Protective Effect of alpha-Tocopherol Against Ochratoxin A in Kidney Cell Line HK-2.

Food safety is a top priority for the protection of infants and young children. Ochratoxin A (OTA) is an emerging concern due to its high toxicity and occurrence in a wide range of agricultural crops and their derived food products including those foods and snacks destined for infants and young children. OTA is considered as a possible human carcinogen, and its main target organ is the kidney. The objective of this study was to investigate the protective effect of α-tocopherol against oxidative stress induced by OTA using human proximal tubule epithelial cells (HK-2). OTA showed dose-dependent increase in cytotoxicity (IC50 = 161 nM, p < 0.05) at 48 h, while treatment up to 2 mM α-tocopherol did not change cell viability. Levels of the reduced form of glutathione (GSH) were decreased with α-tocopherol treatment, although the ratio of the oxidative form (GSSG) to GSH remained the same. Among several genes associated with oxidative stress, expression of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione reductase (GSR), and kidney injury molecule-1 (KIM-1) were significantly up-regulated by OTA treatment. CAT and GSR showed decreased expression at 0.5-2 mM α-tocopherol and OTA at IC50 value, KIM-1 was decreased at 0.5 mM α-tocopherol and OTA at IC50 value, and nuclear factor erythroid 2-related factor 2 (Nrf2) was decreased at 0.5-1 mM α-tocopherol and OTA at IC50 value. In addition, the levels of malondialdehyde (MDA) were increased significantly by OTA while significantly decreased by α-tocopherol. The results show that α-tocopherol may alleviate potential OTA-induced renal damage and oxidative stress through reducing cytotoxicity and enhancing the antioxidant defense systems.

Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress.

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

Reduction of Ochratoxin A during the Preparation of Porridge with Sodium Bicarbonate and Fructose.

Ochratoxin A (OTA) is a potential human carcinogen that poses a significant concern in food safety and public health. OTA has been found in a wide variety of agricultural commodities, including cereal grains. This study investigated the reduction of OTA during the preparation of rice- and oat-based porridge by a simulated indirect steam process. The effects of sodium bicarbonate (NaHCO3) and fructose on the reduction of OTA were also investigated. During the processing, OTA in rice- and oat-porridge was decreased by 59% and 14%, respectively, from initial OTA artificially added at 20 µg/kg (dry weight basis). When 0.5% and 1% of sodium bicarbonate were added to rice porridge, increased reduction of OTA was observed as 78% and 68%, respectively. The same amounts of added sodium bicarbonate also further reduced OTA in oat porridge to 58% and 72%, respectively. In addition, increased reduction of OTA in the presence of fructose was observed. A combination of the two, i.e., 0.5% sodium bicarbonate and 0.5% fructose, resulted in a 79% and 67% reduction in rice porridge and oat porridge, respectively. These results indicate that indirect steaming may effectively reduce OTA in preparation of porridge-type products, particularly when sodium bicarbonate and/or fructose are added.

Ochratoxin A Induces Oxidative Stress in HepG2 Cells by Impairing the Gene Expression of Antioxidant Enzymes.

Ochratoxin A (OTA) is a mycotoxin frequently found in raw and processed foods. While it is considered a possible human carcinogen, the mechanism of action remains unclear. OTA has been shown to be hepatotoxic in both in vitro and in vivo models and oxidative stress may be one of the factors contributing to its toxicity. Hence, the effect of OTA on human hepatocellular carcinoma, HepG2 cells, was investigated on oxidative stress parameters. The cytotoxicity of OTA on HepG2 was time- and dose-dependent within a range between 0.1 and 10 µM; while 100 µM of OTA increased the intracellular reactive oxygen species (ROS) in a time-dependent manner. Additionally, the levels of glutathione (GSH) were increased by 9.7% and 11.3% at 10 and 100 nM of OTA, respectively; while OTA at 100 µM depleted GSH by 40.5% after 24 h exposure compared with the control. Finally, the mRNA level of catalase (CAT) was downregulated by 2.33-, 1.92-, and 1.82-fold after cells were treated with 1, 10, and 10 µM OTA for 24 h, respectively; which was linked to a decrease in CAT enzymatic activity. These results suggest that oxidative stress is involved in OTA-mediated toxicity in HepG2 cells.

