食品中甲醛的细胞毒性及基因表达影响
|
摘要
为了探讨食品中甲醛的细胞毒作用,检测不同浓度下甲醛对细胞生长的抑制情况,并运用基因芯片技术检测甲醛对淋巴细胞基因表达的影响,筛选并分析与甲醛毒性和致癌性相关的差异表达基因。作者进行MTT实验,绘制淋巴细胞的细胞生长曲线及细胞形态观察,然后用5×10-5mol/L甲醛处理人外周血淋巴细胞24 h,提取实验组和对照组细胞m RNA,经逆转录和标记后与人类全基因组芯片(约35 000个点)杂交,芯片扫描后提取并处理数据,筛选出甲醛诱导显著改变的基因,并进行生物信息学分析。发现甲醛与淋巴细胞增殖存在的剂量-效应关系,验证了甲醛对淋巴细胞的细胞毒作用;共筛选出显著上调基因211个,显著下调基因113个,分别参与氧化应激、细胞凋亡、核酸代谢、细胞周期调控、肿瘤生成、肿瘤抑制等途径,推断出OXR1、MLH1、TP53等基因表达的改变可作为甲醛毒性和致癌性的遗传学基础。本研究获得的数据为进一步阐明甲醛的毒性和致癌机理,检测食品中甲醛暴露的早期生物学标志提供了理论依据,证明了基因芯片技术在食品毒理学研究中的可行性和优越性,为建立新的有毒物质暴露的检测和研究方法提供了实验依据。
To investigate the cytotoxic effects of formaldehyde in the food,we detect different concentrations of formaldehyde on the inhibition of cell growth,and use microarray technology to detect the influence of lymphocyte gene expression by formaldehyde in order to screen and analyze the differentially expressed genes related to toxicity and carcinogenicity of formaldehyde. By making MTT experiments,the cytotoxicity of formaldehyde was verified by comparing lymphocytes' growth curve and morphology. Peripheral blood lymphocytes were treated with 5 ×105mol/L formaldehyde for 24 h.The total RNA from cells of the subjects of the treated group and the control group were isolated,then they were reversely transcribed and labeled with fluorescence dyes,and finally hybridized with human whole genome microarray(about 35 000 spots). Microarray data was extracted after microarray scanning,and differentially expressed genes induced by formaldehyde were screened and analyzed by bioinformatics. In MTT experiments,human peripheral blood lymphocytes were treated with various doses of formaldehyde. The dose-effect relationship was also observed after treatment with high doses. 211 up-regulated and 113 down-regulated genes were screened after scanning and data extracting. Genes which significantly altered were represented by several categories,including oxidative stress,DNA repair,apoptosis,nucleic acid metabolism,cell cycle,oncogenesis,tumor suppression and so on. The result suggested the differential expression of these genes of OXR1,MLH1,TP53 may be the genetic basis of formaldehyde toxicity and carcinogenicity. The study provided theoretical evidence for elucidating the toxic and carcinogenic mechanism,detecting of the biomarker for formaldehyde exposure,and preventing the formaldehyde poisoning. It also verified the microarray technique is the feasible and advantageous in food toxicological research. It provides an experimental basis for the establishment of new methods to detect and study exposure of toxic substances.
To investigate the cytotoxic effects of formaldehyde in the food,we detect different concentrations of formaldehyde on the inhibition of cell growth,and use microarray technology to detect the influence of lymphocyte gene expression by formaldehyde in order to screen and analyze the differentially expressed genes related to toxicity and carcinogenicity of formaldehyde. By making MTT experiments,the cytotoxicity of formaldehyde was verified by comparing lymphocytes' growth curve and morphology. Peripheral blood lymphocytes were treated with 5 ×105mol/L formaldehyde for 24 h.The total RNA from cells of the subjects of the treated group and the control group were isolated,then they were reversely transcribed and labeled with fluorescence dyes,and finally hybridized with human whole genome microarray(about 35 000 spots). Microarray data was extracted after microarray scanning,and differentially expressed genes induced by formaldehyde were screened and analyzed by bioinformatics. In MTT experiments,human peripheral blood lymphocytes were treated with various doses of formaldehyde. The dose-effect relationship was also observed after treatment with high doses. 211 up-regulated and 113 down-regulated genes were screened after scanning and data extracting. Genes which significantly altered were represented by several categories,including oxidative stress,DNA repair,apoptosis,nucleic acid metabolism,cell cycle,oncogenesis,tumor suppression and so on. The result suggested the differential expression of these genes of OXR1,MLH1,TP53 may be the genetic basis of formaldehyde toxicity and carcinogenicity. The study provided theoretical evidence for elucidating the toxic and carcinogenic mechanism,detecting of the biomarker for formaldehyde exposure,and preventing the formaldehyde poisoning. It also verified the microarray technique is the feasible and advantageous in food toxicological research. It provides an experimental basis for the establishment of new methods to detect and study exposure of toxic substances.
