黄曲霉毒素M_1与赭曲霉毒素A联合作用诱导分化Caco-2细胞凋亡的机制
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摘要
目的:研究了黄曲霉毒素M_1(Aflatoxin M_1,AFM_1)和赭曲霉毒素(ochratoxin A,OTA)交互作用诱导分化人结肠癌Caco-2细胞凋亡及其作用机制。方法:AFM_1和OTA单独及联合作用于分化的Caco-2细胞后,通过阿尔玛蓝(Alamar-Blue)法测量细胞存活率,研究AFM_1和OTA对细胞增殖的抑制作用;通过流式细胞术检测细胞凋亡率,活性氧荧光探针检测细胞活性氧(ROS)释放量,JC-1线粒体膜电位荧光探针检测细胞线粒体膜电位变化。蛋白免疫印迹法检测凋亡蛋白Caspase-3和Caspase-9的表达水平。结果:AFM_1和OTA可抑制分化的Caco-2细胞增殖并诱导其凋亡,单独使用12μmol/L AFM_1和6μmol/L OTA作用于细胞存活率分别为95.59%和93.14%,联合作用下细胞存活率为81.57%,提示联合作用具有协同作用;AFM_1和OTA单独处理细胞24 h后,细胞的凋亡率分别为25.68%和33.7%,联合作用细胞的凋亡率为37.4%;AFM_1和OTA单独作用下细胞ROS的释放量分别是空白对照组的1.37倍和1.79倍,交互作用是空白对照组的6.21倍,交互作用对活性氧的释放量明显增加。结论:线粒体膜电位和线粒体相关凋亡蛋白的表达水平变化,说明AFM_1和OTA可能通过线粒体途径诱导分化的Caco-2细胞凋亡。
Purpose: To study the combined mechanism of AFM_1(aflatoxin M_1, AFM_1) and OTA(ochratoxin A,OTA) on the apoptosis and mechanism. Method: differentiated Caco-2(human colorectal adenocarcinoma) cells were cultured and treated in the following groups: AFM1(12 μmol/L), OTA(6 μmol/L), AFM_1(12 μmol/L)+OTA(6 μmol/L). Cell viability and proliferation were detected by Alamar-Blue method. Cell apoptotic rate was quantified by AnnexinV-FITC/PI staining through flow cytometry, intracellular ROS(reactive oxygen species, ROS) release was detected by active oxygen fluorescent probe, and the mitochondrial membrane potential was detected by a JC-1 probe. Additionally, the protein of Caspase-3 and Caspase-9 were detected by Western blotting. Results: The results showed that AFM_1 and OTA have a synergistic effect in inhibiting the proliferation of differentiated Caco-2 cells, and inducing cell apoptosis. Cell viability of the three group data was 95.59%, 93.14% and 81.57%, respectively. Apoptotic rate of the three group was 25.68%,33.7% and 37.4%, respectively. What's more, the ROS release in AFM_1 group was 1.37-fold of control group level, in OTA group was 1.79-fold of control group level, and the one in combined group was 6.21-fold of control group level.Conclusion: Expression levels of mitochondrial membrane potential and apoptotic proteins suggested apoptosis of differentiated Caco-2 cells was induced through mitochondrial pathways, moreover, this induction seemed to be more obvious in the combination group.
Purpose: To study the combined mechanism of AFM_1(aflatoxin M_1, AFM_1) and OTA(ochratoxin A,OTA) on the apoptosis and mechanism. Method: differentiated Caco-2(human colorectal adenocarcinoma) cells were cultured and treated in the following groups: AFM1(12 μmol/L), OTA(6 μmol/L), AFM_1(12 μmol/L)+OTA(6 μmol/L). Cell viability and proliferation were detected by Alamar-Blue method. Cell apoptotic rate was quantified by AnnexinV-FITC/PI staining through flow cytometry, intracellular ROS(reactive oxygen species, ROS) release was detected by active oxygen fluorescent probe, and the mitochondrial membrane potential was detected by a JC-1 probe. Additionally, the protein of Caspase-3 and Caspase-9 were detected by Western blotting. Results: The results showed that AFM_1 and OTA have a synergistic effect in inhibiting the proliferation of differentiated Caco-2 cells, and inducing cell apoptosis. Cell viability of the three group data was 95.59%, 93.14% and 81.57%, respectively. Apoptotic rate of the three group was 25.68%,33.7% and 37.4%, respectively. What's more, the ROS release in AFM_1 group was 1.37-fold of control group level, in OTA group was 1.79-fold of control group level, and the one in combined group was 6.21-fold of control group level.Conclusion: Expression levels of mitochondrial membrane potential and apoptotic proteins suggested apoptosis of differentiated Caco-2 cells was induced through mitochondrial pathways, moreover, this induction seemed to be more obvious in the combination group.
