| The worldwide human exposure to Aflatoxins (AFs), particularly in developingcountries, remains to be a serious public health concern. Up to now, twenty more AFswere already identified and separated, among them, there were four AFs occurnaturally: Aflatoxin B1(AFB1), Aflatoxin B2(AFB2), Aflatoxin G1(AFG1), andAflatoxin G2(AFG2). AFB1, classified as a Group I carcinogen, is the mostprevalent and carcinogenic product of Aspergillus flavus and Aspergillus parasiticus,and can contaminate a number of agricultural products and foods. Agricultural foodindustry workers are at risk of ingestion, transmucosal absorption, and inhalation ofAFs released during product preparation or processing. Although the liver is clearlythe principal target organ of AFB1, epidemiological and laboratory studies haveshown a positive association between lung cancer occurrence and exposure to AFB1.In China, epidemiological studies and laboratory evidence had already shown thatdietary AFs contamination (mainly AFG1) may be associated with lung cancer.Virtually almost all of AFs carcinogenic effects are attributable to its metabolicactivation to form epoxide. The epoxides then combined with biomacromolecules, such as DNA, RNA and proteins, forming corresponding adducts, causingcytotoxicity, apoptosis, cell cycle change and DNA damage, which may ultimatelylead to malignant transformation and form tumors in vivo. Studies of AFB1biotransformation have focused primarily on cytochrome P450-mediated metabolism.CYP enzymes, particularly CYP1A2and CYP3A4, are critical in the metabolicactivation of AFB1. However, they are mainly expressed in the human liver andrarely expressed in the lungs. CYP2A13has been reported to be mainly expressed inthe human respiratory system and had a significant activity in metabolizing AFB1tovarieties reactive intermediates including epoxides. Since CYP2A13is predominantlyexpressed in the human respiratory tract, and CYP1A2is almost exclusivelyexpressed in human liver, CYP2A13may be related to AFs activation in human lungand causing tumorigenesis. In this study, we used cell and molecular biologicaltechnologies to foucus on the the comparison of AFs-induced cytotoxicity betweenBEAS-2B cells expressing CYP2A13, CYP2A6and CYP1A2, which might behelpful to provide a possible clue for AFs-induced tumors in human respiratorysystem.Part Ⅰ Critical role of cytochrome P4502A13inAFB1-induced acute damage in human bronchialepithelial cells1. ObjectivesIn the present study, verified monoclonal BEAS-2B cells, stably expressingAFB1metabolism CYP450enzymes (B-CYPs), were used as cell models. Weexplored the CYP2A13-mediated low concentration of AFB1-induced cytotoxicity,apoptosis, cell cycle change, DNA damage and relative molecular mechanism inorder to illustrate the capacity of CYP2A13in metabolic activating AFB1, which willprovide a possible clue to the critical role of CYP2A13in airborne AFB1-inducedrespiratory diseases and lung tumors. 2. Methods2.1Effects of AFB1(or AFB2) on the viability of BEAS-2Bcells that express CYP2A13MTT assay was used to determine cell viability. To evaluate the differenceamong CYP450enzymes in AFB1or AFB2metabolic activation, B-CYPs cells weretreated with0to10,000nM AFB1(or0to100,000nM AFB2) for24or48h. Inaddition, to observe the time-response relationship, B-2A13cells were treated with0,1,10,100,500nM AFB1for6,12,24, or48h. To investigate the effects of nicotineand8-MOP on AFB1-induced cytotoxicity, B-2A13cells were co-treated with100nM AFB1and different concentrations of nicotine (1,10, and100μM) or8-MOP(0.01,0.1, and1μM) for24,48, or72h. Additionally, B-2A13cells were pretreatedwith100μM nicotine or1μM8-MOP for2h, rinsed with1×PBS, and incubated in amedium that contained100nM AFB1for an additional24,48, or72h. The cellstreated with100μM nicotine,100nM AFB1, and1μM8-MOP alone served ascontrols.2.2. Effects of AFB1on apoptosis in BEAS-2B cells thatexpress CYP2A13The Hoechst33258assay and flow cytometry assay were used to investigateAFB1-induced cellular apoptosis. Western blot assay were used to determine theexpression of apoptosis-related proteins. To evaluate the difference among CYP450enzymes in AFB1-induced apoptosis, B-CYPs cells were treated with0,5,10,20,40and80nM