Progress 11/01/15 to 10/31/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students, one visiting scholar, and one undergraduate student were trained to conduct processing studies and analyze OTA in processed oat and rice samples. One graduate student was trained to analyze OTA degradation products in processed samples. Two graduate and one undergraduate students were trained to conduct bioassays. How have the results been disseminated to communities of interest?The results have been disseminated by papers published in the peer-reviewed journals and presentations at the professional meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 3: Determination of residual toxicity of OTA and its degradation products in the infant cereals by rat feeding trials. We have completed the toxicity study of OTA and its degradation products in the infant cereals using in vitro and in vivo models. To determine the toxicity mechanism of OTA in kidney, HK-2 (human kidney cell line) and LLC-PK1 (porcine kidney cell line) as their sensitivity to OTA has been well documented/studied, were hired. We investigated the cytotoxicity effect of OTA on these cell lines using MTT methods, and found a dose- (up to 5 μM) and time- (24hr, 48 hr, and 72 hr) dependence decrease in cell viability. Furthermore, we found that OTA isable to increase intracellular ROS in a time-dependent manner. Additionally, OTA depleted GSH at longer exposure time (> 48 hr) in HK-2 but not in LLC-PK1 cells. Finally, in LLC-PK1 the mRNA levels of G6PD and GPX1 were down-regulated by 0.67- and 0.66-fold, respectively, after 72 hr exposure to OTA. In contrast, CAT, GSR and SOD were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, in HK-2 after 72 hr exposure to OTA. Based on these data, we concluded that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA may be involved in the development of oxidative stress in HK-2 as indicated by changes on the parameters evaluated. As OTA is also known to have hepatotoxicity, we conducted the experiment using HepG2 (human liver cell line) to confirm the oxidative stress induced by OTA. We investigated the cytotoxicity effect of OTA, and found a time- (24 hr, 48 hr, and 72 hr), but not dose-dependent (up to 100 μM) decrease in cell viability. We found that OTA, only at 100 μMwas able to slightly increase the intracellular reactive oxygen species (ROS) concentration in a time-dependent manner. Additionally, at 10 and 100 nM, OTA increased the levels of glutathione (GSH), (9.7% and 11.3%, respectively), while 100 μM depleted GSH by 40.5% after 24 h exposure.Finally, the mRNA level of catalase (CAT) was down-regulated by 2.33-, 1.92-, and 1.82-folds after cells were treated with 1, 10 and 10 μM OTA for 24 h, respectively, which was linked to a decrease in CAT activity. Based on the data, we concluded that oxidative stress is involved in OTA-mediated cytotoxicity in HepG2 cells. While we planned to determine the toxicity of major OTA thermal degradation products such as OTA isomer, OTα, and OTα-amide, they were detected only trace amount in the samples we processed. Nonetheless, we confirmed those OTA thermal degradation products in thermal treated cereal samples using synthesized standards since those OTA thermal degradation products except OTα were not commercially available. To conduct animal studies, batches ofoats were cultured withP. verrucisum until OTA levels reached around 100 mg/kg (DW). Cultured oat grains containing OTA were mixed with clean oat grains to provide a moderately to severely toxic dose of OTA, and then was added to the basal feed (50:50 w/w in the diet). Mice were allocated to control groups and treated groups. Control groups are untreated control (maintained using basal feed without any treatment) and material control (prepared from cleaned oats, added to the diet at the same level used for treated groups). For the OTA treated groups, a daily dose of OTA from 0.2 to 4 mg/kg body weight, were administered to experimental animals. Some randomly selected animals per group were sacrificed at 4 weeks and 8 weeks. At the end of the experimental period the mice were fasted overnight and sacrificed under isoflurane anesthesia.We collected the blood and organs including liver and kidney.We are currently analyzing gene expression in liver and kidney and the pathology/morphology of the affected organs. At the end of the 4- or 8-weeks period, no evidence of renal or hepatic damage was observed when organ weights and serum biochemical parameters were analyzed. However, OTA significantly modulated the mRNA expression levels of catalase (CAT), glutathione peroxidase 1 (GPX1), superoxide dismutase 1 (SOD1), glutathione reductase (GSR), and hexose-6-phosphate dehydrogenase (H6PD) in both the kidney and liver of OTA-treated mice. In the kidney, CAT, GPX1 and GSR mRNA levels were down-regulated after 4 weeks while SOD1 and H6PD mRNA levels were up-regulated. Except for CAT, the mRNA levels of the other enzymes remained down-regulated in the kidney after 8 weeks. In the liver, all the enzymes mRNA levels were down-regulated after 4 weeks but normalized after 8 weeks. Therefore, the reduction of cellular antioxidant defenses may be involved in the nephrotoxicity and hepatotoxicity of OTA in male ICR mice. To evaluate the residual toxicity of OTA by extrusion processing, we conducted another ICR mice feeding study.The extruded and non-extruded (control) samples