Oxidative stress as a mechanism of combined OTA and CTN toxicity in rat plasma,liver and kidney

Abstract

Ochratoxin A (OTA) and citrinin (CTN) commonly coexist in grains. Aiming to evaluate oxidative stress in OTA + CTN toxicity, male Wistar rats were orally treated with two doses of OTA (0.125 and 0.250 mg kg⁻¹ of body weight (b.w.)), CTN (2 mg kg⁻¹ of b.w.) and resveratrol (RSV; 20 mg kg⁻¹ of b.w.) and combined daily during 3 weeks. Protein carbonyl concentrations were measured in kidneys and liver; catalytic activity of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) level in plasma, kidneys and liver, while malondialdehyde (MDA) concentration was measured in plasma, kidneys, liver and urine. Mycotoxin treatment significantly increased MDA concentration in plasma and kidney and decreased SOD activity in the liver. Rats treated with CTN and OTA125 + CTN had lower plasma GPx activity. Concentration of GSH in the kidney and protein carbonyls in the kidney and liver as well as GPx activity in the kidney and liver, SOD activity in the kidney and CAT activity in the liver were not affected. Protective effect of RSV was observed on GSH in the kidney and plasma and MDA in the kidney, plasma and urine. Oxidative stress is involved in OTA + CTN toxicity in vivo because such treatment affects parameters of oxidative stress, particularly in plasma. RSV can reduce but not overcome oxidative stress induced by combined OTA and CTN treatment.

Introduction

Nephrotoxic mycotoxins ochratoxin A (OTA) and citrinin (CTN) are commonly found together in grains in moderate climate regions. ¹ OTA is produced by several mould species of the genera Aspergillus, which mostly contaminate grapes, spices, coffee and cocoa and of Penicillium (P nordicum and P verrucosum) which contaminate cereals, ham, cheese and olives. ^2,3 Mould species of the genera Penicillium and Monascus produce CTN. P verrucosum is able to produce OTA and CTN. ⁴ Biosynthesis of OTA and CTN in P verrucosum is mutually regulated. Low sodium chloride levels and increased oxidative stress caused by blue light irradiation favours biosynthesis of CTN. ^2,3 The co-contamination with two or more mycotoxins was detected in 38% of samples on global level. ⁵ CTN concentration in cereals usually exceeds OTA concentrations. ⁶ Co-contamination with mycotoxins may change their adverse effects due to their interactions.

OTA and CTN are mycotoxins of similar structure, and it can be expected that the mode of their toxicity would be similar with additive toxic effect. Their combined toxicity was mostly studied in vitro, but the results were related to type of cells and their metabolism of xenobiotics. ^{7 –11}

The ultrastructural lesions in the kidneys of New Zealand white rabbits treated with OTA + CTN (0.75 and 15 mg kg⁻¹ feed, respectively) for 60 days were more severe than in animals treated with the same dose of single mycotoxin. ¹² In equally treated rabbits, malondialdehyde (MDA) concentration in the kidneys was significantly increased in OTA + CTN-treated animals compared to controls but not compared to animals given single mycotoxin. ¹³ In the kidneys of 6-week-old male dark Agouti rats treated with OTA and CTN (26 and 100 µg kg⁻¹ feed), it increased the major OTA-DNA adduct, indicating that CTN promotes their formation. ¹⁴

It is known that increased reactive oxygen species (ROS) is involved in the mechanism of OTA and CTN toxicity. ROS in organisms can be neutralized by a small thiol molecule – glutathione (GSH) and enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) ¹⁵ or by antioxidant food compounds. The protective effect of several antioxidants (vitamin E, lycopene, resveratrol (RSV), quercetin) against either OTA or CTN toxicity was studied in vitro. ^11,16,17 There are no studies on protective effect of antioxidants in cells exposed simultaneously to OTA and CTN.

Different antioxidants were used to protect from adverse OTA effects in rats. Vitamin E and selenium administration decreased liver γ-GT activity 24 h after a single OTA dose (2.5 mg kg⁻¹ of body weight (b.w.)). ¹⁸ Levels of lipid peroxidation and SOD activity in the kidneys and serum of rats were modified by melatonin as compared to OTA treatment. ¹⁹ However, there were no changes in activities in antioxidant enzymes and lipid peroxidation in rats’ liver. When OTA (0.5 mg kg⁻¹ of b.w.) was given to rats for 14 days, the DNA damage in lymphocytes, kidney and liver; the decrease of CAT activity and GSH level; and the increase of SOD and apoptosis in the kidney were reversed by lycopene treatment. ^20,21

