Abstract
Melatonin (MEL) and coenzyme Q10 (CoQ10) both display antioxidant and free radical scavenger properties. In the present study, the effect of MEL and CoQ10 on the oxidative stress and fibrosis induced by ochratoxin A (OTA) administration in rats was investigated. Rats were divided into five equal groups, each consisting of seven rats: (1) controls; (2) OTA-treated rats (289 μg/kg/day); (3) OTA+MEL–treated rats (289 μg/kg/day OTA + 10 mg/kg/day MEL); and (4) OTA+CoQ10–treated rats (289 μg/kg/day OTA +1 mg/100 g/day body weight (bw) CoQ10). After 4 weeks of treatment, the level of malondialdehyde (MDA), glutathione peroxidase (GPx), and hydroxyproline (Hyp) were measured in the homogenates of liver and kidney. In the OTA-treated group, the levels of MDA and Hyp in both liver and kidney were significantly increased when compared with the levels of control, whereas GPx activities decreased. In OTA+MEL–treated rats, the levels of MDA and Hyp in both liver and kidney were significantly decreased when compared with the levels of OTA-treated rats; however; GPX activities increased. In the OTA+CoQ10–treated group, the levels of MDA and Hyp were decreased when compared with the levels of OTA-treated rats, whereas GPx activities increased. In the OTA+CoQ 10–treated group, the levels of MDA, Hyp, and GPx were not significantly changed in kidney when compared with OTA-treated group. MEL has a protective effect against OTA toxicity through an inhibition of the oxidative damage and fibrosis both liver and kidney. Although CoQ10 has protective effect against OTA toxicity in liver tissue, it has no effect in kidney tissue.
Ochratoxin A (OTA; 7-carboxyl-5-chloro-8-hydroxyl-3,4-dihydro-3
In essence, all progressive renal diseases are characterized by fibrosis (Klahr and Morrissey 2002, 2003). This is in line with numerous animal studies, showing development of renal disease accompained by proximal tubuler atrophy and cortical interstitial fibrosis after exposition to OTA (Pfohl-Leszkowicz et al. 2002; Aukema et al. 2004).
Melatonin (MEL), the main product of the pineal gland, participates in many physiological functions due to its efficacy as a free radical scavenger and indirect antioxidant (Reiter et al. 2001; Tan et al. 2002; Allegra et al. 2003; Reiter 2003). Because of its small size and lipophilicity, melatonin crosses biological membranes easly, thus reaching all compartments of the cell (El-Sokkary, Abdel-Rahman, and Kamel 2005). Melatonin has also been shown to be an efficient protector of DNA (Lopez-Brillo et al. 2003), protein, and lipids in cellular membrane (Melchioiri et al. 1995; Cuzzocrea and Reiter 2001), as well as antogonist of a number of endogenous and exogenous free radicals generated during cellular processes (Zang et al. 1998)
Coenzyme Q10 (CoQ10) is well located in membranes and act as a primary scavenger of free radicals (Crane 2001). This compound can react with oxygen radicals, preventing direct damage to biomolecules and the initiation and propagation of lipid peroxidation (Crane 2001; Frei, Kim, and Ames 1990; Navarro et al. 1999).
Our aim was to investigate the protective effect of MEL and CoQ10 (as an antioxidant) on oxidative stress induced by OTA in rats. For this purpose, we measured glutathione peroxidase (GPx) activities and malondialdehyde (MDA) levels as markers of oxidative stress and hydroxyproline (Hyp) levels as a marker of fibrotic generation.
MATERIALS AND METHODS
Chemicals
Ochratoxin A from Aspergillus ochraceus, melatonin, and coenzyme Q10 were purcased from Si gma Chemical, Germany.
Experimental Procedures
Thirty-five male Sprague-Dawley rats ages ranging between 7 and 8 weeks and weighting about 200 to 220 g were purchased from the animal house of Medicine Faculty, Eskisehir Osmangazi University. The protocol for the experiment was approved by the appropriate animal care of Osmangazi University.