AFB1for24h. Additional, B-2A13cells were treated with the sameconcentration for12,24or48h in order to observe the time-response relationship.And Hoechst33258assay were used to determine cellular apoptosis. In addition, toconfirm the above experiments, cellular apoptosis was assessed using a flowcytometry assay after the B-CYPs cells were treated with0,5,20, and80nM AFB1for24h. Finally, to determine the expression of apoptosis-related proteins, Westernblot assay was used when the cells were treated with the same concentration (or time) mentioned above. To investigate the effects of nicotine and8-MOP on AFB1-inducedapoptosis, B-2A13cells were treated with80nM AFB1alone or combined with100μM nicotine or1μM8-MOP for24h, and then cellular apoptosis andapoptosis-related proteins were evaluated.2.3. Effects of AFB1on DNA damage in BEAS-2B cells thatexpress CYP2A13The Comet Assay was used to investigate AFB1-induced DNA damage. Westernblot assay was used to determine the expression of DNA damage and repair-relatedproteins. To evaluate the difference among CYP450enzymes in AFB1-induced DNAdamage, B-CYPs cells were treated with0,5,10,20,40and80nM AFB1for24h.To observe the time-response relationship, B-2A13cells were treated with the sameconcentration for6,12or24h. In order to evaluate the effect of DNA damage andrepair, B-2A13cells were treated with40nM AFB1for24h, and then replaced withfresh medium for another6,12or24h. Similarly, to investigate the effects of nicotineand8-MOP on AFB1-induced DNA damage, B-2A13cells were treated with80nMAFB1alone or combined with100μM nicotine or1μM8-MOP for24h. In addition,to determine the expression of DNA damage and repair-related proteins, Western blotassay was used when the cells were treated with the same concentration (or time)mentioned above.2.4. Effects of AFB1on cell cycle in BEAS-2B cells thatexpress CYP2A13Flow cytometry assay was used to investigate AFB1-induced cell cycle change.To evaluate the difference among CYP450enzymes in AFB1-induced cell cyclechange, B-CYPs cells were treated with0and80nM AFB1for24h. To observe theconcentration-response relationship, B-2A13cells were treated with0,5,20and80nM AFB1for24h. In order to investigate the effects of nicotine and8-MOP onAFB1-induced cell cycle change, B-2A13cells were treated with80nM AFB1aloneor combined with100μM nicotine or1μM8-MOP for24h. 2.5. Effect of AFB1on the formation of AFB1-DNA adductsand the expression of8-OHdG and γH2AX protein inBEAS-2B cells that express CYP2A13.Immunofluorescence assay was used to investigate AFB1-induced the formationof AFB1-DNA adducts and the expression of8-OHdG and γH2AX protein. Toevaluate the difference among CYP450enzymes in AFB1-induced the formation ofAFB1-DNA adducts and the express of8-OHdG and γH2AX protein, B-CYPs cellswere treated with0and80nM AFB1for24h. In addition, to investigate the effectsof nicotine and8-MOP on AFB1-induced the formation of AFB1-DNA adducts andthe expression of8-OHdG and γH2AX protein, B-2A13cells were treated with80nM AFB1alone or combined with100μM nicotine or1μM8-MOP for24h.3. Results3.1. Effects of AFB1(or AFB2) on the viability of BEAS-2Bcells that express CYP2A13The results showed that cell viability was decreased in a dose/time-dependentmanner in B-2A13cells. After48h AFB1treatment, the IC50value for B-2A13cellswas140nM, which was one-fifth of the IC50value for B-1A2cells (740nM) andone-twelfth of the IC50value for B-2A6cells (3020nM). B-2A13cells were moresensitive to AFB1than B-1A2cells, whereas B-2A6and B-Vector cells showed notoxicity. Unlike AFB1, AFB2had no cytotoxicity in B-CYPs cells. As predicted, bothnicotine and8-MOP significantly and dose-dependently inhibited AFB1-inducedcytotoxicity in B-2A13cells.100μM nicotine or0.1μM8-MOP could completelyinhibit the AFB1-induced cytotoxicity. These results confirm that CYP2A13plays animportant role in the AFB1-induced cytotoxicity.3.2. Effects of AFB1on apoptosis in BEAS-2B cells thatexpress CYP2A13 Both Hoechst33258assay and flow cytometry assay showed that the apoptoticrate of B-2A13cells dose-dependently increased after24h AFB1treatment, whereasno differences in apoptosis were observed in B-1A2, B-2A6, and B-Vector cells aftertreated with AFB1as compared to