In our previous experiment, which was the first on OTA and CTN toxicity in rats, oxidative stress as a mechanism of subchronic OTA and acute CTN toxicity was studied. Animals were treated with CTN alone (20 mg kg⁻¹) for 2 days, with OTA (0.125 and 0.250 mg kg⁻¹) for 21 days alone or with CTN (20 mg kg⁻¹) in the last 2 days of experiment. ²² Based on those results, another study presented here with subchronic treatment with both mycotoxins was performed. The same experimental schedule was applied for OTA, but the total CTN dose (40 mg kg⁻¹ of b.w.) was divided into 21 daily doses (2 mg kg⁻¹ of b.w.). Concentrations of MDA and GSH, as parameters of oxidative stress, were measured in both studies, but in the present study, oxidative stress parameters were amplified with the activities of SOD, GPx and CAT activity in plasma, kidney and liver tissues and the concentration of protein carbonyls in the kidneys and liver. In the present study, MDA concentration was also measured in urine. The possible protective effect of natural antioxidant RSV in the reduction of their toxicity when given together was also checked either in the first or in the present study because of the different CTN treatment and more measured parameters.

Materials and methods

Chemicals

Anaesthetics (Narketan and Xylapan) were purchased from Chassot AG (Bern, Switzerland). OTA, CTN, RSV, 1,1,3,3-tetramethoxypropane, butylated hydroxytoluene (BHT), 2-thiobarbituric acid (TBA), 5,5′-dithiobis-2-nitrobenzoate (DTNB), 2,4-dinitrophenylhydrazine (DNPH), guanidine hydrochloride, bovine serum albumin (BSA) and methanol were purchased from Sigma (St. Louis, Missouri, USA). Ultrapure water (18 MΩ) was obtained from a Milli-Q Gradient water system (Thermo Scientific Smart2pure 3 UV/UF, Langenselbold, Germany). Other chemicals and reagents were of analytical grade, and their commercial source was indicated under the description of specific methods.

Animals and treatment

Adult male Wistar rats (10 weeks old, 230–270 g of b.w. at the beginning of the experiment) were kept in Makrolon cages at controlled room temperature and day/night cycles (22°C and 12 h, respectively). Before and during experiments, animals had free access to standard pelleted food (4RF21 from Mucedola, Settimo Milanese, Italy) and tap water. Experiments were approved by the Ethic Committee of the Institute for Medical Research and Occupational Health in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU).

Animals were divided into groups (five animals per group) and treated 21 days with (1) water, (2) solvent (51 mM sodium bicarbonate (NaHCO₃)), (3) OTA (0.125 mg kg⁻¹ of b.w.), (4) OTA (0.250 mg kg⁻¹ of b.w.), (5) CTN (2 mg kg⁻¹ of b.w.), (6) OTA (0.125 mg kg⁻¹ of b.w.) + CTN (2 mg kg⁻¹ of b.w.), (7) OTA (0.250 mg kg⁻¹ of b.w.) + CTN (2 mg kg⁻¹ of b.w.) and (8) OTA (0.250 mg kg⁻¹ of b.w.) + CTN (2 mg kg⁻¹ of b.w.) + RSV (20 mg kg⁻¹ of b.w.).

Animals were placed in metabolic cages for 24 h (with free access to water and without access to food) on 14th day of the experiment to collect urine. OTA was dissolved in 51 mM NaHCO₃, CTN in 50 mM sodium carbonate and RSV in 5% ethanol and given by gavage every day between 8 am and 9 am.

Animals were killed under total anaesthesia by Narketan (80 mg kg⁻¹ of body mass (b.m.) and Xylapan (12 mg kg⁻¹ of b.m., intraperitoneal (i.p.)). Blood was taken from the jugular vein into heparinized tubes and centrifuged. Plasma and organs were frozen at −80°C until analysis.

GSH measurement

GSH was analysed using the method of Ellman. ²³ Kidney and liver homogenates (10%) were prepared in 0.3 M phosphate buffer (pH = 7.4) and centrifuged for 10 min at 3500 r min⁻¹. Proteins were removed by adding 100 µL of 5% trichloroacetic acid (TCA) to 300 µL of supernatant. The homogenates were then mixed and centrifuged for 10 min at 3500 r min⁻¹.

To 100 µL H₂O (blank solution), standards or samples (kidney and liver homogenates and plasma), 850 μL phosphate buffer and 50 μL DTNB were added. Absorbance of blank solution, standards and samples was measured spectrophotometrically (Cecil 9000; Cambridge, UK) at 412 nm. GSH concentration was calculated from the calibration curve of standards and expressed as nanomoles per gram of tissue or nanomoles per millilitre of plasma.

