ; Oxidative Stress ; drug effects

Large doses of acetaminophen (APAP) could cause oxidative stress and tissue damage through production of reactive oxygen/nitrogen (ROS/RNS) species and quinone metabolites of APAP. Although ROS/RNS are known to modify DNA, the effect of APAP on DNA modifications has not been studied systematically. In this study, we investigate whether large doses of APAP can modify the nuclear DNA in C6 glioma cells used as a model system, because these cells contain cytochrome P450-related enzymes responsible for APAP metabolism and subsequent toxicity (Geng and Strobel, 1995). Our results revealed that APAP produced ROS and significantly elevated the 8-oxo- deoxyguanosine (8-oxodG) levels in the nucleus of C6 glioma cells in a time and concentration dependent manner. APAP significantly reduced the 8- oxodG incision activity in the nucleus by decreasing the activity and content of a DNA repair enzyme, Ogg1. These results indicate that APAP in large doses can increase the 8-oxodG level partly through significant reduction of Ogg1 DNA repair enzyme.
Acetaminophen/*metabolism ; Analgesics, Non-Narcotic/*metabolism ; Animals ; Cell Line, Tumor ; DNA/metabolism ; DNA Damage ; DNA Glycosylases/*metabolism ; DNA Repair ; Deoxyguanosine/chemistry/*metabolism ; Glioma/*metabolism ; Glutathione/metabolism ; Humans ; Rats ; Reactive Nitrogen Species/metabolism ; Reactive Oxygen Species/metabolism

OBJECTIVETo establish a method for simultaneously determining the urinary concentrations of 8-hydroxy-2'-deoxyguanosine (8-OHdG), trans, trans-muconic acid (tt-MA), and S-phenylmercapturic acid (S-PMA) in subjects exposed to benzene.

METHODSAfter being purified by a solid-phase extraction column, the urine samples were transferred to a liquid chromatography-mass spectrometry system, and the concentrations of 8-OHdG, tt-MA, and S-PMA were determined by external standard method. A C18 reversed-phase column was used as the chromatographic column, and methanol/acidic ammonium formate solution was used as the mobile phase for gradient elution. The mass spectrometer was operated in a multi-reaction monitoring mode.

RESULTSFor tt-MA, the calibration curves were linear in the range of 10-1000 µg/L, and the recovery rates were over 90% (relative standard deviation (RSD) < 3%) at spiked levels of 50 µg/L and 500 µg/L. For S-PMA and 8-OHdG, the calibration curves were linear in the range of 1-100 µg/L, and the recovery rates were over 85% (RSD < 5%) at spiked levels of 5 µg/L and 50 µg/L.

CONCLUSIONThis determination method meets the requirement of Biological materials-

METHODSof monitoring-Guide of development (WS/T 68-1996) and can be used for simultaneous determination of 8-OHdG, tt-MA, and S-PMA in urine.

Acetylcysteine ; analogs & derivatives ; urine ; Benzene ; poisoning ; Chromatography, Liquid ; methods ; Deoxyguanosine ; analogs & derivatives ; urine ; Humans ; Mass Spectrometry ; Occupational Exposure ; prevention & control ; Sorbic Acid ; analogs & derivatives ; metabolism

To synthesize aristolochic acid (AA)-2'-deoxyguanosine 5'-monophosphate (dGp) adducts in vitro and develop a novel method for the characterization of the adducts using multiple mass spectrometric techniques. AA was incubated with dGp in vitro using either enzymatic activation (by xanthine oxidase) or chemical activation (by zinc) to synthesize AA-dGp adducts, and the reaction conditions were optimized. Crude extracts were analyzed by techniques of liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-MS/MS) and high accuracy mass data and isotope pattern of super high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICRMS). The quasi-molecular ion peaks of the AA-dGp adducts were obtained in the negative ion mode. Analysis by electrospray ionization/tandem mass spectrometry (ESI-MS/MS) provided useful structural information about AA-dGp adducts. AA can bind covalently to the exocyclic amino group of deoxyguanosine to form AA-dGp adducts. MS analysis is a powerful tool to detect and identify AA-dGp adducts simply, rapidly and accurately.
Aristolochic Acids ; chemical synthesis ; chemistry ; Chromatography, High Pressure Liquid ; methods ; DNA ; chemistry ; metabolism ; DNA Adducts ; chemical synthesis ; Deoxyguanosine ; chemistry ; Tandem Mass Spectrometry ; methods

