الفهرس 
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Abstract
Diethylnitrosamine is a potent hepatocarcinogenic nitrosamine present in tobacco smoke, cured and fried meats, cosmetics and pharmaceutical products. DENA-induced HCC is an accepted and widely used experimental model of hepatocarcinogenesis.
A two- stage model in which the initiation by a genotoxic compound is followed by a promotion phase is often used for inducing hepatocarcinogenesis. DENA can be used as an initiator and CCl4 as a promoter. Recently, natural products have been used to prevent the hazards induced by chemicals, drugs and carcinogenic xenobiotics.
The present study aims to assess the modulatory effects of hesperidin on DENA/CCl4-induced hepatocarcinogenesis and nephrotoxicity in male Wistar rats. The adult Wistar rats were randomly divided into 5 groups (each comprising 6 rats) as follows:
group I (Control): rats received a single intraperitoneal (ip) injection of physiological saline. After 2 weeks, rats of this group received olive oil subcutaneously (once/week) for 16 weeks. Throughout the experiment, rats received 0.5% CMC as a vehicle via oral gavage.
group II (100 mg Hesp): rats received physiological saline and olive oil as described in group I, and 100 mg/kg hesperidin dissolved in 0.5% CMC via oral gavage daily for 18 weeks.
group III (DENA/CCl4): rats received a single ip injection of DENA (200 mg/kg) dissolved in saline. Two weeks later, the rats received 3 ml/kg CCl4 dissolved in olive oil subcutaneously (once/week) for 16 weeks, and 0.5% CMC via oral gavage daily for 18 weeks.
group IV (DENA/CCl4 + 50 mg Hesp): rats received DENA and CCl4 as described in group III and 50 mg/kg hesperidin dissolved in 0.5% CMC via oral gavage daily for 18 weeks.
group V (DENA/CCl4 + 100 mg Hesp): rats received DENA and CCl4 as described in group III and 100 mg/kg hesperidin dissolved in 0.5% CMC via oral gavage daily for 18 weeks.
In the present study, rats treated with DENA/CCl4 showed marked impairment of liver function as demonstrated by the significant increase in serum AST, ALT, ALP, LDH and γ-GT activities and total bilirubin level as well as the significant decrease in serum level of albumin. Treatment with both 50 mg and 100 mg hesperidin showed marked improvements in these liver function parameters in serum.
The obtained data also revealed a significant increase in AFP, TNF-α and IL-6 levels in DENA/CCl4-administered rats. Co-administration of both tested doses of Hesp significantly ameliorated these changes.
Concerning liver oxidative stress and antioxidant defense system markers, the elevated levels of MDA and NO in DENA/CCl4-administered rats were markedly decreased following Hesp administration. In contrast, administration of two tested doses of Hesp to DENA/CCl4-injected rats potentially increased the declined GSH content and the activities of GPx, GST and SOD.
These biochemical changes in DENA/CCl4-injected group were accompanied with histological alterations including hepatic nodules, focal hepatic necrosis associated with inflammatory cells infiltration, apoptosis, steatosis, oval cells proliferation and heavy accumulation of collagen fibers. Concomitant treatment of these rats with Hesp protected against most of the induced histological alterations and showed nearly normal structures of the hepatic lobules.
DENA/CCl4 administration also down-regulated both PPARγ and Nrf2 gene and protein expression as well as HO-1 protein expression. On the other hand, NF-қB gene and protein expression and iNOS gene expression were markedly increased following DENA/CCl4 administration. Hesp treatment up-regulated PPARγ, Nrf2 and HO-1 expression levels and down-regulated NF-қB and iNOS expression levels.
Immunohistochemically stained sections showed a remarkable increase in COX-2 expression in DENA/CCl4-injected group. This expression was reversed to a great extent in the group treated with 50 mg Hesp and a more potent effect was exerted by 100 mg Hesp.
Nephrotoxicity induced by DENA/CCl4 was evidenced by the elevated concentrations of urea, uric acid and creatinine. Renal damage was further confirmed by the histological changes including congestion or atrophy of glomerular tufts, oedema, cloudy swelling in convoluted tubules and pyknotic nuclei of epithelium lining renal tubules. Masson’s trichrome staining also revealed heavy accumulation of collagen fibers in the interstitium of renal tissue. Treatment of DENA/CCl4-administered rats with both tested doses of Hesp decreased the elevated levels of serum urea, uric acid and creatinine and successfully improved the renal architecture in addition to decreasing collagen deposition.
Regarding kidney oxidative stress, the elevated levels of MDA and NO were potentially decreased in DENA/CCl4-administered rats due to hesperidin treatment. Moreover, a striking decrease in the antioxidants in DENA/CCl4- administered rats elicited a strong evidence for the involvement of oxidative damage in DENA/CCl4-induced nephrotoxicity. The depleted kidney GSH content and activities of GPx, GST and SOD were elevated by treatment with Hesp as compared to DENA/CCl4-administered rats.
The chemopreventive effect of hesperidin against DENA/CCl4-induced nephrotoxicity was also demonstrated through up-regulation of PPARγ and Nrf2 gene and protein expression and HO-1 protein expression as well as down-regulation of NF-қB gene and protein expression and iNOS gene expression. Moreover, treatment with hesperidin decreased COX-2 expression as illustrated by immunohistochemically stained kidney sections.
In conclusion, the possible modulatory effects of hesperidin on DENA/CCl4-induced hepatocarcinogenesis and nephrotoxicity in rats may be explained through attenuation of oxidative stress and inflammation as well as up-regulation of Nrf2/ARE/HO-1 and PPARγ pathways. However, clinical studies are required to assess the efficacy of hesperidin in human beings.