Mycotoxins ochratoxin A (OTA) and citrinin (CTN) are produced by
Target organs of OTA toxicity are the kidney and liver, but it is also immunotoxic, teratogenic, and carcinogenic.
The toxicological properties of CTN have been summarised elsewhere (16), but, generally, it is considered less nephrotoxic than OTA (8). However, CTN could become equally toxicologically important as OTA if the climate change increases CTN production by
The aim of our study was to establish the accumulation of OTA and CTN in the kidney and liver of rats and see how it would be affected in combined exposure. We also wanted to see how resveratrol (RSV) would affect organ accumulation of these mycotoxins, as this antioxidant is known to inhibit the expression of organic anion transporters (OATs) 1 and 3 (22, 23). This study was a part of a larger study investigating the effects of these two mycotoxins on oxidative stress in rat kidney, liver, and plasma (24).
Ketamine hydrochloride and xylazine hydrochloride used in combination to anaesthetise the rats were purchased under brand names Narketan and Xylapan from Chassot AG (Bern, Switzerland). OTA, CTN, and methanol were purchased from Sigma (St. Louis, MO, USA). Ultrapure water (18 MW) was obtained from a Milli-Q Smart2pure 3 UV/UF gradient water purification system (Thermo Fisher Scientific, Waltham, MA, USA). Acetic acid (p. a.) was obtained from Merck (Darmstadt, Germany). Other chemicals and reagents were of analytical grade, and their commercial source is indicated with the description of specific methods.
Adult male Wistar rats (10 weeks old, 230–270 g bw) were kept in makrolon cages at room temperature of 22 °C and 12-hour day/ night cycles and had free access to tap water and standard pelleted food (Mucedola, Settimo Milanese, Italy). Animals were divided into eight groups (N=6 each) as follows: controls (receiving 51 mmol/L NaHCO3), OTA125 (receiving 125 μg/kg bw of OTA alone), OTA250 (receiving 250 μg/kg bw of OTA alone), CTN (receiving 20 mg/ kg bw of CTN alone), OTA125+CTN, OTA250+CTN, OTA125+CTN+RSV (20 mg/kg bw), and OTA250+CTN+RSV. OTA was given dissolved in 51 mmol/L NaHCO3 by gavage every day for 21 days. CTN was dissolved in 50 mmol/L Na2CO3 and given by gavage for two days (CTN alone group), which in combined treatment coincided with the last two days of treatment with OTA and RSV every day between 8 and 9 AM. Animals were sacrificed under general anaesthesia with ketamine and xylazine.
Animal experiments were approved by the Ethics Committee of the Institute for Medical Research and Occupational Health in accordance with the EC Council Directive 2010/63/EU (25).
Organs were taken and kept at -80 °C until analysis. Samples were prepared according to the method described previously and modified for these samples accordingly (26).
Chromatographic analysis of OTA and CTN was run on a tandem quadrupole ultra performance liquid chromatography/ tandem mass spectrometry system (ACQUITY TQD UPLC-MS/ MS, Waters, Milford, MA, USA). Separation was done on a Hibar™ Purospher STAR HR 50x2.1 mm column (Merck, Darmstadt, Germany), 2 μm particle size, and flow rate of 0.53 mL/min. Gradient elution was applied (eluent A – 0.1 % acetic acid; eluent B– methanol) according to the following program: 0–0.61 min – 95 % A; 0.61–4.5 min – 5 % A; 4.5–5 min – 95 % A. The chromatographic run was 7 min per sample. Molecular ions were obtained with electrospray ionisation (positive mode for OTA and negative for CTN). The temperature of the ionisation source was maintained at 115 °C and the temperature of the desolvation gas at 350 °C. Cone gas flow was 60 L/h, and desolvation gas flow 750 L/h. Capillary and cone voltages were maintained at 3.5 kV and ±40 V respectively. Quadrupoles were set to the multiple reaction monitoring (MRM) mode. Each compound was confirmed by the presence of the parent ion and two transitional products. Specific transitions of the precursor ion and product ion were as follows: 249.1 ->177.3 and 249.1->205.4 m/z for CTN and 404->221 and 404->239 m/z for OTA, respectively. Quantification transitions were 249.1->205.4 m/z for CTN and 404->239 m/z for OTA. Optimised collision energy (CE) was 22 and 15 eV for CTN and 20 eV for OTA. Dwell times for each MRM were 0.15 s. Retention times were 3.3 min for CTN and 3.4 min for OTA.
For calibration tissue extracts were spiked with OTA and CTN as follows: 0.1, 1, 10, and 20 μg/kg of the sample for OTA and 0.1, 1, 2, and 10 μg/kg of the sample for CTN. The resulting calibration curves were used for quantification. The established quantification limits of the analytical method were 0.5 μg/kg for OTA, and 0.8 μg/kg for CTN, with a relative standard deviation of reproducibility below 5 % for both compounds. Coefficients of determination (R2) were 0.997 and 0.996 for OTA and CTN, respectively.
Data were analysed and plotted with the GraphPad Prism for Windows version 5 (San Diego, CA, USA) and R statistical software version 3.3.1 (The R Foundation for Statistical Computing, Vienna, Austria). OTA and CTN values are presented as medians and interquartile ranges, and were analysed using a nonparametric version of Tukey’s multiple comparison test. All applied tests were two-tailed. P values of less than or equal to 0.05 were considered statistically significant.
The increase in OTA concentrations in both organs was dose-dependent (Figures 1 and 2). Kidney OTA levels in the OTA125+CTN and OTA250+CTN were significantly lower than in the groups receiving respective doses of OTA alone. This effect was also observed in the liver of animals receiving the higher OTA dose.
RSV did not lower OTA levels in animals receiving OTA+CTN+RSV compared to those receiving OTA+CTN regardless of the OTA dose. In fact, it increased kidney OTA in the OTA250+CTN+RSV group and liver OTA in the OTA125+CTN+RSV group.
Kidney and liver CTN levels in animals receiving OTA+CTN were three to six times higher than in respective tissues of animals receiving CTN alone (Figures 3 and 4).
RSV lowered kidney CTN in the OTA250+CTN+RSV group compared to the OTA250+CTN treatment.
We found that OTA accumulation in the kidney was dose-dependent and comparable with our previous studies (27). Our main finding that OTA levels significantly dropped in both organs in the presence of CTN (Figures 1 and 2) supports
RSV lowered kidney CTN levels in animals treated with OTA250+CTN+RSV compared to OTA250+CTN treatment but increased liver OTA in animals receiving OTA125+CTN. Reports on RSV transport suggest that RSV and its conjugates are the substrates of OAT transporters involved in the transport of OTA and CTN in the kidney (15, 31, 32). RSV was also reported to inhibit OAT1 and 3 when combined with methotrexate (MTX) (33). The same mechanism probably regulates OTA and CTN accumulation in the kidney. However, further mycotoxin interaction studies in doses closer to natural exposure are needed to pinpoint the exact mechanisms of toxicity and their transport through membranes.