Ochratoxin A (OTA) is a mycotoxin produced by some molds belonging to the genera Aspergillus and Penicillium,1 which contaminates foodstuffs for animals and man, such as cereals and wheat derivatives,2 and has recently been found also in coffee, beer, grape juice and wine.3-7 However, epidemiological studies demonstrating that the consumption of these beverages (including contaminated wine) is associated with OTA nephrotoxicity do not exist. OTA is metabolized mainly by isoforms of the P450 cytochrome (CYP450)8 and is converted into both active and inactive metabolites able to generate quinone forms, Fe-porphyrin complexes,9 which determine the production of reactive oxygen species (ROS) with nephrotoxic effects. Indeed, the target organ of OTA is the kidney. As this mycotoxin contaminates large quantities of food in the Balkans, it is believed that it is the cause of Endemic Nephropathy in these regions,10 which manifests with the typical signs and symptoms of Tubulo-interstitial Nephropathies, associated with renal function test changes and interstitial fibrosis.11
In recent years the interest in OTA effects on human health has increased and studies have been performed to assess the activity of the mycotoxin. As grapes and fermented grape juice are contaminated, our group has carried out experimental studies on acute and chronic exposure to assess whether the toxicity of the mycotoxin is influenced by the alcoholic and non alcoholic components of red wine.
The results obtained after acute administration of OTA dissolved in an aqueous solution, a hydro-alcoholic solution and red wine confirm its nephrotoxicity in the rat,12 which is mediated by oxidative damage with tissue peroxidation. It is well known that OTA is able to bind trivalent iron, forming a complex that is promptly reduced by NADPH-CYP450 reductase to OTA-Fe2+, which triggers lipid peroxidation in the presence of oxygen. OTA damages mainly the proximal tubule,13 whose cells are very sensitive to oxidative damage. Our main observation is that the intake of ethanol, both in an aqueous solution and in red wine, is able to prevent OTA-induced acute renal toxicity. This effect of ethanol may be due to alcohol-induced enzyme induction, especially to CYP450-dependent enzyme induction. Indeed, it has been demonstrated that pre-treatment of rats with a CYP450 inducer protects the animals from OTA toxicity, whereas concomitant administration of a reducer causes an exacerbation.8 Furthermore, it has been clearly demonstrated that ethanol induces CYP450 not only in the liver,14 but also in the kidneys.15
Our study also showed that the non-alcoholic components of red wine exert independent antioxidant activity. Indeed, tissue levels of oxidized glutathione (GSSG) were preserved in the group treated with OTA dissolved in wine, whereas ethanol did not exert any effect. Rodrigo et al. (2002) have recently shown that antioxidant polyphenols contained in red wine are able to increase the GSH/GSSG ratio in the kidneys of rats.16 Furthermore, Shaff et al. (2002) have shown that in vitro the covalent bond between OTA and GSH may inactivate the mycotoxin, reducing its nephrotoxicity.17
Based on these observations, we decided to assess the potential interactions between OTA and ethanol/red wine in a 3-month chronic toxicity experimental model.
This study showed that OTA produces renal damage leading mainly to chronic tubulo-interstitial nephropathy that ultimately results in renal failure. It is well known that the renal damage that occurs in tubulo-interstitial nephropathy may be due to increases in ROS as is the case with OTA.18-19
In our experimental model, chronic treatment with a hydro-alcoholic solution produced renal damage similar to that induced by OTA. It is well known that ethanol produces oxidative stress. However, surprisingly, OTA, dissolved in a hydroalcoholic solution, did not cause overt renal damage, except an increase in the renal content in lipoperoxides without a significant reduction in superoxide dismutase tissue activity. Ethanol may increase the formation of ROS via induction of CYP450 2E1, which in turn oxidizes ethanol.20 Numerous papers in the literature document that CYP450 is involved in OTA metabolism, especially CYP2E1, which converts OTA into its less active metabolites. This could explain why the combination of OTA and ethanol does not produce the same damage that occurs when the substances are administered alone: Ethanol may afford protection against OTA, inducing CYP2E1 and competing with OTA as a substrate of this enzymatic system.
