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

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

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

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

OBJECTIVEIxeris chinesis (Thunb.) Ankai has been used as a Chinese folk medicine, but only scanty information is available on the physiological and biochemical functions of the compounds extracted from I. chinesis. In the present study the effects of apigenin-7-glucoside (APIG) isolated from I. chinesis against liver injury caused by carbon tetrachloride (CCl4) were investigated.

METHODSThe contents of malondialdehyde (MDA), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), and reduced glutathione (GSH) were evaluated by spectrophotography. The content of 8-Hydroxydeoxyguanosine (8-OHdG) was measured with high-performance liquid chromatography (HPLC) equipped with electrochemical and UV detection methods. The antioxidant activity of APIG was evaluated using chemiluminescence single photon counting technology.

RESULTSCCl4 significantly increased the enzyme activities of GPT and GOT in blood serum, as well as the level of MDA and 8-OHdG in liver tissue, and decreased the levels of GSH. Pretreatment with APIG was able not only to suppress the elevation of GPT, GOT, MDA and 8-OHdG, and inhibit the reduction of GSH in a dose-dependent manner in vivo, but also to reduce the damage of hepatocytes in vitro. On the other hand, we also found that APIG had strong antioxidant activity against reactive oxygen species (ROS) in vitro in a concentration-dependent manner.

CONCLUSIONThe hepatoprotective activity of APIG is possibly due to its antioxidant properties, acting as scavengers of ROS. These results obtained in vivo and in vitro suggest that APIG has protective effects against hepatic oxidative injury induced by chemicals. Further studies on the pharmaceutical functions and immunological responses of APIG may help its clinical application.

Alanine Transaminase ; blood ; metabolism ; Animals ; Antioxidants ; therapeutic use ; Apigenin ; isolation & purification ; therapeutic use ; Aspartate Aminotransferases ; blood ; metabolism ; Asteraceae ; chemistry ; Carbon Tetrachloride ; Chemical and Drug Induced Liver Injury ; etiology ; prevention & control ; DNA Damage ; Deoxyguanosine ; analogs & derivatives ; analysis ; Drugs, Chinese Herbal ; Glutathione ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Lipid Peroxidation ; Liver ; drug effects ; metabolism ; Male ; Malondialdehyde ; metabolism ; Protective Agents ; therapeutic use ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism

To explore the protective effect of Angelica sinensis polysaccharides(ASP) on subacute renal damages induced by D-galactose in mice and its mechanism. Male C57BL/6J mice were randomly divided into 3 groups, with 10 mice in each group. The D-galactose model group was subcutaneously injected with D-galactose (120 mg x kg(-1)), qd x 42; the ASP + D-galactose model group was intraperitoneally injected with ASP since the 8th day of the replication of the D-galactose model, qd x 35; and the normal control group was subcutaneously injected with saline at the same dose and time. On the 2nd day of after the injection, the peripheral blood was collected to measure the content of BUN, Crea, UA, Cys-C; paraffin sections were made to observe the renal histomorphology by HE staining; senescence-associated β-g-alactosidase (SA-β-Gal) stain was used to observe the relative optical density (ROD) in renal tissues; transmission electron microscopy was assayed to observe the renal ultrastructure; the renal tissue homogenate was prepared to measure the content of SOD, GSH-PX, MDA; the content of AGEs and 8-OH-dG were measured by ELISA. According to the result, compared with the D-galactose model group, the ASP + D-galactose model group showed obviously decreases in the content of BUN, Crea, UA, Cysc, AGES, 8-OH-dG, the number of hardening renal corpuscle, renal capsular space and renal tubular lumen, ROD of SA-β-Gal staining positive kidney cells, mesangial cells, basement membrane thickness, podocyte secondary processes fusion and MDA and increases in the number of normal renal corpuscle, ribosome and rough endoplasmic reticulum in podocytes, the activity of SOD and GSH-PX. In Conclusion, A. sinensis polysaccharides can antagonize kidney subacute damages induced by D-galactose in mice. Its protective mechanism may be correlated with the inhibition of the oxidative stress injury.
Angelica sinensis ; chemistry ; Animals ; Deoxyguanosine ; analogs & derivatives ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Galactose ; adverse effects ; Humans ; Kidney ; anatomy & histology ; drug effects ; injuries ; Kidney Diseases ; chemically induced ; drug therapy ; metabolism ; prevention & control ; Male ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; drug effects ; Polysaccharides ; administration & dosage ; Protective Agents ; administration & dosage

CpG-Oligodeoxynucleotide (ODN) has two backbones. Phosphorothioate backbone (PS) shows a strong immunostimulating effect while phosphodiester (PE) shows little in vivo. 3' hexameric deoxyriboguanosine-run (3' dG6-run) conjugation to PE CpG-ODN has been reported to enhance immunostimulation and to protect against asthma when injected at the time of sensitization in mice. We evaluated the treatment effects of PE and PS CpG-ODN with or without 3' dG6-run on asthma in presensitized mice. BALB/c mice sensitized with ovalbumin and alum were challenged with 1% ovalbumin on three days. CpG-ODNs (100 microgram) or PBS were injected 4 times; 27 hr before challenge and 3 hr before each challenge (CpG-dG6: CpG-ODN with 3' dG6-run, PE*-CpG-dG6: PE-CpG-dG6 with two PS backbones at the 5' terminus). PE-CpG showed no treatment effect. PE-CpG-dG6 only increased ovalbumin-specific IgG2a. PE*-CpG-dG6 increased ovalbumin-specific IgG2a but also reduced BAL fluid eosinophils and airway hyperresponsiveness. PS-CpG increased ovalbumin-specific IgG2a, reduced airway inflammation and airway hyperresponsiveness. PS-CpG-dG6 was less effective than PS-CpG on airway inflammation and airway hyperresponsiveness. In pre-sensitized mice, PE-CpG required not only 3' dG6-run but also the modification of two PS linkages at 5' terminus to inhibit features of asthma. PS-CpG was strong enough to inhibit asthma but PS-CpG-dG6 was less effective.
Animals ; Anti-Asthmatic Agents/*therapeutic use ; Asthma/*drug therapy/physiopathology ; Bronchial Hyperreactivity/drug therapy ; Bronchoalveolar Lavage Fluid/immunology ; Deoxyguanosine/*analogs & derivatives/*chemistry ; Female ; Immunoglobulin G/metabolism ; Interleukin-12/analysis ; Interleukin-4/analysis ; Interleukin-5/analysis ; Lung/pathology ; Mice ; Mice, Inbred BALB C ; Oligodeoxyribonucleotides/*therapeutic use ; Phosphorothioate Oligonucleotides/therapeutic use ; Splenomegaly/pathology