The Protective Effect of Melatonin against Fumonisin-Induced Renal Damage in Rats

Abstract

The present study was designed to investigate the potential protective effect of melatonin against the renal toxicity of fumonisin in female rats. Six groups of animals were used in this study. The first group served as control. The second group was given melatonin only at a dose level of 10 mg/kg. The third group was fed ration contaminated with fumonisin (100 mg/kg diet). The fourth group was fed ration contaminated with fumonisin (200 mg/kg diet). The fifth group was given daily interperitoneal injection (IP) 10 mg/kg melatonin and fed ration contaminated with fumonisin (100 mg/kg diet). The sixth group was given daily interperitoneal injection of 10 mg/kg melatonin and fed ration contaminated with fumonisin (200 mg/kg diet). The rats were treated for 1 month. Histopathological and histochemical changes in the kidney were investigated. In addition, DNA ploidy was measured in the kidney. Fumonisin administration (100 or 200 mg/Kg diet) to unpretreated control rats caused extensive renal damage as evaluated by histopathology, histochemistry, and/or DNA ploidy measurement. No apparent changes following administration of melatonin. Melatonin coadministration to the fumonisin-administered rats reduced kidney damage and the tissues appeared more or less like the normal. The present study indicates that melatonin has a protective effect in fumonisin-induced renal damage.

Fumonisins are classes of mycotoxins produced by Fusarium moniliforme and other Fusarium spp (Pepelinjak et al. 2003; Flaherty and Woloshuk 2004). These compounds are commonly found in corn (He et al. 2002).

Fumonisins induce cardiovascular toxicity (Constable et al. 2003), and cause equine leukoencephalomalacia (Gelderblom et al. 2004), porcine pulmonary edema, liver tumors, and chronic nephritis in rats (Gumprecht et al. 2001; Desai et al. 2002). Fumonisins cause erythrocyte membrane defect and interferes with carp’s respiratory process (Pepeljnjak et al. 2003). Fumonisins may also possess potential risk to humans, having been correlated with human esophageal cancer (Sydenham et al. 1990). There is no evidence that fumonisins are genotoxic, mutagenic, and unscheduled DNA synthesis both in vitro and in vivo (Creppy 2002).

Melatonin is the chief secretory product of the pineal gland. It was recently found to be a potent free radical scavenger and antioxidant (Sener et al. 2004a). Melatonin is widely available, relatively free of side effects, rapidly active after oral administration, and commonly used in humans in the treatment of insomnia (Brzezinski 1997). Melatonin has been used as anti-inflammatory agent (Reiter et al. 1994), and provides protection against cyclophosphamide-induced tissue damage (Manda and Bhatia 2003) and nephrotoxicity induced by Adriamycin (Tunez et al. 2003). Melatonin may exhibit a protective effect on free radical–mediated oxidative damage induced by extracorporeal shock wave lithotripsy (ESWI) in rabbit kidney (Serel, Ozgune, and Soyupek 2004), and improvement of endothelial-mediated relaxation in blood vessels of diabetic rats Reyes-Toso et al. 2004).

Therefore, the present work was planned to study the effect of melatonin in preventing renal damage induced by fumonisin. For this purpose the histological and histochemical changes and DNA ploidy were evaluated.

MATERIALS AND METHODS

Chemicals

Fumonisin (standard) and melatonin were obtained from Sigma Chemical, St. Louis, Mo., USA.

