| Aflatoxin is a strongly carcinogenic mold-produced contaminant ofdietary staples in Asia and Africa. Epidemiological studies suggest a causativerelationship between exposure to aflatoxin and elevated risk for primary lungcancer. The human respiratory tract is a target for aflatoxin carcinogenicitythrough inhalation or ingestion. Our previous studies showed intragastricadministration of Aflatoxin G1(AFG1), one of the most frequently detectedcontaminating mycotoxins in grains and foodstuffs in north China, induceslung adenocarcinoma in NIH mice, and the cancer cells arise from Alveolartype II cells (AT-II). We recently showed that a single intratrachealadministration of AFG1causes chronic inflammatory changes and AT-II cellsdamage in the alveolar septum. Chronic inflammation is now being recognizedas a major driving force in the development of about one-third of all theknown cancers, including lung cancer. Cigarette smoke-producedinflammation and activation of NF-κB pathway is linked to lung cancer inboth genetic and carcinogen-induced models. Long-term use ofanti-inflammatory compounds reduces the incidence of urethane-induced lungcancer. Additionally, chronic inflammation and loss of protective mucus mayincrease intestinal permeability for environmental toxins, which inducemutations in stem cells that give rise to cancer. In inflammatorymicroenvironment, the activated AT-II cells expressing high levels of MHC-IIand COX-2exhibit altered phenotypes, and likely inhibit antitumor immunityby triggering regulatory T cells (Treg). Chronic inflammation often leads tooxidative stress in the form of reactive oxygen species (ROS) formation tocause DNA damage and mutations. Oxidative DNA damage is also animportant mechanism involved in the cytotoxicity and carcinogenic effect of AFB1. Hence, it is tempting to hypothesize that an etiology of AFG1-inducedlung carcinogenesis through the diet may be chronic inflammation, whichinduces oxidative DNA damage on AT-II cells, and causes the phenotypicalterations of AT-II cells associated with an immunosuppression of anti-tumorimmunity.The primary objective of this investigation is to evaluate whether longterm of AFG1exposure through ingestion induces lung chronic inflammationand lung adenocarcinoma in Balb/c mice. Then, we will investigate thephenotypic alterations of AT-II cells in response to AFG1-inducedinflammation in vivo and vitro to evaluate the immune function of AT-II cellsinvolved in AFG1-induced pulmonary tumorigenesis. Furthermore, we will usethe A549cell line as a model of human AT-II cells and treated it with AFG1and TNF-α together to mimic an AFG1-induced inflammatory response in vitro.We will explore the effect of AFG1and TNF-α on oxidative stress and DNAdamage in A549cells. Our results will provide a new mechanism ofAflatoxin-induced lung cancer and will have useful implications in humanhealth risk assessment strategies of contamination of Aflatoxin in food.PartⅠ Long term oral administration of Aflatoxin G1induces chronicalveolar inflammation associated with lung tumorigenesisObjective: The primary objective of this investigation was to evaluatewhether chronic AFG1exposure through ingestion induces lung chronicinflammation, as well as whether the lung chronic inflammation contributes toAFG1-induced lung tumorigenesis.Methods: Balb/c mice were treated by repeated oral administration ofAFG1(three times weekly) for1,3and6months, and inflammatory cellsinfiltration, cytokines production, activation of NF-κB and STAT3, oxidativestress, cell proliferation, angiogenesis as well as COX-2expression weremeasured in mice lung tissues. After a6-month AFG1treatment as that in theabove model, we also observed the mice for another6months (without AFG1treatment). At the end of twelve months we examined the mice forAFG1-induced lung tumorigenesis and how this correlates with AFG1-induced chronic inflammation by measuring NF-κB, STAT3and COX-2in aAFG1-induced lung adenocarcinoma.Results:1AFG1treatment resulted in pathological alterations in