All animals were kept under the same laboratory conditions, temperature (25°C ± 2°C), and lighting (12:12-h light:dark cycle) for 1 week before the start of the experiment and were given free acsess to standart laboratory chow and tap water ad libitium.
The animals were divided into five groups (each group consisted of seven animals). The first group served as controls and received 0.5 mol/L NaHCO3 and ethanol (1/1000 ν/ν) for 4 weeks. The second group received OTA at a dose of 289 μg/kg per day for 4 weeks (OTA in 0.5 mol/L NaHCO3, pH 7.4) (Atroshi et al. 2000; Creppy, Baudrimont, and Betbeder 1995). The third group received melatonin at a dose of 10 mg/kg per day (in ethanol:distilled water 1:1000, ν/ν) (Ohta et al. 2000), coadministered with OTA, for 4 weeks. The fourth group received 0.4 ml 0.9 % NaCl + coin oil for 4 weeks and served as controls. The fifth group received CoQ10 (1 mg/100 g body weight [bw] in 0.4 ml of 0.9 % NaCl + corn oil), coadministered with OTA (Genova et al. 1999; Portakal and Inal 1999), daily for 4 weeks.
The daily water consumption of each animal was measured prior to the experiment. During the measured time window, the rats consumed 20 ml of water. The rats were then submitted to a regime that restricted their access to drinking water at a fixed time of the day. The regime of the drinking water timing was controlled manually by the researcher. OTA, MEL, and CoQ10 were added to the drinking water so that the ingested amount would be approximately 289 μg/kg for OTA, 10 mg/kg for MEL, and 1 mg/100 g bw for CoQ10 per day.
All animals were sacrificed by anesthesia administering xylasine (5 mg/kg intraperitoneally) and ketamine (30 mg/kg intraperitoneally) (Serdaroglu, Islamoglu, and Ozgentas 2005). Livers and kidneys were excised immediately and were homogenized in ice-cold 10 mM phosphate buffer (pH 7.4) using a Ultra Turrax (125-Janke Kunkol) homogenizer. After homogenization, homogenates were sonicated for 30 s. Then homegenates were centrifuged at 3000 × g for 5 min and stored at −70°C until measured.
Analytical Procedures
Lipid peroxidation (LPO) was assayed by quantifying and MDA in the form of thiobarbituric acid reaction (Ohkawa, Ohishi, and Yagi 1979). Thiobarbituric acid–reactive substances (TBARS) were measured spectrophotometrically in homogenate of the kidney and liver at 532 nm. GPx activity was determined spectrophotometrically at 340 nm by the method of Paglia and Valentine (1967). The reaction mixture consisted of phosphate buffer (pH 7), EDTA, NaN3, NADPH, GSSGR, GSH, and H2O2. All ingredients, except the enzyme source and H2O2, were combined at the beginning of each day. Blank reactions with the enzyme source replaced by distilled water were substracted from each assay for nonenzymatic oxidation of NADPH by the peroxides. Liver and kidney Hyp were measured by a modification of the method of Bergman and Loxey (1969, 1970). Hyp was measured by chemical assay based on the oxidation of Hyp to a derivated pyrole that produces chromogen with Ehrlich reagent. Liver and kidney tissue (100 mg) was hydrolyzed in a volume of 4 ml in 7 N HCI at 110°C for 6 h in a glass tube with a Teflon stopper. The hydrolysate was filtered and centrifuged; 1 ml of hydrolysate was neutralized with 4 ml of 4.8% LiOH and 0.1 ml of 7 N HCl, a 0.5-ml portion of the neutral or sightly acidic solution to be analyzed was pipetted into a clean 30-ml test tube, and 1 ml of isopropanol was added to each test tube. Chloramine-T solution (0.5 ml) was added to the samples and standard. This was done after it was added to the sample blanks of the Ehrlich’s reagent. After 4 ± 1 min, Ehrlich’s solution (1 ml) was added to each test tube. The mixture was incubated at 60°C for 20 min. Having reached room temperature, the samples, sample blanks, and standarts were at measured at 562 nm against a reagent blank that contained the complete system without added tissue. Each tube was assayed in duplicate. The concentration of hydroxyproline in each sample was determined from a standard curve generated from known quantities of hydroxyproline.
