OBJECTIVETo observe protective effects of Schisandra extract (SE) on embryotoxicity and reproductive toxicity of early pregnant rats exposed to Benzo[a]pyrene (Bap).

METHODSPregnant rat model was prepared using periodic screening cage method. Totally 50 female pregnant SD rats were divided into five groups by randomized block design according to the weight, i.e., the BaP model group, the low dose SE group, the middle dose SE group, the high dose SE group, the normal control group, 10 rats in each group. Rats in the BaP model group were administered with BaP at a daily dose of 2 mg/kg by gastrogavage. Rats in low, middle, and high dose SE groups were administered by gastrogavage with BaP (at a daily dose of 2 mg/kg) plus SE at a daily dose of 40, 200, and 1 000 mg/kg, respectively. Equal volume of olive oil was administered to rats in the normal control group by gastrogavage. All medication was performed for 8 successive days. Changes of rat body weight in each period were observed. The uterus embryonic total quality and ovary quality were measured, and organ index calculated. The number of corpus luteum, the number of embryo implantation, and the number of absorbed embryo were statistically calculated respectively. The implantation rate and the absorbed embryos rate were calculated. Serum levels of human chorionic gonadotrophin β (β-HCG) and progesterone (PROG) were detected by ELISA.

RESULTSCompared with the normal control group, the weight of 9-day pregnant rats, the number of embryo implantation, the uterus embryonic total index, ovary index, serum levels of β-HCG and PROG all decreased in the Bap model group with significant difference (P < 0.05, P < 0.01). Compared with the Bap model group, body weight, the uterus embryonic total index, and the PROG level increased in 3 dose SE groups (P < 0.05, P < 0.01). Ovary index and serum β-HCG increased in middle and high dose SE groups (P < 0.05, P < 0.01). The number of implantation obviously increased in the high dose SE groups (P < 0.01).

CONCLUSIONSE could reduce the embryotoxicity and reproductive toxicity of early pregnant rats exposed to Benzo[a]pyrene.

Animals ; Benzo(a)pyrene ; toxicity ; Chorionic Gonadotropin ; blood ; Embryo Implantation ; drug effects ; Female ; Ovary ; drug effects ; Plant Extracts ; pharmacology ; Pregnancy ; Progesterone ; blood ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reproduction ; drug effects ; Schisandra ; chemistry ; Uterus ; drug effects

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

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 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

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that is easily introduced to humans via consumption of grilled or smoked meat. BaP causes harmful oxidative effects on cell development, growth and survival through an increase in membrane lipid peroxidation, oxidative DNA damage and mutagenesis. Therefore, the present study was conducted to evaluate the synergistic effects of BaP on oxidative stress in hepatic tumors. In this study, we established a hepatic tumor model by injecting rat hepatoma N1-S1 cells into healthy rats. Changes in the abundance of heat shock proteins (HSPs), antioxidant enzymes and pro-inflammatory cytokines were then investigated by western blot analysis. In addition, we examined changes in oxidative stress levels. Injection of N1-S1 cells or concomitant injection of BaP and N1-S1 cells resulted in the formation of hepatic tumors at the injection site. Evaluation of rat plasma reveals that hepatic tumors induced by BaP and N1-S1 cells expresses higher levels of Hsp27, superoxide dismutase (SOD), and tumor necrosis factor-alpha (TNF-alpha) when compared to those induced by N1-S1 cells only. The collective results of this study suggest that BaP exerts synergistic effects on the expression of HSP, cytokines and antioxidant enzymes in hepatic tumors induced by rat hepatoma N1-S1 cells.
Animals ; Antioxidants/*metabolism ; Benzo(a)pyrene/*pharmacology ; Carcinoma, Hepatocellular/metabolism/pathology ; Cell Line, Tumor/*drug effects ; Cytokines/*metabolism ; Heat-Shock Proteins/*metabolism ; Humans ; Liver Neoplasms/*enzymology/*metabolism/pathology ; Male ; Neoplasms, Experimental/metabolism/pathology ; Oxidative Stress/drug effects ; Rats ; Rats, Sprague-Dawley

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

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

OBJECTIVETo investigate the role of mitogen activated protein kinases (MAPKs) signaling pathways in the regulation of benzo(a)pyrene (B(a)P)-induced c-Jun activation in human embryo lung fibroblasts (HELFs).

METHODSHELFs were cultured with 2.0 micromol/L B(a)P for various time (0, 3, 6, 12, 24 h) or with various concentration of B(a)P (0.0, 0.5, 1.0, 2.0 micromol/L) for 12 h. Western blot was performed to examine the effect of B(a)P on c-Jun activation. The dominant negative mutants of p38, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) were applied to establish stable transfectant, and to detect the relationship of MAPK signal molecules and c-Jun activation in B (a) P-treated cells.

