Effects Of Aluminum Exposure On The Level Of Breast Cancer Susceptibility Gene 1 And Its Regulatory Factors In Primary Neurons

Objective: Aluminum is an extremely abundant metal element in the earth's crust,and its content is second only to oxygen and silicon.In daily life,aluminum can be transmitted into body through breathing,diet,skin contact,etc.and then accumulate.Brain is the main accumulation site and an important target organ of aluminum.Neurons cannot be renewed after injury due to the characteristics of the neurons which can't proliferate any more,which is the reason why neurons are particularly vulnerable to aluminum toxicity.Excessive exposure to aluminum can cause neuropathological,physiological,and neurochemical changes,resulting in neurobehavioral abnormalities and decreased learning and memory in mammals.Aluminum could strongly hasten oxidation in cells.Studies indicates that aluminum can reduce the activity of antioxidant enzymes such as glutathione peroxidase(GSH-Px),catalase(CAT)and superoxide dismutase(SOD)in brain tissue,leading to reduced scavenging of oxygen free radicals,the imbalance of the assisted oxidation and antioxidant system,and accelerated cellular oxidative damage.Mitochondria are very sensitive to oxidative stress and mitochondrial DNA is also highly susceptible to oxidative damage.Breast cancer susceptibility gene 1(BRCA1)is an important tumor suppressor gene which can reduce intracellular ROS and lessen DNA oxidative damage.BRCA1 might be a new mechanism of regulating oxidative damage.The level of BRCA1 in hippocampus reduced in AD model animals.Aluminum can decrease BRCA1 mRNA and protein levels in normal mammary epithelial cells.Another study found that BRCA1 is mediated by the PKA/CREB signaling pathway which promotes the expression of BRCA1.The PKA inhibitor H89 reduced BRCA1 mRNA levels.Our previous studies confirmed that aluminum exposure can down-regulate the PKA/CREB pathway.For the above researches,we hypothesized that aluminum may eventually cause neurotoxicity by down-regulating the PKA/CREB signaling pathway and thereby inhibiting the expression of BRCA1.Based on the existing work,this study selected in vitro cell experiments via cell biology,biochemistry and molecular biology techniques to observe the effects of aluminum on neuronal general oxidation,DNA oxidative damage,BRCA1 mRNA and protein levels.Intervention substances including antioxidant QUE,pro-oxidant BSO and cAMP signal agonist forskolin and PKA inhibitor H89 were used to observe the changes of the above indicators to explore the possible mechanism of aluminum exposure-induced neuronal damage.Hopefully,our study could provide evidence and a new clue for the early prevention and treatment of aluminum-induced neurotoxicity.Methods: In this study,primary neurons were isolated from newborn rats within 24 h and cultured to observe the changes in neuronal viability after exposure to different doses of aluminum(Aluminum maltolate),reduced glutathione(GSH)contents,nuclear DNA(n DNA)damage,mitochondria 8-OHdG level,mitochondria ATP level,BRCA1 mRNA level,BRCA1 protein content were detected.CCK-8 kit was used to detect the vitality of primary neurons.Micro-reduced glutathione kit was used to detect GSH contents.Comet assay were used to detect n DNA damage.Rat 8-OHdG Elisa kit was used to determine mt DNA damage.ATP Elisa kit was used to detect ATP levels.Real-time PCR method was used to investigate BRCA1 mRNA level.BRCA1 Elisa kit was used to detect BRCA1 protein content.In the meanwhile,anti-oxidant quercetin(QUE),pro-oxidant buthionine sulphoximine(BSO),cAMP signal enhancer Forskolin,PKA inhibitor H89 were used to confirm the possible role of BRCA1 and regulatory factors in primary neuronal damage induced by aluminum.Results: With the dosage of aluminum increased,the neuronal viability decreased and the cell survival rate reduced in the CCK-8 experiment.The GSH level of primary neurons decreased.The n DNA damage increased in the comet assay,and the mitochondrial 8-OHdG content elevated.The ATP level decreased.BRCA1 mRNA levels lowered and the BRCA1 protein contents decreased.After intervention of QUE,the injuries in primary neurons showed different degrees of alleviation,while after treatment of BSO,the injuries deteriorated.BRCA1 mRNA levels and protein levels increased after intervention of FSK,PKA/CREB pathway agonist,whereas these levels decreased after treatment of H89,PKA/CREB pathway inhibitor.Conclusion: 1.Aluminum exposure can cause morphological damages of primary neurons;2.Aluminum exposure can cause DNA oxidative damage,reduce mitochondrial function;3.Aluminum exposure can decrease BRCA1 mRNA and protein levels;4.QUE intervention can alleviate the injuries,while BSO intervention can aggravate the injuries;5.FSK treatment can alleviate the decrease of BRCA1 mRNA and protein levels,while H89 intervention can aggravate the decrease of these levels.