引文
[1]JIN Hongguo,LIU Hualin,ZHANG Rui,et al.Review of analytical methods for determination of formaldehyde in food[J]Science and Technology of Food Industry,2013(19):373-377.(in Chinese)
[2]LIN Chaopeng,XU Xiaochun,ZHONG Ruimin,et al.Rapid determination of trace amounts of formaldehyde based on chromati aberration in aquatic product logistics[J].Journal of Food Science and Biotechnology,2009,28(6):795-798.(in Chinese)
[3]YANG Yuhua,XI Zhuge,ZHAO Fuhuan.Research progress in formaldehyde pollution and human health[J].Journal of Preventive Medicine of Chinese People’s Liberation Army,2005,23(1):68-71.(in Chinese)
[4]WANG X J,LI Y,LUO L,et al.Oxaliplatin activates the Keapl/Nrf2 antioxidant system conferring protection against th cytotoxicity of anticancer drugs[J].Free Radic Biol Med,2014,70:68-77.
[5]ZHU Jieli,LV Aihua,ZHANG Rui,et al.Research progress in detection of formaldehyde in food[J].Jiangsu Agricultural Sciences,2013,41(1):280-282.(in Chinese)
[6]WANG Yanli,WANG Yuguang,LIANG Qiande,et al.using c DAN microarray to screen differently expressed genes in SH-SY5Y treated by veratridine[J].Chinese Pharmacological Bulletin,2013,29(5):643-647.(in Chinese)
[7]TYIHAK E,BOCSI J,TIMAR F,et al.Formaldehyde promotes and inhibits the proliferation of cultured tumour and endothelia cells[J].Cell Proliferation,2001,34(3):135-141.
[8]ELLIOTT N A,VOLKERT M R.Stress induction and mitochondrial localization of Oxr1 proteins in yeast and humans[J]Molecular&Cellular Biology,2004,24(8):3180-3187.
[9]QIAO Yan,HE Hujun,NIU Dandan,et al.Effect of formaldehyde inhalationon glutathione level in various organs of mice[J]Hubei Journal of Preventive Medicine,2004,15(5):12-14.(in Chinese)
[10]LI X,ZHOU M,WANG X,et al.Quantitative determina-tion of high-temperature requirement protein A1 and its possible associated molecules during induced reparative dentin formation[J].Journal of Endodontics,2012,38(6):814-820.
[11]QIAN H,ZHAO Y,PENG Y,et al.Activation of cannabi-noid receptor CB2 regulates osteogenic and osteoclastogenic gene expression in human periodontal ligament cells[J].Journal of Periodontal Research,2010,45(4):504-511.
[12]SOUSSI T,LOZANO G.p53 mutation heterogeneity in cancer[J].Biochemical&Biophysical Research Communications,2005,331(3):834-842.
[13]OLIVIER M,LANGEROD A,CARRIERI P,et al.The clinical value of somatic TP53 gene mutations in 1 794 patients with breast cancer[J].Clinical Cancer Research An Official Journal of the American Association for Cancer Research,2006,12(4):1157-1167.
[14]MORENO M,PIMENTEL F,GAZDAR A F,et al.TP53 abnormalities are frequent and early events in the sequential pathogenesis of gallbladder carcinoma[J].Ann Hepatol,2005,4(3):192-199.
[15]TODOROVA T A,JORDANOV S H,STANCHEVA G S,et al.Mutational status of CDKN2A and TP53 Genes in laryngeal squamous cell carcinoma[J].Pathology&Oncology Research,2015,21(2):413-421.