lung tissuesDuring the length of our experiments, mice did not show any clinicalsigns of sickness or respiratory distress and there was no mortality or bodyweight loss in AFG1-treated mice. After treatment for1,3and6months,AFG1-treated mice exhibited enlarged lung alveolar septum due toinflammatory infiltrates, including macrophages, mononuclear cells and a fewlymphocytes, as well as alveolar epithelial cells proliferation, as compared tocontrol mice. The histological alterations of alveolar septum were mostprominent and peaked at3and6months after AFG1-treatment.Further immunohistological examination revealed that macrophages andlymphocytes were the dominant infiltrating inflammatory cells and mainlylocalized in alveolar sac area. However, we did not observe signs of airwayinflammation as evidenced by the absence of inflammatory cells infiltrationaround the respiratory and terminal bronchioles and proximal alveolar ducts.These findings indicate that long-term repeated oral administration of AFG1causes chronic inflammation in lung alveolar septum.2AFG1treatment results in increased cytokine and chemokine expression inthe lungSignificant changes of cytokine and chemokine gene expression levelsappeared starting one month (TNF-α), three months (CXCL1, IL-6, CCL-2,and CXCL2) and six months (IL-1β) after AFG1-treatment initiation. Theexpression of TNF-α, CCL-2and CXCL-2peaked at3months, while IL-6expression in AFG1-treated mice remained in high expression levels from3and up to6months post treatment. In addition, we also found that AFG1increased IL-1β expression at6months post treatment. TNF-α is a key factorthat regulates the production of cytokines involved in chronic inflammation.Increased TNF-α expression was found in both alveolar epithelial cells andmacrophages of AFG1-treated mice. In accordance with the TNF-α mRNA expression, increased TNF-α expression at protein levels was detected from1month to6months in lung tissues of AFG1-treated as compared to controls.These findings confirm that long-term oral administration of AFG1induces chronic inflammation in lung tissues evidenced by increased cytokineand chemokine production.3Activation of NF-κB and STAT3pathways involved in AFG1-inducedchronic inflammationThe p65subunit of NF-κB translocated from the cytoplasm to the nucleusof alveolar epithelial and inflammatory cells in AFG1-treated mice.Phosphorylated STAT3was detected in the nucleus of both alveolar epithelialand inflammatory cells. Consistent with the changes of inflammatorycytokines, higher levels of p65and p-STAT3expression were induced in thealveolar epithelium of AFG1-treated mice compared to control group. Theincreased number of NF-κB positive cells in AFG1-treated mice peaked at3and6months post treatment. p-STAT3positive cells appeared at3months andpeaked at6months post AFG1treatment. These results indicate that NF-κBand STAT3pathways are activated in alveolar epithelium in AFG1-inducedchronic inflammatory environment.4Enhanced SOD-2and HO-1expression is associated with AFG1-inducedlung chronic inflammationPrevious studies have shown that enhanced expression of superoxidedismutase (SOD-2) and/or hemoxygenase-1(HO-1) is an established markerfor oxidative stress in vivo. After AFG1treatment, alveolar epithelial cellswere strongly immunostained for SOD-2and HO-1. Increased SOD-2positivecells were detected in alveolar epithelium of AFG1-treated mice at1,3and6months post-treatment. The increased expression of SOD-2at protein level inAFG1-treated mice was also verified by western blot. Additionally, higherlevel of HO-1expression was detected in alveolar epithelium ofAFG1-treatment mice at3and6months post-treatment, compared with that incontrol mice. In combination, these results indicate that AFG1-induced chronicinflammation enhances oxidative stress in alveolar epithelial cells as evidenced by incasing SOD-2and HO-1activity.5AFG1-induced lung chronic inflammation results in enhanced alveolarepithelial cells proliferation and angiogenesisKi67is extensively used as a