[Study of combined effects of DES and EV on the proliferation of MCF-7 cells by two experimental designs].

The single toxicity of diethylstilbestrol (DES) and beta-estradiol 17-valerate (EV) and the joint toxicity of their binary mixtures in equiconcentration to the proliferation of MCF-7 cells were investigated, respectively. Additive index (AI) method was adopted to evaluate the joint toxicity effect. At the same time, 3 x 3 factorial experimental design was used to verify the joint toxiciy types derived from equiconcentration of DES and EV. The results show that the EC50 values of single EV and DES for 24, 48 and 72 h are 6.02, 0.40 and 0.33 nmol x L(-1) and 5.90, 6.98 and 2.90 nmol x L(-1), respectively. The EC50 values of the binary mixtures of DES and EV for 24, 48 and 72 h are 2.33, 0.71 and 0.39 nmol x L(-1). The binary joint effects of DES and EV for 24 h were synergistic, and the joint effects of DES and EV for 48 and 72 h were antagonistic. But synergistic and antagonistic effects are not strong; their values can be found close to the values of additive effects. Factorial experiment results show that combined effects of DES and EV to proliferation of MCF-7 cells for 24, 48 and 72 h three exposure periods are additive effect types. The consistent joint combined effect types can be drawn from both factorial experimental design and equiconcentration ratio of DES and EV to the proliferation of MCF-7 cells. However, the factorial experimental design is simpler and more convenient, and can avoid unnecessary mistakes due to the derivation of EC50 values.

Formation of fused-ring 2'-deoxycytidine adducts from 1-chloro-3-buten-2-one, an in vitro 1,3-butadiene metabolite, under in vitro physiological conditions.

1-Chloro-3-buten-2-one (CBO) is a potential metabolite of 1,3-butadiene (BD), a carcinogenic air pollutant. CBO is a bifunctional alkylating agent that readily reacts with glutathione (GSH) to form mono-GSH and di-GSH adducts. Recently, CBO and its precursor 1-chloro-2-hydroxy-3-butene (CHB) were found to be cytotoxic and genotoxic in human liver cells in culture with CBO being approximately 100-fold more potent than CHB. In the present study, CBO was shown to react readily with 2'-deoxycytidine (dC) under in vitro physiological conditions (pH 7.4, 37 °C) to form four dC adducts with the CBO moieties forming fused rings with the N3 and N(4) atoms of dC. The four products were structurally characterized as 2-hydroxy-2-hydroxymethyl-7-(2-deoxy-ß-d-erythro-pentofuranosyl)-1,2,3,4-tetrahydro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-1 and dC-2, a pair of diastereomers), 4-chloromethyl-4-hydroxy-7-(2-deoxy-ß-d-erythro-pentofuranosyl)-1,2,3,4-tetrahydro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-3), and 2-chloromethyl-2-hydroxy-7-(2-deoxy-ß-d-erythro-pentofuranosyl)-1,2,3,4-tetrahydro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-4). Interestingly, dC-1 and dC-2 were stable under our experimental conditions (pH 7.4, 37 °C, and 6 h) and existed in equilibrium as indicated by HPLC analysis, whereas dC-3 and dC-4 were labile with the half-lives being 3.0 ± 0.36 and 1.7 ± 0.06 h, respectively. Decomposition of dC-4 produced both dC-1 and dC-2, whereas acid hydrolysis of dC-1/dC-2 and dC-4 in 1 M HCl at 100 °C for 30 min yielded the deribosylated adducts dC-1H/dC-2H and dC-4H, respectively. Because fused-ring dC adducts of other chemicals are mutagenic, the characterized CBO-dC adducts could be mutagenic and play a role in the cytotoxicity and genotoxicity of CBO and its precursors, CHB and BD. The CBO-dC adducts may also be used as standards to characterize CBO-DNA adducts and to develop potential biomarkers for CBO formation in vivo.

[Effect of deltamethrin on production of reactive oxygen species in PC12 cells].

OBJECTIVE: To investigate the effect of deltamethrin (DM) on production of reactive oxygen species (ROS) of rat pheochromocytoma (PC12) cells and its mechanism. METHODS: PC12 cells were treated with various dose of DM (0, 10 or 100 micromol/L) for 1, 6 or 12 h respectively. Furthermore, PC12 cells were treated with various dose of DM (0, 10 or 100 micromol/L) for 24 or 48 h, respectively. PC12 cells were pre-incubated with 10 mmol/L N-acetyl-L-cysteine (NAC) for 2 h, or with 500 micromol/L DL-Buthionine-[S, R]-Sulfoximine (BSO) for 16 h, or with 40 micromol/L tertiary butylhydroquinone (tBHQ) for 16 h, prior to exposure to DM and then with 10 micromol/L DM for 6 h. After treatment, ROS production in PC12 cells were measured by a molecular probe, 2', 7'-dichlorofluorescein diacetate (DCFH-DA). RESULTS: DM induced a dose-time dependent increase in ROS production (indicated by DCF fluorescence intensity). 10 micromol/L DM treatment for 6 h enhanced DCF fluorescence intensity that reached approximately 2.24 times of values of control group. Furthermore, a pretreatment with NAC, BSO or tBHQ significantly reduced the DM-enhanced DCF fluorescence intensity that reached approximately 22%, 62% or 38% of values of DM treatment, respectively (P < 0.05), indicating that all these pretreatments attenuate ROS production. CONCLUSION: The in vitro studies demonstrate that DM could enhance ROS production, and may be the influential factor for the decreased mercapto level and antioxidative function.

