The Role Of NF-E2 Related Factor 2 (Nrf2) /Antioxidative Response Elements (ARE) Pathway In Deltamethrin Neurotoxicity And Its Mechanism

Pyrethroids (Py) are artificial synthetic pesticides based on the chemical structure of natural pyrethrins. Due to their properties of broad spectrum, highly insecticide and low residual, Py has been widely used in agriculture, forestry, home pest control, protection of stored foodstuffs etc. Py is a neurotoxic agent. But the exact mechanism underlying for neurotoxicity is not clear.Some results of studies showed the involvement of reactive oxygen species (ROS) in Py toxicity as it increased lipid peroxidation (LPO) and altered the antioxidant system. Nrf2, a member of the CNC family of basic region-leucine zipper transcription factors, has been shown to bind to anlioxidant-responsive element (ARE). ARE has been found in the promoter region of several detoxifying and antioxidant stress genes such as γ-glutamylcysteine synthetase (γ-GCS) and hemeoxygenase 1 (HO-1). NF-E2 related factor 2 (Nrf2) has been identified as an antioxidant response element (ARE)-mediated positive regulator of detoxifying enzyme genes for protecting cells against electrophile toxicity, oxidative stress, and carcinogenicity. Recently, there are several lines of evidences that Nrf2 plays an important role in protecting neurons from toxic insult.Molecular mechanisms of DM-induced oxidative injure are not established. These data raise the question as to whether oxidative stress involves in neurotoxicity induced by DM, and disruptions in Nrf2-mediated gene expression also occurs in neurotoxicity induced by DM. Thus, the aims of this study were to determine whether the DM poisoning was associated with oxidative stress and disruption in Nrf2/ARE pathway and to explore their possible mechanisms, to test the hypothesis that if Nrf2/ARE pathway protects neuronal cells from DM-induced oxidative impairment. The present study will provide new insights to molecular mechanisms of oxidative neurotoxicity induced DM. Part I Deltamethrin induced oxidative stress in nerve tissue or neuronal cells and its mechanisms.Section I Effect of deltamethrin on lipid peroxidation and antioxidant system in nerve tissue or neuronal cells.Objective To explore the lipid peroxidation induced by deltamethrin (DM) in rats and PC12 cells , and the effect of DM on the antioxidant enzyme, content of glutathione in the cerebral cortex and hippocampus of rat administrated for short term in order to provide the scientific support for the study on DM toxic mechanism for oxidative stress.Methods Wistar male rats were randomly assigned to three groups (n=6) and administrated with i.p.(daily dose is respectively 0,3.13,12.50 mg/Kg BW) for one day or five day. The content of Malondialdehyde(MDA) and the activity of total-superoxide dismutase(SOD)(a manganese containing SOD(Mn-SOD) and a copper-zinc dependent SOD(CuZn-SOD) ),Catalase(CAT), Glutathione-S-transferase(GST), Glutathione peroxidase(GP_X) and Glutathione reductase(GR) in the cytosolic supernatant of cerebral cortex and hippocampus tissue were determined. The cytosolic fraction of cerebral cortex and hippocampus tissue was used to determine reduced glutathione (GSH) content and gamma-glutamylcysteine synthetase (γ-GCS) activity by reversed-phase high performance liquid chromatographic assay with o-phthalaldehyde pre-column derivatization. The relative amount of mRNA expression of CuZn-SOD and GR genes was measured by the method of reverse transcription polymerase chain reaction (RT-PCR)Results (1) In rats administrated for one day, there was no effect on MDA content in both cerebral cortex and hippocampus tissue. However, in rats administrated for 5 day, MDA content in cerebral cortex of the high dose group was significantly higher than that in the low dose group, and MDA content in hippocampus tissue of the high dose group was significantly higher than that in both the control and the low dose group. (2) There was no effect on the activity of T-SOD and CAT in both cerebral cortex and hippocampus tissue of rats administrated for one day and in hippocampus tissue of rat administrated for 5 day. However, in rats administrated for 5 