Fumonisin was produced through the fermentation of corn by Fusarium moniliforme obtained from Department of Plant Pathology, Agriculture Research Center, as described by Visconti and Doko (1994). The fermented corn was autoclaved, and ground to a powder. The toxins were extracted with acetonitrile:water (1:1 v/v) by shaking the grains and solvent for 30 min on an orbital shaker and then the extracts were filtered through filter paper (Whatman no. 4). An aliquot of the extracts (1000 ml) was taken and diluted with acetonitrile water as necessary for high-performance liquid chromatography (HPLC) analysis. Fumonisin content was measured by HPLC according to Shephard et al. (1990). Briefly, an aliquot (50 ml) of the diluted extract was derivatized with 200 ml of an o-phthaldialdehyde solution obtained by adding 5 ml of 0.1 mol/L sodium tetraborate and 50 ml of 2-mercaptoethanol to 1 ml of methanol containing 40 mg of ophthaldialdehyde. Fumonisin B1 (FB1) was detected and quantified with HPLC (Waters) equipped with fluorescence detector. The wavelengths used were 335 and 440 nm for excitation and emission of fluorescence, respectively. An analytical reverse-phase C18 column (150 mm by 4.6 mm [internal diameter]; 5 mm particle size), connected to a C18 precolumn (20 mm by 4.6 mm; 5 mm particle size), was used. The mobile phase was methanol (0.1 mol/L) and NaH₂PO₄ in a 75:25 ratio (v/v); the pH was set at 3.35 ± 0.2 with orthophosphoric acid, and a flow rate of 1.5 ml/min was used. Fumonisin quantification was performed by peak height measurements and comparison with reference standard solutions. The standard solution was obtained by dissolving pure fumonisin B1 in acetonitrile:water (1:1) at concentrations of 100 mg/ml. The detection limit of the analytical method for the fumonisins was 1 mg/g. (Millennium program was used for calculation of fumonisin B1 concentrations.)

Animals

Forty-eight female albino rats, weighing approximately 150 g, purchased from the Animal House Laboratory, National Research Center, were used in this study. The animals were kept under the same laboratory conditions of temperature (25°C ± 2°C) and lighting (12:12-h light:dark cycle) for 1 week prior to the commencement of the treatment and were given a standard lab diet and water ad libitum.

Experimental Design

Animals were randomly divided into six groups (eight rats each). The first group served as control. The second group was given intraperitoneal (IP) dose of 10 mg/kg melatonin only. The third group was fed ration contaminated with fumonisin (100 mg/kg diet). The fourth group was fed ration contaminated with fumonisin (200 mg/kg diet). The fifth group was given daily IP injection of 10 mg/kg melatonin and fed ration contaminated with fumonisin (100 mg/kg diet). The sixth group was given daily IP injection of 10 mg/kg melatonin and fed ration contaminated with fumonisin (200 mg/kg diet). The rats were treated daily for 1 month.

Histopathological and Histochemical Studies

At the end of the experiment, the rats were anaesthetized with ether and then sacrificed. The kidneys of different groups were dissected and fixed in 10% formolin and embedded in paraffin; sections 5 μm thick were stained with hematoxylin and eosin (Drury and Wallington 1980). Sections were stained with Feulgen (Feulgen and Rosenbeck 1942) for DNA analysis. DNA ploidy was performed at the Department of Pathology, National Research Center, using Leica Qwin 500 image analyzer (LEICA Imaging Systems, Cambridge, England). Image analyzer automatically gave the DNA content of each individual nucleus measured, then gave the percentage of the total number of nuclei examined. It classified the cells into four groups, namely diploid (2c), S-phase cells (3c) (proliferation index), tetraploid (4c), and cells with more than 5c DNA content (>5c), which indicates aneuploidy. Gomori stain was used for detection of alkaline phosphatase activity (Gomori 1951).

RESULTS

Histological Results

The normal histological structure of the kidney was observed in (Figure 1A ). No histological changes could be noticed in the kidney of rats treated with melatonin only (Figure 1B ). The histopathological examination of the kidney of fumonisin (100 mg/kg diet) fed rats showed remarkable changes as compared to control rats. These changes include vacuolar degeneration in the epithelial cells of the renal tubules and hyaline casts scattered in the tubular lumina. Degenerative and pyknotic nuclei were noticed. Some degenerative glomeruli and cellular infiltration in interstitial tissue were also detected (Figure 1C ).

The kidney of rats fed with fumonisin contaminated diet (200 mg/kg diet) showed markedly degenerated tubules with pyknotic nuclei, focal tubular necrosis, and disruption of the tubular basement membrane. Extreme hydropic change in the renal tubules, focal areas of cloudy swelling and hyaline casts in tubular lumina could be observed. Glomerular degeneration with increased thickening of Bowman capsules and interstitial haemorrhages were also noticed (Figure 1D ).

In rats treated with melatonin at dose level of 10 mg/kg along with fumonisin (100 mg/kg diet), examination of kidney sections showed diminution of degenerated or pyknotic nuclei in tubular epithelial cells. No inflammatory cells could be noticed. Moderate vacuolar degeneration in tubular epithelial cells, interstitial hemorrhage, and glomerular degeneration with wide urinary space could be observed (Figure 1E ).