NF-E2 related factor 2 activation and heme oxygenase-1 induction by tert-butylhydroquinone protect against deltamethrin-mediated oxidative stress in PC12 cells.

Recent findings suggest that oxidative stress caused by pyrethroid pesticides could be closely involved in the neurotoxicity. tert-Butylhydroquinone ( tBHQ) is a known inducer of Nrf2-mediated transcription, and treatment of cells with tBHQ can confer protection against H 2O 2 and 6-hydroxydopamine (6-OHDA). In this study, we investigated the neuroprotective effect of tBHQ against deltamethrin (DM)-induced oxidative stress using rat PC12 adrenal pheochromocytoma cells. The pretreatment of PC12 cells with tBHQ significantly reduced DM-induced generation of reactive oxygen species (ROS) and increased intracellular ionized calcium ([Ca (2+)] i). We also observed that DM or tBHQ induced the expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), a Nrf2-regulated gene. In addition, the Nrf2 antioxidant responsive element (ARE) pathways activated by tBHQ caused a partial inhibition of the DM-induced Nrf2 and HO-1 expression. Altogether, our data clearly indicate that an activation of Nrf2/ARE pathways in PC12 cells by tBHQ treatment protects cells from DM-induced oxidative stress and regulates DM- mediated adaptive responses in PC12 cells via translocation of Nrf2.

[Protective effect of melatonin on oxidative damage by deltamethrin in rat brain].

OBJECTIVE: To study the potential protective effect of melatonin on the oxidative damage induced by deltamethrin in cerebral cortex, hippocampus and cerebellum of rats. METHODS: 35 male wistar rats were randomly divided into five groups(seven rats per group): olive oil control, deltamethrin-treated (12.5 mg/kg), melatonin(25.0 mg/kg) and deltamethrin plus melatonin (25.0 mg/kg , 2.5 mg/kg respectively) group. Levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione (GSH) in cerebral cortex, hippocampus and cerebellum were determined after 5 days of DM treatments. RESULTS: MDA content in cerebral cortex, hippocampus and cerebellum tissue of the DM-treated rats were significantly higher than those in control group, and compared with DM-treated group, MDA content in those tissue of MT + DM-treated group have significantly decreased after 5 days of DM exposure (P < 0.05). Activities of GSH-Px in DM-treated group were significantly lower than those in control group, and those in the MT + DM group were significantly higher than DM group(P < 0.05). CONCLUSION: DM can induce the oxidative damage in rat brain and melatonin has protective effects on deltamethrin-induced oxidative damage in hippocampus, cerebral cortex and cerebellum of rats.

[Time course of gene expression of gamma-glutamylcysteine synthetase subunit and Nrf2 in brain tissues of rats exposed by deltamethrin].

OBJECTIVE: To investigate the time course of mRNA expression of gamma- glutamylcysteine synthetase light (GCSl) and heavy subunit (GCSh), as well as protein expression of both GCSh and NF-E2 related factor 2 (Nrf2) in cerebral cortex and hippocampus in rats exposed at single dose of deltamethrin (DM). METHODS: Wistar male rats were exposed at single dose of DM (12.50 mg/kg bw) with i.p. At various time points of post-exposure, the rats were sacrificed at indicated time, then their cerebral cortex and hippocampus were isolated. The relative amount of mRNA expression of these genes was measured by the method of reverse transcription polymerase chain reaction (RT-PCR). The protein level was detected by the method of immunohistochemistry and image analysis system. RESULTS: (1) At 5 and 48h after DM exposure, GCSh mRNA relative levels in cerebral cortex in rats were significantly decreased to 83.9% and 86.0% of mRNA level of corresponding tissue of control group at Oh, respectively (P < 0.05). At 5, 24 and 48h after DM exposure, transcriptional factors Nrf2 mRNA relative levels were more higher than those of both control and 72 h group and increased to 146.4%, 145.2% and 147.9% of those of control group at Oh,respectively (P < 0.05). (2) At 5 and 48h after DM exposure, GCSh mRNA relative levels in hippocampus were significantly increased to 118.4% and 118.4% of those of control group at 0h, respectively (P < 0.05). At 5h after DM exposure, GCSl mRNA relative levels in hippocampus were more higher than those of control,24h and 48h group and increased to 121.4% of those of control group at Oh (P < 0.05). CONCLUSION: After single dose of DM exposure, there are up-regulation of mRNA expression of both GCSh and GCSl gene in rats hippocampus, down-regulation of mRNA expression of GCSh gene and up-regulation of mRNA expression of Nrf2. gene in rats cerebral cortex under the experimental condition.