Histological examination proceeded as follows: Liver and kidney tissues were fixed in neutral-buffered formalin for histopathological examination in light microscopy. Then tissue blocks were processed by routine techniques. Furthermore, serial sections (5 μm) were prepared for each block. Moreover sections were stained with hematoxylineosin (H&E). Finally digital images were obtained by Olympus PM10 ADS photomicroscope with DP70 digital camera.
Statistical Evaluation
The data are presented as means ± standard error (SE). Kolmogorov-Smirnov normalite test was applied to MDA, GPx, and Hyd for liver and kidney and it was found that all of them were non-normally distributed. The result were analyzed with regard to statistical significance using the Kruskal-Wallis test and the Mann-Whitney U test. The level of significance was accepted with p < .05.
RESULTS
In the OTA-treated rats, the levels of MDA and hydroxyproline had significantly increased in liver and kidney when compared with control, although GPx activities were lower than in the control group (Tables 1, 2). In OTA+MEL treated rats, significantly reduced MDA and Hyp levels were found in the liver and kidney compared with the OTA-treated group, whereas GPx activities increased (Table 1). In OTA+MEL–treated group, the levels of MDA, Hyp, and GPx in liver were not changed significantly in comparison with control (Table 1). In the OTA+MEL–treated rats, the levels of MDA and Hyp were significantly increased in kidney when compared with control, although GPx activities were lower than in control (Table 1).
In OTA+CoQ10–treated rats significantly reduced MDA and Hyp levels were found in the liver when compared with the OTA-treated group; however, GPx activities were increased (Table 2). In the OTA+CoQ 10–treated rats, the levels of MDA and Hyp were significantly increased in liver when compared with control; however, GPx activities were lower than in control (Table 2). In OTA+CoQ10–treated rats the levels of MDA, GPx, and Hyp were not significantly changed in the kidney when compared with the OTA-treated group (Table 2).
HISTOLOGIC EVALUATIONS
The examination of the liver in the control and the kidney revealed normal histologic appearence (Figures 1, 2).
Kidney section from a OTA-treated rat showed mild degenerative changes in the tubular epithelium, with prominent dilated tubules and interstitial fibrosis and tubular atrophy. The tubular epithelial nuclei became enlarged and necrotic. The necrotic cells have no nuclei and the remaining cytoplasm is red (eosinophilic) (Figure 3). Reduced proximal tubular degeneration were seen in kidney section from OTA+MEL–treated group (Figure 4). In OTA+CoQ10–treated group, cell necrosis, karyomegali, and desquamation in proximal tubules were observed (Figure 5).
The liver of OTA-treated rat showed focal area of necrosis infiltrated with mononuclear cells (Figure 6), the nuclei of hepatocytes became smaller in size and their chromatin were condensed. Furthermore, the cytoplasm of the cell was detectable. Some hepatic cell showed dark stained cytoplasm. The liver of the OTA+MEL–treated group showed marked congestion in the sinusoids, with slight vacuolar degeneration of the hepatocyte (Figure 7). Melatonin, at a dosage of 10 mg/kg, reduced parenchymal and stromal degeneration in the liver and kidney. The liver section from the OTA+CoQ 10–treated group decreased mononuclear cell inflammation and congestion in sinuzoids was observed (Figure 8).
