Cyp2a13 In Metabolic Activation Of Afb1 And Caused By The Role Of Beas - 2 B Cell Malignant Transformation

Aflatoxins, the most toxical metabolites of Aspergillus flavus and Aspergillusparasiticus fungi, which occur mostly in tropical regions with high humidity andtemperature, are naturally occurring contaminants of food. Consequently,>5billionpeople in developing countries worldwide are at risk of chronic exposure to aflatoxinsthrough contaminated foods. In1976, the IARC placed naturally occurring mixturesof aflatoxins on the list of human carcinogens, and in2002aflatoxin b1（AFB1） hasbeen concluded as a class1human carcinogen. Since AFB1is a major etiologicalagent of human liver cancer, extensive studies have been focused on dietary exposureto AFB1and AFB1-induced liver cancer. However, human respiratory system is alsoa target for AFB1carcinogenesis. Small particle-like AFB1can be inhaled intohuman respiratory system through breathing during food harvesting, transporting, andhandling. Both epidemiological and laboratory evidence suggests that AFB1plays animportant role in human lung cancer.Metabolic activation is required for AFB1to exert its carcinogenicity andtoxicity, the major carcinogenic and mutagenic metabolites of AFB1areAFB1-8,9-epoxides, which can bind to DNA to form the predominant AFB1-N7-Guaadduct and induce genetic injury. Among human CYPs, CYP1A2and CYP3A4arethe major AFB1-metabolizing enzymes which almost exclusively expressed in humanliver but not in lung. Previous studies revealed that CYP2A13, predominantlyexpressed in human respiratory tract, has an high capacity to activate AFB1to8,9-epoxides. But the types of AFB1metabolites metabolized by CYP2A13and themechanisms of AFB1been actived to human lung carcinogenesis by CYP2A13remain to be clearly demonstrated.Expression of CYP450s in heterologous systems is the chief method to obtainsingle ingredient and functional enzymes for reconstituting metabolic systems in vitro. Insect cells used in baculovirus/sf9system have a similar mode to mammalian cellsfor post-transcription and post-translation modification of products, it can yield highlevel of unmodified, native CYP450proteins. But this system have a noted deficiencyin heme incorporation for CYP450expression, systematical studies should be takento find the favourable conditions for stable and efficient expression of CYP450s.Reconstitute catalytically active systems in vitro by cloning and expression ofhuman CYP450s in heterologous systems is now a well established and widely useddrug metabolism investigating system. This method can be used to performcharacteristic studies of AFB1metabolized by CYP2A13.BEAS-2B cells were isolated from normal human bronchial epithelium obtainedfrom autopsy of non-cancerous individuals, which free of CYP450s for AFB1metabolic and can be used to screen chemical and biological agents for ability toinduce or affect differentiation and/or carcinogenesis.To determine the detail metabolites of AFB1metabolized by CYP2A13andillustrate the mechanisms of AFB1been actived to human lung carcinogenesis byCYP2A13:（1） we investigated the effects of different heme precursors on theexpression of CYP2A13, POR and their co-expression in baculovirus/sf9system;（2）we reconstructed a metabolism system in vitro by AFB1-metabolizing enzymesexpressed in optimal baculovirus/sf9system, then HPLC and HPLC-Q-TOF analysiswas used to detect the epoxide metabolites of AFB1and the characteristic activitiesof CYP2A13;（3） we used a lentiviral system to construct monoclonal BEAS-2B cellswhich can express CYP2A13（B-2A13） stably, then B-2A13cells were exposed toDMEM medium containing0,0.1,1,10nM AFB1for40passages（about17weeks）,then differences of cellular protein levels between differ passages and the transformedB-2A13（T-B-2A13） cells were detected by Western blot. We chosen the proteins ofDNA damage responses and repairs as well as apoptosis pathways to reveal the mechanisms of AFB1malignant transformation after metabolic activatied byCYP2A13.Part Ⅰ Optimization of heme precursors for the expression ofhuman CYP2A13in baculovirus/sf9system5-ALA is the first committed intermediate of the heme biosynthesis