| The worldwide human exposure to aflatoxin B1 (AFB1), particularly in developing countries, remains to be a serious public health concern. AFB1, classified as a Group I carcinogen, is the most prevalent and carcinogenic product of Aspergillus flavus and Aspergillus parasiticus, and can contamate a number of agricultural products and foods. Although the liver is clearly the principal target organ of AFB1, a lot of epidemiological studies have shown a positive association between human lung cancer occurrence and inhalation exposure to AFB1. Cytochrome P450 (CYP) are the major enzymes for metabolic activation of AFB1 to epoxides, a reactive electrophilic intermediate associated with AFB1's carcinogenicity. Some CYP isforms are capable of bioactivating AFB1, such as CYP1A2, CYP3A4 and CYP2A6, among them, CYP1A2 is known as the major enzyme for AFB1 activation in human liver, but it rare expresses in human lung. However, some recent studies demostrated that CYP2A13 had a significant activity in metabolizing AFB1 to varieties reactive intermediates including epoxides. The epoxides then formate DNA adducts in the body, and subsequently attack the biomacromolecules, causing cytotoxicities, as well as DNA damage, cell apoptosis, and so on. Finally, cell carcinogenesis was happened. Considering CYP2A13 is predomantly expressed in the human respiratory tract, while CYP1A2 is almost exclusively expressed in human liver, suggesting that CYP2A13 may be related to AFB1 activation in human lung and inducing tumorigenesis. In this study, we used cell and molecular biological technologies to foucus on the the comparison of AFB1-induced cytotoxicity between BEAS-2B cells expressing CYP2A13, CYP2A6 and CYP1A2, which might be helpful to provide the clues for AFB1-induced tumors in human respiratory system.ObjectivesBy comparing the differences of AFB1-induced cytotoxicities among the established BEAS-2B cells expressing CYP2A13, CYP1A2 and CYP2A6, the study aims to illustrate the capacity of CYP2A13 in metabolic activating AFB1, which will provide us the clues of CYP2A13 function and its mechanism in AFB1-induced tumors in human respiratory system.MethodsPCR amplification was used to add the restriction site Nheâ… and Xhoâ… to the upstream and downstream of CYP2A13 cDNA in order to insert it in pLJM1 vector via double digestion. Restriction enzyme EcoRâ… was used to digest CYP1A2 cDNA to construct the pLJM1-CUP1A2 plasmid. After verified by PCR and sequencing, the recombinant pLJM1-CYP2A13, pLJM1-CYP2A6, pLJM1-CYP1A2 plasmids and pLJM1 vector plasmid were all transfected into SV40 Immortalized Human Bronchial Epithelial cells (BEAS-2B) via lentivirus transfection system to construct the BEAS-2B cell highly expressing CYP2A13, CYP2A6, CYP1A2 (BEAS-2A13, BEAS-2A6 and BEAS-1A2 cells). The transfected positive cells with green fluorescent screen were sorted using flow cytometry, and verified using Western Blot method. The BEAS-CYP cells were then exposed to 0 nM,10 nM, 100 nM, 1000 nM and 10,000 nM AFB1 for 24 h to determ the cell viability using MTT assay. Also the cell viability was conduced after 10 nM and 100nM AFB1 treatment for 24 h, 48 h and 72 h. Similarly, the cell apoptosis was detected by both Hoechst 33258 assay and flow cytometry after the BEAS-CYP cells were treated with 0 nM, 10 nM and 100 nM of AFB1 for 24 h, or 100 nM AFB1 for 6 h, 12 h, 24 h, 48 h and 72 h.Results1. Recombinant lentivirus plasmids of pLJM1-CYPs were gotten via restriction enzymes and Quick T4 DNA ligase by choosing suitable and correct restriction enzymes, then they were vrified by PCR and sequencing. No extra mutations were detected.2. Lentivirus transfection system was used to transfect the recombinant pLJM1-CYPs lentivirus plasmids and pLJM1 vector plasmid