Rats treated with melatonin at dose level of 10 mg/kg in combination with fumonisin contaminated diet (200 mg/kg diet) showed some histological changes, but these changes were less in comparison with group of rats fed with fumonisin only. The kidney sections showed mild cloudy swelling in tubular epithelial cells (Figure 1F ).

DNA Ploidy

Table 1 showed normal DNA content in kidney of control rats with 73% cell diploid (2c), 15.8% triploid cells (3c) (medium proliferation index), 1% tetraploid cells (4c), and no aneuploid cells.

Table 2 showed DNA content in kidney of rats with melatonin 74% cell diploid (2c), 12.8% triploid cells (3c) (medium proliferation index), 1.8% tetraploid cells (4c), and no aneuploid cells.

In rats treated with fumonisin (100 mg/kg diet), DNA ploidy analysis showed 72% diploid cells (2c), 10% of the cells contained 3c DNA value (medium proliferation index), 3.7% tetraploid cells (4c), and no aneuploid cells (Table 3).

Rats fed ration contaminated with fumonisin (200 mg/kg diet) showed kidneys containing 12.15% aneuploid cells (>5c), 35.5% of the cells at 4c area (tetraploid), 38.3% of the cells contained 3c DNA value (high proliferation index), and only 5.6% of the examined cells contained diploid DNA value (2c) (Table 4).

The rats treated with melatonin along with fumonisin (100 mg/kg diet) showed no aneuploid cells, 54% of the examined cells contained diploid DNA value (2c), 6.8% of the cells at 3c area (low proliferation index), and 1.9% of the cells were at 4c area (tetroploid) (Table 5).

The rats treated with melatonin along with fumonisin (200 mg/kg diet) showed no aneuploid cells, 50.5% of the examined cells contained diploid DNA value (2c), 1.9% of the cells at 3c area (low proliferation index), and 1% of the cells were at 4c area (tetroploid) (Table 6).

Histochemical Result

In control rats and melatonin group, alkaline phosphatase was located in the brush border and basement membrane of renal tubules (Figure 2A and B ). The renal tubules showed increase in alkaline phosphatase enzyme activity in the group of rats fed ration contaminated with fumonisin only for 1 month (100 and 200 mg/kg diet) (Figure 2C and D ). Moderate decrease in alkaline phosphatase enzyme activity was recorded in the group of rats treated with melatonin in combination with fumonisin (100 and 200 mg/kg diet) as compared to group of rats fed with fumonisin only (Figure 2E and F ).

DISCUSSION

In the present work, the effect of fumonisin B₁ on kidney was pronounced at different doses (100 and 200 mg/kg diet). The drastic effect was dose dependent.

These effects demonstrated as vacuolar degeneration, fatty change in tubular epithelial cells, and degenerative or pyknotic nuclei in animals fed ration contaminated with fumonisin (100 mg/kg diet). These results were in agreement with (Mathur et al. 2001; Voss et al. 2001; Abdel Wahhab, Nada, and Arbid 2002). They noticed that fumonisin induced cytoplasmic vacuolation and fatty degeneration of the renal tubules and pyknotic nuclei.

Vacuolation observed in epithelial cells of tubules after supplementation of fumonisin may be due to swelling of mitochondria (Isobe et al. 2003). Another study indicated that vacuoles are formed because of lactate accumulation in the tubules, resulting in increased osmotic pressure and subsequent water influx (Lannergren, Westerblad, and Bruton 2002).

In the present study, the intensity of histological damage was more marked in rats fed ration contaminated with fumonisin (200 mg/kg diet). The kidney tissues showed markedly tubular degeneration, necrosis, and cloudy swelling. Glomerular degeneration associated with increase of Bowman capsule thickness was also noticed. These results agreed with previous observations of Pfohl-Leszkowicz et al. (2002); and Stoev et al. (2002) reported that the mycotoxin caused focal tubular epithelial cell proliferation, tubular degeneration, tubulonephrosis, and glomerulonephrosis in kidney of chicken. Also Theumer et al. (2002) showed that the treatment of rats with fumonisin caused necrosis and apoptosis of tubular epithelial cells in kidney, with increased mitotic figures.