proliferation marker. Lung tissues of miceexposed to DMSO alone (control group) had only a few Ki67-positive cells,representing the basal proliferation levels. On the other hand, AFG1treatmentresulted in an increase of Ki67-positive cells in alveolar septum starting from3months and up to6months post-treatment. These results further support ourhistologic analysis, showing that alveolar epithelial cells proliferation isenhanced by AFG1-induced chronic inflammation.We examined early signs of uncontrolled cell growth in lung tissues bydetecting the AT-II cell specific markers, SP-C, and airway Clara cells specificmarker, CC-10, expression in the proliferating lung epithelial cells. In normallung tissues, SP-C staining is located in the cytoplasm of alveolar epithelialcells, and CC-10is located in the cytoplasm of airway epithelial cells. Therewere increased numbers of SP-C positive cells in alveolar septum parallel tothe increased Ki-67expression in AFG1-treated mice. CC-10positive cellswere not observed in the proliferated alveolar epithelium of AFG1-treatedmice. These findings indicate that alveolar AT-II cell proliferation is inducedin alveolar septum in AFG1-induced chronic inflammatory microenvironment.In a chronically inflamed microenvironment, vascular endothelial growthfactor (VEGF) mainly acts as an important pro-angiogenic factor. Expressionof the VEGF protein levels was increased in AFG1-treated mice comparedwith that of the control mice at3and6months post-treatment.Immunohistological results showed that both alveolar epithelial cells andmacrophages are activated to express VEGF in AFG1-treated mice. CD34is amarker for hematopoietic progenitor cells and endothelial cells. CD34positivecells were detected frequently in AFG1-treated mice but only sparsely incontrol mice, suggesting that AFG1-induced chronic inflammation increasesmicrovessel density in lung tissues.These results indicate alveolar type II cell proliferation and angiogenesis are enhanced by AFG1-induced lung chronic inflammation.6Increased COX-2expression in alveolar epithelial cells by AFG1-inducedchronic inflammationIn chronic inflammation, activation of NF-κB pathway also controls theexpression of COX-2, which is an important mediator of inflammation andinflammation-associated tumorgenesis. COX-2expression was upreragulatedin alveolar epithelial cells from AFG1-treated mice. Increased numbers ofCOX-2positive cells were found in lung alveolar epithelium of AFG1-treatedmice compared with that in normal mice at3and6months post-treatment.Higher level of COX-2is associated with increased SP-C expression inAFG1-treated mice, indicating that COX-2positive cell exhibits lung AT-II cellphenotype. AFG1-treated mice showed increased COX2at protein levels inAFG1-treated mice at3and6months after treatment. These results indicatethat AFG1-induced chronic inflammation enhances COX-2expression in lungAT-II cells.7AFG1-induced alveolar neoplastic lesions linked to chronic inflammationFifteen mice were observed over a period of additional6months after thesix months oral AFG1treatment period. All mice survived and did not showsigns of sickness evidenced by body weight loss and overall behavior.No hyperplastic and/or neoplastic lesions were detected in any of thecontrol mice. Pulmonary lesions were detected in8of15AFG1-inducedBalb/c mice and were classified as alveolar epithelial hyperplasia and lungadenocarcinoma. The remaining seven AFG1-treated mice did not exhibit anysigns of epithelial hyperplasia or lung adenocarcinoma. The frequency ofalveolar hyperplasia was26.7%(4/15). Epithelial hyperplasia lesions in lungsfrom mice treated with AFG1were localized to the alveolar area and werecharachterized by focal proliferation and papillary hyperplasia along thealveolar septae, but not in bronchioles. To further determine the cell-specificdifferentiation of alveolar epithelial hyperplasia, we also examined theexpression patterns of the AT-II cell marker SP-C and airway Clara cellmarker CC-10by immunohistochemistry. Increased