were mixed to the basal feed (50:50, w/w in the diet). The OTA concentration in extruded oats (cultured with P. verrucosum) was higher than that of non-extruded cultured oats. When OTA was added to clean oats, however, OTA concentration was reduced by the same extrusion process, suggesting bound form of OTA may exist in cultured oats. These results also suggest that some OTA may be trapped or bound with fiber of oats while P. verrucossum produce OTA in oats. Additional work was conducted - 8 wk ICR male mice were fed with cultured oats (PG, equivalent to 3 mg OTA/kg body weight) or extruded-cultured oats (EG). Other groups served as control groups (normal control, non-processed oat control, processed oat control), and fed the basal diet and non-processed/processed oats from sound oats using the same dosing scheme for 6 wk. Significantly lower body weight was observed in PG and EG from the first week with increased weight of liver and spleen and decreased weight of kidney in PG compared with control groups. Meanwhile, decreased liver and spleen weight of EG was observed. The histopathologic changes in the liver and kidney tissues of mice were examined using H&E staining.There were no significant changed in the kidney tissues of the control groups while significant histopathologic changes were found in the liver tissues.Macro steatosis and severe inflammation near central vein in liver tissue of PG were observed, and the severity of the lesions in liver tissue was significantly reduced in EG indicating reduced toxicity of OTA by extrusion processing. We performed one more experiment to determine the residual toxicity of OTA using ICR mice feeding study and cultured and extruded rice samples. Because when we repeated same food processing using P. verrucosum cultured rice, OTA concentration in extruded P. verrucosum cultured rice determined OTA reduction compared with nonextruded P. verrucosum cultured rice. The feed was made the sample way with the oat samples and fed to 8 wk male ICR mice for 6 wk. Significant decrease in body weights and kidney weights were observed with dose-dependent manner. ALT, AST, and BUN in blood also were decreased dose-dependent manner. According to histopathology with H&E staining, lympho-monocyte infiltration and severe inflammation near central vein in liver tissue of high dose (3.5 mg/kg) of extruded rice group were observed, while those finding were reduced at low dose (2.5 mg/kg) of extruded rice group. Moreover, based on histopathology with PAS staining for kidney damage, the damage degree was increased by dose. These results suggest that dietary OTA exposure can cause oxidative stress in the liver and kidney. Reduced toxicity of OTA was observed in the thermally processed samples. It should also be noted that masked form of OTA may be present in oat contaminated by P. verrucosum.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
H.J. Lee, S. Kim, H.J. Suh, and D. Ryu. 2019. Effects of explosive puffing process on the reduction of ochratoxin A in rice and oats. Food Control 95:334-338.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
D. Ryu, R.J. Kowalski, G. Ganjyal, and H.J. Lee. 2019. Reduction of ochratoxin A in oats and rice by twin-screw extrusion processing with baking soda. Food Control 105:21-28.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
H.J. Lee, C. Lee, and D. Ryu. 2020. Effects of baking soda and fructose in reduction of ochratoxin A in rice and oat porridge during retorting process. Food Control 116:107325
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
H.J. Lee. 2020. Stability of ochratoxin A in oats during roasting with reducing sugar. Food Control 118:107382
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
H.J. Lee, S. Lee, K. Gu, D. Ryu. 2021. Reduction of ochratoxin A during the preparation of porridge with sodium bicarbonate and fructose. Mycotoxin Research
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
H.J. Lee, K. Gu, D. Ryu, H.J. Lee. 2021. Ochratoxin A and its reaction products affected by sugars during heat processing. Food Chemistry 348, 129038
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
H.J. Lee, D. Ryu. Optimization of enzyme assisted extraction of masked form of Ochratoxin A from Penicillium verrucosum cultured oats. J. Agric. Food Chem. (In preparation)
- Type:
Journal Articles
Status:
Other
Year Published:
2021
Citation:
H.J. Lee, E.G. Perez, and D. Ryu. Oxidative stress induced by Ochratoxin A in kidney an in vitro and in vivo study. Food & Chem. Toxicol. (In preparation)
|
Progress 11/01/18 to 10/31/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate and one undergraduate studentswere trained to conduct processing studies and analyze OTA in processed oat samples as well as bioassays. How have the results been disseminated to communities of interest?The results were disseminated via peer reviewed paper published in scientific journal. What do you plan to do during the next reporting period to accomplish the goals?We are planning to complete optimization and verification of methods for detecting and quantifying analogs of OTA in OTA producer cultured grains to obtain reliable data using new extraction method with HPLC and LC-MS/MS. At the same time, we are going to finish analyzing animal organs and evaluate the reduced toxicity by processing for Objective 3. And then, we are going to submit/publish obtained data to a peer reviewed journal.
