PURPOSE: We wanted to demonstrate the anti-cancer effect and interaction between belotecan and cisplatin on gastric cancer cell line and we evaluated the mechanisms of this synergistic effect in vitro. MATERIALS AND METHODS: The growth inhibitory effect of belotocan and cisplatin against several gastric cancer cell lines (SNU-5, SNU-16 and SNU-601) was estimated by tetrazolium dye assay. The effect of a combination treatment was evaluated by the isobologram method. The biochemical mechanisms for the interaction between the drugs were analyzed by measuring the formation of DNA interstrand cross-links (ICLs) and DNA topo-I activity. RESULTS: Belotecan showed synergism with cisplatin for growth inhibitory effect on the gastric cancer cell lines SNU-5, and SNU-16, but this was subadditive on the SNU-601 cell line. The formation of DNA ICLs in SNU-16 cells by cisplatin was increased by combination with belotecan, but this was not affected in SNU-601 cells. The topo-I inhibition by belotecan was enhanced at high concentrations of cisplatin in SNU-16, but not in SNU-601 cells. CONCLUSION: Belotecan and cisplatin show various combination effect against gastric cancer cells. The synergism between cisplatin and belotecan could be the result of one of the following mechanisms: the modulating effect of belotecan on the repair of cisplatin-induced DNA adducts and the enhancing effect of cisplatin on the belotecan-induced topo-I inhibitory effect.
Cell Line ; Cisplatin* ; DNA ; DNA Adducts ; Stomach Neoplasms*

Aldehydes ; chemistry ; pharmacology ; Chromatography, High Pressure Liquid ; DNA Adducts

OBJECTIVETo explore the adduct characteristics of styrene and DNA.

METHODSThe adduct reactions between styrene, urinary mandalic acid(MA), phenylglyoxalic acid(PGA), mercapturic acid of styrene (UMA) and DNA were studied by ultraviolet spectral analysis. The SO-DNA adducts by 32P-post labeled method, the chemical structures of SO-DNA adducts by GC-MS and NMR were also studied.

RESULTSSO combined with DNA at O6, N2 positions of dGMP to form six adducts, but styrene, urinary mandalic acid, phenylglyoxalic acid and mercapturic acid of styrene did not react with DNA to form adduct.

CONCLUSIONSStyrene formed adduct with DNA through its active center metabolite--SO after entering the body. SO combined with DNA at O6, N2 positions of dGMP to form adducts. If these DNA adducts are not repaired or are mis-repaired before cell duplication, the gene mutation and chemical damage would happen. No adduct reactions are seen among other metabolites of styrene.

Acetylcysteine ; metabolism ; DNA ; metabolism ; DNA Adducts ; metabolism ; DNA Repair ; Glyoxylates ; metabolism ; Humans ; Mandelic Acids ; metabolism ; Styrene ; metabolism

OBJECTIVETo design and develop a novel, sensitive and versatile method for in vivo foot printing and studies of DNA damage, such as DNA adducts and strand breaks.

METHODSStarting with mammalian genomic DNA, single-stranded products were made by repeated primer extension, these products were ligated to a double-stranded linker having a randomized 3' overhang, and used for PCR. DNA breaks in p53 gene produced by restriction endonuclease AfaI were detected by using this new method followed by Southern hybridization with DIG-labeled probe.

RESULTSThis randomized terminal linker-dependent PCR (RDPCR) method could generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR), and it was more rapid, convenient and accurate than the terminal transferase-dependent PCR (TDPCR).

CONCLUSIONDNA strand breakage can be detected sensitively in the gene level by RDPCR. Any lesion that blocks primer extension should be detectable.

Animals ; DNA Adducts ; DNA Damage ; DNA Primers ; Genes, p53 ; genetics ; Humans ; Mammals ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVEThe oxidation of benzo (a) pyrene mediated by 5-lipoxygenase (5-LOX) were investigated in HBE cells in order to provide further proof that lipoxygenase is the alternative pathway for the oxidation of xenobiotics.

METHODSEnzymic experiment: Soybean lipoxygenase (SLO), substrate (benzo[a] pyrene) and other component react in the enzymic system and the reaction product are detected by spectrophotometry. At the same time, in vitro detect of benzo (a) pyrene-DNA adducts with a UV spectrophotometer and HPLC. Cellular experiment: After HBE cells exposure to different poison (B[a]P 4, 8, 16, 32, 64, 128µmol/L, AA-861, naproxen or α- naphthoflavone 0.1, 1, 10 µmol/L) for 24 hours, the effect of benzo (a) -pyrene on cell survival rate were assessed by reductions of tetrazolium dye (MTT) and flow cytometry in cultured HBE cells, and the protein expressions of 5-lipoxygenase in the cells are tested by western-blot, and the DNA damages by the single cell gel electrophoresis. And then, the effect of the specific inhibitor of 5-lipoxygenase (AA-861) on 5-lipoxygenase protein expression and DNA damage in the cells are detected.