引文
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[16]计成.霉菌毒素对家禽的危害及降解技术[J].中国家禽, 2014, 36(2):40-42.
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[23]CLARKE R, CONNOLLY L, FRIZZELL C, et al.High content analysis:A sensitive tool to detect and quantify the cytotoxic, synergistic and antagonistic effects of chemical contaminants in foods[J]. Toxicology Letters, 2015, 233(3):278-286.
[24]LI S J, EDIAGE E N, SAEGER S D, et al. Occurrence and pathology of mycotoxins in commercial parrot feeds[J]. World Mycotoxin Journal, 2013, 6(4):449-453.
[25]ZHANG J, ZHENG N, LIU J, et al. Aflatoxin B1and aflatoxin M1 induced cytotoxicity and DNA damage in differentiated and undifferentiated Caco-2cells[J]. Food&Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association, 2015, 83(2):54-60.
[26]MARESCA M, MAHFOUD R, PFOHL-LESZKOWICZ A, et al. The mycotoxin ochratoxin a alters intestinal barrier and absorption functions but has no effect on chloride secretion[J]. Toxicology&Applied Pharmacology, 2001, 176(1):54-63.
[27]GONZ LEZ-ARIAS C A, CRESPO-SEMPERE A,MAR N S, et al. Modulation of the xenobiotic transformation system and inflammatory response by ochratoxin A exposure using a co-culture system of Caco-2 and HepG2 cells[J]. Food&Chemical Toxicology, 2015, 86(2):245-252.
[28]高薇,侯微,李伟,等.细胞凋亡机制研究进展[J].中国畜牧兽医, 2014, 41(10):150-156.
[29]COSTA S, UTAN A, SPERONI E, et al. Carnosic acid from rosemary extracts:a potential chemoprotective agent against aflatoxin B1. An in vitro study[J]. Journal of Ap plied Toxicology, 2007, 27(2):152-159.
[30]SHEN H, JING L, YUAN W, et al. Aflatoxin G 1-induced oxidative stress causes DNA damage and triggers apoptosis through MAPK signaling pathway in A549 cells[J]. Food&Chemical Toxicology,2013, 62(12):661-669.
[31]中华人民共和国卫生部. GB 2761-2011食品安全国家标准,食品中真菌毒素限量[S].北京:中国标准出版社, 2011.
[2]IQBAL S Z, NISAR S, ASI M R, et al. Natural incidence of aflatoxins, ochratoxin A and zearalenone in chicken meat and eggs[J]. Food Control,2014, 43(3):98-103.
[3]IQBAL S Z, PATERSON R R, BHATTI I A, et al. Survey of aflatoxins in chillies from Pakistan produced in rural, semi-rural and urban environments[J]. Food Additives&Contaminants Part B Surveillance, 2010, 3(4):268-274.
[4]IQBAL S. A survey of Aflatoxin M1 contamination in milk from urban and rural farmhouses of Punjab,Pakistan[J]. Food Additives&Contaminants Part B,2014, 7(1):17-20.
[5]EGMOND H P V. Aflatoxin M1:occurrence, toxicity, regulation[J]. Food and Chemical Toxicology,2009, 47(2):984-991.
[6]ASI M R, IQBAL S Z, ARI O A, et al. Effect of seasonal variations and lactation times on aflatoxin M1 contamination in milk of different species from Punjab, Pakistan[J]. Food Control, 2012, 25(1):34-38.
[7]ORUC H H, CIBIK R, YILMAZ E, et al. Distribution and stability of Aflatoxin M1 during processing and ripening of traditional white pickled cheese[J]. Food Additives&Contaminants, 2006, 23(2):190-195.
[8]KJ V D M, STEYN P S, FOURIE L, et al. Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus Wilh[J]. Nature, 1965, 205(976):1112-1113.
[9]CLARK H A, SNEDEKER S M. Ochratoxin a:its cancer risk and potential for exposure[J]. Journal of Toxicology&Environmental Health Part B Critical Reviews, 2006, 9(3):265-296.
[10]O'BRIEN E, DIETRICH D R. Ochratoxin A:the continuing enigma[J]. Critical Reviews in Toxicology,2005, 35(1):33-60.
[11]SHUNDO L, ALMEIDA A P D, ALABURDA J, et al. Aflatoxins and ochratoxin A in Brazilian paprika[J]. Food Control, 2009, 20(12):1099-1102.
