Ochratoxin A potentiates citrinin accumulation in kidney and liver of rats

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.

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 in vitro findings that CTN competes with OTA for human OAT1 and 3 with different K_i values (3080 and 15.4 μmol/L, respectively) and that these two transporters have higher affinity for CTN than for OTA (28). Similar findings have also been reported in immortalised human proximal tubule cells in the presence of CTN (29) in which OTA dropped by over 60 %. In another study (30) in which rats received ten times lower doses than in ours for 21 days, CTN levels did not change and CTN did not affect Oat1 and Oat3 protein expression, but OTA₂₅₀ significantly downregulated Oat2 protein expression in the kidney. In addition, both mycotoxins downregulated Oat5 protein expression. All this suggests that high doses of OTA or both mycotoxins together inhibit kidney transporters involved in the excretion of CTN. There are no reports of transporters responsible for CTN excretion from the liver.

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.