| Mycotoxin (MYO) belongs to toxical and harmful secondary metabolites of mold or fungi. In the soil, on plants, grains, forage and silage can be found mycotoxins. Mycotoxins can formate in crop harvest, mycotoxin or post-harvest crop formation. About 25% grain to suffer from various mold contaminations in the world each year. Because of different levels of pollution or regional disparities, China is one of the hardest hit of the mycotoxin contamination.Deoxynivalenol (DON) also known as vomitoxin, it can bring great harm to human health. DON is mainly produced by Fusarium species of bacteria, chemical structure and biological activity of toxic metabolites-the single trichothecenes compounds. The DON comes from producing strains of Fusarium graminearum(Fusarium graminearum). It is reported that there are some other Fusarium can produce. DON belongs to highly poison. DON could accumulate in the human body and it has strong cytotoxicity, no specific target organ. People and livestock intake of DON contamination of food/feed will lead to anorexia, vomiting, diarrhea, fever, staggering and unresponsive symptoms of acute poisoning. And it could damage on the hematopoietic system resulting in death. But, different animals have different sensitivities to DON, the pig is the most sensitive animal to DON. Other researching also illustrated that DON may affect the immune system, and it has embryotoxicity, teratogenesis or genotoxicity. But, it has no Carcinogenic or mutagenic effects.Currently, in many food safty and basic toxicology studies, DON can detect very low levels in many foods. For instance, barley and wheat products can be detected in the 1 ppm DON content, DON content of 10 ppm can be detected in 4-month-old chicken, pork can be detected in 5 ppm content and alcohol could be detected in the DON. One hundred ng/mL of DON can induce cell viability decreasing, overexpression of inflammatory substances and cell apoptosis. Meantime, low dose of DON also made micronucleus increasing and chromosome damage in V79 cells.1000 mg/kg of DON can induce skeletal deformity in fetal rat and chicken or can induce derm cells decreasing and seformity. In sum, the different dose of DON can lead damage on animal organs or cells. Especially, DON can induce number of germ cells decreasing and cell viability decreasing. These data indicated that DON has potential cytotoxicity and genotoxicity. However, literatures on human primary cells genotoxicity or toxicity of molecular toxicology from DON are still limited.Therefore, our research aims that human peripheral blood lymphocytes were used as experimental subjects for observing cytotoxicity and genotoxicity from DON. And we use molecular biotechnology to illuminate molecular level studies on the cytotoxic and genotoxic.Part. IThe basis of toxicological studies of DON on human peripheral blood lymphocytesObjective: This study aims human peripheral blood lymphocytes as experimental subject to observe that DON inhibited cell vability, DON broken DNA strand (s) and DON induced instability of chromosome.Methods:This study used a panel of classic methods to prove cyototoxicity and genotoxicity in human lymphocytes. (1) Human blood sample collected from 60 healthy people. Sixty volunteeres have to write the imformed consents including:genetic diseases, infectious diseases, smoking or alcoholism etc. (2) The different blood samples were extracted by Ficoll. The lymphocytes used PRMI 1640 resuspended. The cell density was cultured into 24,72 and 96 wells. (3) The cell viability checked by CCK-8 assay. (4) The DNA strand (s) damage illustrated by single cell gel electrophoresis (Comet assay). (5) Micronucleus in lymphocytes was demonstrated by cytochalasin-B block assay. (6) DON induced chromosome damage was illuminated by sister chromatid exchange method.Results:(1) DON can induce obviously cytotoxicity in human lymphocytes, the cell viability clearly decreasing in DON groups (98.07±0.10%,0 ng/mL DON vs 14.03±1.78 %500 ng/mL,24 h). The IC50 values in two genders are 103.7±2.83 ng/mL for males and 101.4±3.15 ng/mL for females. (2) In the comet assay, DON can siginificantly induce DNA damage in human lymphocytes. Compared with negative control group, the values of Tail length, Tail DNA% and Tail moment were obviously increasing in DON groups (P< 0.01). (3) In micronucleus assay, the micronuleus in DON groups were obviously more than negative control group (62±13.01/30 cells,0 ng/mL DON vs 206.17±18.40/30 cells,50 ng/mL DON,24 h, P< 0.01). This result could depicted that DON can stagnate genome duplication or can induce genomic instability. (4). DON can break chromosome strctures in S phase. The chromosomes were missing or deformity. The deformity number of chromosome increasing showed as a clearly time-dose dependency (62±13.01/30 cells,0 ng/mL DON vs 206.17±18.40/30 cells,50 ng/mL DON,24 h, P< 0.01).Conclusion:(1) Human lymphocytes exposure in DON that obviously induced DNA damage, micronucleus number increasing and chromosome deformity increasing. These toxical effects indicated DON had potential genotoxicity in human pymphocytes. (2) DON obviously inhibited cell viability in lymphocytes and samples of two genders have no differences. The cytotoxicity has a siginificant relationship of time and dose dependency.Part. IIDON induced oxidative stress in human peripheral blood lymphocytesObjective:Due to DON contains hydroxyl group that can produce free radicals when exposed in vitro. These free radicals probably related with genotoxicity. Therefore, oxidative stress is important pathway for researching on DNA damage and chromosome damage.Methods:(1) The different blood samples collected from healthy persons (n=36). Human lymphocytes samples exposed in DON with 0,6.25,12.5,25 and 50 ng/mL for 6, 12 and 24 h. (2) The