Impacts
What was accomplished under these goals?
Objective 1 - We conducted indirect steaming and direct steaming to optimize processing conditions to maximize the reduction of OTA in infant cereals. In addition, we added two additives, such as baking soda and fructose, to enhance OTA reduction during food processing. Roasting - Moisture content of oat grains were adjusted to 16% (wet weight basis, wb) and OTA was spiked at 100 µg/kg. And then the grains were roasted at 120°C and 180°C for 30 min and 60 min. The reduction of OTA was observed in the ranges of 2 - 18% and the OTA reduction was increased with increasing roasting temperature and time. Greater reduction of OTA was observed when oat grains were roasted with reducing sugars, i.e. glucose (11%) and fructose (15%), compared with oat-based cereal samples with no added sugar (10%). Retorting - In this study, the effects of retorting process on the stability of OTA in spiked (20 μg/kg of dry weight basis) rice and oat porridge (10% solid content; w/v) in the absence and presence of baking soda (0.5% and 1%; w/w), fructose (0.5% and 5%; w/w), or combination of them (baking soda 0.5% + fructose 0.5%; w/w) were investigated using a laboratory horizontal steam retort system. The reduction of OTA in retorted rice and oat porridge were 53.8% and 17.2%, respectively, while greater reduction of OTA was observed at increased amount of baking soda. The reduction of OTA in retorted rice porridge with 0.5% and 1% baking soda were 55.5% and 66.4%, respectively. In the retorted oat porridge, reduction of OTA was also evident to result in 30.3% and 47.9% with 0.5% and 1.0% of added baking soda, respectively. While greater reduction of OTA in oat porridge was observed by adding fructose (40.8% and 35.5%, respectively), decreased reduction of OTA in rice porridge was observed at increased amount of fructose (38.7% and 18.2%, respectively). The reduction of OTA in retorted rice and oat porridges with combination 0.5% baking soda and 0.5% fructose were 35.8% and 39.8%, respectively. Objective 3 - To conduct animal studies, batches of naturally contaminated oats grain were first obtained by culturing Penicillum verrucosum in clean oat grains until OTA levels reached around 100 mg/kg. For the culture, 1 kg of oat grains were weighed into a gallon size glass jars, autoclaved for 1 hour at 121°C. The autoclaved oat grains were inoculated with P. verrucosum spores with autoclave water to adjust moisture content (about 20-22% MC), and then incubated at 25°C the desired OTA concentration is achieved. Cultured oat grains were autoclaved and stored at -20°C until used for analysis. OTA contaminated and OTA non-contaminated oat were extruded to preparing animal feed. Cultured oat grains containing OTA were mixed with clean oat grains to provide a moderately to severely toxic doses of OTA, and then was added to the basal feed (50:50 w/w in the diet). 8-weeks old ICR male mice were used for this study and treated for 8 weeks with OTA contaminated or OTA non-contaminated diet. The animals were sacrificed, and blood, kidney, and liver were harvested for further analysis. Body weight and food intake were measured every week and there were no significant differences in feed intake between groups. The significant difference in body weight were observed between OTA contaminated diet treated groups and OTA non-contaminated diet treated groups but no significant difference among OTA contaminated diet treated groups or OTA non-contaminated diet treated groups. We measured several organs such as lung, heart, liver, spleen, testis, epididymis+fat, and kidney to evaluated toxicity and no significant difference was observed except kidney. We are currently analyzing serum biochemical parameters, mRNA levels in kidney and liver, and histopathology slides of kidney and liver.