into 293T cells successfully, virus particles were gained and used to infect BEAS-2B cells. The transfected positive cells were selected by flow cytometry sorting successfully.3. Western blot was used to detect the transfected BEAS-2B cells. A single protein band of each protein was detected, the bands of CYP2A13 and CP2A6 were 45 KD, the band of CYP1A2 was 55 KD, and they were the same as the positive controls separately. No bands of pLJM1 high expressing BEAS-2B cells or normal BEAS-2B Cells were detected. The high expressing CYPs cell models were successfully established. 4. Each BEAS-2B cell model was treated with 0 nM , 10 nM, 100 nM, 1000 nM and 10,000 nM for 24 h, and the results showed cell viability of all the cells decreased in dose-dependent manner, after the treatment of 10,000 nM AFB1, all BEAS-CYPs cell viabilities decreased significantly compared with the control cells (P<0.01). From 100 nM AFB1, the viability of BEAS-2A13 was significantly lower than BEAS-1A2 and BEAS-2A6 (P<0.01). The IC50 of BEAS-2A13 was 350 nM, and BEAS-1A2 was 2200 nM, the IC50 of BEAS-2A6 and BEAS-vector was more than 10000 nM. When the transfected BEAS-2B cell were treated with 100 nM AFB1 for 24 h, 48 hand 72 h, the cell viability of all the cells decreased in time-dependent manner, from 72 h treatment, all the BEAS-CYPs cell viabilities decreased significantly (P<0.01). At any treatment time, the viability of BEAS-2A13 was significantly lower than any other cells (P<0.01).These reaults revealed that BEAS-2A13 cells were the most sensitive towards the treatment of AFB1, then BEAS-1A2 cells, BEAS-2A6 cells was not more sensitive.5. Each cell model was induced apoptosis by 10 nM and 100 nM AFB1 for 24 h. The cell apoptosis was detected by Hoechst 33258 assay. The cell apoptosis of all the cell models increased following the concentration of AFB1 increased. At the treatment of 10 nM and 100 nM AFB1, the apoptosis rates of all BEAS-CYPs cell models increased significantly when compared with the control cell model ( P<0.01), and the apoptosis rate of BEAS-2A13 was significantly more than BEAS-1A2 and BEAS-2A6. The results from Flow cytometry assay showed that, the death rates and the late apoptosis rates of the three BEAS-CYPs increased following the concentration of AFB1 increased. At the same concentration of AFB1, the death rate and the late apoptosis rate of BEAS-2A13 was the highest. The results of these two experiments suggested that at the same treatment conditions, AFB1 could induce the more apoptosis of BEAS-2A13, followed by BEAS-1A2 and BEAS-2A6. In addition, each cell model was induced apoptosis by 100 nM AFB1 for 6 h, 12 h, 24 h, 48 h and 72 h. The apoptosis rates of BEAS-2A13 and BEAS-1A2 were increased significantly, but the apoptosis rates of BEAS-2A6 and BEAS-vector didn't change obviously. From 12 h treatment, the apoptosis rate of BEAS-1A2 increased when compared with the control cell model. At any treatment time, the apoptosis rates of BEAS-2A13 were more than any other cell models (P<0.01). There were no significant differences between BEAS-2A6 and BEAS-vector. The results also showed that, the death rates of all BEAS-CYPs increased following the treatment time increased, the death rate of BEAS-2A13 increased fast. As the treatment time increased, the death rate of BEAS-1A2 had a certain increase, while the death rate of BEAS-2A6 didn't change significantlyConclusions1. BEAS-2B cells expressing CYP2A13, CYP2A6, CYP1A2 were established successfully.2. Compared with the CYP1A2 and CYP2A6, CYP2A13 showed higher metabolic activation to AFB1, which should be helpful to provide the evidence for the study on the critical role of CYP2A13 in AFB1 induced respiratory tumours.
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