[Study on activation of benzo(a)pyrene and DNA damage mediated by lipoxygenase in human bronchial epithelial cells].
OBJECTIVE
The 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.


METHODS
Enzymic 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.


RESULTS
SLO 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).


CONCLUSIONS
The 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.
Department of Occupational and Environmental Health, School of Public Health, Central South University, Changsha 410078, China.
Chang-ke
CK
Chen
Yun
Y
Huang
Yue
Y
Wu
Jing
J
Wang
Jian-an
JA
Hu
D001564:Benzo(a)pyrene / Q000378:metabolism*; D002478:Cells, Cultured; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008084:Lipoxygenase / Q000494:pharmacology*
D018736:DNA Adducts; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D008084:Lipoxygenase