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 effects of selenium on DNA damage induced by benzo[a] pyrene (BaP) in mouse lung cells.

METHODSSodium selenite was given to Kunming male mice by i.p. and BaP was given by oral gavage. The control group was given solvent only with the same method. DNA damage was detected by single cell gel electrophoresis (or comet assay).

RESULTSThe damage degrees in mice treated with 125, 250 and 500 mg/kg of BaP were more severe than that of control (P < 0.01). The rates of comet cells in the BaP-treated groups (43.50%, 84.00%, 95.63%) were significantly higher than that of control (9.75%, P < 0.01), and there was obvious dose-response relationship. 0.75, 1.50 and 3.00 mg/kg of sodium selenite presented antagonistic effects against DNA damage induced by 250 mg/kg of BaP in mouse's lung cells. The antagonistic effect of sodium selenite at the dose of 1.50 mg/kg was better than those of sodium selenite at the doses of 0.75, 3.00 mg/kg.

CONCLUSIONBaP at the doses of 125 approximately 500 mg/kg could significantly induce DNA damage of lung cells in mice. 0.75 approximately 3.00 mg/kg of sodium selenite could inhibit DNA damage of lung cells in mice induced by 250 mg/kg of BaP.

Animals ; Benzo(a)pyrene ; toxicity ; DNA Damage ; drug effects ; Lung ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Selenium ; pharmacology

OBJECTIVETo evaluate the possible vascular effects of an environment carcinogen benzo(a)pyrene (BaP).

METHODSThe cytotoxicit of BaP and rat liver S9 (0.25 mg/mL)-activated BaP were examined by MTT assay. Thoracic aortic rings were dissected from Sprague-Dawley rats. Contraction of aortic rings was induced by 60 mmol/L KCl or 10(-6) mol/L phenylephrine (PE) in an ex-vivo perfusion system after BaP (100 μmol/L) incubation for 6 h. [Ca(2+)](i) was measured using Fluo-4/AM. For in-vivo treatment, rats were injected with BaP for 4 weeks (10 mg/kg, weekly, i.p.).

RESULTSBaP (1-500 μm) did not significantly affect cell viability; S9-activated BaP stimulated cell proliferation. BaP did not affect the contractile function of endothelium-intact or -denuded aortic rings. BaP did not affect ATP-induced ([Ca(2+)](i)) increases in human umbilical vein endothelial cells. In BaP-treated rats, heart rate and the number of circulating inflammatory cells were not affected. Body weight decreased while blood pressure increased significantly. The maximum aortic contractile responses to PE and KCl and the maximum aortic relaxation response to acetylcholine were significantly decreased by 25.0%, 34.2%, and 10.4%, respectively.

CONCLUSIONThese results suggest, in accordance with its DNA-damaging properties, that metabolic activation is a prerequisite for BaP-induced cardiovascular toxicity.

Animals ; Aorta ; drug effects ; Benzo(a)pyrene ; pharmacology ; Calcium ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Humans ; Male ; Rats ; Vasoconstriction ; drug effects

OBJECTIVETo investigate the effect of benzo(a)pyrene (BaP) on platelet aggregation and expression of P-selectin.

METHODSBlood samples were collected from healthy volunteers and the platelets was washed. Platelet aggregation was monitored by aggregometer and the expression of P-seletin was detected by whole blood flow cytometry.

RESULTBaP (10 μmol/L, 1 μmol/L and 0.1 μmol/L) did not induce platelet aggregation; however, preincubation with BaP (10 μmol/L) significantly enhanced ADP-induced platelet aggregation (P < 0.01) and platelet aggregation was (80 ± 10)%, while BaP-preincubation failed to enhance platelet aggregation under collagen and thrombin stimulation. Flow cytometry showed that preincubation with BaP increased ADP-induced, but not thrombin-induced P-selectin expression (P < 0.01).

CONCLUSIONBaP can stimulate ADP-induced platelet aggregation and P-selectin expression, probably through the interaction with ADP-mediated signal pathway.