Protein carbonyls

Protein oxidation was measured in the kidneys and liver tissue homogenates by the method of Mercier et al. ²⁴ Briefly, proteins were precipitated in two aliquots by 10% TCA (w/v) and centrifuged at 2000g for 10 min. One pellet was treated with 1 mL of 2 M hydrochloric acid (HCl) and the other with an equal volume of 0.2% (w/v) DNPH. Samples were incubated for 1 h at room temperature and stirred regularly. The samples were again precipitated with 10% TCA (w/v), centrifuged and then washed twice with 1 mL of ethanol:ethyl acetate (1:1) to eliminate traces of DNPH and to make soluble residual lipids. Proteins were finally dissolved in 2 mL of 6 M guanidine hydrochloride and centrifuged to remove insoluble fragments. Protein concentration was measured at 280 nm using BSA as standard. Carbonyl concentration was measured at 370 nm using 22.0 L mmol⁻¹ cm⁻¹ as a molar absorption coefficient. The results were expressed as nanomoles of 2,4-DNPH bound per milligram of protein.

Enzyme activities

The enzyme activity assays were performed in the 96-well plates on the Tecan Infinite M200PRO plate reader, Tecan Austria GmbH (Grodig, Austria) and calculated from the calibration curve of standards using template from the Cayman website.

GPx activity

Enzyme activity of GPx was measured using GPx Assay Kit (Cayman Chemical Company, Ann Arbor, Michigan, USA) according to manufacturer’s instruction. Oxidized GSH, which is a product of reduction of hydroperoxides by GPx, is reduced to GSH by GSH reductase and NADPH which is oxidized to NADP⁺ is accompanied by a decrease in absorbance. GPx activity was measured every 30 s during 6 min at 340 nm, and the rate of absorbance decrease was directly proportional to the GPx activity in the sample. According to manufacturer’s instructions, kidney and liver tissues were homogenized in 50 mM Tris-HCl buffer, containing 5 mM EDTA and 1 mM DTT (pH 7.5). Homogenized tissues were centrifuged at 10,000g for 15 min at 4°C. Different dilutions were tested and 1:7, 1:30 and 1:80 dilutions were selected as optimal for plasma, kidney and liver tissues, respectively. The results were expressed as nanomoles per millilitre per minute for GPx activity in plasma and nanomoles per millilitre per milligram of proteins in the kidney and liver tissues.

SOD activity

SOD activity was measured using SOD Assay Kit (Cayman Chemical Company) according to manufacturer’s instruction. The SOD assay measures all three types of SOD (copper (Cu)/zinc (Zn), manganese and iron SOD) generated by xanthine oxidase and hypoxanthine using tetrazolium salt for detection. Kidney and liver homogenates were prepared in 20 mM HEPES buffer (pH 7.2), containing 1 mM EGTA, 210 mM mannitol and 70 mM sucrose. Homogenized tissues were centrifuged at 10,000g for 5 min at 4°C. Different plasma and tissue dilutions and absorbance at different wavelengths were examined. Optimal wavelength was 435 nm, and dilution for plasma, kidney and liver tissues was 1:5, 1:200 and 1:400, respectively. The results of SOD activity were expressed as units per millilitre in plasma and units per milligram of proteins in kidney and liver tissues.

CAT activity

CAT activity was measured using CAT Assay Kit (Cayman Chemical Company) according to the manufacturer instructions. The method is based on the reaction of the enzyme with methanol in the presence of hydrogen peroxide in the sample. The produced formaldehyde is measured colorimetrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole as the chromogen which upon oxidation changes from colourless to a purple colour. Kidney and liver tissues were homogenized in potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and centrifuged at 10,000g for 15 min at 4°C. Kidney and liver tissue samples were diluted at 1:600 and 1:1200. CAT concentration was measured at 540 nm. The results were expressed as nanomoles per millilitre per minute in plasma and micromoles per milligram of proteins in the kidneys and liver.

MDA measurement

Methods of MDA analysis in the kidneys, liver, plasma and urine were based on the method by Drury et al. ²⁵ Kidneys and liver homogenates (10%) were prepared in 0.3 M phosphate buffer (pH = 7.4). To 50 μL sample (plasma or 10% tissue homogenate) or standard (2.5 μM 1,1,3,3-tetraethoxy propane), 5 μL BHT (0.2%, w/v), 750 µL phosphoric acid (1%, v/v), 250 µL TBA (0.6%, w/v) and 445 µL water were added. To 600 μL of urine, 60 μL BHT (0.2%, w/v), 3 mL phosphoric acid (1%, v/v), 1 mL TBA (0.6%, w/v) and 340 µL water were added. Samples were mixed and incubated in a boiling water bath for 30 min.

