Analysis Of Ochratoxin A Contamination In Grains From Different Areas Of Hebei Province And Study Of The Cellular Injury Effects Of Ochratoxin A

Ochratoxins (OT)is the toxic metabolites of Asperegillus alutacells, Penicillium viridicatum etc, including 7 structurally similar compounds (Ochratoxin A, B, C, D, etc). Ochratoxin A (OA) is the most common and toxic one among the 7 Ochratoxins. Chemically, OA is a colorless crystal with molecular weight of 404, fusion point of 169℃, solvable in both organic solvents (chloroform and methanol) and NaHCO3 solution, while minimally solvable in water. Under ultraviolet exposure, OA shows green fluorescence with peak absorption wavelength at 333nm. Being quite stable in nature, OA could only be partly destroyed during cooking and grain processing. Animal experiment showed that OA was of nephrotoxicity, immunotoxicity and was carcinogenic, teratogenic and mutagenic. In 1993, International Cancer Research Center anounced OA was a possible human carcinogen. OA was detected in human blood and milk in some countries. Studies showed that the main route for human OA exposure was via grain contamination of OA. Thus, the evaluation of the contamination of OA in the grain is very important for food safety and human health. The detection and monitoring of OA contamination in the foodstuff and grains have been widely reported abroad. Several countries have set the maximal limitation for human OA intake. Up to now, few works have been done in the evaluation of OA contamination in grain and foodstuffs and the background of OA contamination in grains and human exposure is still not clear enough in our country. The methods used currently for OA analysis in grain and foodstuff include thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), enzyme-linked immunosorbnent assay (ELISA), etc. The authorized method in Examination Methods of Ochratoxin A(OA) in Grains and Beans (NSSn GB/T 13111-1991) issued by China authority in 1991 is thin layer chromatography(TLC), which is a semi-quantitative method with relatively poor accuracy, the lowest detectable limit is only 10μg/kg. Thus,it is not a reliable analysis method for the accurate evaluation of human exposure to OA via food intake.In order to accurately evaluate the contamination of OA in the grain and other foodstuffs in our country and to further explore the putative biological significance of OA contamination, we conducted the following studies. The method for OA analysis was improved and a new sensitive, repeatable and quantitative HPLC analysis method of OA was established. The contamination of OA in different grains from three different geographic areas in Hebei province was analyzed with HPLC method. At the same time, the effects of OA on the proliferation and apoptosis of human peripheral blood mononuclear cells in vitro were studied with cell culture, FCM methods. The injury effects of OA on human kidney tubular epithelial cell line- HKC and human lung AT-II cell line -A549 was also studied. On the basis of the above study, the putative role of JNK pathway on the apoptosis of HKC induced by OA was explored at protein level with immunocytochemical method, Western Blot, etc.The study included five parts:1 Establishment of method for the analysis of ochratoxin AObjective: To establish a sensitive method of high-performance liquid chromatography (HPLC) for the analysis of ochratoxin A in grains and wines.Methods: The wheat and barley samples for determination of OA were first extracted with chloroform. OA was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 333 nm, emission wavelength 460 nm) using acetonitrile-water-acetic acid (99:99:2) as mobile phase; 30 ml wine samples were recovered from activated C18 column with 2ml methy1 cyanide and 2ml water (250mg/3ml). The flowing velocity was 30drops/min. OA was eluted with methanol, dryed by nitrogen gas at 45℃and quantified by HPLC with fluorometric detection using mobile phase with 20μl OA samples. 2 Detection of the contamination of OA in grains from three different geographic areas in Hebei provinceObjective: To analyze OA contamination in grains from three different geographic areas in Hebei province and to evaluate the OA exposure level of local residents in Hebei province.Method: Grain samples were randomly taken from household in the south, middle and north part of in Hebei province with different geographic feature. Wheat samples were taken from Cixian County in the south Hebei province and Zanhuang County in the middle part of Hebei province while the samples of barley were taken from Cicheng County in the north of Hebei province with quite cold climate. The species of grain sample was selected on the bases of the amount of consumption. OA was exacted with chloroform from wheat and barley, eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 333 nm, emission wavelength 460 nm) using acetonitrile-water-acetic acid (99:99:2) as mobile phase.Results: Reversed-phase HPLC analysis showed that of the 31 barley samples of Chicheng county, OA could be detected in 11 (35.5%) with the mean content 6.96ug/kg. The highest contamination level reached 35.36ug/kg. The positive detection rates of ochratoxin A, the mean content and highest contents in wheat samples from Zanhuang country and Cixian county were 45.16%, 2.41μg/kg, 14.25μg/kg and 33.33%, 0.59μg/kg, 