day, the activity of T-SOD and CuZn-SOD in cerebral cortex of both the high and the low dose group was significantly lower than that in the control group, and there was no effect on CAT activity in cerebral cortex. (3) In rats administrated for 5 day, GSH content in cerebral cortex of the high dose group was significantly higher than that in the control group (15.13±4.49 vs 7.61±3.27 nmol/mg pro,P<0.05) , and that in hippocampus tissue of high dose was significantly lower than that in both the control and low dose group(5.38±1.78 vs 10.29±3.65,10.32±2.70 nmol/mg pro, P<0.05). In rats administrated for 5 day, there was no effect on γ-GCS activity in cerebral cortex, and the GR activity of low dose group in cerebral cortex was significantly lower than that in both the control and the high group(11.80±5.15 vs 18.98±3.68,17.35±2.47 U/mg pro, P<0.01). γ-GCS activity in hippocampus tissue of the high dose group was significantly lower than that in both the control and the low dose group (1.75±0.60 vs 3.17±0.79, 2.72±0.75 nmol.mg pro^-1 .min^-1,P<0.01 ) . GR activity in hippocampus tissue of both high and low dose group was significantly lower than that in the control group (21.63±4.92, 21.46±8.89 vs 31.22±6.97 U/mg pro, P<0.05). There was no effect on the activity of GST and GP_X in both cerebral cortex and hippocampus tissue of rats administrated for 1 or 5 day. (4) There was no change on mRNA expression level of CuZn-SOD and GR gene in both cerebral cortex and hippocampus tissue in rats administrated with DM.Conclusion The oxidative stress in nerve tissue, which can be resulted from effect of DM on the activity of SOD, γ-GCS and GR and GSH content, is one of mechanisms of nerve toxicity induced by DM; The decreased activity of γ-GCS and GR could be the primary mechanism that GSH content in hippocampus tissue was decreased by DM. The mechanism by which DM influences activities of both GR and CuZn-SOD is post-transcriptional mechanism.Section II Deltamethrin-induced reactive oxygen species in PC12 cells and rats:role of NAC or BSO or tBHQ.Objective To investigate whether in vitro or in vivo exposure to DM produce reactive oxygen species (ROS), and to explore the effect of pretreatment PC12 cells with an electrophilic antioxidant inducer-tertiary butylhydroquinone (tBHQ), or a well-known bioprecursor of GSH-N-acetyl-L-cysteine (NAC), or a well-known special irreversible blocker of GCS-DL-Buthionine-[S,R]-Sulfoximine (BSO), on the increased ROS induced by DM.Methods Rat pheochromocytoma (PC12) cells were treated with various dose of DM(0, 10 or 100 μ mol/L) for various time (1, 6 or12h) respectively. Furthermore,PC12 cells were treated with various dose of DM(0, 10 or 100 μ mol/L) for 24 or 48h, respectively. PC12 cells were pre-incubated with 10mmol/L NAC for 2 h, or with 500 μ mol/L BSO for 16h,or with 40 μ mol/L tBHQ for 16h,prior to exposure to DM and then with 10 μ mol/L DM for 6 h .After treatment, ROS production in PC 12 cells were measured by a molecular probe, 2' ,7' -dichlorofiuoresceindiacetate (DCFH-DA) and ROS production in hippocampus of Sprague-Dawley rats were measured by electron spin resonance (ESR).Results The results showed that DM induced a dose- and time-dependent increase in ROS production (indicated by DCF fluorescence intensity) and an increase in ROS production in hippocampus of Sprague-Dawley rats. DM treatment for 6h enhanced DCF fluorescence intensity that reached approximately 224% 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, indicating that all these pretreatments attenuate ROS production. Conclusion The studies of in vitro and in vivo demonstrate that oxidative stress, evidenced by enhanced ROS production, is a mechanism involved in DM neurotoxicity. Moreover, NAC, BSO or tBHQ is effective in preventing DM-induced oxidative stress.Part II Effect of DM on the Nrf2/ARE pathway in nerve tissue or neuronal cell and its mechanismObjective To determine the effect of DM on Nrf2 activation and expression of Nrf2 targeted gene such as HO-1, GCS in nervous tissue in vivo or in neuronal cells in vitro, as well as to explore the effect of pre-treatment with NAC or BSO or tBHQ on the changes in Nrf2/ARE pathway induced by DM.Methods Wistarmale rats were administrated (daily dose is respectively 0,3.13,12.50mg/Kg BWT, i.e. control group, low and high dose group, respectively) with i.p. for consecutive five days. Wistar male rats were exposed to single dose of DM (12.50mg/Kg BWT) 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. Rat primary astrocytes were treated with DM (10μmol/L), tBHQ (40μmol/L), or both for 1 or 6h, respectively. PC 12 cells were pre-incubated with NAC for 2 h, or with BSO for 16h,or with tBHQ for 16h,prior to exposure to DM and then with DM for 1 h . 