numbers of SP-C positive cells were observed in the areas of epithelial hyperplasia associated withhigher level of Ki67in AFG1-treated mice. However, we did not observe anyCC-10positive cells in AFG1-treated mice.These results show that oral AFG1treatment induces AT-II cells hyperplasia. Four of15AFG1-induced micedeveloped lung adenocarcinomas close to the pleural surface, and effaced andreplaced the alveolar parenchyma, causing compression to the adjacentalveolar tissues. Immunohistochemical analysis confirms that AFG1-inducedlung adenocarcinoma demonstrates an AT-II cell phenotype. These datatogether with the previous result presented here that AFG1-induced chronicinflammation enhances AT-II cells proliferation, suggest that proliferation andmalignant transformation of AT-II cells along the alveolar septae play a criticalrole in the initiation of AFG1-induced lung adenocarcinoma.Several reports have shown that NF-κB and STAT3are importanttranscription factors sensitive to inflammation, contributing to lungtumorigenesis. We examined the expression of NF-κB p65and p-STAT3in alllung adenocarcinoma by immunohistological staining. All lungadenocarcinoma expressed higher levels of NF-κB p65and p-STAT3,suggesting that up-regulation of NF-κB and STAT3in AT-II cells byAFG1-induced inflammation may contribute to lung tumorigenesis. In additionto high expression in AT-II cells in chronic inflammatory stage (3-6months),COX-2was also up-regulated in hyperplastic AT-II cells and lungadenocarcinoma in the mouse model of tumor-development. These resultsindicate that overexpression of COX-2in AFG1-induced chronic inflammationmay contribute to lung tumorigenesis.Taken together, our results confirm that long-term oral administration ofAFG1could induce AT-II cells hyperplasia and lung adenocarcinoma, and alsosupport that chronic inflammation induced by AFG1may contribute to lungtumorigenesis. PartⅡ Enhanced phenotypic alterations of Alveolar type Ⅱ cells inresponses to Aflatoxin G1-induced chronic lung inflammationObjective: In inflammatory microenvironment, the activated AT-II cellsexpressing high levels of MHC-II and COX-2exhibit altered phenotypes, andlikely inhibit antitumor immunity by triggering regulatory T cells (Treg). Inthe previous study, we found high level of COX-2expression in AT-II cells inAFG1-induced inflammatory microenvironment as well as AFG1-inducedadenocarcinoma. However, whether and how the phenotypic alterations ofAT-II cells are induced in AFG1-induced inflammatory microenvironmentremains unknown. Understanding these phenotypic alterations of AT-II cellsmay provide new insight into the immune function of AT-II cells involved inAFG1-induced pulmonary tumorigenesis. In this study, we aim to explore thephenotypic alteration of AT-II cells as well as its mechanism in response toAFG1-induced chronic inflammation in vivo and in vitro.Methods: First, we will explore MHC-II, CD74and Tregs specificmarker (FoxP3) expression in mice lung tissues with AFG1-inducedinflammation and lung adenocarcinoma. Second, we will detect the effect ofAFG1on human major histocompatibility complex (MHC) class II molecules(HLA-DR), CD80and CD86expression on freshly isolated human alveolartype II cells and A549cells. Furthermore, we used the A549cell line as amodel of human AT-II cells and treated it with AFG1and TNF-α together tomimic an AFG1-induced inflammatory response in vitro. We investigated theexpression of HLA-DR, CD80, CD86, CD54(ICAM-I), inflammatorycytokines, and COX-2, as well as the regulation of MAPK and NF-κBpathway on549cells treated with AFG1and TNF-α coordinately.Results:1Increased MHC-II and CD74expression associated with Treg infiltration inAFG1-induced inflammatory microenvironment and lung adenocarcinomaWe collected the lung tissues from mice with AFG1-inducedinflammation (6months) or lung adenocarcinoma (12months) in the previousstudy, and stained with MHC-II, CD74, and Treg cell-specific transcriptionfactor, Foxp3.We found increased numbers of MHC-II and CD74positive