Gelderblom et al. (1994, 2004) reported that the de novo interruption of lipid and fatty acid biosynthesis at different levels affected the structure of cellular membrane. This appeared to be an important event determining alterations in growth-related response induced by fumonisin in primary rat tissues. In addition, the accumulation of polyunsaturated fatty acids in tissues treated with high doses of fumonisin B1 could be an important determinant in the induction of cell death. Lipid peroxidation was stimulated by mycotoxin (Abdel Wahhab, Nada, and Arbid 1999). The drastic changes observed in kidney of rats due to feeding ration contaminated with fumonisin may be attributed to lipid peroxidation and glutathione depletion (Kang and Alexander 1996).

In the present work the treatment of rats with melatonin along with fumonisin showed some improvement in histological changes in comparison with group of rats fed with fumonisin only. These results were in agreement with those of Ferraz et al. (2002) and Esrefoglu et al. (2003); these authors stated that no histological changes occurred in kidney of rats treated with glycerol or cyclosporin in combination with melatonin, consistent with Aydin et al. (2003) who reported that ochratoxin A caused severe histopathological changes in liver and kidney of albino rats and the severity of lesions was significantly reduced by administration of melatonin. According to Koc, Buyukokuroglu, and Taysi (2002), melatonin participates in the regulation of a number of important physiological and pathological processes.

In vivo and in vitro melatonin has been found to protect tissue against oxidative damage generated by a variety of toxic agents and metabolic processes including chemotherapy-induced toxicity (Lissoni et al. 1997). The mechanism of the protective effects of melatonin seems to consist particularly of scavenging OH and other free radicals and reactive oxygen intermediates, and its ability to modify the activity of enzymes participating in oxidative stress. Melatonin has been found to protect cells from oxidative stress by a variety of processes (Kunduzova et al. 2003; Sener et al 2004a). Melatonin has recently been found to protect against chronic renal failure. It also has the ability to decrease lipid peroxides and permit a recovery of reduced glutathione, scavenger enzyme, and parameters of renal function (Tunez et al. 2002; Sener et al. 2004b). According to Kunduzova et al. (2003), melatonin inhibits caspase-3 activation and prevents postreperfusion apoptotic and necrotic cell death in kidney.

Concerning DNA ploidy results, the rats fed ration contaminated with fumonisin (200 mg/kg diet) had kidneys that contain 12.15% aneuploid cells (>5c), 5.6% of the examined cells contain diploid DNA (2c) and high proliferation index. According to Marrero et al. (1996), increasing value of abnormal DNA content are related to severity of dysplasia. This agreed with Johnson et al. (2003), who found that cells treated with fumonisin B1 showed increased DNA fragmentation and terminal uridine nucleotide end labeling in response to tumor necrosis factor alpha (TNFα) treatment. Fumonisin B2 increased DNA synthesis and resulted in cell cycle arrest in the G2-M phase of the cell cycle. Also, Kim et al. (2001) demonstrated that fumonisin induced DNA damage and an enhancement of caspase-3 activity.

For rats treated with fumonisin (200 mg/kg diet) along with melatonin, no aneuploid cells could be noticed, 50.48% of the examined cells contain diploid DNA value and low proliferation index. These results were in agreement with several reports (Liu, Zhao, and Zheng 2003; Lei et al. 2004; Underger et al. 2004) that melatonin may facilitate the repair of the DNA damage. Melatonin led to a significant decrease in DNA strand breakage and lipid peroxidation.

In the present study the rats fed ration contaminated with fumonisin (200 mg/kg diet) resulted in increase of alkaline phosphatase activity. These results were in agreement with (Edrington et al. 1995) who found that increased alkaline phosphatase activity in liver and kidney was detected in fumonisin-intoxicated lambs.

The increased alkaline phosphatase activity noticed in the tubules of kidney may be due to increase in the functional load in the kidney. According to Mayer and William (1954) and Zomborszky-Kovacs et al. (2000), increased alkaline phophatase activity in the cytoplasm of degenerated liver cell may be attributed to failure of cells to release the enzyme.

On the contrary, Espada et al. (1994) demonstrated a decrease of alkaline phosphatase activity in kidney of chickens after treatment of fumonisin at dose level of 300 mg/kg at total period of 8 days.

In conclusion, fumonisin treatment induces renal tubular damage as indicated by histological and histochemical changes. The treatment of melatonin along with fumonisin B1 was found to diminish the harmful effect of fumonisin.