PURPOSE: To compare oxidative stress status in the aqueous humor of highly myopic eyes and control eyes. METHODS: Aqueous humor samples were collected from 15 highly myopic eyes (high myopia group) and 23 cataractous eyes (control group) during cataract surgery. Central corneal thickness, corneal endothelial cell density, hexagonality of corneal endothelial cells, and cell area of corneal endothelial cells were measured using specular microscopy. Axial length was measured using ultrasound biometry. 8-Hydroxydeoxyguanosine (8-OHdG) and malondialdehyde levels were measured using enzyme-linked immunosorbent assay. RESULTS: 8-OHdG level was lower in the aqueous humor of myopic patients than in that of control group (p = 0.014) and was positively correlated with central corneal thickness and negatively correlated with axial length (r = 0.511, p = 0.02; r = -0.382, p < 0.001). There was no correlation between 8-OHdG level and corneal endothelial cell density, hexagonality, or cell area. Malondialdehyde level did not show any correlation with any parameters evaluated. CONCLUSIONS: 8-OHdG might be a sensitive biomarker for evaluating oxidative stress status in the eye. Oxidative stress level was lower in the aqueous humor of highly myopic eyes compared to that in control eyes, which indicates lower metabolic activity in these eyes.
Aged ; Aqueous Humor/*metabolism ; Deoxyguanosine/*analogs & derivatives/metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Malondialdehyde/*metabolism ; Middle Aged ; Myopia/*metabolism/physiopathology ; *Oxidative Stress ; Refraction, Ocular/*physiology ; Severity of Illness Index

Parkinson's disease (PD) is an age-related progressive neurodegenerative disease associated with selective loss of dopaminergic neurons. The characteristic hallmark of the disease is intracytoplasmic proteinacious inclusion bodies called Lewy bodies, primarily consisting of a presynaptic protein alpha-synuclein. Oxidative stress-mediated damage to macromolecules have been shown to occur frequently in PD. Oxidative damage to DNA in the form of oxidized guanine (8-oxodG) accumulates in both the mitochondrial and nuclear DNA of dopaminergic neurons of the substantia nigra in PD. 8-oxodG-mediated transcriptional mutagenesis has been shown to have the potential to alter phenotype of cells through production of mutant pool of proteins. This review comprehensively summarizes the role of oxidative stress-mediated damage incurred during neurodegeneration, and highlights the scope of transcriptional mutagenesis event in leading to alpha-synuclein aggregation as seen in PD.
Amino Acid Sequence ; Animals ; Deoxyguanosine/*analogs & derivatives/metabolism ; Humans ; Molecular Sequence Data ; Mutagenesis ; *Oxidative Stress ; Parkinson Disease/*genetics/metabolism/pathology ; Protein Aggregation, Pathological/*genetics/metabolism/pathology ; Substantia Nigra/metabolism/*pathology ; Transcription, Genetic ; alpha-Synuclein/chemistry/*genetics

Cigarette smoke is associated with the development of several diseases, such as chronic obstructive pulmonary disease (COPD). The purpose of this study was to investigate genotoxicity and heat shock protein 70 (Hsp70) in human airway smooth muscle cells (HASMCs) exposed to cigarette smoke extract (CSE). HASMCs was exposed to CSE with different doses for 24 h. The level of 8-hydroxydeoxyguanosine (8-OHdG) was determined by using HPLC-ECD, the DNA damage was analyzed by using comet assay, and apoptosis was examined by using Annexin-FITC/PI staining. The production of Hsp70 after CSE stimulation was tested. Results indicated that CSE significantly increased the level of 8-OHdG, DNA damage and cell apoptosis, and reduced the production of Hsp70. In particular, levels of Hsp70 were inversely correlated with 8-OHdG, DNA damage and cell apoptosis. It was concluded that cigarette smoke induced genotoxicity and decreased the production of cell protective protein Hsp70, which may contribute to the development of some airway diseases.
Apoptosis ; DNA Damage ; Deoxyguanosine ; analogs & derivatives ; metabolism ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Lung ; cytology ; drug effects ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Smoke ; adverse effects ; Tobacco ; toxicity ; Tumor Cells, Cultured

OBJECTIVETo study the genotoxicity effect of environmental tobacco side-stream smokes (ETSS) on oxidative DNA damage and its molecular mechanism.

METHODSDNA adduct 8-hydroxydeoxyguanosine (8-OHdG) was used as a biomarker of oxidative DNA damage. The level of 8-OHdG in DNA exposed to ETSS was detected by high performance liquid chromatography with electrochemical detection. Organic and inorganic components in ETSS were analyzed by gas chromatography-mass spectrum and atomic absorption spectrum respectively.

RESULTSParticle matters (PMs) and volatile organic compounds (VOCs) in ETSS could directly induce oxidative DNA damage and formation of 8-OHdG. There were 123 and 84 kinds of organic components in PMs and VOCs respectively, and 7 kinds of inorganic components in ETSS. Some components, especially quinones and polyphenols in ETSS, could produce free radicals in vitro by auto-oxidation without any biological activity systems, and with the catalytic reaction of metals, the DNA adduct 8-OHdG was produced.

CONCLUSIONETSS have biological oxidative effect on DNA in vitro and in vivo, and expressed direct genotoxicity. 8-OHdG is a valuable biomarker of oxidative DNA damage.

Animals ; Biomarkers ; analysis ; Cattle ; DNA ; drug effects ; metabolism ; DNA Adducts ; analysis ; DNA Damage ; Deoxyguanosine ; analogs & derivatives ; analysis ; Female ; Lung ; chemistry ; metabolism ; Metals, Heavy ; analysis ; Organic Chemicals ; analysis ; Oxidation-Reduction ; Rats ; Tobacco Smoke Pollution ; adverse effects ; analysis