vehicle control. The time-dependently increased ofapoptotic rate was found in B-2A13cells, treated with the same concentration for12,24or48h. The Hoechst33258assay and flow cytometry assay showed that both100μM nicotine and1μM8-MOP markedly inhibited AFB1-induced cellular apoptosis.These results confirmed that CYP2A13plays an important role in the AFB1-inducedcellular apoptosis.Based on the analysis in the expression of apoptosis-related proteins, C-PARPand C-Caspase-3expression dose-dependently increased, with a marked differencebetween20nM AFB1(P<0.001). Bcl-2expression dose-dependently decreased, andBax expression dose-dependently increased. The results showed that C-PARP andC-Caspase-3were expressed in a time-dependent manner (P<0.001). Compare to80nM AFB1, both100μM nicotine and1μM8-MOP significantly blocked theAFB1-induced expression of C-PARP and C-Caspase-3(P<0.001or0.05,respectively) and reduced the AFB1-induced inhibition of Bcl-2expression, whichwas consistent with their effects on AFB1-induced cytotoxicity and apoptosis. Theresults demonstrated that apoptosis mediated by CYP2A13metabolic activity afterAFB1treatment is associated with the mitochondrial signaling pathway.3.3. Effects of AFB1on DNA damage in BEAS-2B cells thatexpress CYP2A13Comet assay showed that the DNA damage of B-2A13cells dose-dependentlyincreased after24h AFB1treatment. The DNA damage in B-2A13cells was almostthree times stronger than B-1A2cells, whereas no differences in DNA damage wereobserved in B-2A6and B-Vector cells after treated with AFB1as compared to vehiclecontrol. After treated with the same concentration for6,12or24h, atime-dependently increased of DNA damage was found in B-2A13cells. Compared to20nM AFB1for24h, the DNA damage continued to exist in a time-dependent increased manner (P<0.001). Additionally, both100μM nicotine and1μM8-MOPmarkedly inhibited AFB1-induced DNA damage (P<0.001). These results confirmthat CYP2A13plays an important role in the AFB1-induced DNA damage.Western blot assay showed that there was a dose-dependently increased in theprotein lever of p-BRCA1, p-ATM, p-Chk1, p-Chk2, p-p95, p-p53and γH2AX. Andthere was almost no change in the expression of p-ATR, Mre11, Ku-80, XLF, Rad50and Rad52. Compared to20nM AFB1for24h, there was a time-dependentlyincreased in the protein lever of DNA-PK, p-BRCA1, p-ATM, p-Chk1, p-Chk2,p-p95, p-p53and γH2AX, and the expression of Mre11, Ku-80, XLF, Rad50andRad52remained essentially unchanged. The protein lever of CYP2A13and GFPremained unchanged, suggesting that the cells had a good stability. In addition,compare to80nM AFB1, both100μM nicotine and1μM8-MOP significantlyblocked the AFB1-induced expression of p-BRCA1, p-ATM, p-Chk1, p-Chk2, p-p95,p-p53and γH2AX, which was consistent with their effects on AFB1-induced DNAdamage. The results demonstrated that DNA damage mediated by CYP2A13metabolic activity after AFB1treatment is associated with the expression of DNAdamage and repair-relatived proteins.3.4. Effects of AFB1on cell cycle in BEAS-2B cells thatexpress CYP2A13Flow cytometry assay showed that the S arrest in B-2A13and B-1A2cells after80nM AFB1treatment (P<0.001). Compared to B-1A2cells, the S arrest in B-2A13was much stronger and with a dose-dependently increased after24h AFB1treatment(P<0.001). In addition, compare to80nM AFB1, both100μM nicotine and1μM8-MOP significantly blocked the AFB1-induced cell cycle change (P<0.001). Theresults showed that there was mainly the S arrest in cell cycle changed after AFB1treatment.3.5. Effect of AFB1on the formation of AFB1-DNA adductsand the expression of8-OHdG and γH2AX protein inBEAS-2B cells that express CYP2A13. Compared to DMSO, there was apparently AFB1-DNA adducts in the nuclei ofB-2A13and B-1A2cells (P<0.001). And compared to B-1A2cells, the relativefluorescence intensity was probably nine times more in B-2A13cells (311vs36). Aspredicted, no significant AFB1-DNA adducts were found in either B-2A6or B-Vectorcells. Similarly, there was apparently8-OHdG in the nuclei of B-2A13and B-1A2cells (P<0.001), and compared to B-1A2cells, the relative fluorescence intensity wasalmost three times more in B-2A13cells (160vs55), no significant8-OHdG wasfound in either B-2A6or B-Vector cells. In