[12]MEETING J F W E C O F A, ORGANIZATION W H. Evaluation of certain food additives and contaminants:fifty-third report of the Joint FAO/WHO Expert Committee on Food Additives[J]. Technical Report, 2000, 901(2):189-979.
[13]KUMAGAI S, AIBARA K. Intestinal absorption and secretion of ochratoxin A in the rat[J]. Toxicology&Applied Pharmacology, 1982, 64(1):94-102.
[14]GROLLMAN A P, SHIBUTANI S, MORIYA M, et al. Aristolochic acid and the etiology of endemic(Balkan)nephropathy[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(29):12129-12134.
[15]MARESCA M, YAHI N, YOUN S-SAKR L, et al.Both direct and indirect effects account for the proinflammatory activity of enteropathogenic mycotoxins on the human intestinal epithelium:Stimulation of interleukin-8 secretion, potentiation of interleukin-1βeffect and increase in the transepithelial[J]. Toxicology&Applied Pharmacology, 2008, 228(1):84-92.
[16]计成.霉菌毒素对家禽的危害及降解技术[J].中国家禽, 2014, 36(2):40-42.
[17]AKBARI P, BRABER S, GREMMELS H, et al.Deoxynivalenol:a trigger for intestinal integrity breakdow n[J]. Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology, 2014, 28(6):2414-2429.
[18]PINTON P, TSYBULSKYY D, LUCIOLI J, et al.Toxicity of deoxynivalenol and its acetylated derivatives on the intestine:differential effects on morphology, barrier function, tight junction proteins,and mitogen-activated protein kinases[J]. Toxicological Sciences, 2012, 130(1):180-190.
[19]KOUADIO J H, DANO S D, MOUKHA S, et al.Effects of combinations of Fusarium mycotoxins on the inhibition of macromolecular synthesis, malondialdehyde levels, DNA methylation and fragmentation, and viability in Caco-2 cells[J]. Toxicon,2007, 49(3):306-317.
[20]HENGARTNER M O. The biochemistry of apoptosis[J]. Nature, 2000, 407(6805):770-776.
[21]IGNEY F H, KRAMMER P H. Death and antideath:tumour resistance to apoptosis[J]. Nature Reviews Cancer, 2002, 2(4):277-288.
[22]LIU J, ZHANG W, JING H, et al. Bog bilberry(Vaccinium uliginosum L.)extract reduces cultured Hep-G2, Caco-2, and 3T3-L1 cell viability, affects cell cycle progression, and has variable effects on membrane permeability[J]. Journal of Food Science, 2010, 75(3):103-107.
[23]CLARKE R, CONNOLLY L, FRIZZELL C, et al.High content analysis:A sensitive tool to detect and quantify the cytotoxic, synergistic and antagonistic effects of chemical contaminants in foods[J]. Toxicology Letters, 2015, 233(3):278-286.
[24]LI S J, EDIAGE E N, SAEGER S D, et al. Occurrence and pathology of mycotoxins in commercial parrot feeds[J]. World Mycotoxin Journal, 2013, 6(4):449-453.
[25]ZHANG J, ZHENG N, LIU J, et al. Aflatoxin B1and aflatoxin M1 induced cytotoxicity and DNA damage in differentiated and undifferentiated Caco-2cells[J]. Food&Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association, 2015, 83(2):54-60.
[26]MARESCA M, MAHFOUD R, PFOHL-LESZKOWICZ A, et al. The mycotoxin ochratoxin a alters intestinal barrier and absorption functions but has no effect on chloride secretion[J]. Toxicology&Applied Pharmacology, 2001, 176(1):54-63.
[27]GONZ LEZ-ARIAS C A, CRESPO-SEMPERE A,MAR N S, et al. Modulation of the xenobiotic transformation system and inflammatory response by ochratoxin A exposure using a co-culture system of Caco-2 and HepG2 cells[J]. Food&Chemical Toxicology, 2015, 86(2):245-252.
[28]高薇,侯微,李伟,等.细胞凋亡机制研究进展[J].中国畜牧兽医, 2014, 41(10):150-156.
[29]COSTA S, UTAN A, SPERONI E, et al. Carnosic acid from rosemary extracts:a potential chemoprotective agent against aflatoxin B1. An in vitro study[J]. Journal of Ap plied Toxicology, 2007, 27(2):152-159.
[30]SHEN H, JING L, YUAN W, et al. Aflatoxin G 1-induced oxidative stress causes DNA damage and triggers apoptosis through MAPK signaling pathway in A549 cells[J]. Food&Chemical Toxicology,2013, 62(12):661-669.
[31]中华人民共和国卫生部. GB 2761-2011食品安全国家标准,食品中真菌毒素限量[S].北京:中国标准出版社, 2011.
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