pathway invivo, ferric citrate has an iron necessary for the heme biosynthesis pathway, andhemin is an oxidized form [Fe³⁺] of heme, which is defined as ferrous iron [Fe2+]bound to tetrapyrrole, a macrocyclic porphyrin. Previous studies showed functionalCYP450proteins could be obtained by adding these cofactors. The electron transferchain, usually NADPH and POR, plays an important role in catalyticallyreconstituting active CYP450systems for metabolic activation.To obtain functional characterization and highly active CYP2A13proteins inbaculovirus/sf9system, we first constructed and verified recombinantAc-Bacmid-CYPs （POR）; To investigated the effects of different heme precursors onthe expression of CYP2A13, POR and their co-expression, different concentrationsand combinations of5-ALA, Fe³⁺, or hemin were added to the culture medium aftersf9cells were infected with bvCYP2A13or bvPOR; Then we tried co-expression ofCYP2A13and POR by co-infection of bvCYP2A13and bvPOR in the system.Our results revealed that differ concentrations and combinations can effectexpression of CYP2A13and POR largely, while both of CYP2A13and POR wererepresent the highest expression levels or activity with0.2mM5-ALA,0.02mM Fe³⁺,and0.5-1.0μg/ml hemin. The combination of0.2mM5-ALA and0.02mM Fe³⁺significantly improved CYP2A13expression and activity compared with the hemeprecursors alone （P <0.01）. A MOI value of5pfu/cell for bvCYP2A13induced veryhigh CYP2A13expression. When co-infected with different bvPOR MOI values, aviral ratio of5:2was associated with the highest CYP2A13activity （P <0.01）. In conclusion, the combine addition of0.2mM5-ALA and0.02mM Fe³⁺, andco-expression of the virul ratio5:2（bvCYP2A13:bvPOR） may best fit for CYP2A13expression in baculovirus/sf9system, and our results provide an efficient approach forexpressing functionally characterized, highly active, and homogeneous CYP450proteins.Part ⅡStudy on the metabolic activity of CYP2A13in AFB1metabolism in vitroReconstructed models in vitro are widely used to discribe the interrelationshipbetween single CYP450enzyme and specifical substrates, enzyme conditions can beoptimized and manipulated in ways that are impossible in vivo by using these models.Besides the preferences for substrate of enzymes, the types of metabolitesmetabolized by enzymes can be clearly demonstrated in these models. High levels ofmetabolites are available by using reactive CYP450s in recombinant systems, this canmade determination of qualitative and quantitative for metabolites much convenient.Prvious studies tentativly confirmed that AFB1can be metabolized to AFB1-diol andAFM1-diol by CYP2A13, but still some of them have been undetermined. Especiallythe genotoxicity and carcinogenic activity epoxides as well as its isomer exo-8,9-epoxide and endo-8,9-epoxide are unclear. In addition, the metabolic efficiencyand epoxide metabolites of CYP2A13for AFB1need to be evaluated compared withCYP1A2and CYP3A4, the major AFB1-metabolizing enzymes in human liver.To evaluated the characteristic of CYP2A13for AFB1in vitro systems, firstlywe co-expressed POR and major AFB1activation-related enzymes in baculovirus/sf9system under the optimal conditions for expression which obtained in partⅠ;Commercial products of CYP1A2and CYP3A4were used as positive controls toevaluate the reconstructed metabolizing system, then optimal conditions ofderivatization and HPLC analysis were explored for detecting AFB1and its metabolites （such as AFM1）; Finally the metabolic clearances and epoxidesmetabolites of AFB1metabolized by heterologous expression proteins weremeasured.Our results revealed that highly active CYP1A2, CYP3A4, CYP2A6andCYP2A13proteins were successfully expressed in baculovirus/sf9system by usingthe optimal conditions for expression obtained in part Ⅰ; Our reconstructedmetabolizing system can successfully acquire AFB1metabolizing, and our methodsof HPLC analysis obtained a good linearity for AFB1and AFM1. A linearrelationship between time and AFB1remaining was established and our resultsrepresented the highest clearance of5μM AFB1for CYP2A13in60minutes whichachieved to80%; and the exo-8,9-epoxide metabolites of AFB1by