[Effects of deltamethrin on gene expression of some antioxidase, gamma glutamylcysteine synthetase and NFE2 related factor 2 (Nrf2) in brain tissue].

OBJECTIVE: To study the effects of deltamethrin (DM) on the mRNA expression of copper-zinc dependent SOD (CuZn-SOD), glutathione reductase (GR) and gamma glutamylcysteine synthetase (gamma-GCS) light subunit (GCSl), as well as on expression of both mRNA and protein of gamma-GCS heavy subunit (GCSh) and NFE2 related factor 2 (Nrf2) in cerebral cortex and hippocampus of rats. METHODS: Eighteen Wistar male rats were randomizedly divided into three groups, six for each group. The low dosage and high dosage DM treated groups were administrated intraperitoneally with DM (the daily dosage was 3.125, 12.500 mg/kg BWT respectively) for five consecutive days while the control group was administered intraperitoneally with olive oil. The relative amount of mRNA expression of these genes was measured by the method of reverse transcription polymerase chain reaction (RT-PCR) (n = 6). The protein level was detected by the method of immunohistochemistry and image analysis system (n = 4). RESULTS: There was no change in mRNA expression level of CuZn-SOD, GR, GCSh and Nrf2 gene in both cerebral cortex and hippocampus tissue in rats administrated with DM. However, the mRNA level of GCSl gene in cerebral cortex of high dosage group as well as in both cerebral cortex and hippocampus of the low dosage group was significantly lower than that in corresponding tissue in the control group, and was decreased to 71.1%, 63.6% and 75.2% of mRNA level of corresponding tissue in the control group (P < 0.01). There was no obvious effect on protein level of both GCSh and Nrf2 in CA1, CA2, CA3 and dentate gyrus (DG) of hippocampus as well as on that in cerebral cortex in rats treated with DM. CONCLUSION: Under the experimental conditions, there is no obvious effect in the mRNA expression level of CuZn-SOD, GR gene, as well as on expression of both mRNA and protein of Nrf2 gene in both cerebral cortex and hippocampus tissue in rats administered with DM. DM depresses the mRNA expression of GCSl gene, but does not affect the mRNA expression of GCSh gene.

[Effects of deltamethrin on the apoptosis and the expression of caspase-3 in rat neural cells].

OBJECTIVE: To study the effect of deltamethrin on the apoptotic rate and the expression of caspase-3 in rat neural cells. METHODS: Male Wistar rats were randomly divided into 5 groups: control, 5 h, 24 h, 48 h and 5 d exposed groups. Apoptotic rate and the expression of caspase-3 were measured by FACS420 Flow Cytometer; Ac-DEVD-pNa was used as a substrate to detect the activity of caspase-3. RESULTS: Apoptotic rates in 24 h, 48 h and 5 d exposed groups in hippocampus and cerebral cortex [hippocampus: (8.45 +/- 1.02)%, (9.44 +/- 1.14)%, (7.58 +/- 0.75)%; cerebral cortex: (7.90 +/- 0.49)%, (8.01 +/- 0.87)%, (7.97 +/- 0.41)% respectively] were higher than those in the control [hippocampus: (2.97 +/- 0.36)%; cerebral cortex: (3.50 +/- 0.48)%] (P < 0.01); the activity of caspase-3 in 5 h, 24 h and 48 h exposed groups (A(405) nm in hippocampus: 0.389 +/- 0.038, 0.472 +/- 0.041, 0.295 +/- 0.049; A(405) nm in cerebral cortex: 0.321 +/- 0.068, 0.429 +/- 0.077, 0.344 +/- 0.047) and 5 d group of hippocampus (0.246 +/- 0.065) were all higher than those of the control (hippocampus: 0.184 +/- 0.054; cerebral cortex: 0.198 +/- 0.049) (P < 0.05, P < 0.01); the expression of caspase-3 in 5 h, 24 h and 48 h exposed groups increased apparently while 5 d group did not. CONCLUSION: Exposure to high dose of deltamethrin would affect the apoptosis, the activity and expression of caspase-3 in rat neural cells. The increase in caspase-3 activity and expression occurred before the rising of neuronal apoptotic rate may be the upstream event of apoptosis.