1.63μg/kg, respectively.3 Studies of the effects of Ochratoxin A on the apoptosis and proliferation of human peripheral blood mononuclear cells in vitroObjective: To explore the effects of Ochratoxin A (OA) on the apoptosis and proliferation of human peripheral blood mononuclear cells in vitro and to evaluate the putative effects of OA exposure on human immune system.Methods: Human Peripheral blood mononuclear cells (HPBMc), isolated from 200ml heparinic acid antiagglutinated fresh venous blood of healthy Donors by Ficolly-Hypaque density gradient centrifugation, were initially cultured in RPMI 1640 medium supplemented with 10% new-born calf serum (NBCS), streptomycin (100μg/mL), penicillin (100U/mL) and phytohemagglutinin (PHA, 30μg/mL) at 37℃, 5% CO₂ for 24h. After initial culture, the medium was replaced by new medium without PHA and then the flasks were randomly divided into 4 groups: control, solvent control, OA 1μmol/L and OA 5μmol/L. OA was added to the medium at final concentrations of 1μmol/L and OA 5μmol/L in OA treated groups, alcohol was added to the medium of solvent control group and saline was added to the medium of control group. The cells were cultured for 24 h after treatment and harvested for detection. The cultured cells were centrifuged and fixed in 70% ethanol for preparation of single cell suspension and used for flow cytometric analysis of apoptosis rates, proliferation index (PI) and the expression of Caspase-3.Results: FCM analysis showed that the apoptosis rates of HPBMc in 1μmol/L and 5μmol/L OA-treated groups were 10.90±0.85% and 16.03±0.37%, respectively, which were significantly higher than that in control group (8.17±1.45%,P<0.05). No significant difference in PI value was seen between OA-treated groups and control group (35.77±4.74% and 36.89±7.63% vs 33.53±1.97%,P>0.05). The results suggest OA may induce apoptosis of HPBMc, but have no effect on the proliferation of HPBMc.FI of Caspase-3 protein expression in HPBMc treated with 1μmol/L and 5μmol/L OA for 24 h was 1.13±0.02 and 1.14±0.06 respectively, which was significantly higher than that in control group (1.00±0.04,P < 0.05). The results indicated that exposure of OA could increase the expression of Caspase-3 protein in HPBMc in vitro.4 Effects of Ochratoxin A on the cell injury of A549 and HKC cells in vitroObjective: To explore the effects and mechanism of Ochratoxin A (OA) on the apoptosis and proliferation of A549 and HKC cells and the effects on the injury of DNA induced by OA.Methods: A549 and HKC cells were harvested, centrifuged and resuspended in DMEM medium supplemented with 10% FCS at the concentration of (1²)×10⁸ cells·L^-1. Flasks of the two cell lines were both randomly divided into 4 groups: control, solvent control, OA 1μmol/L and OA 5μmol/L. A549 and HKC cells in OA groups were treated with OA at the final concentration of 1μmol/L and 5μmol/L, respectively, while these in solvent control and control group were incubated with DMSO and saline, respectively. The cells were cultured for 24h after treatment and harvested for detection. Cell viability for A549 cells was assessed with MTT assay. Then A549 and HKC cells were centrifuged and fixed in 70% ethanol for preparation of single cell suspension respectively and used for flow cytometric analysis of apoptosis rates, proliferation index (PI) and the expression of Caspase-3. The salmine DNA and calf thymus DNA was coadminstrated with different concentrations of OA, DNA-adduct was detected by the Ultraviolet spectra and Fluorescence spectra.Results: MTT assay showed that 24h after OA treatment, the survival rates of A549 cells in OA 1μmol/L and OA 5μmol/L groups were 80.71% and 70.85% ,respectively, significantly lower than that in control group (100%,P<0.05), while the survival rates of HKC cells in OA 1μmol/L and OA 5μmol/L groups were also significantly lower than that in control group( 51.09% and 54.69% vs 100%, P<0.05).The results showed that OA could cause proliferation inhibition and injuries in A549 and HKC cells. FCM analysis showed that the apoptosis rate of A549 cells in 5μmol/L OA-treated groups was significantly higher than that in control group (2.68±0.55% vs 1.53±0.23%,P<0.05). The apoptosis rates of HKC cells in 1μmol/L and 5μmol/L OA-treated groups were 3.85±0.29% and 4.86±0.51%, respectively, which were significantly higher than that in control group(1.23±0.05%,P<0.05). The results showed that OA could induce apoptosis of A549 and HKC cells. A significant increase in Caspase-3 protein expression of A549 and HKC cells after OA treatment revealed that OA could increase the expression of Caspase-3 protein to modulate the apoptosis of A549 and HKC cells. NO significant DNA-adduct was found in the mixture of DNA and OA measured by the Ultraviolet spectra and Fluorescence spectra. 5 Effects of JNK signaling pathways on the apoptosis of HKC cells induced by OAObjective: To explore the effects of JNK signaling pathways on the apoptosis of HKC cells induced by OA.Methods:5.1 Determination of the effects on activation of JNK and the expression of Caspase-3 protein in HKC cells treated with OA for 24 hThe culture, grouping and treatment of HKC were the same as that described in part 4 of the study. The HKC cells in OA groups were treated with OA for 24h. The cells were cultured for 24h after treatment and harvested for further study. The phospho-JNK (p-JNK) levels and the Caspase-3 protein expression were determined by immunocytochemical staining and Western blot.5.2 The effects of JNK inhibitor, SP600125 on OA induced activation of JNK, apoptosis, proliferation inhibition of HKC cells in vitroHKC cells seeded at (1²)×10⁸ cells·L^-1 in culture flasks were randomly divided into 4 groups: control, solvent control, JNK inhibitor pretreatment and OA 1μM mg·L^-1. 