cells in lung alveolar epithelium in AFG1-induced chronic inflammatory tissues. Increasedexpression of Treg cell-specific transcription factor, Foxp3, was also observedin infiltrating lymphocytes of lung tissues with AFG1-induced inflammation.There were more regulatory T cells (Foxp3positive cells) in lymphnode closeto lung blood vessels of AFG1-treated mice. The results indicate thatAFG1-induced chronic inflammation may enhance MHC-II expression inalveolar epithelium and Treg infiltration in lung tissues.In4cases of AFG1-induced lung adenocarcinoma samples, we foundincreased regulatory T cells infiltrated around lung adenocarcinoma associatedwith increased MHC-II and CD74expression. It has been reported that thecancer cells in AFG1-induced lung adenocarcinoma all express AT-II cellsspecific marker SP-C. The results indicate that AT-II cells expressing highlevel of MHC-II and CD74may contribute to Tregs infiltration inAFG1-induced lung adenocarcinoma.Thus, those results suggest that AFG1-induced inflammation increasesMHC-II expression on AT-II cells, and the phenotypic altered AT-II cells maytrigger Treg infiltration to contribute to lung tumorigenesis.2AFG1induces phenotypic maturation of A549cells by upregulatingHLA-DR and CD54expressionAT-II cells have the ability to take up, process, and then present antigensto T cells. During the maturation process, immature APCs lose their ability totake up antigens and acquire the capacity to present antigens to T cells. Thepositive FITC-dextran histogram of AFG1-treated A549cells shifted leftcompared to that of control cells, suggesting a decreased cellular uptakeability of AFG1-treated A549cell. Thus, the results suggest that AFG1mayinduce AT-II cells to mature into functional APCs.Mature APCs, upregulating MHC-II and costimulatory molecule, canpresent antigen to na ve CD4+T cells triggering an adaptive immune response.After AFG1treatment, the cells were stained with anti-human HLA-DR, themost strongly expressed class II locus, as well as CD80, CD86, and CD54. Wefound that AFG1treatment increased HLA-DR expression on A549cells, and that the level of HLA-DR expression in the4mg/L AFG1group was similar tothe expression on cells stimulated by IFN-γ.Co-stimulatory molecules from the B7family and ICAM-1are alsoessential for effective activation of T cells. We measured the expression of thecostimulatory molecules CD80and CD86. Both AFG1and IFN-γ treated A549cells showed no detectable CD80expression and a low level of CD86expression. The expression of CD54was higher in AFG1-treated A549cellsthan that in controls, though still lower than that in IFN-γ-treated cells. Ourresults indicate that AFG1induces phenotypic maturation of AT-II cells byup-regulating HLA-DR and CD54expression, while expression of CD80andCD86is low or undetectable.Our previous study showed that AFG1could activate JNK, ERK and p38pathways on A549cells. To further explore whether MAPK pathwayscontribute to AFG1-induced AT-II cell maturation, we measured HLA-DR andCD54expression on A549cells pretreated with inhibitors for JNK, ERK andp38(SP600125, PD98059and SB203580). We found that upregulation ofHLA-DR and CD54in AFG1-treated A549cells were significantly inhibitedby the p38inhibitor SB203580. Whereas, blocking the JNK pathway withSP600125or the ERK pathway with PD98059did not influenceAFG1-induced AT-II cell maturation. These results indicate that AFG1up-regulates HLA-DR and CD54expression on AT-II cells through p38signaling pathway.3Up-regulation of HLA-DR in freshly isolated human AT-II cells treated withAFG1Since AFG1induced MHC class II-restricted phenotypic maturation inA549cells along with low or undetectable expression of CD86and CD80, weisolated human primary AT-II cells to further validate the effect of AFG1onHLA-DR, CD80and CD86expression on fresh AT-II cells. Compared to A549cells, the freshly isolated human AT-II cells expressed a high level of HLA-DR,and the expression of CD80and CD86was still low. We found AFG1significantly increased HLA-DR expression on