RESULTSB(a)P treatment resulted in a marked activation of c-Jun in time-dependent manner with a peak at 12 h (the densitometric ratios of phosphorylated c-Jun Ser63, Ser73 to actin were 20.1, 15.2 times for control respectively) and in dose-dependent manner. However, there was no evident change on total c-Jun expression in B(a)P-treated HELFs. Moreover, B(a)P-induced activation of c-Jun was inhibited by stable expression of dominant negative mutants of JNK or ERK, but not by dominant negative mutant of p38.

CONCLUSIONJNK and ERK signaling pathways, but not p38 pathway regulate B(a)P-induced c-Jun activation in HELFs.

Benzo(a)pyrene ; pharmacology ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; cytology ; metabolism ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-jun ; metabolism ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo study the role of mitogen activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway in benzo(a)pyrene (B(a)P)-induced changes of cell cycle in human embryo lung fibroblasts (HELF).

METHODSAP-1 luciferase activity was determined by the Luciferase reporter gene assay using a luminometer. The expression levels and activity of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. The dominant negative mutant of ERK2, JNK1 and p38 were applied to detect the upstream or downstream relationship of signaling pathways.

RESULTSB(a)P treatment resulted in a marked activation of AP-1 and its upstream MAPK, including ERK, JNK and p38 in human embryo lung fibroblasts (HELF). B(a)P exposure also led to increase the population of cells at S phase compared to control (P < 0.01) with a concomitant decline of cells at G(1) phase. B(a)P-induced cell cycle alternation was markedly impaired by stable expression of a dominant negative mutant of ERK2 or JNK1, but not p38. B(a)P-induced AP-1 transactivation was inhibited by the overexpression of dominant-negative mutant of ERK2 or JNK1, but not p38. Inhibition of the activation of AP-1 by curcumin, a chemical inhibitor of AP-1, significantly inhibited the cell cycle changes in response to B(a)P treatment.

CONCLUSIONERK and JNK, but not p38, mediated benzo(a)pyrene-induced cell cycle changes by AP-1 transactivation in HELF.

Benzo(a)pyrene ; pharmacology ; Blotting, Western ; Cell Cycle ; drug effects ; Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Lung ; cytology ; embryology ; Mitogen-Activated Protein Kinase 1 ; metabolism ; physiology ; Mitogen-Activated Protein Kinase 8 ; metabolism ; physiology ; Phosphorylation ; Transcription Factor AP-1 ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the roles of cyclin D1/CDK4-E2F-1/4 pathway in cell cycle changes of human embryo lung fibroblasts (HELF) induced by two different benzo(a)pyrene [B(a)P] treatment models.

METHODSTwo B(a)P treatment models: HELF were treated by 2 micromol/L B(a)P for 24 hours; HELF were treated by 100 micromol/L B(a)P three times 24 hours each and provide with some characteristics of transformed cells (T-HELF). Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometry and Western bolt analysis.

RESULTSAfter 24 hours 2 microml/L B(a)P treatment, the HELFs in the G(1) phase was decreased. In HELF transfected with antisense cyclin Dl (A-Dl) and antisense CDK4 (A-K4), the expression of cyclin Dl and CDK4 blocked the cell cycle changes from the G(1) phase to the S phase induced by B(a)P. The overexpression of cyclin Dl and E2F-1 in HELF was induced by B(a)P. The E2F-1 overexpression in A-D1 induced B(a)P was inhibited. The E2F-4 expression was decreased and the CDK4 expression was increased significantly in A-K4 after B(a)P treatment. Most of T-HELF transfected with antisense cyclin Dl (T-A-Dl) and antisense CDK4 (T-A-K4) were retained in G(1) phase. The cyclin Dl expression in T-HELF was increased significantly compared with that in HELF. The E2F-4 expression in T-A-Dl and T-A-K4 was increased significantly compared with that in T-HELF.

CONCLUSIONB(a)P induces the cell cycle changes through cyclin D1/CDK4-E2F-1/4 pathway in HELF treated by 2 micromol/L B(a)P while it induces cell cycle changes through cyclin D1/CDK4-E2F-4 pathway in T-HELF.

Benzo(a)pyrene ; pharmacology ; Blotting, Western ; Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin D1 ; biosynthesis ; Cyclin-Dependent Kinase 4 ; biosynthesis ; Dose-Response Relationship, Drug ; E2F1 Transcription Factor ; biosynthesis ; E2F4 Transcription Factor ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Lung ; cytology ; embryology