The HPLC apparatus consisted of a degasser, isocratic pump, column oven and ultraviolet (UV) detector (Shimadzu Corporation, Kyoto, Japan). The guard and analytical columns were C-18 reverse-phase (Zorbax Eclipse Plus; Agilent) with 5 μm particles (4.6 × 12.5 and 4.6 × 100 mm, respectively). The mobile phase consisted of 50 mM monopotassium phosphate and methanol (60:40, v/v, pH 6.8). Analysis conditions were the injection volume 20 μm, flow rate 1 mL min⁻¹, absorbance at UV detector 532 nm and the temperature in the column oven 32°C. Under these conditions, the retention time of MDA was about 2.5 min. The MDA concentration was calculated by software from the calibration curve and expressed as nanomoles per gram of tissue or micromoles per millilitre.

Statistic

Data analysis and plotting were done using GraphPad Prism for Windows, version 5 and R statistical software, version 3.3.1. Values of all measured parameters are presented as the mean and standard deviations and were analysed using analysis of variance followed by Tukey’s multiple comparison test. ²⁶ All applied tests were two-tailed. The p values of less than or equal to 0.05 were considered statistically significant. ²⁷

Results

GSH

The effect of OTA, CTN and RSV treatment on the GSH concentration in the plasma, kidney and liver is shown in Figure 1(a) to (c), respectively. Liver GSH was not affected with the toxins treatment. In the kidney, GSH level was lower compared to control, but this was not statistically significant. Only in plasma higher OTA dose significantly increased GSH concentration (0.31 ± 0.02 nmol mL⁻¹) as compared to control (0.23 ± 0.03).

Figure 1.

GSH concentration in plasma (a), kidney (b) and liver (c) of rats treated with water, solvent, OTA 0.125 mg kg⁻¹ of b.w. (O125), OTA 0.250 mg kg⁻¹ of b.w. (O250), CTN 2 mg kg⁻¹ of b.w. (c) and combined treatment of both OTA doses with CTN (O125 + C and O250 + C) and higher OTA dose with CTN and RSV (20 mg kg⁻¹ of b.w.) (OCR). b: different from solvent, c: different from O125, d: different from O250, e: different from C, g: different from O250 + C, h: different from OCR (p < 0.05). GSH: glutathione; OTA; ochratoxin A; CTN: citrinin; RSV: resveratrol; b.w.: body weight.

RSV treatment significantly increased GSH concentration (3.43 ± 1.03 nmol g⁻¹ tissue) in the kidneys of animals treated with OTA (0.250 mg kg⁻¹ of b.w.) + CTN + RSV compared to animals treated with the same OTA dose given alone (1.82 ± 0.64) or with CTN (2.15 ± 0.43). RSV also increased GSH concentration in plasma of OCR-treated rats (0.32 ± 0.04 nmol mL⁻¹) compared to CTN-treated rats (0.25 ± 0.04).

Protein carbonyls

Mycotoxin treatment did not affect the concentration of protein carbonyls in kidney and liver tissues (Figure 2).

Figure 2.

Protein carbonyls in kidneys (a) and liver (b) of rats treated with water, solvent, OTA 0.125 mg kg⁻¹ of b.w. (O125), OTA 0.250 mg kg⁻¹ of b.w. (O250), CTN 2 mg kg⁻¹ of b.w. (C) and combined treatment of both OTA doses with CTN (O125 + C and O250 + C) and higher OTA dose with CTN and RSV (20 mg kg⁻¹ of b.w.) (OCR). OTA; ochratoxin A; CTN: citrinin; RSV: resveratrol; b.w.: body weight.

GPx

GPx activities (Figure 3) were significantly elevated in plasma of rats treated with solvent (3426.45 ± 352.18 nmol mL⁻¹ min⁻¹) compared to water-treated animals (2566.30 ± 207.65). Rats treated with CTN and OTA (0.125 mg kg⁻¹ of b.w.) + CTN had lower GPx activity in plasma (2963.25 ± 279.56 and 2580.22 ± 210.05, respectively) than rats treated with solvent. RSV treatment reduced GPx activity in plasma (2683.92 ± 294.04) compared to solvent-treated animals.

Figure 3.