Publications
|
Progress 11/01/17 to 10/31/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student and visiting scholar were trained to conduct processing studies and analyze OTA in processed oat samples. How have the results been disseminated to communities of interest?The results were disseminated via published paper. What do you plan to do during the next reporting period to accomplish the goals?We are planning conduct and complete optimization and verification of methods for detecting and quantifying "Masked form of OTA" in OTA producer cultured grains to obtain reliable data using new extraction method with HPLC and LC-MS/MS, and we are planning to complete it in 6 months. And then, we are going to do animal study to determine the reduced toxicity effect by processing for Objective 3 in 1 year.
Impacts
What was accomplished under these goals?
Objective 1 - We conducted indirect steaming and direct steaming to optimize processing conditions to maximize the reduction of OTA in infant cereals. In addition, we added two additives, such as baking soda and fructose, to enhance OTA reduction during food processing. Our previous study confirmed that OTA is very heat stable, particularly under acidic and neutral conditions, as no significant reduction of OTA was observed after heating at 100°C for 60 min. Under alkaline conditions (pH 10), however, resulted in about 50% reduction of OTA. In the same study, a substantial reduction of OTA was observed at temperatures above 150°C Lee, et al. (2017). Therefore, certain food processes using high temperature with alkaline condition may warrant reduction of OTA during processing. So we decided to use baking soda to elevate the pH during processing. Sugar - To study the formation of OTA artifacts and the binding of OTA to matrix components such as saccharides in thermal-treated food, model experiment was performed. OTA in water was heated at 200°C with glucose, fructose, and sucrose (mono- and disaccharide models). Reduction of OTA was carried out using HPLC. Among three different sugar types, only fructose showed synergetic effect with thermal treatment on reduction of OTA. We developed HPLC method for analyzing OTA thermal degradation products such as OTA-isomer, OTα, and OTα-amide. We observed different pattern in forming OTA thermal degradation products by different sugars with OTA standard solution. Especially, more OTα-amide was formed when OTA standard solution was heated with fructose compare to glucose, sucrose, or just DI water, and more OTα was formed at lower concentration of fructose (1 mg/mL) compare to 3 and 5 mg/mL of fructose. We are currently figuring out the exact mechanism of it, but assuming that it is because of OTA is more stable at lower pH from higher fructose concentration. And we demonstrated that the presence of fructose may not big change the reduction of OTA but at lease it can affect formation of OTA thermal degradation products. In other word, fructose can affect the kind of OTA thermal degradation products formed nontoxic instead of toxic. When OTA standard solution heated in water, the major OTA thermal degradation products was OTA-isomer which is known to have similar toxicity as OTA, while OTA standard solution heated with fructose, the major OTA thermal degradation products was OTα-amide which is virtually nontoxic. Indirect steaming - Oat flour at 5% solid content was used in this study. We used the pilot scale steam jacketed kettle as well as smaller lab scale with tow pots. For both set up, we heated till the temperature at the center reaches 85% then kept for 10 min. While indirect steaming of oat flours alone reduced OTA by 14%, the reduction of OTA increased significantly in oat flour and topped around 73% when baking soda was added (0.5% and 1%, w/w of oat flour). And then, we tested varying concentrations of fructose from 0.5% to 10% (w/w of oat flour) during indirect steaming of oat flour as we observed the highest reduction of OTA (69%) was at 1% of fructose. Direct steaming - In this study, the effect of direct steam injection (DSI) process along with the absence and presence baking soda (0.5% and 1% of solid contents) on the reduction of OTA in oat-based infant cereals was investigated. The solid contents were adjusted to 5% (wet weight basis, wb) and OTA was spiked to 100 μg/kg. Then the 5% solids content oat flour porridge were processed by direct steam injection at 85°C and 121°C and then spray dried at 210oC. Reduction of OTA in DSI processed oat-based infant cereals increased with increasing process temperature to result in 20-28% reduction. In addition, the reduction of OTA increased with increasing amounts of baking soda. The reduction of OTA by added 1% of fructose was not significant. Objective 2 & Objective 3 - To conduct animal studies, batches of naturally contaminated oats grain were first obtained by culturing Penicillum verrucosum in clean oat grains until OTA levels reached around 100 mg/kg. For the culture, 1 kg of oat grains were weighed into a gallon size glass jars, autoclaved for 1 hour at 121°C. The autoclaved oat grains were