Ochratoxin A-Induced Hepatotoxicity through Phase I and Phase II Reactions Regulated by AhR in Liver Cells.

Ochratoxin A (OTA) is a widespread mycotoxin produced by several species of the genera Aspergillus and Penicillium. OTA exists in a variety of foods, including rice, oats, and coffee and is hepatotoxic, with a similar mode of action as aflatoxin B1. The precise mechanism of cytotoxicity is not yet known, but oxidative damage is suspected to contribute to its cytotoxic effects. In this study, human hepatocyte HepG2 cells were treated with various concentrations of OTA (5-500 nM) for 48 h. OTA triggered oxidative stress as demonstrated by glutathione depletion and increased reactive oxygen species, malondialdehyde level, and nitric oxide production. Apoptosis was observed with 500 nM OTA treatment. OTA increased both the mRNA and protein expression of phase I and II enzymes. The same results were observed in an in vivo study using ICR mice. Furthermore, the relationship between phase I and II enzymes was demonstrated by the knockdown of the aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) with siRNA. Taken together, our results show that OTA induces oxidative stress through the phase I reaction regulated by AhR and induces apoptosis, and that the phase II reaction is activated by Nrf2 in the presence of oxidative stress.

Renal toxicity through AhR, PXR, and Nrf2 signaling pathway activation of ochratoxin A-induced oxidative stress in kidney cells.

Because ochratoxin A (OTA) is widely found in foods, people are susceptible to OTA exposure. The mechanism leading to renal toxicity induced by OTA remains unclear. The aim of this study was to investigate OTA-induced toxicity in human proximal tubule HK-2 cells. OTA decreased cell viability, and the expression of kidney injury molecule-1 (KIM-1), a kidney damage marker, was increased when HK-2 cells were exposed to OTA. Additionally, OTA treatment of cells increased intracellular reactive oxygen species and malondialdehyde and decreased glutathione levels. OTA-treated cells induced the aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) genes followed by induction of the cytochrome P450 1A1 (CYP1A1), CYP1A2, and CYP3A4 genes representing phase I enzyme. The mRNA expression of phase II enzymes such as heme oxygenase-1, nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase 1, and glutamate cysteine ligase catalytic subunit were upregulated by activation of NF-E2-related factor 2 (Nrf2) translocation by treatment with OTA. The response of OTA-orally administered mice also showed marked increases in these enzymes as well as KIM-1. These results indicate that OTA induces phase I and II enzymes through the AhR, PXR, and Nrf2 signaling pathways in HK-2 cells, which may lead to modulation of proximal tubule injury.

Reduction of Ochratoxin A in Oat Flakes by Twin-Screw Extrusion Processing.

Ochratoxin A (OTA) is one of the most important mycotoxins owing to its widespread occurrence and toxicity, including nephrotoxicity and potential carcinogenicity to humans. OTA has been detected in a wide range of agricultural commodities, including cereal grains and their processed products. In particular, oat-based products show a higher incidence and level of contamination. Extrusion cooking is widely used in the manufacturing of breakfast cereals and snacks and may reduce mycotoxins to varying degrees. Hence, the effects of extrusion cooking on the stability of OTA in spiked (100 µg/kg) oat flake was investigated by using a laboratory-scale twin-screw extruder with a central composite design. Factors examined were moisture content (20, 25, and 30% dry weight basis), temperature (140, 160, and 180°C), screw speed (150, 200, and 250 rpm), and die size (1.5, 2, and 3 mm). Both nonextruded and extruded samples were analyzed for reductions of OTA by high-performance liquid chromatography, coupled with fluorescence detection. The percentage of reductions in OTA in the contaminated oat flakes upon extrusion processing were in the range of 0 to 28%. OTA was partially stable during extrusion, with only screw speed and die size having significant effect on reduction (P < 0.005). 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.

Reduction of Fumonisin Toxicity by Extrusion and Nixtamalization (Alkaline Cooking).