Activity of GSH peroxidase in plasma (a), kidney (b) and liver (c) of rats treated with water, solvent, OTA 0.125 mg kg⁻¹ of b.w. (O125), OTA 0.250 mg kg⁻¹ of b.w. (O250), CTN 2 mg kg⁻¹ of b.w. (C) and combined treatment of both OTA doses with CTN (O125 + C and O250 + C) and higher OTA dose with CTN and RSV (20 mg kg⁻¹ of b.w.) (OCR). a: different from water, b: different from solvent. GSH: glutathione; OTA; ochratoxin A; CTN: citrinin; RSV: resveratrol; b.w.: body weight.

SOD

There was no difference in SOD activity in plasma of rats treated with single mycotoxins compared to water and solvent treatment (Figure 4). Combined treatment with OTA (both doses) and CTN caused a significant increase of SOD activity in plasma of treated rats (11.64 ± 0.94 and 12.39 ± 1.08 U mL⁻¹, respectively) compared to treatment with water (7.71 ± 0.71) and solvent (7.63 ± 0.80). In such treatment, SOD activity was also higher than in treatment with both OTA doses (7.57 ± 1.55 and 7.77 ± 1.69) and CTN (7.92 ± 0.47). RSV did not reduce SOD concentration in combined treatment with higher OTA dose and CTN in plasma. SOD activity in the kidneys was not significantly affected by mycotoxin treatment. On the contrary, in liver tissue, SOD activity significantly decreased in all groups treated with single and combined mycotoxins compared to controls. RSV treatment not further affected the SOD activity.

Figure 4.

Activity of SOD in plasma (a), kidney (b) and liver (c) of rats treated with water, solvent, OTA 0.125 mg kg⁻¹ of b.w. (O125), OTA 0.250 mg kg⁻¹ of b.w. (O250), CTN 2 mg kg⁻¹ of b.w. (C) and combined treatment of both OTA doses with CTN (O125 + C and O250 + C) and higher OTA dose with CTN and RSV (20 mg kg⁻¹ of b.w.) (OCR). a: different from water, b: different from solvent, c: different from O125, d: different from O250, e: different from C, h: different from OCR (p < 0.05). SOD: superoxide dismutase; OTA; ochratoxin A; CTN: citrinin; RSV: resveratrol; b.w.: body weight.

CAT

CAT activity of plasma, kidney and liver tissues showed increasing pattern depending on OTA dosage in animals treated with single OTA as well as in animals treated with combinations of OTA and CTN (Figure 5). However, a significant increase of CAT activity was obtained after combined treatment with both mycotoxins and RSV in plasma (99.08 ± 28.04 nmol mL⁻¹ min⁻¹) compared to water (43.06 ± 26.84), solvent (38.47 ± 10.54) and CTN (52.90 ± 19.02). In the kidneys, CAT activity was significantly increased in animals treated with OTA (0.125 mg kg⁻¹ of b.w.), OTA (0.250 mg kg⁻¹ of b.w.) + CTN and OTA (0.250 mg kg⁻¹ of b.w.) + CTN + RSV compared to water-treated animals (92.66 ± 41.75, 96.82 ± 28.16 and 103.20 ± 44.03, respectively, vs 25.73 ± 4.39).

Figure 5.

Activity of catalase in plasma (a), kidney (b) and liver (c) of rats treated with water, solvent, OTA 0.125 mg kg⁻¹ of b.w. (O125), OTA 0.250 mg kg⁻¹ of b.w. (O250), CTN 2 mg kg⁻¹ of b.w. (C) and combined treatment of both OTA doses with CTN (O125 + C and O250 + C) and higher OTA dose with CTN and RSV (20 mg kg⁻¹ of b.w.) (OCR). a: different from water, b: different from solvent, e: different from C. OTA; ochratoxin A; CTN: citrinin; RSV: resveratrol; b.w.: body weight.

MDA

The results of MDA concentrations are presented in Figures 6 and 7.

Figure 6.

MDA concentration in kidney (a) and liver (b) of rats treated with water, solvent, OTA 0.125 mg kg⁻¹ of b.w. (O125), OTA 0.250 mg kg⁻¹ of b.w. (O250), CTN 2 mg kg⁻¹ of b.w. (C) and combined treatment of both OTA doses with CTN (O125 + C and O250 + C) and higher OTA dose with CTN and RSV (20 mg kg⁻¹ of b.w) (OCR). a: different from water, b: different from solvent, d: different from O250, e: different from C. MDA: malondialdehyde; OTA; ochratoxin A; CTN: citrinin; RSV: resveratrol; b.w.: body weight.

Figure 7.