inoculated with P. verrucosum spores, and then incubated at 25°C the desired OTA concentration is achieved. Cultured oat grains were autoclaved and stored at -20°C until used for analysis. We tried to prepare processed oat samples containing OTA to feed animals and determined OTA concentration in extruded P. verrucosum cultured oats was increased compared with nonextruded P. verrucosum cultured oats while OTA spiked oats showed OTA reduction by extrusion. When we repeated same food processing using P. verrucosum cultured rice, OTA concentration in extruded P. verrucosum cultured rice determined OTA reduction compared with nonextruded P. verrucosum cultured Rice. We are currently figuring out how the matter stands, but we are assuming that some of OTA was trapped or bound with fiber of oats while P. verrucosum produced OTA in oats. Therefore, we are going to extract "Masked form of OTA" by cellulase enzyme treatment. And then, optimization and verification of methods for detecting and quantifying "Masked form of OTA" in OTA producer cultured grains to obtain reliable data will be conducted and completed using HPLC and LC-MS/MS.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
HJ Lee, BJ Gu, G Ganjyal, D Ryu, 2018. Reduction of ochratoxin A in direct steam injected oat-based infant cereals with baking soda. Food Control. Food Control 96:441-444.
|
Progress 11/01/16 to 10/31/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One full-time graduate student was trained to analyze OTA in steamed or retorted oats, and two undergraduate students were trained to conduct bioassays. How have the results been disseminated to communities of interest?The results were disseminated via published paper and poster presentations. What do you plan to do during the next reporting period to accomplish the goals?We are planning to complete the processing studies followed by the determination of OTA reduction. The effects of additives on OTA reduction for Objective 1 will also be concluded in 6 months. And we will complete analyzing OTA thermal degradation products formed during the processing using HPLC or LC-MS/MS by next reporting period for Objective 2. We are going to analyze animal study data for Objective 3 in 6 months.
Impacts
What was accomplished under these goals?
Objective 1 - We conducted extrusion, indirect steaming, and direct steaming to optimize processing conditions to maximize the reduction of OTA in infant cereals. In addition, we added two additives to enhance OTA reduction during processing. Prior to processing, effect of common food additives, i.e. baking soda and sugar, that may affect the heat stability of OTA were tested in an aqueous buffered model system. Since OTA is stable under most food processing conditions including higher-temperature, we tried to find factors affecting its stability or reduction during thermal processes. The reduction of OTA was measured during various heating times (up to 60 min) at different temperatures (100, 125, 150, 175, and 200°C) in aqueous buffer solutions at different pHs (pH 4, 7, and 10). Quantification of OTA was carried out using HPLC. The results showed that the rate and extent of OTA reduction were dependent on pH, processing time, and temperature; greater than 90% OTA reduction was achieved at 200°C for all treatments except pH 4. After processing under an alkaline condition (pH 10) at 100°C for 60 min, about 50% of the OTA was lost, while after 60 min under neutral and acidic conditions at 100°C. So we decided to use baking soda to elevate the pH during processing. Sugar - To study the formation of OTA artifacts and the binding of OTA to matrix components such as saccharides in thermal-treated food, model experiment was performed. OTA in water was heated at 200°C with glucose, fructose, and sucrose (mono- and disaccharide models). Reduction of OTA was carried out using HPLC. Among three different sugar types, only fructose showed synergetic effect with thermal treatment on reduction of OTA. We are currently underway to analyze the reaction products formed by LC-MS/MS. Extrusion - The effects of extrusion cooking on the stability of OTA in oat flake were investigated using a laboratory scale twin screw extruder with a central composite design. Factors examined were moisture content (20, 25, and 30% dry basis), temperature (140, 160, and 180°C), screw speed (150, 200, and 250 rpm) and die size (1.5, 2, and 3 mm). Both unextruded and extruded samples were analyzed for reductions of OTA by HPLC. The percent reductions of OTA in oat flakes artificially contaminated at 100 µg/kg were in the range of 0 - 28% depending on the combination of factors. OTA was very heat stable during extrusion; with only screw speed and die size having significant effect on reduction. The highest reduction of 28% was achieved at 180°C, 20% moisture, 250 rpm screw speed, and a 3-mm die with 193 kJ/kg specific mechanical energy. According to the central composite design analyses, up to 28% of OTA can be reduced by a combination of 162°C, 30% moisture, and 221 rpm, with a 3-mm die. We also added baking soda during