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 or western blot. Nrf2 subcellular distribution and translocation was detected by immunocytochemistry combined with laser scan confocal microscopy (LSCM) or western blot. Reduced glutathione (GSH) content and gamma-glutamylcysteine synthetase (γ-GCS) activity in cells, which were pre-incubated with NAC for 2 h, or with BSO for 16h,or with tBHQ for 16h,prior to exposure to DM and then with DM for 6 h or 12h, respectively, were determined by reversed-phase high performance liquid chromatographic assay with o-phthalaldehyde pre-column derivatization.Results (1) Effect of deltamethrin on gene expression of gamma- glutamylcysteine synthetase and Nrf2 in brain tissueThere was no change on mRNA expression level of GCSh and Nrf2 gene in both cerebral cortex and hippocampus tissue in rats administrated with DM. However, the mRNA level of GCS1 gene in cerebral cortex of high dose group as well as in both cerebral cortex and hippocampus of low dose group was significantly lower than that in corresponding tissue of control group, and decreased to 71 .1%, 63.6% and 75.2% of mRNA level of corresponding tissue of control group (P<0.001). 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 rat treated with DM. DM induced the expression of Nrf2 protein and activcted Nrf2 in bothcortex and hippocampus.(2) Time course of gene expression of gamma- glutamylcysteine synthetase subunit and Nrf2 in brain tissues of rats exposed by deltamethrin.â‘ 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 levels 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 72h 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 Oh, respectively (P< 0.05) . At 5h after DM exposure, GCS1 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 0h (P<0.05) .(3) Deltamethrin activates transcriptional factors Nrf2 but does not increase GCS-HS protein expression in primary astrocytes.Analysis using immunofluorescent confocal microscopy revealed that tBHQ treatment led to a dramatic increase in the protein level of Nrf2. The ratio of cytoplasmic to nuclear Nrf2 protein was markedly reduced in astrocytes by either tBHQ or DM, indicating activation and nuclear translocation of Nrf2 by the treatments. Co-treatment with tBHQ and DM for 1h decreased the protein level of Nrf2, but increased the ratio of cytoplasmic to nuclear Nrf2 protein compared with that of the tBHQ only group, indicating an inhibitory effect of DM on the activation of Nrf2 by tBHQ. The expression of GCS-HS in astrocytes was not altered by DM or tBHQ or both in astrocytes.(4) Effect of DM on the Nrf2/ARE pathway in PC12 cells and its possible mechanisms.â‘ The present study, for the first time, showed that DM enhanced cellular expression of Nrf2 at the transcriptional and ptotein level expression. In addition, DM treatment caused nuclear accumulation of Nrf2 in association with downstream activation of Nrf2 mediated oxidative response genes,such as GCSh and HO-1. tBHQ treatment exhibited the similar effects.â‘¡Furthermore, the tBHQ enhanced expression and activation of Nrf2 were reduced by the treatment of DM. The tBHQ induced expression of GCS-HS was also suppressed by the treatment of DM.â‘¢ NAC treatment could decreased the protein expression of Nrf2,did not change the Nrf2 protein in cytoplasm or nucleus. NAC pretreatment suppressed the enhanced expression and activation of Nrf2 ,as well as the enhanced expression of HO-1 mRNA and protein and GCSh mRNA in PC12 cells induced by DM. Whereas it did not change the expression of Nrf2 mRNA.