MDA concentration in plasma (a) and urine (b) of rats treated with water, solvent, OTA 0.125 mg kg⁻¹ of b.w. (O125), OTA 0.250 mg kg⁻¹ of b.w. (O250), CTN 2 mg kg⁻¹ of b.w. (C) and combined treatment of both OTA doses with CTN (O125 + C and O250 + C) and higher OTA dose with CTN and RSV (20 mg kg⁻¹ of b.w.) (OCR). a: different from water, b: different from solvent, d: different from O250, g: different from O250 + C, h: different from OCR (p < 0.05). MDA: malondialdehyde; OTA; ochratoxin A; CTN: citrinin; RSV: resveratrol; b.w.: body weight.

Both OTA doses significantly increased kidney MDA concentration (34.21 ± 3.90 and 41.81 ± 5.51 nmol g⁻¹ tissue, respectively) as compared to water-treated rats (18.19 ± 5.45). In animals treated with higher OTA dose, MDA level was significantly higher than in animals receiving solvent (23.17 ± 7.01). Animals treated with OTA (0.125 mg kg⁻¹ of b.w.) + CTN had significantly higher MDA concentrations in the kidney (45.07 ± 12.59) compared to animals treated with CTN alone (30.33 ± 5.35). RSV reversed these effects in the kidney (23.96 ± 1.83) compared to OTA applied at higher dose.

MDA concentrations in the liver showed increasing OTA dose-related pattern in single and combined treatment. MDa concentrations were higher in the liver of OTA-treated animals, but these results were not statistically significant. Significantly higher MDA concentration was found in the liver of animals treated with OTA (0.250 mg kg⁻¹ of b.w.) + CTN (30.01 ± 14.52 nmol g⁻¹ tissue) compared to solvent treatment (13.63 ± 2.89). RSV treatment decreased MDA concentration, but this decrease was not statistically significant.

In plasma of OTA-treated animals, the MDA concentration was increased significantly (0.68 ± 0.08 and 0.65 ± 0.05 μmol mL⁻¹, respectively) as compared to solvent (0.34 ± 0.03). Higher MDA concentrations were also found in animals treated with CTN (0.65 ± 0.09), as well as combined mycotoxins treatment (0.75 ± 0.12 and 0.69 ± 0.09, respectively). RSV significantly decreased MDA level (0.43 ± 0.03) compared to matching toxins treatment (0.69 ± 0.09).

MDA was significantly elevated in urine of rats treated with higher OTA dose (2.94 ± 1.45 μmol mL⁻¹) compared to solvent treatment (1.43 ± 0.19 μmol mL⁻¹). RSV significantly lowered MDA level (1.46 ± 0.47 μmol mL⁻¹) compared to OTA-treated animals (0.250 mg kg⁻¹ of b.w.).

Discussion

Although many studies on OTA toxicity have been published, the mechanism of its toxicity is still not clear. The mechanism of CTN toxicity is much less studied due to its lower nephrotoxicity. Although their co-occurrence in food and feed is high, studies on their common toxicity on experimental animals are rare and not conclusive. ^6,28 This study was performed as continuation of our previous study ²² to clarify the mechanism of subchronic CTN toxicity in vivo and to further clarify the involvement of oxidative stress in mechanism of toxicity of simultaneous exposure to OTA and CTN.

The present study was performed on male rats because of their higher sensitivity to OTA than in females. ²⁹ In our experiment, two different doses of OTA were chosen according to our previous studies ^30,31 and RSV according to literature. ³² The total CTN daily doses in this subchronic study were calculated to be equal to the total CTN dose from our previous study, thus enabling their comparison. In the present study, OTA and CTN were given daily alone or together to study their common effect on parameters of oxidative stress in the kidney, liver and plasma. In experimental animals, the kidney is the target organ of both mycotoxins and the liver is also the target organ of OTA toxicity in some animal species. CTN may also cause liver lesions when high doses are applied. ³³

The effects of OTA on GSH concentration in the kidney and liver in other studies are not consistent, and it seems that they are related to the dose and length of treatment. Thus, in the study of Palabiyik et al., ²⁰ oral OTA treatment (0.5 mg kg⁻¹ × 2 weeks) caused a significant reduction in the kidney on GSH concentration (44%). The GSH concentration was also significantly reduced either in the kidney or in the liver of rats treated with OTA (0.250 mg kg⁻¹ × 4 weeks). ³⁴ Opposite to that, two groups of authors have found that renal GSH level of male F344 rats was not affected by oral treatment with OTA (0.210 mg kg⁻¹ × 4 weeks). ^35,36 The length of OTA treatment and doses in our experiment were similar to the later studies with no significant effect in the kidney and liver on GSH concentration. In the present study, CTN treatment for 3 weeks did not affect GSH concentration in the kidney and liver of experimental animals the same as 2-days CTN treatment in our previous study. ²²

The increased GSH concentration in the kidney but not in the liver of OTA + CTN + RSV-treated animals is probably due to higher concentration of RSV and its metabolites in the kidneys than in the liver. ³⁷ Thus, the effectiveness of RSV may be organ-specific. In our previous study in combined treatment, RSV showed protective effects by increasing GSH in the kidney and liver, but animals were CTN-treated only for 2 days.