RESULTSSLO can catalyze the co-oxidation of benzo (a) pyrene to generate benzo (a) pyrene-7,8-epoxide in the presence of hydrogen peroxide. GTP can inhibit the reaction , the IC50 value is 0.46 mg/L, the model equation is Probit (P) = 0.8985+2.6824 Log (dose). SLO can catalyze the co-oxidation of benzo (a) pyrene to generate a new product, but fail to form DNA adducts in vitro. HBE cell viability decreased with the benzo (a) pyrene concentration increased , but AA-861 and naproxen can inhibit it. Flow cytometry and single cell gel electrophoresis experiments show, Benzo (a) pyrene can induce 5-lipoxygenase protein expression, but AA-861 cannot in HBE. Benzo (a) pyrene causes toxic action and DNA damage in HBE, which can significantly inhibit by AA-861, the difference is statistically significant (P < 0.05).

CONCLUSIONSThe co-oxidate of benzo (a) pyrene by 5-LOX turns into electrophiles that covalently bind to DNA and induce DNA damage, which can be significantly inhibited by AA-861.

Benzo(a)pyrene ; metabolism ; Cells, Cultured ; DNA Adducts ; metabolism ; DNA Damage ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Lipoxygenase ; pharmacology

OBJECTIVETo explore the relationship of GSTT1, GSTM1 gene polymorphisms and PAH-DNA adduct with pathogenesis of multiple myeloma.

METHODSThe bone marrow samples from 37 newly-diagnosed MM patients and 52 healthy peoples as controls were collected; the PCR-based restriction fragment length polymorphism (PCR-RFLP) method was used to detecte the polymorphism of GSTT1 and GSTM1, and to analysis their relationship with clinical characters of MM patients; the engyme linked immunosorbent assay (ELISA) was performed to detect the concentration of PAH-DNA adducts.

RESULTSGSTT1 null and GSTM1 null genotypes increased the risk of multiple myeloma with OR 2.57 (P=0.035) and 1.37 (P>0.05) respectively. In MM patients group, GSTT1 null genotype in stage III was significantly higher than that in stages I, II (P=0.038). However, no statistically significant association was found between GSTT1 gene polymorphism and clinical characters, such as age, type, hemoglobin, β2-MG, albumin. Compared with Hb≥85 g/L of the newly-diagnosed MM patients, MM patients with Hb<85 g/L had significantly higher incidence of GSTM1 null genetype (P<0.05). The level of PAH-DNA adducts in MM patients was higher than that in controls (2358±1182 pg/ml vs 1853±996 pg/ml) (P<0.05). GSTT1 null genotype with PAH-DNA level≥2100 pg/ml showed a risk index of MM (OR=4.500, P<0.05).

CONCLUSIONGSTT1 gene may have a critical function in the development of MM, and correlates with staging of MM. GSTM1 gene polymorphism correlates with hemoglobin levels of patients with MM. The content of PAH-DNA adducts may play an important role in the pathogenesis of multiple myeloma.

DNA Adducts ; Genotype ; Glutathione Transferase ; Humans ; Incidence ; Multiple Myeloma ; Polycyclic Aromatic Hydrocarbons ; Polymerase Chain Reaction ; Polymorphism, Genetic

OBJECTIVETo detect the structure and chemical characteristics of the adduct from the reaction of p-benzoquinone (BQ) with deoxyguanoside (dGMP).

METHODSdGMP and calf thymus DNA were reacted with BQ in buffered solutions with neutral pH, the reaction products were separated and purified by high performance liquid chromatograph (HPLC), and then characterized by UV spectroscopy and mass spectrometry.

RESULTSThe reaction of BQ with dGMP yielded two adduct products (Ad(1) and Ad(2) respectively). The characterized results of Ad(1) suggested that BQ reacted at the N-1 and N(2) position of dGMP by losing one H(2)O molecule, the molecular weight of Ad(1) was 437, and the molecular formula was C(16)H(16)O(8)N(5)P. Ad(1) could also be detected from calf thymus DNA reacted with BQ in vitro, which possessed the same elution profile by HPLC analysis. Meanwhile, Ad(2) was detected in the experimental condition. It was proposed that Ad(2) was formed by BQ reacted at the N-9 position of dGMP by losing one molecule of deoxyribose, the molecular weight was 241, and the molecular formula was C(11)H(7)O(2)N(5).

CONCLUSIONThe structure of one major adduct from reaction of BQ with DNA is (3'-OH)-1, N(2)-C(6)H(5)CH-2'-deoxyguanosine-5'-monophosphate.