extrusion processing with two different concentrations (0.5% and 1%, w/w). The reduction of OTA by added baking soda was not significant at 0.5% (36 - 40% of OTA reduction) while 3-7% additional reduction was observed at 1% of baking soda. We are planning to conduct OTA reduction effect with sugars, especially fructose. Direct steaming - Oat flour at moisture content of 13 % w.b. (wet basis) was used in this study. The experimental setup is composed from: a processing reactor, where the sample was treated at steam pressure/temperature, a vacuum system which comprises mainly a stainless steel vacuum tank, a vacuum pump, and a steam generator supplying steam into the reactor. The hydrothermal treatments were performed in pilot scale at fixed steam pressure (137°C, 35 psi). Oat flour with or without baking soda (1%, w/w) or fructose (10%, w/w) were processed without any hydration step followed by drying in oven at 50°C overnight. The OTA reduction by direct steaming showed around 75%. And the OTA reduction showed decreased values by adding baking soda (72%) and fructose (70%). Objective 2 - Optimization and verification of methods for detecting and quantifying OTA thermal degradation products in foods to obtain reliable data in varying types of food matrices were conducted and completed using HPLC and LC-MS/MS. Detection methods of HPLC and LC-MS/MS will be used depend on the type of matrices. Objective 3 - We conducted bioassay studies using cell culture and animal models to verify the range of toxicities and endpoints of OTA in the liver and kidney. In the cell culture studies, HepG2 (human origin liver hepatocellular cell), HK-2 (human origin kidney proximal tubule epithelial cell), and LLC-PK1 (porcine origin kidney proximal tubule epithelial cell) were used to investigate the OTA-mediated cytotoxicity and oxidative stress response. OTA exposure of these cells resulted on time or dose-dependent elevation of cytotoxicity. We also determined OTA is able to increase intracellular ROS in a time-dependent manner. GSH concentration was depleted by OTA in HK2 and HepG2, but not in LLC-PK1. In LLC-PK1, the mRNA levels of G6PD (glucose-6-phosphate-dehydrogenase) and GPX1 (glutathione peroxidase 1) were down-regulated by OTA treatment, but CAT (catalase), GSR (glutathione reductase) and SOD (superoxide dismutase) did not show differences between groups. In contrast, CAT, GSR and SOD were up regulated by OTA treatment in HK-2. However, the mRNA levels of G6PD and GPX1 were shown different pattern by OTA exposure time in HK-2. OTA was able to up-regulated the mRNA level of CAT, G6PD, GXP1, GSR, and SOD in a time-dependent manner in HepG2. To conduct animal studies, batches of naturally contaminated oats grain were first obtained by culturing Penicillum verrucosum in clean oat grains until OTA levels reached around 100 mg/kg. For the culture, 1 kg of oat grains were weighed into a gallon size glass jars, autoclaved for 1 hour at 121°C. The autoclaved oat grains were inoculated with P. verrucosum spores, and then incubated at 25°C the desired OTA concentration is achieved. Cultured oat grains were autoclaved and stored at -20°C until used for animal diet. Cultured oat grains containing OTA were mixed with clean oat grains to provide a moderately to severely toxic doses of OTA, and then was added to the basal feed (50:50 w/w in the diet). The prepared diets were stored frozen and dispensed fresh 2-3 times/week. For this study, 11 weeks old male ICR mice were purchased from Envigo. Mice were allocated to control groups and treated groups. Control groups are untreated control (maintained using basal feed without any treatment) and material control (prepared from cleaned oats, added to the diet at the same level used for treated groups). For the OTA treated groups, a daily dose of OTA from 0.2 to 4 mg/kg body weight, were administered to experimental animals. Some randomly selected animals per group were sacrificed at 4 weeks and 8 weeks. At the end of the experimental period the mice were fasted overnight and sacrificed under isoflurane anesthesia. We collected the blood and organs including liver and kidney. At the end of the 4- or 8-weeks period, no evidence of renal or hepatic damage was observed when organ weights and serum biochemical parameters were analyzed. However, OTA significantly modulated the mRNA expression levels of CAT, GPX1, SOD1, GSR, and H6PD (hexose-6-phosphate dehydrogenase) in both the kidney and liver of OTA-treated mice. In the kidney, CAT, GPX1 and GSR mRNA levels were down-regulated after 4 weeks while SOD1 and H6PD mRNA levels were up-regulated. Except for CAT, the mRNA levels of the other enzymes remained down-regulated in the kidney after 8 weeks. In the liver, mRNA levels of all the enzymes were down-regulated after 4 weeks. We are currently analyzing the pathology/morphology of the affected organs.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
HJ Lee, S Dahal, EG Perez, RJ Kowalski, R Ganjyal, D Ryu. 2017. Reduction of ochratoxin A in oat flakes by twin-screw extrusion processing. J. Food Prot. 80(10): 1628-1634.