It seems that the production of protein carbonyls is the most resistant parameter of oxidative stress caused by mycotoxins and that it depends on toxin concentration and route of exposure. In our previous study, the levels of protein carbonyls in the kidney and liver were increased after 21 days of i.p. treatment with higher OTA dose (0.5 mg kg⁻¹). ³⁸ In the present study, oral treatment with OTA (0.125 and 0.250 mg kg⁻¹) did not affect protein carbonyls concentration. So far, there are no data on CTN or OTA + CTN effect on protein carbonyls in experimental animals.

GPx activity was significantly decreased in mycotoxin-treated animals compared to vehicle treatment. Such decrease was seen in animals treated with CTN, OTA (0.125 mg kg⁻¹) + CTN and OTA (0.250 mg kg⁻¹) + CTN + RSV. In rats treated with 0.289 mg kg⁻¹ t.m. for 4 weeks, reduced GPx activity in the kidney was measured. ¹⁹ The similar phenomenon was observed in the present study in the liver of rats treated with higher OTA dose, although this result was not statistically significant. The observed decrease in GPx activity in plasma together with higher GPx activity in plasma of animals treated with vehicle (NaHCO₃) could be linked to higher affinity of free radicals to oxidation of HCO₃ ^– (Liochev and Fridovich, 2010).

In various studies, the activity of SOD was measured in tissue homogenates and in plasma of OTA-treated animals, but the results are not conclusive. In OTA-treated rats (0.07–0.50 mg kg⁻¹) for 2–13 weeks, SOD activity in the kidney was decreased, unchanged or increased and unchanged in the liver. ^19,36,39,40 When rats were treated with OTA at 0.289 mg kg⁻¹ for 4 weeks, SOD activity in serum was decreased. ⁴¹ In the present study, shorter treatment period with lower doses of OTA as well as with CTN did not affect SOD activity in plasma, but combined toxin treatment increased it significantly. In the present study, SOD activity was not significantly changed in the kidney, but in the liver, it was decreased in all mycotoxin-treated groups compared to controls. Interaction between OTA, CTN and Cu and Zn in SOD molecules could inhibit SOD activity. ³⁴

It seems that catalytic activity of CAT is the most expressed in kidney tissue of rats. Lower OTA dose increased the catalytic activity of CAT, while combined treatment with the same OTA dose + CTN did not increase it further probably due to lower CTN toxicity. In the literature, CAT activity was not affected with OTA treatment ^19,20 or it was slightly decreased in the kidney. ³⁴ There are no data of OTA + CTN treatment on CAT activity in vivo. CAT is the most abundant in the liver and it works in conjunction with SOD. ⁴²

In the present study, MDA concentration in plasma was significantly increased in mycotoxin-treated animals. GPx can reduce lipid hydroperoxides and other soluble hydroperoxides after their release from membrane lipids. ⁴³ To the best of our knowledge, there are no studies on plasma antioxidant enzymes activity upon combined OTA and CTN treatments in experimental animals.

In other studies, activity of CAT was increased in serum of OTA-treated rats (0.289 mg kg⁻¹ × 4 weeks), while in the kidney, it was unchanged. ^19,41 In the present study, CAT activity in plasma had increasing trend with the increase of OTA dose, but the significant increase was seen only in the plasma of animals treated with OTA + CTN + RSV. We do not have plausible explanation for this effect.

In acute toxicity studies, it was found that single dose of OTA 2 mg kg⁻¹ does not increase MDA concentration in rat kidney, ⁴⁴ but a very high dose (10 mg kg⁻¹) increases it significantly. ⁴⁵ In 4-week treatment studies, the results are controversial because the increased kidney MDA concentrations were found in rats treated with 0.2, 0.250 and 0.289 mg kg⁻¹ OTA ^19,34,46 but not in rats treated with 0.4 mg kg⁻¹. ⁴⁰ In the present study, lower OTA dose (0.125 mg kg⁻¹ of b.w.) significantly increased kidney MDA concentration, while double dose did not cause further significant increase, the same as in our previous research. ²² Although kidney MDA concentration increased in parallel with increasing OTA dose, such pattern was not seen in the liver and plasma. It seems that MDA concentration is organ-specific and that OTA affects more kidney than liver due to the liver higher antioxidative potential.