The administration of red wine, Barbera d’Asti, did not damage the kidney and no alterations of oxidative stress indices were recorded, probably because wine is a beverage rich in polyphenols, antioxidant substances that reduce renal damage due to oxidative stress.21
Neither renal damage nor oxidative stress occurred in the group of animals to which OTA was administered in red wine. Consequently, the claim can be made that wine affords protection against OTA induced damage, thanks to its content in polyphenols. As these compounds are metabolized by the same enzymatic system that metabolizes OTA and are able to also modulate its activity,22,23 we have put forth three hypotheses to explain the protection against oxidative damage. Starting from the assumption that in the case of OTA-wine the metabolic substrates of CYP450 are both polyphenols and OTA:
1. In the first hypothesis CYP450 prefers polyphenols as substrates. These molecules are converted from an active form with antioxidant properties into an inactive form; however, OTA is metabolized in negligible quantities or not at all, with consequent reduction in the formation of ROS, whose quantity is then too low to produce damage.
2. In the second hypothesis OTA is the preferred substrate: OTA is metabolized both into active and inactive forms. However, the latter are counterbalanced by the high levels of polyphenols that are not metabolized and that are therefore “active”.
3. Finally, as some polyphenols, such as resveratrol, inhibit CYP450 inactivation of this enzymatic system reduces both the metabolization of other polyphenols,24 which maintain their antioxidant properties, and the metabolization of OTA, which in this way does not cause any harm.
It has been shown that moderate consumption of red wine, thanks to its components, reduces the increase in activity of renal CYP induced by ethanol and prevents oxidative stress.25 Data in the literature indicate that the typical histo-pathological findings in OTA induced nephropathy consist in tubulo-interstitial fibrosis. This suggests that OTA may alter the turnover of collagen, producing an imbalance between synthesis and degradation that leads to the development of fibrosis. However, the molecular mechanisms underlying this event have not been fully elucidated.
Histological examinations of the kidneys in our studies have shown that prolonged treatment with OTA induces significant morphological changes in the kidney in terms of collagen content in the tubular interstitium and in the expression of genes related to fibrogenesis.26 Indeed, the morphological analysis showed that OTA produced the appearance of severe tubulointerstitial fibrosis in the kidney of treated rats as compared to controls, whereas glomeruli did not appear to be involved. Furthermore, the accumulation of collagen was significantly higher in the renal cortex, suggesting therefore that the cortex is the area that is most involved in the fibrotic process induced by OTA: The levels of mRNA of the main interstitial collagens were not significantly modified by OTA treatment. However, there was a trend towards an increase in COL-I and COL-III gene expression in treated rats as compared to controls. After treatment with OTA-wine, COL-I and COL-III gene expression tended to return to the levels recorded in controls. There were no evident and important changes also in the rats treated with ethanol, wine or OTA-ethanol. This finding suggests that red wine may partly counteract the upregulation of interstitial collagen induced by OTA.
Interesting findings that emerge from this study are the significant increase in the levels of pro-matrix metalloproteinase-9 (proMMP-9) and of a Smooth Muscle Actin (aSMA) in rats treated with OTA as compared to controls and their reduction after treatment with OTA-wine, but not after administration of OTA-ethanol.
MMP-9 and aSMA play a key role in the promotion of the tubular epitelio-mesenchymal transition (EMT), a process in which epithelial cells lose their unique characteristics and take on a phenotype that resembles the one of fibroblasts, which are the main effector cells in the production of the components of the extracellular matrix. In vitro OTA has proved to possess high tropism for the epithelial cells of the renal tubule (especially the proximal tract) and carriers actually transport OTA into them. Thus, OTA converts tubular epithelial cells into myofibroblasts (expressing aSMA), which migrate into the interstitium in-between tubules, where they synthesize and deposit interstitial collagen, inducing the appearance of tubulo-interstitial fibrosis. This event occurs thanks to the proteolytic action of MMP-9, which is able to degrade basement membrane collagen. Consequently, the over-expression of proMMP-9 in the kidneys of rats treated with OTA, in compliance with the pattern of aSMA expression, could be one of the molecular mechanisms that play a key role in the pathogenesis of the tubulointerstitial fibrosis induced by chronic treatment with OTA. The concomitant administration of OTA and wine is able to modulate the levels of proMMP-9 and aSMA, reducing them, and may therefore exert a protective effect on the kidneys. The modulation does not occur after treatment with OTA-ethanol, suggesting that it is likely that the polyphenolic rather than the alcoholic component of wine exerts protective effects on the kidneys.
Considering all the data together, our findings indicate that the toxicity of this mycotoxin may be positively modified by the properties of both alcoholic and non alcoholic compounds contained in red wine; consequently, caution is required in the transposition of results when different matrices are used.
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