[16]JI P,AGRAWAL S,DIEDERICHS S,et al.Cyclin A1,the alternative A-type cyclin,contributes to G1/S cell cycle progression in somatic cells[J].Oncogene,2005,24(16):2739-2744.
[17]PALLOCCA G,FABBRI M,SACCO M G,et al.mi RNA expression profiling in a human stem cell-based model as a tool for developmental neurotoxicity testing[J].Cell Biology and Toxicology,2013,29(4):239-257.
[18]GIGLIO S,CIROMBELLA R,AMODEO R,et al.Micro RNA mi R-24 promotes cell proliferation by targeting the CDKs inhibitors p27Kip1 and p16INK4a[J].Journal of Cellular Physiology,2013,228(10):2015-2023.
[19]CORY S,HUANG D C,ADAMS J M.The Bcl-2 family:roles in cell survival and oncogenesis[J].Oncogene,2003,22(53):8590-8607.
[20]JIANG Y J,POLINA I,JIN J L,et al.FXR inhibits gankyrin in mouse livers and prevents development of liver cancer[J].Hepatology,2013,57(3):1098-1106.
[21]ZHANG J,YANG Y.Gankyrin plays an essential role in estrogen-driven and GPR30-mediated endometrial carcinoma cell proliferation via the PTEN/PI3K/AKT signaling pathway[J].Cancer Letters,2013,339(2):279-287.
[22]YOKOI S,YASUI K,IIZASA T,et al.Down-regulation of SKP2 induces apoptosis in lung-cancer cells[J].Cancer Science,2003,94(4):344-349.
[23]PONZIELLI R,KATZ S,BARSYTE L D,et al.Cancer therapeutics:targeting the dark side of Myc[J].European Journal of Cancer,2005,41(16):2485-2501.
[24]CORY S,ADAMS J M.Killing cancer cells by flipping the Bcl-2/Baxswitch[J].Cancer Cell,2005,8(1):5-6.
[25]RUBIO M F,BLESA D,MESTRE C,et al.Characterization of 8p21.3 chromosomal deletions in B-cell lymphoma:TRAIL-R1and TRAIL-R2 as candidate dosage-dependent tumor suppressor genes[J].Blood,2005,106(9):3214-3222.
[26]DUMON K R,ISHII H,VECCHIONE A,et al.Fragile histidine triad expression delays tumor development and induces apoptosis in human pancreatic cancer[J].Cancer Research,2001,61(12):4827-4836.
[27]METE F,KILIC E,SOMAY A,et al.Effects of heat stress on endocrine functions&behaviour in the pre-pubertal rat[J].Indian Journal of Medical Research,2012,135(2):233-236.
[2]LIN Chaopeng,XU Xiaochun,ZHONG Ruimin,et al.Rapid determination of trace amounts of formaldehyde based on chromati aberration in aquatic product logistics[J].Journal of Food Science and Biotechnology,2009,28(6):795-798.(in Chinese)
[3]YANG Yuhua,XI Zhuge,ZHAO Fuhuan.Research progress in formaldehyde pollution and human health[J].Journal of Preventive Medicine of Chinese People’s Liberation Army,2005,23(1):68-71.(in Chinese)
[4]WANG X J,LI Y,LUO L,et al.Oxaliplatin activates the Keapl/Nrf2 antioxidant system conferring protection against th cytotoxicity of anticancer drugs[J].Free Radic Biol Med,2014,70:68-77.
[5]ZHU Jieli,LV Aihua,ZHANG Rui,et al.Research progress in detection of formaldehyde in food[J].Jiangsu Agricultural Sciences,2013,41(1):280-282.(in Chinese)
[6]WANG Yanli,WANG Yuguang,LIANG Qiande,et al.using c DAN microarray to screen differently expressed genes in SH-SY5Y treated by veratridine[J].Chinese Pharmacological Bulletin,2013,29(5):643-647.(in Chinese)
[7]TYIHAK E,BOCSI J,TIMAR F,et al.Formaldehyde promotes and inhibits the proliferation of cultured tumour and endothelia cells[J].Cell Proliferation,2001,34(3):135-141.
[8]ELLIOTT N A,VOLKERT M R.Stress induction and mitochondrial localization of Oxr1 proteins in yeast and humans[J]Molecular&Cellular Biology,2004,24(8):3180-3187.