The importance of treatment period was also seen in rabbits exposed to OTA (0.75 mg kg⁻¹) + CTN (15 mg kg⁻¹) in feed. ¹³ No differences in MDA concentration in the kidney after 30 days of exposure to OTA, CTN or OTA + CTN were found, but after 60 days of treatment, MDA increased significantly in OTA-treated animals. When OTA was applied together with CTN, kidney MDA concentration was significantly higher than in animals treated with CTN only. However, it was not higher than in animals treated with OTA only. Similarly, in the present study on rats when OTA was applied together with CTN, kidney MDA concentration was significantly higher than in animals treated with CTN only. MDA concentration in kidney in the present study is in accordance with our previous research ²² except when rats were treated with lower dose of OTA + CTN. Different accumulations of OTA and CTN in the kidney and liver may explain this phenomenon. In our previous study, ⁴⁷ it was found that when OTA is given continuously for 21 days and CTN in the last 2 days of experiment, CTN concentration increases and OTA concentration decreases in the kidney as compared to animals given the same dose of single mycotoxin. From in vitro studies, it is known that both mycotoxins are transported to the kidney by organic anion transporters hOAT1 and hOAT3 and that CTN competes with OTA with higher affinity than OTA, ⁴⁸ which may explain the decreased OTA and the increased CTN concentration. In our study, it was found that OTA increases lipid peroxidation more than CTN, but in combined treatment, probably due to the decreased OTA concentration in the kidney, MDA concentration is also decreased. This phenomenon was particularly seen in our previous study when CTN treatment was applied only 2 days but the doses were much higher.

In the liver, MDA concentration in rats treated with both OTA doses was higher than control but it was not statistically significant. In our previous study, the same treatment caused a significant elevation of MDA. ²² There is a trend of increasing MDA concentration in animals treated with combined mycotoxins which is in accordance with our previous research, and the increase is significant in animals treated with OTA (0.250 mg kg⁻¹) + CTN as compared with animals treated with vehicle. The other authors reported the increased liver MDA concentration after 4 weeks treatment with OTA 0.250 mg kg⁻¹. ³⁴ However, in another study, similar OTA dose (0.210 mg kg⁻¹) after 4 and 13 weeks did not affect liver MDA concentration. ³⁶

OTA or CTN probably induced lipid peroxidation in some other organs but kidneys and liver, because MDA concentrations in plasma reflect the lipid peroxidation not only in these organs. MDA concentration in plasma depends on length of treatment because single treatment with OTA up to 2 mg kg⁻¹ and 24 h of exposure did not increase MDA concentration in rat plasma, ⁴⁴ but the treatment with OTA (0.250 mg kg⁻¹) for 4 weeks significantly increased it. ³⁴ In the present study, even lower OTA concentration (0.125 mg kg⁻¹) significantly increased MDA concentration in plasma. It seems that CTN follows the same pattern because in our previous study treatment with two high doses of CTN MDA concentration was not higher than in controls, while in the present study, CTN significantly increased MDA concentration. Combined treatment did not additionally increase MDA concentration. In the available literature, there are no data on MDA in plasma of animals treated with CTN or with OTA + CTN.

There is a trend of decreasing MDA concentration caused by RSV in the kidney, liver and plasma, but this effect is significant only in plasma if results are compared with OTA (0.250 mg kg⁻¹) + CTN. RSV concentrates more in the kidney than in the liver, and it seems that this exogenous antioxidant cannot overcome the lipid peroxidation caused by both mycotoxins. ³⁷

Conclusions

Oxidative stress plays a significant role in single and combined OTA and CTN toxicity mechanism; mycotoxin treatments in male Wistar rats evoked different effects in plasma, kidney and liver which are not surprising because the organization of antioxidative defence is tissue- and cell-specific. ¹⁵ Effect of mycotoxins on oxidative stress is less pronounced in the liver because of its elaborate antioxidant defence system. ¹⁸ Oxidative stress was best shown by an increase of lipid peroxidation particularly in plasma of treated rats. Single OTA treatment has higher pro-oxidative potential than CTN and combined mycotoxins evoked higher increase in MDA production than OTA and CTN given alone. RSV as exogenous antioxidant showed organ-specific protective effect which was most pronounced in the kidney. However, RSV can reduce but not overcome oxidative stress induced by single and combined OTA and CTN.