|
Progress 11/01/15 to 10/31/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two full time graduate students were trained to analyze OTA in extruded oats and to conduct bioassays. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We are planning to complete analyzing cell culture data and animal study data to development of bioassay designs for Objective 3 in 6 months.
Impacts
What was accomplished under these goals?
Objective 1 - The effects of extrusion coking on the stability of OTA in spike (100 µg/kg) oat flake were investigated using a laboratory scale twin screw extruder with a central composite design. Factors examined were moisture content (20, 25, and 30% dry basis), temperature (140, 160, and 180°C), screw speed (150, 200, and 250 rpm) and die size (1.5, 2, and 3 mm). Both unextruded and extruded samples were analyzed for reductions of OTA by high performance liquid chromatography coupled with fluorescence detection. The percent reductions of OTA in the contaminated oat flakes upon extrusion processing were in the range of 0 - 30% depending on the combination of factors. OTA was very heat stable during extrusion; with only screw speed and die size having significant effect on reduction. Objective 3 - We conducted bioassay studies using cell culture and animal models to verify the range of toxicities and endpoints of OTA in the liver and kidney. (i) In the cell culture studies, HepG2 (human origin liver hepatocellular cell), HK-2 (human origin kidney proximal tubule epithelial cell), and LLC-PK1 (porcine origin kidney proximal tubule epithelial cell) were used to investigate the OTA-mediated cytotoxicity and oxidative stress response. OTA exposure of these cells resulted on a concentration-dependent elevation of cytotoxicity. There are interesting differences on cell cytotoxicity between cell lines. We are currently underway to analyze OTA-mediated oxidative stress response using ROS generation, GSH concentration, and gene expression of anti-oxidative enzymes. ii) The objective of this animal study was to validate the in vivo bioassay system. Batches of naturally contaminated oats grain were obtained by culturing Penicillum verrucosum in clean oat grains until OTA levels reached around 100 mg/kg. For the culture, 1 kg of oat grains were weighed into a gallon size glass jars, autoclaved for 1 hours at 121°C. The autoclaved oat grains were inoculated with P. verrucosum spores, and then incubated at 25°C. The production of OTA was monitored by HPLC analysis, and cultured were allowed to proceed until the desired OTA concentration is achieved. Cultured oat grains were autoclaved and stored at -20°C until used for animal diet. Cultured oat grains containing OTA were mixed with clean oat grains to provide a moderately to severely toxic dose of OTA, and then was added to the basal feed (50:50 w/w in the diet). The prepared diets were stored frozen and dispensed fresh 2 - 3 times/week. For this study, 11 weeks old male ICR mice were purchased from Envigo. Mice were allocated to control groups and treated groups. Control groups are untreated control (maintained using basal feed without any treatment) and material control (prepared from cleaned oats, added to the diet at the same level used for treated groups). For the OTA treated groups, a daily dose of OTA from 0.2 to 4 mg/kg body weight, were administered to experimental animals. Some randomly selected animals per group were sacrificed at 4 weeks, 8 weeks, and 12 weeks. At the end of the experimental period the mice were fasted overnight and sacrificed under isoflurane anesthesia. We collected the blood and organs including liver and kidney. We are currently analyzing gene expression in liver and kidney and the pathology/morphology of the affected organs.
Publications
|
|