[9]QIAO Yan,HE Hujun,NIU Dandan,et al.Effect of formaldehyde inhalationon glutathione level in various organs of mice[J]Hubei Journal of Preventive Medicine,2004,15(5):12-14.(in Chinese)
[10]LI X,ZHOU M,WANG X,et al.Quantitative determina-tion of high-temperature requirement protein A1 and its possible associated molecules during induced reparative dentin formation[J].Journal of Endodontics,2012,38(6):814-820.
[11]QIAN H,ZHAO Y,PENG Y,et al.Activation of cannabi-noid receptor CB2 regulates osteogenic and osteoclastogenic gene expression in human periodontal ligament cells[J].Journal of Periodontal Research,2010,45(4):504-511.
[12]SOUSSI T,LOZANO G.p53 mutation heterogeneity in cancer[J].Biochemical&Biophysical Research Communications,2005,331(3):834-842.
[13]OLIVIER M,LANGEROD A,CARRIERI P,et al.The clinical value of somatic TP53 gene mutations in 1 794 patients with breast cancer[J].Clinical Cancer Research An Official Journal of the American Association for Cancer Research,2006,12(4):1157-1167.
[14]MORENO M,PIMENTEL F,GAZDAR A F,et al.TP53 abnormalities are frequent and early events in the sequential pathogenesis of gallbladder carcinoma[J].Ann Hepatol,2005,4(3):192-199.
[15]TODOROVA T A,JORDANOV S H,STANCHEVA G S,et al.Mutational status of CDKN2A and TP53 Genes in laryngeal squamous cell carcinoma[J].Pathology&Oncology Research,2015,21(2):413-421.
[16]JI P,AGRAWAL S,DIEDERICHS S,et al.Cyclin A1,the alternative A-type cyclin,contributes to G1/S cell cycle progression in somatic cells[J].Oncogene,2005,24(16):2739-2744.
[17]PALLOCCA G,FABBRI M,SACCO M G,et al.mi RNA expression profiling in a human stem cell-based model as a tool for developmental neurotoxicity testing[J].Cell Biology and Toxicology,2013,29(4):239-257.
[18]GIGLIO S,CIROMBELLA R,AMODEO R,et al.Micro RNA mi R-24 promotes cell proliferation by targeting the CDKs inhibitors p27Kip1 and p16INK4a[J].Journal of Cellular Physiology,2013,228(10):2015-2023.
[19]CORY S,HUANG D C,ADAMS J M.The Bcl-2 family:roles in cell survival and oncogenesis[J].Oncogene,2003,22(53):8590-8607.
[20]JIANG Y J,POLINA I,JIN J L,et al.FXR inhibits gankyrin in mouse livers and prevents development of liver cancer[J].Hepatology,2013,57(3):1098-1106.
[21]ZHANG J,YANG Y.Gankyrin plays an essential role in estrogen-driven and GPR30-mediated endometrial carcinoma cell proliferation via the PTEN/PI3K/AKT signaling pathway[J].Cancer Letters,2013,339(2):279-287.
[22]YOKOI S,YASUI K,IIZASA T,et al.Down-regulation of SKP2 induces apoptosis in lung-cancer cells[J].Cancer Science,2003,94(4):344-349.
[23]PONZIELLI R,KATZ S,BARSYTE L D,et al.Cancer therapeutics:targeting the dark side of Myc[J].European Journal of Cancer,2005,41(16):2485-2501.
[24]CORY S,ADAMS J M.Killing cancer cells by flipping the Bcl-2/Baxswitch[J].Cancer Cell,2005,8(1):5-6.
[25]RUBIO M F,BLESA D,MESTRE C,et al.Characterization of 8p21.3 chromosomal deletions in B-cell lymphoma:TRAIL-R1and TRAIL-R2 as candidate dosage-dependent tumor suppressor genes[J].Blood,2005,106(9):3214-3222.
[26]DUMON K R,ISHII H,VECCHIONE A,et al.Fragile histidine triad expression delays tumor development and induces apoptosis in human pancreatic cancer[J].Cancer Research,2001,61(12):4827-4836.
[27]METE F,KILIC E,SOMAY A,et al.Effects of heat stress on endocrine functions&behaviour in the pre-pubertal rat[J].Indian Journal of Medical Research,2012,135(2):233-236.