Adenosine Diphosphate ; pharmacology ; Benzo(a)pyrene ; pharmacology ; Blood Platelets ; drug effects ; metabolism ; Collagen ; pharmacology ; Humans ; P-Selectin ; blood ; drug effects ; Platelet Aggregation ; drug effects ; Thrombin ; pharmacology

OBJECTIVE: To investigate the protective effects and the potential mechanisms of vitamin E (VE) on benzo(a)pryene (B[a]P)-induced toxicity in the reproductive system of male rats. 
 METHODS: A total of 60 male Sprague Dawley (SD) rats, weighted 70-90 g, were randomly assigned to 6 groups: a control group, a vehicle group, a B[a]P group (5 mg/kg), a VE (10 mg/kg)+ B[a]P (5 mg/kg) group, a VE (50 mg/kg) + B[a]P (5 mg/kg) group and a VE (100 mg/kg)+B[a]P (5 mg/kg) group (n=10 per group). The rats were treated with B[a]P and/or VE once a day for 30 days via intragastric administration. The sperm quality and the levels of SOD, GSH-Px, 8-OHdG and MDA were detected, respectively. The testicular tissue morphology and DNA damage were observed by HE staining and comet assay.
 RESULTS: The sperm count, the rate of sperm deformation, the content of MDA and 8-OHdG were all significantly increased in single B[a]P-treated group in comparison to the control groups. The activities of SOD and GSH-Px were markedly decreased by B[a]P as compared with the control groups (P<0.05). The injury of testicular tissue in B[a]P-treated rats was remarkably improved after VE treatment. The levels of oxidative stress and DNA damage indicators in the B[a]P-treated group were all attenuated by VE. These protective effects of VE were in a dose-dependent manner (P<0.05).
 CONCLUSION Vitamin E can protect the male SD rats against the B[a]P-induced reproductive toxicity.
Animals ; Benzo(a)pyrene ; toxicity ; DNA Damage ; Male ; Oxidative Stress ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatozoa ; drug effects ; Testis ; drug effects ; pathology ; Vitamin E ; pharmacology

OBJECTIVETo investigate the effect of ERCC1 gene on the repair capability of damaged DNA in lung cancer A549 cells induced by benzo[a]pyrene.

METHODSRecombinant plasmid expressing ERCC1 antisense RNA was constructed and transfected into A549 cells by Lipofectin reagent. The stable-transfected cell colonies were selected by hygromycin. Cell viability was determined by the MTT assay. The level of ERCC1 mRNA was measured by Northern Blot analysis. Single cell gel electrophoresis assay was applied to determine the cellular DNA damage and fifty cells for each group were counted.

RESULTSSeven positive colonies expressing ERCC1 antisense RNA were screened. There was no growth rate difference between the antisense-transfected cells and the parental cells. The endogenous mRNA level in transfected colonies decreased in varied degrees, i.e. 12% approximately 86% of that of the parental cells in Northern Blot assay. After 24 h treatment of 10 micro mol/l benzo[a]pyrene, the repair capability for DNA damage in transfected colonies was reduced to 29% approximately 71% of that of the parental cells. Also, a statistically significant correlation was observed between expression of ERCC1 mRNA and repair capability (r = 0.84).

CONCLUSIONAntisense ERCC1 RNA decreased the repair capability for damaged DNA in lung cancer cells induced by benzo[a]pyrene.

Benzo(a)pyrene ; toxicity ; Cell Line, Tumor ; DNA Damage ; drug effects ; DNA Repair ; drug effects ; DNA-Binding Proteins ; genetics ; metabolism ; pharmacology ; Endonucleases ; genetics ; metabolism ; pharmacology ; Humans ; Lung Neoplasms ; pathology ; Plasmids ; RNA, Antisense ; pharmacology ; RNA, Messenger ; metabolism ; Repressor Proteins ; Transfection

Biochanin A, an isoflavone compound, is reported to have an inhibitory effect on benzo(a)pyrene [B(a)P] metabolism. We examined the modifying effect of biochanin A on in vivo carcinogenesis using a mouse lung tumor model. As carcinogens, a single subcutaneous injection of 0.5mg of B(a)P was given within 24 hours after birth. The test groups were injected with 0.125mg of biochanin A in 0.1ml DMSO by i.p. 3 times a week for 6 weeks after weaning. All mice were sacrificed at week 9 and the incidence and multiplicity of lung tumors were examined. Concomitant administration of biochanin A showed a significant inhibitory effect on the incidence of tumor-bearing mice (12.5%, P<0.01), as well as the mean number of tumors (0.13, P<0.001), compared with the group treated with B(a)P alone in which the incidence was 57.1% and the mean number was 1.0. These results suggest that biochanin A has inhibitory potential on the development of mouse lung tumor induced by B(a)P.
Animals ; Animals, Newborn ; Anticarcinogenic Agents/*pharmacology ; Benzo(a)pyrene ; Body Weight/drug effects ; Female ; *Genistein ; Incidence ; Isoflavones/*pharmacology ; Lung Neoplasms/chemically induced/*prevention & control ; Mice ; Neoplasms, Multiple Primary/chemically induced/prevention & control ; Survival Analysis

OBJECTIVETo investigate the expressions of JWA gene and heat shock protein 70 (hsp70) in human embryonic lung (cccHPF-1) cells after exposure to activated benzo(a)pyrene (B(a)P), and to explore the role and the possible mechanism of JWA gene involved in B(a)P-induced DNA damage and repair.