Fumonisins are mycotoxins found in corn. They are toxic to animals and cause cancer in rodents and neural tube defects in LM/Bc mice. Reducing their concentrations in corn-based foods is therefore desirable. Chemical analysis or in vitro bioassays of food extracts might not detect toxic fumonisin reaction products that are unknown or unextractable from food matrices, thus potentially underestimating in vivo toxicity. The effectiveness of two common cooking methods, extrusion and nixtamalization (alkaline cooking), to reduce the toxicity of fumonisin-contaminated corn grits (extrusion) and whole kernel corn (nixtamalization) was shown by means of rat feeding bioassays using fumonisin-specific kidney effects as indicators of potential toxicity. A third bioassay showed that in contrast to fumonisin B1 (FB1), hydrolyzed fumonisin B1 (HFB1; formed from FB1 during nixtamalization) did not cause neural tube defects in LM/Bc mice. The findings indicate that extrusion and nixtamalization reduce the potential toxicity of FB1-contaminated corn.

Occurrence of Ochratoxin A in Infant Foods in the United States.

Ochratoxin A (OTA) is a possible human carcinogen and occurs frequently in cereal grain, soy, and other agricultural commodities. Infants and young children may be more susceptible to contaminants than adults because of their lower body weight, higher metabolic rate, reduced ability to detoxify food toxicants, and more restricted diet. The purpose of this study was to investigate the occurrence and levels of OTA in infant formula and infant cereal products available in the U.S. market. In the present study, 98 powdered infant formula (milk- and soy-based) samples and 155 infant cereal (barley-, rice-, oat-, wheat-, and mixed grain-based) products were collected from different retail locations in the United States over a 2-year period. OTA levels were determined by liquid chromatography-tandem mass spectrometry. Although OTA was not detected in any of the infant formula samples, 47 (30%) of 155 infant cereals were contaminated with OTA in the range of 0.6 to 22.1 ng/g. At present, there is no regulatory limit for OTA in the United States. However, all of the positive samples were above the maximum level set by the European Commission (0.5 ng/g) for OTA in baby foods. OTA was detected in all types of infant cereals, but the highest incidence and concentrations were found in oat-based infant cereals (59%), followed by mixed grain cereals (34%). Increased surveillance and monitoring of OTA levels in grains used in infant foods may be needed to reduce exposure of infants and young children to OTA from cereal products.

Polyvinylpolypyrrolidone reduces cross-reactions between antibodies and phenolic compounds in an enzyme-linked immunosorbent assay for the detection of ochratoxin A.

Ochratoxin A (OTA) is a fungal metabolite and putative carcinogen which can contaminate a variety of foods such as cereals, wine, and nuts. Commercial ELISA kits are known to give false-positive results for OTA concentrations when phenolic compounds are present. Pistachios represent a food matrix rich in phenolic compounds potentially contaminated with OTA, and were used to model OTA cross-reactivity. Polyvinylpolypyrrolidone (PVPP) was incorporated during extraction of OTA using a commercial ELISA protocol. HPLC methods were used to confirm that PVPP does not interact with OTA and levels of gallic acid and catechin remaining in pistachio extracts decreased with increasing PVPP application. Cross-reactivity of extracts also decreased with increasing PVPP application, and color loss was used as an indicator of anthocyanin removal. Incorporating PVPP into ELISA protocols allows for the continued use of rapid immunological methods in food matrices containing phenolic compounds.

Heat Stability of Ochratoxin A in an Aqueous Buffered Model System.

Ochratoxin A (OTA) represents one of the most widespread mycotoxins in agricultural commodities in the world and is considered a possible human carcinogen with its potent nephrotoxicity. OTA is stable under most food processing conditions; however, higher-temperature treatment may reduce OTA content in foods. Since OTA can be found in processed products destined for both human and animal consumption, factors affecting its stability or reduction during thermal processes were investigated here. 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 high-performance liquid chromatography with fluorescence detection. 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, significant reductions of OTA were not shown.

Extrusion cooking with glucose supplementation of fumonisin-contaminated corn grits protects against nephrotoxicity and disrupted sphingolipid metabolism in rats.