the primary AT-II cells and had no effect on CD80and CD86expression. These results support the conclusionthat AFG1induces AT-II cell phenotypic maturation by up-regulating MHC-IIexpression. Because AT-II cells treated with AFG1or IFN-γ still express low orundetectable levels of CD86and CD80, we propose that activated AT-II cellsbecome unable to activate effector CD4+T cells due to lacking of necessarycostimulatory molecule expression.4AFG1and TNF-α coordinately enhanced HLA-DR and CD54expression onA549cellsTNF-α is a key factor that regulates the production of cytokines involvedin chronic inflammation. Increased TNF-α expression was also found in bothalveolar epithelial cells and macrophageso f AFG1-treated mice in my previousstudy. Thus, we treated A549cells with AFG1and TNF-α to mimic anAFG1-induced inflammatory microenvironment in vitro. The combination ofAFG1and TNF-α significantly enhanced HLA-DR and CD54expression onA549cells, compared to AFG1treatment alone. The results indicate that thecombination of AFG1and TNF-α could work together to enhance HLA-DRand CD54expression on AT-II cells in AFG1-induced chronic inflammatorymicroenvironment.5NF-κB and p38pathways contribute to the enhanced expression of HLA-DRand CD54on A549cells treated with AFG1and TNF-α coordinatelyWe explore whether p38and NF-κB pathways contribute to increasedexpression of HLA-DR and CD54on A549cells treated with AFG1andTNF-α coordinately. Western blot analysis demonstrate that the combinationof AFG1and TNF-α down-regulated cytoplasmic levels of IκB-α and IκB-βand up-regulated p-NF-κB expression at0.5,1and3h after treatment.Following stimuli that elicit the NF-κB pathway, the IB inhibitory subunit ofthe NF-κB complex is degraded, exposing nuclear targeting signals in thetranscription factor subunits of the NF-κB complex. We also found AFG1andTNF-α coordinately induced nuclear translocation of the p65component ofNF-κB since the levels of the NF-κB p65subunit protein in the nuclearfractions of A549cells were also significant increased at3-6h after treatment. However, we did not found AFG1activated NF-κB pathways because therewere no differences in IκB-α, IκB-β, p-NF-κB, and NF-κB expression afterAFG1treatment for0.5,1,3and6hours. The results indicate that TNF-α notAFG1, contribute to the activation of NF-κB pathway in AFG1-inducedinflammatory microenvironment. TNF-α-activated NF-κB pathway mayregulate the phenotypic alteration of AT-II cells.Our previous study showed that p38pathway was activated from12to24h after AFG1-treatment to contribute to phenotypic maturation of AT-II cells.However, we found the combination of AFG1and TNF-α together acceleratedp38phosphorylation from3h, exhibiting a rapid activation of p38pathwaycompared to AFG1alone. Thus, p38signaling pathway may also play a role inphenotypic alterations of AT-II cells in response to AFG1-inducedinflammation.To further explore the mechanism responsible for the phenotypicalterations of A549cells induced by AFG1and TNF-α coordinately, weinvestigated HLA-DR and CD54expression on A549cells by blocking p38and NF-κB pathways. Pre-treatment with the p38specific inhibitor SB203580or NF-κB inhibitor PDTC inhibited the phosphorylation of p38or nuclearNF-κB p65expression respectively in AFG1+TNF-α-treated cells.Furthermore, we found blocking of p38or NF-κB pathway inhibited theupregulation of HLA-DR and CD54in AFG1+TNF-α-treated cells. The resultsindicate that the combination of AFG1and TNF-α coordinately enhancedHLA-DR and CD54expression through p38and NF-κB pathways. The resultssuggest that p38pathway activated by AFG1and TNF-α coordinately andNF-κB pathways activated by TNF-α may contribute to phenotypic alterationsof A549cells in AFG1-induced chronic inflammatory environment.6The combination of AFG1and TNF-α coordinately enhanced COX-2expression on A549cellsCOX-2underlies an immunosuppressive network by regulatingregulatory T cells (Treg) activity, to promote tumorigenesis. COX-2expression was also enhanced in lung alveolar AT-II cells in both