â‘£ BSO alone could not induce Nrf2 nuclear translocation and its expression,but could induce the expression of both HO-1 protein and GCSh mRNA. BSO pretreatment potentially suppressed the induction of Nrf2 mRNA and protein and the activation of Nrf2 by DM, and suppressed the enhanced mRNA level of HO-1 and GCSh by DM.⑤ 6-OHDA,as control substance in the present study,could induce the Nrf2 expression of both mRNA and protein and activate Nrf2. 6-OHDA could also induce the HO-1 or GCSh expression of both mRNA and protein.(5) Effect of DM, BSO, NAC or tBHQ on the reduced glutathione (GSH) content and gamma-glutamylcysteine synthetase (γ-GCS) activity in cellsγ-GCS activities in PC12 cells were inhibited by the high dose of DM treatment for a long time, BSO,or NAC treatment. BSO decreased the GSH content, whereas NAC and tBHQ increased it.Conclusion After multiple dose of DM exposure, there is down-regulation of the mRNA expression of GCS1 gene in nerve tissue of rats. After single dose of DM exposure, there are up-regulation of mRNA expression of both GCSh and GCS1 gene in hippocampus, down-regulation of mRNA expression of GCSh gene and up-regulation of mRNA expression of Nrf2 gene in rats' cerebral cortex. These findings demonstrate, for the first time, that Nrf2 is activated and induced by DM in vivo and in vitro such as in cultured astrocytes or PC12 cells, and Nrf2 target genes such as HO-1 and GCS were induced by DM. The possible mechanisms for activation of Nrf2 are dependent on ROS, not on GSH depletion, are at least in part due to an increase in Nrf2 gene transcription. It is complicated mechanism for DM effecting HO-1 or GCS-HS gene expression. There are differences in the mechanism of different gene influenced by DM. Nrf2 may contribute to the regulatory expression of these gene induced by DM, as well as the other multiple redox sensitive transcriptional factors may do. Treatment cell with high dose of DM for 12h slightly inhibited the activity of GCS. This indicates that Nrf2 gene may be a novel target for prevention and cure of DM poisoning and provides new insights to the research of prevention and cure for pesticide intoxication.Part III Effect of Nrf2/ARE pathway on neurotoxicity induced by DM: effect of pretreatment PC12 cells with tBHQ on the DM-enhanced Intracellular calciumlevel.Objective To investigate the effect of pretreatment PC12 cells with tBHQ, a inducer of the Nrf2/ARE pathway, on the DM-enhanced Intracellular calcium level (as a index of cellular toxicity), in order to explore the significance of Nrf2/ARE pathway on neurotoxicity induced by DM.Methods PC12 cells were exposed to 40μmol/L tBHQ for 16h following treatment with 100μmol/L DM for 6h. [Ca²⁺], in PC12 cells was detected with the application of fluorescence spectrophotometer using the Ca²⁺ indicator Fura-2/AM.Results DM induced a significant increase in the intracellular calcium. Pretreatment of the neuronal cell line PC12 with the antioxidant enzyme inducer tBHQ reduced the enhanced [Ca^2-] i elicited by DM.Conclusion Pretreatment of the neuronal cell line PC12 with tBHQ, the Nrf2/ARE pathway inducer, attenuated the enhanced [Ca²⁺] i and cellular toxicity induced by DM. These finding suggests that tBHQ may in part confer protection by altering DM-induced the disorder of [Ca²⁺], homeostasis and Nrf2/ARE pathway contribute to neuroprotective effect in DM-induced neurotoxcity.Taken together, these findings demonstrate for the first time that DM induces oxidative stress through stimulating an increase in ROS and damaging the antioxidant system. DM induces an activation of the transcriptional factor NrO, Nrf2 nucleus translocation, as well as the expression of Nrf2 target genes such as HO-1 and GCS gene, which confers cytoprotection against oxidative stress. The inducer of Nrf2/ARE pathway, tBHQ, may in part confer protection by altering DM-induced [Ca²⁺], signals. These findings imply that NrO is a potential target molecular for prevention and cure of pesticide poisoning. The proposed theory concerning the effect of deltamethrin on the Nrf2/ARE pathway in neurotoxicity induced by deltamethrin (DM) is shown in figure 1. Fig.1 The proposed theory concerning the effect of deltamethrin on the Nrf2/ARE pathway in neurotoxicity induced by deltamethrin (DM) in the present research.