SCOPE: Fumonisin B1 (FB1) is a mycotoxin found in maize and maize-based foods. It causes animal diseases and is a suspected risk factor for cancer and birth defects in humans. Extrusion cooking reduces FB1 concentrations in maize however toxicity caused by unknown degradation or FB1-matrix reaction products might persist. METHODS AND RESULTS: To test the efficacy of extrusion to reduce FB1 toxicity, Fusarium verticillioides fermented corn (= maize) grits (Batch-1= 9.7 ppm FB1; Batch-2= 50 ppm FB1) were extruded without (Batch-1E; Batch-2E) or with 10% glucose supplementation (Batch-1EG; Batch-2EG). FB1 concentrations were reduced 64% (Batch-2E) to 94% (Batch-1EG) after cooking. When the uncooked and processed grits were fed (50% w/w in rodent chow) to rats for up to 8 weeks, FB1 intakes averaged 354, 103, and 25.1 çg/kg body weight/day for Batch-1, Batch-1E and Batch-1EG and 1804, 698, and 222 çg/kg body weight/day for the Batch-2, Batch-2E and Batch-2EG, respectively. Nephrotoxicity including apoptotic lesions and elevated sphingoid base concentrations decreased in a dose-dependent manner in groups fed Batch-1, Batch-1E, Batch-2, Batch-2E, or Batch-2EG and was absent in the Batch-1EG group. CONCLUSION: Extrusion cooking, especially with glucose supplementation, is potentially useful to reduce FB1 concentrations and toxicity of FB1-contaminated maize.

Reduction of fumonisin B1 in corn grits by twin-screw extrusion.

UNLABELLED: This study was designed to investigate the fate of fumonisins in flaking corn grits during twin-screw extrusion by measuring fumonisin B1 (FB1) and its analogs with a mass balance approach. Food grade corn grits and 2 batches of grits contaminated with FB1 at 10 and 50 µg/g by Fusarium verticillioides M-2552 were processed with or without glucose supplementation (10%, w/w) with a twin-screw extruder. Extrusion reduced FB1 in contaminated grits by 64% to 72% without glucose and 89% to 94% with added glucose. In addition, extrusion alone resulted in 26% to 73% reduction in the levels of fumonisin B2 and fumonisin B3, while levels of both mycotoxins were reduced by >89% in extruded corn grits containing 10% glucose. Mass balance analysis showed that 38% to 46% of the FB1 species detected in corn extruded with glucose was N-(deoxy-D-fructos-1-yl)-FB1, while 23% to 37% of FB1 species detected in extruded corn grits with and without added glucose was bound to the matrix. It was also found that the hydrolyzed form of FB1 was a minor species in extruded corn grits with or without added glucose, representing <15% of the total FB1 species present. Less than 46% of FB1 originally present in corn grits could be detected in the fumonisin analogues measured in this study. Research is needed to identify the reaction products resulting from extrusion processing of fumonisin-contaminated corn products. PRACTICAL APPLICATION: Twin-screw extrusion is widely used in food industry for its versatility. This technology may reduce the level of fumonisins in corn particularly with added glucose.

Antibacterial effects of long-chain polyphosphates on selected spoilage and pathogenic bacteria.

The antimicrobial activities of four long-chain food-grade polyphosphates were studied at concentrations allowed in the food industry (<5,000 ppm) in defined basal media by determining the inhibition of growth of three gram-negative and four gram-positive spoilage and pathogenic bacteria. Both generation time and lag phase of Escherichia coli K-12, E. coli O157: H7, and Salmonella Typhimurium were increased with all of the polyphosphates tested. Bacillus subtilis and Staphylococcus aureus were more sensitive to polyphosphates, but not in all cases, with multiphased growth. The growth of Lactobacillus plantarum was inhibited by polyphosphates at concentrations above 750 ppm, but the lag time of Listeria monocytogenes was shortened by the presence of polyphosphates. No single polyphosphate was maximally inhibitory against all bacteria. Polyphosphates with chain lengths of 12 to 15 were significantly different from those with chain lengths of 18 to 21 depending on the organism and concentrations of polyphosphate used. Overall, higher polyphosphate concentrations resulted in greater inhibition of bacterial growth.