The present in vivo sub-chronic experiment demonstrated that OTA exposition significantly increased free radical formation in the liver and kidney. OTA distributed into different tissues more or less equally after oral intake [39
], therefore its effects were possibly the same at the same time in both tissues. Oxidative stress is proposed as the effect of OTA toxicity both in the liver [40
] and kidney [41
], and the oxidative reactions activate lipid peroxidation [42
]. These findings were partly supported by the results of the present study because the initial phase of lipid peroxidation, amount of CD and CT, did not increase. However, it reached the termination phase, as proven by the higher MDA content, possibly due to the long-term period of exposure. The changes were more marked in the liver than in the kidney, but in both tissues only the highest OTA dose (1126 μg/kg feed) revealed significant changes. These results suggested that the cellular defence mechanism was unable to inhibit the lipid peroxidation processes at that dose.
These results can be explained with the changes of the glutathione redox parameters, because neither GSH content nor GPx activity increased systematically during the period of OTA exposure. This meant that the amount or activity of the glutathione redox system was not adequate for the elimination of oxygen free radicals, in particular at high OTA exposure.
On the other hand, the expression of the redox sensitive gene, KEAP1
, was overexpressed in the liver and downregulated in the kidney. This meant that release of Nrf2, as a transcription activator of the Antioxidant Response Element (ARE) gene cluster [43
], might have been released from the binding with Keap1 in the kidney, but much less efficiently in the liver. However, at the same time the relative expression of the NRF2
gene increased in both the liver and kidney, as a response to oxidative stress, but hypothetically Nrf2 remains bounded to Keap1 in the liver, if the same changes occurred also at the protein expression level, however, it was not determined in this study.
A higher level of unbound Nrf2 activates the ARE containing genes encoding glutathione metabolism enzymes, such as GSS
and glutathione peroxidases (in this study GPX3
). Non-significant changes in GSH concentration together with downregulation of the gene expression of GSS
suggests that the cellular concentration of GSH did not change, because GSS protein is required for the synthesis of GSH. This result was supported by previous studies where OTA exposure down-regulated the genes involved in GSH metabolism [9
], but contradictory with another in rats [44
], where a significant decrease of GSH concentration was found in the kidney. The possible cause of this contradiction would be the higher OTA exposure applied in the previous study, and probably another mechanism, for instance, the conjugation of OTA with GSH [45
] which may also result in a lower concentration. GSR
expression also did not increase as an effect of OTA exposure, which meant that the reduction of glutathione-disulphide (GSSG) to GSH was also not effective, which also supported the insufficient antioxidant response in the liver and kidney, therefore induction of oxidative stress and consequently lipid peroxidation. However, it should be noted that GSH homeostasis in the cell is regulated by its synthesis and/or recycling, and also by the rate of utilization and efflux [46
encodes the extracellular glutathione peroxidase (GPx3), which mainly originates from kidney tubular cells [47
]. The overexpression in the kidney proves the importance of the kidney as a source of GPx3, but an opposite tendency, downregulation, occurred in the liver, which has less importance in the synthesis of this GPx isoenzyme. This difference in the gene expression also suggests that regulation of glutathione peroxidase genes, in particular GPX3
, in the kidney is more sensitive to oxidative stress caused by OTA exposure than in the liver. However, higher relative gene expression did not manifest at the protein level because there were no significant changes in GPx activity in the blood, even at overexpression of kidney-origin GPX3,
which meant that such an increase did not cause a significantly higher amount of enzymatically active proteins. The other glutathione peroxidase enzyme, GPX4,
expression at both gene and protein levels occurred in most of the cell types, due to its effect on the inhibition of membrane phospholipid oxidation [48
]. However, its gene expression did not change in the liver, and was downregulated in the kidney. At the protein level, the activity of GPx did not change as an effect of OTA, which suggested that even in the case of downregulation of gene expression the enzyme activity remains stable, possibly due to the post-translation modification of the preformed enzyme proteins [49
In conclusion, the results of the present study have revealed that OTA initiates free radical formation both in the liver and kidney, but not in blood. The results showed that lipid peroxidation depended on the length of exposure and the dose applied. Lack of ARE activation, which was suggested by the low mRNA level of most of the Nrf2 dependent genes, resulted in improper antioxidant defence, thus mild oxidative stress, even at the regulatory dose proposed by the European Commission for poultry diets. Based on the results, defining lower values for poultry diets can be proposed and attention should be placed on the use of feed additives, such as phytobiotics, which are useful for activation of the antioxidant gene cluster [50
], possibly even in the case of OTA exposure.