AFG1-induced chronic inflammatory microenvironment and lung cancer. Here,we treated A549cells with AFG1and TNF-α to mimic an AFG1-inducedinflammatory response and explore the mechanism of increased COX-2expression. We found TNF-α upregulated COX-2expression on A549cells atmRNA and protein levels, while AFG1alone did not. The combination ofAFG1and TNF-α significantly enhanced COX-2expression on A549cells,compared to TNF-α alone. The results suggest the combination of AFG1andTNF-α coordinately enhanced COX-2expression in AFG1-inducedinflammatory microenvironment.To explore the mechanism responsible for increased COX-2expressioninduced by the combination of AFG1and TNF-α coordinately, we investigatedCOX-2expression on A549cells by inhibiting p38or NF-κB pathways. Wefound that blocking of NF-κB pathway inhibited COX-2upregulation onAFG1+TNF-α-treated cells, while blocking of p38did not change COX-2expression. The results suggest TNF-α-activated NF-κB pathway maycontribute to the increased COX-2expression on AT-II cells in AFG1-inducedchronic inflammatory microenvironment.7Combination of AFG1and TNF-α significantly enhanced cytokinesexpression on A549cellsWe found that AFG1alone increased TNF-α, IL-1β, IL-6, MCP-1, IL-12,IL-10, and TGF-α mRNA expression on A549cells. After24h treatment, thecombination of AFG1and TNF-α significantly enhanced TNF-α, IL-6, MCP-1,MIP-2, and IL-10expression on A549cells, compared to AFG1alone. Theresults suggest AT-II cells could be activated by AFG1and TNF-α to producecytokines, playing an important role in lung inflammatory responses. Inaddition, we found that blocking of NF-κB or p38pathway all partly inhibitedenhanced TNF-α, IL-6, MCP-1, and MIP-2mRNA expression onAFG1+TNF-α-treated cells. Blocking of NF-κB pathway also inhibited IL-10upregulation on AFG1+TNF-α-treated cells, while blocking of p38did not.Dramatic inhibitory impact of cytokines expression was achieved by blockingNF-κB pathway, suggesting TNF-α-activated NF-κB pathway plays key roles in mediating cytokines production of AT-II cells. Though TNF-α-activatedNF-κB pathway did not contribute to the increased TGF-β expression,AFG1-activated p38pathway plays a critical role in upregulating TGF-βexpression on AT-II cells. The results suggest that p38pathway activated byAFG1and TNF-α coordinately and NF-κB pathways activated by TNF-α mayalso contribute to cytokine production of A549cells in AFG1-induced chronicinflammatory environment. Upregulation of IL-10and TGF-β may drive AT-IIcells to trigger Treg activity.PartⅢ Combination of AFG1and TNF-α enhanced oxidative stress andDNA damage in A549cellsObjective: In the previous study, we found that AFG1-induced chronicinflammation enhances oxidative stress in alveolar epithelial cells asevidenced by incasing SOD-2and HO-1activity in Balb/C mice. However, itis not clear whether AFG1or AFG1-triggered cytokine induces oxidative stressto cause DNA damage in AT-II cells in AFG1-induced chronic inflammatorymicroenvironment. We used the A549cell line as a model of human AT-IIcells and explored the effect of the combination of AFG1and TNF-α onoxidative stress and DNA damage in A549cells.Method: Oxidative stress in the form of reactive oxygen species (ROS)formation is a major risk factor in the induction of lung epithelial damage anddysfunction. First, we used the human tumor cell line A549as an AT-II cellmodel and examined the effects of AFG1alone on ROS generation andoxidative DNA damage in A549cells. Second, we compared ROS generationin A549cells on different time points upon AFG1-treatment or AFG1+TNF-α-treatment. Third, we detected the effect of combination of AFG1and TNF-α onCYP450expression on A549cells, as well as the regulation of NF-κBpathway.Results:1AFG1induces oxidative stress by increasing ROS generation in A549cellsROS mainly include superoxide anion (O2ˉ), hydrogen peroxide (H2O2),and the hydroxyl radical (OH). To investigate the effect of AFG1on the generation of intracellular ROS, we measured the levels of intracellularsuperoxide and