addition, there was significant γH2AX inB-2A13cells (P<0.001), but neither in B-1A2, B-2A6or B-Vector cells. Additional,compare to80nM AFB1, both100μM nicotine and1μM8-MOP significantlyinhibited the AFB1-induced AFB1-DNA adducts,8-OHdG and γH2AX proteinexpression in B-2A13cells (P<0.001). The results showed that CYP2A13canmetabolism AFB1to AFB1-exo-8,9-epoxide, the epoxides then combined with DNAto form DNA adducts. Furthermore, AFB1can induce oxidative damage to form8-OHdG and cause DNA double strand breaks (DSBs).4. Conclusions4.1. AFB1was efficiently stronger than AFB2in cytotoxicity. The acute damage inB-2A13cells was more sensitive than B-1A2cells, even far stronger than B-2A6and B-Vector cells. The results confirmed that CYP2A13was more sensitive toAFB1and played an important role in the metabolism of AFB1.4.2. The AFB1-induced apoptosis may be associated with the mitochondria signalpathways, and the DNA damage and repair signal may be involved in theAFB1-induced DNA damage.4.3. The results preliminary confirmed that AFB1can induce B-2A13cells to formAFB1-DNA adducts, and express the protein of8-OHdG and γH2AX, which maycause oxidative damage and DSBs, inducing cytotoxity, apoptosis, DNA damageand cell cycle change in molecular mechanism. Part Ⅱ Critical role of cytochrome P4502A13inAFG1-induced acute damage in human bronchialepithelial cells1. ObjectivesBy comparing AFG1-induced acute damage and the related molecularmechanism in the B-CYPs cells, the study aim to investigate the capacity ofCYP2A13in metabolic activating AFG1. We explored the CYP2A13-mediated lowconcentration of AFG1-induced cytotoxicity, apoptosis, cell cycle change and DNAdamage in order to illustrate the capacity of CYP2A13in metabolic activating AFB1and AFG1mixed pollution, which will provide a possible clue to the critical role ofCYP2A13in airborne AFB1/AFG1pollution-induced respiratory diseases and lungtumors.2. Methods2.1. Effects of AFG1(or AFG2) on the viability of BEAS-2Bcells that express CYP2A13MTT assay was used to determine cell viability. To evaluate the differenceamong CYP450enzymes in AFG1or AFG2metabolic activation, B-CYPs cells weretreated with0to10,000nM AFG1(or0to100,000nM AFG2) for24or48h. Toinvestigate the effects of nicotine and8-MOP on AFG1-induced cytotoxicity, B-2A13cells were co-treated with100nM AFG1and different concentrations of nicotine (1,10, and100μM) or8-MOP (0.01,0.1, and1μM) for24,48, or72h. Additional,B-2A13cells were pretreated with100μM nicotine or1μM8-MOP for2h, rinsedwith1×PBS, and incubated in a medium that contained100nM AFG1for anadditional24,48, or72h. The cells treated with100μM nicotine,100nM AFG1, and1μM8-MOP alone served as controls. 2.2. Effects of AFG1on apoptosis in BEAS-2B cells thatexpress CYP2A13B-CYPs cells were treated with0,5,20and80nM AFG1,80nM AFB1,10,000nM AFB2and10,000nM AFG2for24h, Hoechst33258assay was used todetermine cellular apoptosis. B-2A13and B-Vector cells were treated with0,5,20and80nM AFG1,80nM AFB1and10,000nM AFG2for24h, flow cytometry assaywas used to determine cellular apoptosis. To investigate the effects of nicotine and8-MOP on AFG1-induced apoptosis, B-2A13cells were treated with80nM AFG1alone or combined with100μM nicotine or1μM8-MOP for24h, Hoechst33258assay and flow cytometry assay were used to determine cellular apoptosis.2.3. Effects of AFG1on DNA damage in BEAS-2B cells thatexpress CYP2A13B-2A13and B-Vector cells were treated with0,5,20and80nM AFG1,80nMAFB1and10,000nM AFG2for24h, DNA damage was determined using cometassay. To investigate the effects of nicotine and8-MOP on AFG1-induced DNAdamage, B-2A13cells were treated with80nM AFG1alone or combined with100μM nicotine or1μM8-MOP for24h, DNAdamage was detected using comet assay.2.4. Effects of AFG1on cell cycle in BEAS-2B cells thatexpress CYP2A13Flow cytometry assay was used to investigate AFG1-induced cell cycle change.To evaluate the difference among CYP450enzymes in AFG1-induced cell cyclechange, B-CYPs cells were treated with0and80nM AFG1for24h. To observe thedose-response relationship, B-2A13cells were treated with0,5,20and80nM AFG1for24h. In order to investigate the effects of nicotine and8-MOP on AFG1-inducedcell cycle change, B-2A13cells were treated with80nM