METHODSThe models of DNA damage of ccc-HPF-1 cells were established by treatment of cells with B(a)P plus S9 at 10 to 100 micromol/L for 3 hours with or without 24 hours recovery for DNA repairing. The DNA damage was detected by single cell gel electrophoresis (SCGE) assay (comet assay). And the immuno-blotting assay was used for detecting expressions of JWA and hsp70.

RESULTSJWA expression was actively modulated by B(a)P exposure. The expressions of both JWA and hsp70 were increased greatly at 50 micromol/L and 100 micromol/L B(a)P treated cells and maintained at over expressed levels treated by 10-100 micromol/L B(a)P during the restored time. In addition, JWA expression pattern was similar to that of hsp70.

CONCLUSIONJWA is determined in this study by its functioning as an effective environmental responsive gene and actively participating in the signal pathways of DNA damage and repair which might be associated with excision repair.

Benzo(a)pyrene ; pharmacology ; Cell Line ; Cell Survival ; drug effects ; Comet Assay ; DNA Damage ; DNA Repair ; Heat-Shock Proteins ; metabolism ; Humans ; Immunoblotting ; Intracellular Signaling Peptides and Proteins ; metabolism ; Lung ; cytology ; embryology ; metabolism ; Signal Transduction ; drug effects

OBJECTIVETo investigate the reverse effect of all-trans retinoic acid (ATRA) on Benzo (a) pyrene (B (a) P)-induced cyclin D1, CDK4, E2F-1 and E2F-4 expression and cell cycle progression in human embryo lung fibroblast (HELF).

METHODSAfter HELF cells was treated with ATRA, they were exposed to 2 micromol/L of B (a) P. Western blotting was employed to detect protein expression level; the RNA transfection techniques was used to investigate ATRA-induced signal pathway; flow cytometry was used to detect cell cycle progression.

RESULTAfter treatment with 2 micromol/L B (a) P for 24 h, the expression of cyclin D1 and E2F-1 were both increased significantly in HELF; the expression of E2F-4 and CDK4 were not changed markedly; pretreatment with 0.1 micromol/L ATRA for 24 h could efficiently decrease B (a) P-induced overexpression of cyclin D1 and E2F-1; stimulation to antisense cyclin D1 or antisense CDK4 by B (a) P could significantly impair E2F-1 up-regulation; pretreatment with ATRA, cells with antisense cyclin D1 or antisense CDK4 showed a less decrease in B (a) P-induced overexpression of E2F-1 compared to similarly treated control cells; flow cytometry analysis showed B (a) P promoted cell cycle progression from G(1) phase to S phase, while pretreatment with ATRA could inhibit B (a) P-induced cell cycle progression by an accumulation of cells in the G(1) phase.

CONCLUSIONATRA could block B (a) P-induced cell cycle promotion through cyclin D1/E2F-1 pathway in HELF.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Lung ; cytology ; metabolism ; Signal Transduction ; drug effects ; Tretinoin ; pharmacology

OBJECTIVETo investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]P.

METHODSHuman embryo lung fibroblasts (HELFs) were treated with 2 micromol/L or 100 micromol/L B[a]P which were provided with some characteristics of transformed cells (T-HELFs). Cyclin D1, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle.

RESULTSAfter B[a]P treatment, the proportion of the first gap (G1) phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 micromol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs (A-D1 and A-K4) and T-HELFs (T-A-D1 and T-A-K4). After 2 micromol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4.

CONCLUSIONSCyclin D1/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 micromol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 micromol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.

Benzo(a)pyrene ; pharmacology ; Cell Cycle ; drug effects ; Cell Line ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; E2F4 Transcription Factor ; metabolism ; Fibroblasts ; drug effects ; enzymology ; metabolism ; Humans ; Lung ; cytology ; drug effects ; embryology ; enzymology ; metabolism