DISCUSSION
In the current study, OTA treatment produces significant alterations of the cellular antioxidant defense mechanism. Several reports reveal that oxidative stres can result from the excess occurrence in the cells of free radicals, which are induced directly or indirectly by OTA (Abdel-Wahhap, Abdel-Galil, and El-Lithey 2005). Initiation of oxidant stress mechanism by OTA-induced depletion of GPx activities was reported (Meki and Hussein 2001). GPx is an antioxidant enzyme catalyzing the detoxification of lipid peroxides and hydrogen peroxide. Selenium is an essential cofactor for GPx and its deficiency may lead to a decrease in GPx activity (Smith-Kielland, Aaseth, and Thomassen 1986). Our results showed a reduction in the activity of GPx in liver and kidney of OTA-treated rats. This decline in GPx activity may be attributable to decreased levels of selenium due to the interference of OTA with the absorption of selenium; however, Soyoz et al. (2004) reported increase in the blood and liver GPx activities in rats treatment with OTA.
Oxygen free radicals (OFRs) have been suggested to exert their cytotoxic effects by causing peroxidation of membrane phospholipids. This increases membrane fluidity, membrane permeability, and loss of membrane integrity. End products produced during the lipid peroxidation process, including MDA, are very reactive and capable of cross-linking membrane proteins containing amino groups (Prasad and Karla 1993; Kehrer 1993). Omar et al. demonstrated that OTA induced LPO product by chelating Fe+3, and the resulting OTA-Fe+3 chelate was more readily reductible by the flavoprotein NADPH-P450 reductase to an OTA-Fe+3 complex that, in the presence of oxygen, provides the active species that initiated LPO products (Omar, Rahimtula, and Bartsch 1991). Hydrogen peroxide, which is produced by toxic effect of OTA and Fe+2, was increased via this mechanism and converted to the more potent hydroxyl radical. At the end of this process, the level of LPO products were increased (Van Ginkel and Sevanian 1994; Soyoz et al. 2004). In our results, the levels of MDA were significantly increased in the liver and kidney in OTA-treated rats when compared with controls.
In our study, rats treated with OTA+MEL had reduced levels of MDA and increased GPx activities in liver and kidney compared with OTA-treated rats (Baundrimont et al. 1994; Soyoz et al. 2004; Oz and Ilhan 2006). It is most likely that the ability of MEL to stimulate the synthesis of antioxidants enzyme may lead to reduction of MDA levels and increase the GPx.
CoQ10 can react with oxygen radicals, preventing direct damage to biomolecules and the initiation and propagation of lipid peroxidation (Dallner and Sindelar 2000; Navarro et al. 1999; Frei, Kim, and Ames 1990).
According to our related study, we found decreased levels of MDA in liver tissues in OTA+ CoQ10–treated rats when compared with OTA-treated rats, whereas GPx activities increased. Our results correlate with the finding of Farswan et al. (2005) and Upaganlawar et al. (2006) that the levels of MDA and GPx were not significantly changed in kidney. These results indicate that CoQ10 plays an important role in the antioxidant defense system for liver tissues.
Another purpose of this study is to determine the formation of liver and kidney fibrosis caused by OTA and evaluate to change during treatment with MEL and CoQ10. We measured Hyd levels as a marker of fibrotic degeneration. In the OTA+MEL–treated group, the levels of Hyd were decreased as compared to the OTA-treated group in the liver. MEL seems to be part of a defense mechanism that saves the organelles from the oxidative damage produced by OTA. In the OTA+CoQ10–treated rats, the levels of Hyd were not significantly changed in the kidney when compared with OTA-treated rats. In order for our group to explain in more detail the reasons why CoQ10 was not effective, more research is needed.
In conclusion, our study shows that an increase in MDA and hydroxyproline was associated with a decrease of GPx activities and significant histopathologic changes were found in the kidney and liver tissue of rats treated with OTA. From the above observations we can conclude OTA induced structural tissue damage in liver and kidney and administration of melatonin together with OTA significantly reversed the effects caused by the toxin alone, supporting the hypothesis that OTA toxicity may be reduced by using antioxidant agents such as melatonin. Melatonin therapy may be more effective than CoQ10 therapy alone. Thus, clinical application of melatonin as therapy should be considered in cases of ochratoxicolosis.