24h later, the cells in JNK inhibitor group were pretreated for 30 min with 0.5μM SP600125. Then the cells in JNK inhibitor group and AFG1 group were treated with AFG₁ 1.0 mg·L^-1, while these in solvent control and control groups were incubated with alcohol and saline respectively. The cells were cultured for 24 h after treatment and harvested for further analysis. The phospho-JNK (p-JNK) levels were detected by Western blot. The apoptosis rates and the expression of Caspase-3 were determined by FCM. Cell viability for HKC cells was assessed with MTT assay.Results:5.1 Effects of OA on the expression of Caspase-3 protein in HKC cellsThe results of immunocytochemical staining showed the expression of Caspase-3 in 1μM OA and 5μM OA group was 54.03±9.21% and 60.75±9.95%, both were significantly higher than that in control group (14.31±4.24%,P<0.05). Western blot results showed the expression of Caspase-3 in 1μM OA and 5μM OA group was increased in HKC cells treated with OA.5.2 Effects of OA on the phospho-JNK levels of HKC cellsImmunocytochemical staining results showed that p-JNK level in 1μM OA and 5μM OA group was significantly higher than that in control group (53.31±11.01% and 58.53±11.11% vs 10.19±6.85%,P<0.05). Western blot results showed p-JNK level in 1μM OA and 5μM OA group was increased in HKC cells treated with OA. The results suggest that OA could cause the activation of JNK in HKC cells.5.3 Effects of OA on the expression of JNK protein in HKC cellsNo difference in the expression of JNK among OA treatment groups and the control group was found by Western blot and immunocytochemical staining.5.4 Effects of SP600125 pretreatment on OA induced increase of the p-JNK levels in HKC cellsImmunocytochemical staining and Western blot showed that pretreatment of OA cell with 0.5μM SP600125 could significantly decrease the relative p-JNK level as compared with that of OA treatment alone. The effect of activation of JNK in OA-treated cells was inhibited in the presence of SP600125.5.5 Effects of SP600125 pretreatment on OA induced apoptosis in HKC cellsFCM analysis showed apoptosis rate of JNK inhibitor group was significantly lower than that of OA group (2.44±0.38% vs 4.24±0.38%, P<0.05) and higher than that of control group (2.44±0.38% vs 1.06±0.14%, P<0.05). The results showed that SP600125 pre-treatment might partly inhibit the apoptosis of cultured HKC induced by OA in vitro.5.6 Effects of SP600125 pretreatment on OA induced changes in the expression of Caspase-3 at protein level in HKC cellsImmunocytochemical staining results showed the expression of Caspase-3 in JNK inhibitor group was lower than that in 1μM OA group (31.42±4.67% vs 55.86±6.51%, P<0.05), and higher than that in control group (31.42±4.67% vs13.97±1.59%, P<0.05). Similar to the results with immunocytochemical methods, Western blot also confirmed activation of the JNK pathway in OA-treated cells was involved in the modulation of Caspase-3.5.7 Effects of SP600125 pretreatment on OA induced survival rate inhibition of HKC cellsThe survival rates of OA treated HKC cells pretreated with 0.5μM SP600 125 were 82.09%, which significantly higher than that in only OA treated group (66.83%,P<0.05), but lower than that in control group (98.58%,P<0.05). The results suggest the pretreatment with low concentration of SP600125 may partly reduce the cell injury of OA and the activation of JNK pathway in OA-treated cells may partly modulate the OA related cell injury.Conclusion:1 A sensitive, repeatable and quantitative reversed-phrase HPLC method for the analysis of OA in grain was established. The method was successfully used in the detection of OA contamination in wine and grains.2 Analysis of the contamination of OA in the consumption grains in three different geographic areas of Hebei province showed that OA contamination rate in the wheat samples from south and middle part of Hebei province reached 39.34% and that of barley from north of Hebei province was 35.48%, which were all significantly higher that reported in our country. Bases on the contents of OA contamination, the daily OA exposure level for the residents in south, middle and north part of Hebei province was 0.31μg/ kg, 1.17μg/ kg and 3.38μg/ kg respectively, significantly over the permitted intake limit by FAO/WHO JECFA.3 OA could induce apoptosis and increase the expression of Caspase-3 protein of HPBMc in vitro, but have no effect on the proliferation of the cells.4 OA could cause proliferation inhibition and injuries in A549 and HKC cells, and increase the expression of Caspase-3 protein and induce apoptosis of A549 and HKC cells.5 OA could increase the level of p-JNK and cause the activation of JNK.6 JNK inhibitor, SP600125 pretreatment could significantly decrease the apoptosis, up-regulation of Caspase-3 and cell injury caused by OA. The results revealed that JNK pathway may partly be involved in the injury of HKC by OA.