Benzoquinones ; metabolism ; DNA Adducts ; chemistry ; Deoxyguanine Nucleotides ; metabolism ; Hydrogen-Ion Concentration ; Mass Spectrometry ; Molecular Weight ; Spectrophotometry, Ultraviolet

OBJECTIVETo develop bioluminescence method to measure DNA adducts in human peripheral lymphocytes and study the factors that affect the level of DNA adduct, in order to use it in studying the relationship between water pollution and human DNA adduct levels.

METHODThe measurement of DNA adduct in peripheral lymphocytes was carried out in 234 normal persons, and the relationship between DNA adducts levels and age, smoking and eating habit etc. was analyzed.

RESULTS(1) The level of DNA adduct in males was higher than that in females, but without significance (P > 0.05); (2) The level of DNA adduct was significantly correlated with age, smoking, tea drinking, eating habit and alcohol drinking. Among them, age plus smoking were the most important factors affected the DNA adduct levels (P < 0.01). The level of DNA adduct increased with age. The more cigarette smoked, the higher was the level of DNA adduct.

CONCLUSIONThe level of DNA adduct is affected by many factors, so it needs further research to apply it in the biomonitoring and risk assessment of environment chemicals.

Adult ; Age Factors ; Aged ; DNA Adducts ; analysis ; Female ; Humans ; Luminescent Measurements ; Lymphocytes ; chemistry ; Male ; Middle Aged ; Sex Factors ; Smoking ; metabolism

OBJECTIVETo study albumin adduct with naphthalene metabolites, namely 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ), as a potential biomarker for intermediate/long-term exposure to polycyclic aromatic hydrocarbons (PAH) in coke oven workers.

METHODSTwenty-eight coke oven workers and 22 control workers were recruited from a cokery. Spot urine and venous blood samples were collected from the workers after four continuously working days and personal information was obtained by questionnaire. Plasma albumin adduct was detected with gas chromatography-mass spectrometry.

RESULTSAlbumin adduct with 1,2- & 1,4-NPQ (1,2-NPQ and 1,4-NPQ), respectively, were detected in all coke oven workers and controls. Median plasma level of 1,2-NPQ-Alb in coke oven workers was significantly higher than that in controls (76.6 pmol/g vs. 44.9 pmol/g, P < 0.01). However, there was no significant difference in plasma median level of 1,4-NPQ-Alb between the two groups (48.6 pmol/g vs. 44.2 pmol/g, P > 0.05). Plasma level of 1,2-NPQ-Alb was significantly higher than that of 1,4-NPQ-Alb in coke oven workers. Urine levels of naphthalene, 1-naphthol, 2-naphthol and 1-pyrenol in coke oven workers correlated significantly with their plasma level of 1,2-NPQ-Alb (Pearson coefficient of correlation greater than 0.371, P < 0.01), but did not do significantly with 1,4-NPQ-Alb.

CONCLUSIONPlasma level of 1,2-NPQ-Alb could effectively reflect their magnitude of personal internal dose of exposure to air PAH, so it could be used as a potential biomarker to evaluate their intermediate/long-term exposure to PAH in coke oven workers.

Air Pollutants, Occupational ; adverse effects ; Albumins ; Biomarkers ; blood ; Coke ; DNA Adducts ; Humans ; Male ; Naphthalenes ; metabolism ; Naphthoquinones ; blood ; Occupational Exposure

This study aims to establish a quantitative method of 4 aristolochic acids-DNA adducts in mice kidney and liver based on high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) for monitoring the content changes of aristolochic acids-DNA adducts. A Shiseido Capcellpak AQ C_(18) column(3 mm×100 mm, 3 μm) was used, with a mixture of 0.2% acetic acid-5 mmol·L~(-1) ammonium acetate as the aqueous phase and methanol as the organic phase for gradient elution. The multiple reaction monitoring(MRM) scanning method under positive mode by electrospray ionization(ESI) was performed for the detection of the aristolochic acids-DNA adducts which formed by combining aristolochic acid Ⅰ/Ⅱ with deoxyadenosine, deoxyguanosine, and deoxycytidine, respectively. Balb/c mice were given Guanmutong extract by gavage, and the relative content of aristolochic acids-DNA adducts in liver and kidney samples were analyzed within 60 days. It was found that the concentration of 4 aristolochic acids-DNA adducts in the kidney was significantly higher than that in the liver, and there were about 15.87 adducts in per 1×10~6 normal deoxynucleosides, which was 4.5-7.5 times than that of the liver. What's more, some adducts can still be detected on the 30 th day after administration. The concentration of the adducts in the liver was highest on the first day after administration, and a second peak appeared during the 7 th to 14 th days. The results indicated that aristolochic acids-DNA adducts are difficult to eliminate in vivo, and it is of great significance to study the mechanism of liver and kidney injury of aristolochic acid.
Animals ; Aristolochic Acids ; Chromatography, High Pressure Liquid ; DNA Adducts ; Liver ; Mice ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry