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Ochratoxin a Potentiates Citrinin Accumulation in Kidney and Liver of Rats

Abstract Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic mycotoxins often found together in grain. The aim of this study was to measure their accumulation in the kidney and liver of adult male Wistar rats, see how it would be affected by combined treatment, and to determine if resveratrol (RSV) would decrease their levels in these organs. The rats received 125 or 250 mg/kg bw of OTA by gavage every day for 21 days and/or 20 mg/kg bw of CTN a day for two days. Two groups of rats treated with OTA+CTN were also receiving 20 mg/kg bw of RSV a day for 21 days. In animals receiving OTA alone, its accumulation in both organs was dose-dependent. OTA+CTN treatment resulted in lower OTA but higher CTN accumulation in both organs at both OTA doses. RSV treatment increased OTA levels in the kidney and liver and decreased CTN levels in the kidney. Our findings point to the competition between CTN and OTA for organic anion transporters 1 and 3.

[Received in October 2021; Similarity Check in October 2021; Accepted in February 2022] Ochratoxin A (OTA) and citrinin (CTN) are nephrotoxic mycotoxins often found together in grain. The aim of this study was to measure their accumulation in the kidney and liver of adult male Wistar rats, see how it would be affected by combined treatment, and to determine if resveratrol (RSV) would decrease their levels in these organs. The rats received 125 or 250 mg/kg bw of OTA by gavage every day for 21 days and/or 20 mg/kg bw of CTN a day for two days. Two groups of rats treated with OTA+CTN were also receiving 20 mg/kg bw of RSV a day for 21 days. In animals receiving OTA alone, its accumulation in both organs was dose-dependent. OTA+CTN treatment resulted in lower OTA but higher CTN accumulation in both organs at both OTA doses. RSV treatment increased OTA levels in the kidney and liver and decreased CTN levels in the kidney. Our findings point to the competition between CTN and OTA for organic anion transporters 1 and 3.
KEY WORDS: experimental rats; mycotoxins; organic anion transporters; resveratrol; toxicity Mycotoxins ochratoxin A (OTA) and citrinin (CTN) are produced by Aspergillus and Penicillium strains and are often found together in grain. Their co-occurrence in food and feed may be the consequence of contamination with several moulds or one mould species producing both mycotoxins such as P. verrucosum (1)(2)(3)(4). Mandatory OTA monitoring in various commodities in the EU has given a rather good insight into human exposure to this mycotoxin (5). CTN monitoring, in contrast, is not mandatory, despite the recommendation issued by the European Food Safety Authority (6), and data on its levels in food and feed are limited. Grain contamination with CTN is usually low (<10 % of positive samples), but its levels tend to be high (1.1-33 times higher than those of OTA) (7). In Croatia contamination is high (50-75 % samples), with a considerably large level range (0-400 µg/kg) (8,9). In Bangladesh (10) and Germany (11) a very high percentage of CTN-positive samples was found in human urine, indicating substantial exposure to this mycotoxin (10,11).The biological half-life of OTA is long, which increases the possibility of OTA interaction with other mycotoxins. In rat plasma it is 120 h and in human plasma 35.5 days (12,13). Being a small molecule, OTA enters the enterohepatic circulation, gets redistributed across organs, and accumulates in the kidney (14). Its renal excretion by glomerular filtration is limited and involves organic anion transporters (OATs) either on the basolateral or apical side of cells in kidney tubules (15).
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 P. verrucosum. Studies of its mutagenicity are inconclusive and studies of its carcinogenicity are lacking. Some have found it genotoxic (17)(18)(19), and others not (20,21).
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).

MATERIALS AND METHODS
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 NaHCO 3 ), OTA 125 (receiving 125 µg/kg bw of OTA alone), OTA 250 (receiving 250 µg/kg bw of OTA alone), CTN (receiving 20 mg/ kg bw of CTN alone), OTA 125 +CTN, OTA 250 +CTN, OTA 125 +CTN+RSV (20 mg/kg bw), and OTA 250 +CTN+RSV. OTA was given dissolved in 51 mmol/L NaHCO 3 by gavage every day for 21 days. CTN was dissolved in 50 mmol/L Na 2 CO 3 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. 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 (R 2 ) were 0.997 and 0.996 for OTA and CTN, respectively.

Statistical analysis
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 twotailed. P values of less than or equal to 0.05 were considered statistically significant.

OTA and CTN concentrations
The increase in OTA concentrations in both organs was dosedependent (Figures 1 and 2). Kidney OTA levels in the OTA 125 +CTN and OTA 250 +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 OTA 250 +CTN+RSV g roup and liver OTA in the OTA 125 +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 OTA 250 +CTN+RSV group compared to the OTA 250 +CTN treatment.

DISCUSSION
We found that OTA accumulation in the kidney was dosedependent 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 OTA 250 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 OTA 250 +CTN+RSV compared to OTA 250 +CTN treatment but increased liver OTA in animals receiving OTA 125 +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.

Conflicts of interest
None to declare.