AFG1alone or combinedwith100μM nicotine or1μM8-MOP for24h.2.5. Effect of AFG1on the expression of8-OHdG andγH2AX protein in BEAS-2B cells that express CYP2A13. To investigate the effects of AFG1-induced the expression of8-OHdG andγH2AX protein, B-2A13cells were treated with80nM AFG1alone or combined with100μM nicotine or1μM8-MOP for24h. Immunofluorescence assay was used toinvestigate AFG1-induced the expression of8-OHdG and γH2AX protein.3. Results3.1. Effects of AFG1(or AFG2) on the viability of BEAS-2Bcells that express CYP2A13The results showed that cell viability was decreased in a dose/time-dependentmanner in B-2A13cells, which was similar to AFB1. B-2A13cells were moresensitive to AFG1than B-1A2cells, whereas B-2A6and B-Vector cells showed notoxicity. After48h AFG1treatment, the IC50value for B-2A13cells was140nM,which was one-thirtieth of the IC50value for B-1A2cells (4320nM) andone-forty-fifth of the IC50value for B-2A6cells (6290nM). Unlike AFG1, AFG2hadno cytotoxicity in B-CYPs cells. As predicted, both nicotine and8-MOP significantlyand dose-dependently inhibited AFG1-induced cytotoxicity in B-2A13cells.100μMnicotine or0.1μM8-MOP could completely inhibit the AFG1-induced cytotoxicity.These results confirmed that CYP2A13played an important role in theAFG1-induced cytotoxicity.3.2. Effects of AFG1on apoptosis in BEAS-2B cells thatexpress CYP2A13Both Hoechst33258assay and flow cytometry assay showed that the apoptoticrate of B-2A13cells dose-dependently increased after24h AFG1treatment, whereasno differences in apoptosis were observed in B-1A2, B-2A6, and B-Vector cells aftertreated with AFG1as compared to vehicle control. Compare to80nM AFB1, theapoptotic rate of B-2A13cells was little lower in80nM AFG1, while there was nodifferences in apoptosis in10μM AFB2or10μM AFG2. The Hoechst33258assayand flow cytometry assay showed that both100μM nicotine and1μM8-MOP markedly inhibited AFG1-induced cellular apoptosis (P<0.001). These resultsconfirmed that CYP2A13was more sensitive to AFG1than AFG2, and played animportant role in the AFG1-induced cellular apoptosis.3.3. Effects of AFG1on DNA damage in BEAS-2B cells thatexpress CYP2A13Comet assay showed that the DNA damage of B-2A13cells dose-dependentlyincreased after24h AFG1treatment, whereas no differences in apoptosis wereobserved in B-Vector cells after treated with AFG1as compared to vehicle control.Compare to80nM AFB1, the DNA damage in B-2A13cells was little lower in80nM AFG1, while there was no differences in DNA damage in10μM AFB2or10μMAFG2. In addition, both100μM nicotine and1μM8-MOP markedly inhibitedAFG1-induced DNA damage (P<0.001). These results confirmed that CYP2A13wasmore sensitive to AFG1than AFG2, and played an important role in theAFG1-induced DNA damage.3.4. Effects of AFG1on cell cycle in BEAS-2B cells thatexpress CYP2A13Flow cytometry assay showed that the S arrest in B-2A13after80nM AFG1treatment (P<0.001), and with a dose-dependently increased after24h AFG1treatment. Compare to80nM AFG1, both100μM nicotine and1μM8-MOPsignificantly blocked the AFG1-induced cell cycle change (P<0.001). The resultsshowed that there was mainly the S arrest in cell cycle change after AFG1treatment.3.5. Effect of AFG1on the expression of8-OHdG andγH2AX protein in BEAS-2B cells that express CYP2A13.Compared to DMSO, there was apparently8-OHdG and γH2AX in the nuclei ofB-2A13cells (P<0.001). Compare to80nM AFG1, both100μM nicotine and1μM8-MOP significantly inhibited the AFG1-induced8-OHdG and γH2AX proteinexpression in B-2A13cells (P<0.001). The results showed that CYP2A13canefficiently metabolism AFG1and cause oxidative damage and DSBs. 4. Conclusions4.1. The cytotoxicity in AFG1was similar to AFB1, and stronger than AFG2. Theacute damage in B-2A13cells was more sensitive than B-1A2cells, even farstronger than B-2A6and B-Vector cells. The results firstly confirmed thatCYP2A13can active AFG1and played an important role in the metabolism ofAFG1.4.2. The results preliminary confirmed that AFG1can induce B-2A13cells to expressthe protein of8-OHdG and γH2AX, which may cause oxidative damage andDSBs, induce cytotoxity, apoptosis, DNA damage and cell cycle change inmolecular mechanism. |
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