lipids peroxidation checked by binding adducts of thiobarbituric acid (TBA) and malondialdehyde (MDA) in boiling water. Then, levels of TBA-MDA adducts demonstrated by high performance liquid chromatography. The mitomycin served as positive control group (1μg/mL). (3) The levels of GSH and GSSG were checked by high performance liquid chromatography-electrospray ionization mass spectrometry. (4) The ROS increasing levels were demonstrated by DCFH-DA fluorescent probe and characterizations of ROS in lymphocytes were illustrated by laser scanning confocal microscope. (5) Annexin V-FITC and flow cytometry were used to illustrate symmetry of the double layer of phospholipids and liquidity. (6) The 8-Hydroxy-2'-deoxyguanosine is maker of oxidative DNA damage that was checked by ELISA method for evaluating DNA damage.Results:(1) The human pymphocytes exposed in DON with 6,12 and 24 h that induced MDA levels significantly higher than negative control group (19.64 nmol/mL,0 ng/mL DON vs 110.44±10.81 nmol/mL,50 ng/mL DON,24 h, P< 0.01). And MDA levels in positive group also were higher than negative control group (19.64 nmol/mL,0 ng/mL DON vs 171.64±13.38 nmol/mL,1μg/mL/Mitomycin C,24 h, P< 0.01). This result indicated that DON could produce free radical leading membrane to peroxidation. (2) The human lymphocytes exposed in DON with five concentrations for three times that induced GSH clearly decreasing (104.84±7.46 nmol/mg protein,0 ng/mL DON vs 32.66±2.02 nmol/mg protein,50 ng/mL DON,24 h, P<0.01) and GSSG elevating compared with negative control group (53.1±5.06 nmol/mg protein,50 ng/mL DON vs 2.98±0.20 nmol/mg protein,0 ng/mL DON,24 h, P<0.01). This result indicated that DON produced many free radicals leading depletion of antioxidants. (3) The human lymphocytes cultured with DON for several doses and three times that induced intensity of ROS fluorescent probe higher than negative control group (3.68±0.18%,0 ng/mL DON vs 69.83±4.26%, 50 ng/mL DON,24 h, P<0.01). Meantime, in the figures of laser scanning confocal microscope, ROS fluorescent probes were pretty brightness in DON group and the lymphocytes became to deformity. (4) The contents of 8-OHdG demonstrated as a time-dependent and dose-dependent. In DON groups, the contents of 8-OHdG were gradually increasing compared with negative control group in 6,12 and 24 h (27.30±2.18 pg/mL,0 ng/mL DON vs 410.35±12.88 pg/mL,50 ng/mL DON,24 h, P< 0.01).Conclusions:(1) DON could induce membrane to lipids peroxdation and gradually impair capacity of antioxidase by free radicals increasing. These results indicated that DNA danmage and chromosome instability of human lymphocytes were probably related with oxidative stress. (2) Combining basis toxicological results and onxidative stress, these data depicted that deformity of membrane, cell viability dereasing and cell necrosis increasing were related with free radicals increasing. DON has potential cytotoxicity for human lymphocytes. (3) The DON could induce 8-OHdG increasing and the micronucleus or chromosomes damage also were increasing. These phenomenons indicated that DON induced cytotoxicity and genotoxicity in human lymphocytes cause DNA oxidative damamge (DNA oxidative adducts).Part.ⅢMolecular toxicology studies of DON induced to cytotoxicity and genotoxicity in human peripheral blood lymphocytesObjective:Molecular biology techniques were used as researching method that human lymphocytes exposed with DON as an in vitro model for further investigating cytotoxicity and genotoxicity in cells.Methods:(1) The different samples collected from healthy people who did not receive any drug therapy, genetic diseases and infectious diseases (n=36). Human lymphocytes cultured DON with five concentrations (0,6.25,12.5 and 50 ng/mL) and 6,12 and 24 h. (2) The DNA repairing genes mRNA expressions were checked by RT-PCR method after exposed sereval doses DON and three times. DNA repairing genes include PARP1, PARP2, RAD51, APE-1, ERCC-1, HO-1, hOGG1, PCNA, XRCC1, XRCC3, XRCC4 and TP53. (3) The protein expressions of hOGG-1 and XRCC-1 in human lymphocytes in one DON dose (12.5 ng/mL) and three times were checked by Western-Blot method. (4) The protein expression of HO-1 with one DON dose (12.5 ng/mL) and three times in human lymphocytes was checked by Western-Blot method. Results:(1) The mRNA related expressions of DNA repairing genes and HO-1 in 6 (P <0.01) or 12 h (P<0.05) were higher than negative control group. However, the mRNA related expressions of DNA repairing genes and HO-1 in the 24 h were no significantly difference with negative control group. (2) The hOGG-1 and XRCC-1 related protein levels were higher than negative control group in 6 (P<0.01) or 12 h (P<0.05). But, the hOGG-1 and XRCC-1 related protein levels were without significant difference with 0 ng/mL when DON exposed in 12 or 24 h. (3) The HO-1 related protein levels was higher than negative control group in 6 h (P< 0.05). Nevertheless, the HO-1 related protein levels were without significant difference with 0 ng/mL when DON exposed in 12 or 24 h.Conclusions:(1) The mRNA expressions of DNA repairing genes were up-regulation in 6 h and were down-regulation in 12 or 24 h. These results indicated that DNA repairing efficiency gradually was lower than the rate of DNA damage by doses and time increasing. DON could enhance DNA repairing ability in short time and inhibit with time prolonging, and showed a time -dependent and dose-dependent. (2) The results of Western-Blot indicated that DON stimulated HO-1,hOGG-1 and XRCC-1 espression peak of protein in 6 h and inhibited expression in 12 or 24 h. These results depicted an important molecular mechanism of the genotoxicity that DON induced oxidative stress indirectly HO-1 expression to inhibit expression of hOGG-1 and XRCC-1.
|
PDF Full Text Request