CYP2A13wasconfirmed after HPLC-Q-TOF analysis.In conclusion, CYP2A13represented a high clearance of low level AFB1in vitro,while even higher than CYP1A2, which known to be a major metabolizing enzymefor low AFB1concentrations, and its major activity metabolite was AFB1exo-8,9-epoxide.Part III Effects of AFB1in the malignant transformation in theBEAS-2B cells stably expressing CYP2A13Both epidemiological and laboratory evidence suggest that AFB1plays animportant role in human lung cancer following dietary and inhalational exposure.AFB1exo-8,9-epoxide plays an important role in AFB1toxicity for its ability tobinding proteins and causing acute toxicity （aflatoxicosis） or binding DNA to form acovalent adduct with guanine yielding AFB1-N7-guanine （the major AFB1-DNAadduct in the liver）, these adducts may then cause DNA strand break, DNA basedamage, and oxidative damage, these lesions may lead to mutation of oncogenes andanti-oncogenes, which may ultimately lead to cancer. In human liver, oncogenic activation of Ras and inactive of P53can promote cell poliferation and differentiationwhich lead to HCC. However, the mechanisms of AFB1tumorigenesis to respiratorysystem are still unclear.To get close to the tumorigenesis mechanisms of AFB1to lung, we first purifycell lines by monoclonal cell chosing to get monoclonal BEAS-2B cells whichexpress CYP2A13（B-2A13） stably; Cell viabilities and sensibility to AFB1weredetermined by MTT assay, then an low concentration of AFB1without overt toxicitywas used to conduct cell malignant transformation; Cell capacity of soft-agar coloneformation and tumorigenicity in nude mice were adopted to confirm the neoplastictransformation; Finally differences of cellular protein levels between differ passagesand the transformed B-2A13（T-B-2A13） cells were detected by Western blot. Wechoose the proteins of DNA damage responses and repairs as well as apoptosispathways.Our results revealed that CYP450protein levels in each cell line were close aftermonoclonal cell chosing; MTT assay showed that the toxicity of AFB1for differentcell lines were differ much, while B-2A13displayed the most sensibility and toxicityto AFB1. B-2A13cells were transformed by chronic exposure to low levels of AFB1for40passages （about17weeks）. Immunofluorescence analysis found thatAFB1-DNA adducts were formed in the nuclei of B-2A13; Finally differences ofcellular protein levels between differ passages and the transformedB-2A13（T-B-2A13） cells were evaluated by Western blot. Our results revealed thatproteins of DNA damage responses were actived at early period of AFB1treatment,and with exposue time increasing, protein levels of homologous recombination andnonhomologous end-joining repair pathways were up-expression. And proteins ofpro-apoptotic members of Bcl-2family were induced at early period while aftertransformation were inhibited and down expression. Besides pro-oncogenes of Ras， Raf，ERK and so on were actived during the transformation.In conclusion, AFB1-DNA adducts can be formed in the nuclei of B-2A13whenexposed to low levels of AFB1, these adducts can lead to double strand break byaccumulation, which then active and recuit proteins in homologous recombinationand nonhomologous end-joining pathways for repairation; When damages can not berepaired correctly in early period, pro-apoptotic proteins were activated and leading toapoptosis; But obviously, activation of pro-oncogenes can improve the ability ofproliferation and anti-apoptosis, at the same time, activation of Raf/MEK/ERK andPI3K/Akt pathways may promote the malignant transformation by improve the abilityof proliferation and anti-apoptosis.In summary, our results demonstrated the key role of CYP2A13in metabolizelow levels of AFB1, and determined the mechanisms for AFB1induced malignanttransformation of B-2A13cells in cellular and protein levels. This study provided areliable research model for further identify key toxic metabolites of AFB1aftermetabolized by CYP2A13and will help to illustrate the mechanism of AFB1-inducedtumorigenesis in respiratory system. It is still useful for chemoprevention ofAFB1-induced lung cancer by finding specific inhibitor of CYP450s.