hydrogen peroxide in A549cells using FCM assay(fluorescent dyes DHE for O2ˉand DCFH for H2O2respectively). AFG1notably stimulated A549cells to increase both mean fluorescence intensity(MFI) of DHE and DCFH (P<0.05), suggesting that AFG1induces an increasein steady state levels of superoxide and hydrogen peroxides in A549cells.To further confirm that AFG1could increase ROS generation in AT-IIcells, we measured the effect of NAC on ROS generation in AFG1-treatedA549cells. FCM analysis showed that the higher levels of DHE and DCFH inAFG1-treated A549cells were significantly attenuated by NAC pretreatment(P<0.05). Thus, our results indicate that AFG1induces oxidative stress in AT-IIcells by increasing intracellular ROS generation.2AFG1induces oxidative DNA damage in A549cellsWe detected DNA damage after AFG1treatment using the alkaline cometassay. The assay can detect strand DNA breaks and alkalile-labile DNA adductby the presence of comet “tails”. We found that2,4and10mg/L AFG1induced a significant increase in damaged DNA fragments in A549cells byproducing typical comet tails. Pre-treatment with NAC inhibited DNA damagein4mg/L AFG1-treated A549cells. These results suggest that AFG1caninduce oxidative DNA damage in AT-II cells.Among different types of DNA damage, double-strand DNA breaks(DSBs) are arguably one of the most serious. DSB marker γ-H2AX isaccepted as a reliable and sensitive signal for the existence of DNAdouble-strand breaks. We detected the effect of AFG1on γ-H2AX expressionin A549cells with or without NAC pre-treatment to further determine whetherAFG1induces oxidative DNA DSBs. Western blot results showed that2,4and10mg/L AFG1treatment resulted in the up-regulation of γ-H2AX protein inA549cells, while NAC reduced γ-H2AX expression in4mg/L AFG1-treatedA549cells. Taken together, these results suggest that AFG1-induced oxidativestress results in DNA DSBs in AT-II cells. 3The combination of AFG1and TNF-α enhanced oxidative DNA damage inA549cellsWe found the combination of AFG1and TNF-α i ncreased intracellularROS generation at3h after treatment in A549cells, while AFG1alone startedto increase ROS generation at6h after treatment. After6h treatment,combination of AFG1and TNF-α significantly increase γ-H2AX and SOD-2expression on A549cells, while AFG1alone did not affect γ-H2AX andSOD-2expression (P<0.05). After24h treatment, we found combination ofAFG1and TNF-α significantly enhanced γ-H2AX and SOD-2expression thanAFG1alone. The results clearly suggest that TNF-α could collaborate withAFG1, and then enhance AFG1-induced oxidative stress to trigger DNAdouble-strand breaks in AT-II cells.4The combination of AFG1and TNF-α i ncreased CYP450expression onA549cellsWe found AFG1alone only increased CYP2A13expression at mRNAand protein level, but did not affect CYP2A6expression. However, thecombination of AFG1and TNF-α significantly enhanced both2A13and2A6expression on A549cells than AFG1alone. The results suggest TNF-α mayenhance the metabolism of AFG1in A549cells by up-regulating CYP450expression in AFG1-induced chronic inflammatory environment, which playan important role in enhancing AFG1-induced oxidative stress.5NF-κB pathway contributed to the enhanced oxidative DNA damage inA549cells triggered by AFG1and TNF-α coordinatelyTo further explore that TNF-α-activated NF-κB contributed to theenhanced oxidative DNA damage induced by AFG1and TNF-α coordinately,we investigated CYP2A13and γ-H2AX expression on A549cells by blockingNF-κB pathways. We found blocking of NF-κB pathway significantlyinhibited the enhanced expression of2A13and γ-H2AX on AFG1+TNF-α-treated cells (P<0.05). The results suggest that TNF-α-activated NF-κBpathway upregulates CYP450to promote the metabolism of AFG1in cells,and then enhances oxidative DNA damage in AT-II cells. Conclusions:1Repeated oral administration of AFG1induces chronic lunginflammation in the alveolar septum, which significantly promotes oxidativestress, cell proliferation and angiogenesis, and enhances COX-2... |
PDF Full Text Request