الفهرس 
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Abstract
Hepatocellular carcinoma is a primary hepatic neoplasia that ranks the second among cancer-related mortality worldwide. Nitrates and nitrites that are usually used as food preservatives and coloring agents are the source for human exposure to the genotoxic nitrosamine compounds. DENA is one of these nitrosamine compounds that trigger HCC progression on prolonged exposure. DENA induces HCC development either directly by DNA damage or indirectly by increasing oxidative damage. Therefore, the use of antioxidants is one of the chemoprevention strategies against DENA induced HCC. Tiopronin is a drug with free sulfhydryl group that has been used to control cystinuria. It showed antioxidant hepatoprotective against nonalcoholic steatohepatitis and isoniazid-induced hepatotoxicity. Hesperidin is a flavanone glycoside that is naturally found in citrus fruits. It showed antioxidant hepatoprotective effects against several liver diseases. It also has anticancer effect against colon cancer and renal carcinoma.
This study aimed to investigate the hepatoprotective effects of tiopronin and hesperidin against DENA-induced hepatocellular carcinoma. It also aimed to investigate the possible signaling pathways targeted by tiopronin and hesperidin to induce their effects. The liver function tests and oxidative stress markers were evaluated. Additionally, the protein levels of phospho-ASK1, phospho- P38, phospho- P53, PI3K, phospho-Akt and CDK2 were measured in the liver tissues by western blotting analysis. Alpha fetoprotein concentration was measured by an ELISA technique. Histopathological examination of the liver tissues was also performed.
This study showed that DENA induced HCC in rats and that was evidenced by elevation of liver function enzymes such as serum ALT, AST, ALP activities and total bilirubin level, elevation of serum alpha fetoprotein level as well as the pathological alterations in liver tissues macroscopically and microscopically. It also caused oxidative damage of liver tissues that was evidenced by the elevated levels of malondialdhyde and nitric oxide as well as the decreased activities of catalase and glutathione peroxidase. Both tiopronin and hesperidin possess antioxidant hepatoprotective effects against DENA-induced hepatocellular carcinoma. Tiopronin and hesperidin improved liver function tests and oxidative stress markers that were deteriorated by DENA. Moreover, both tiopronin and hesperidin prevented the pathological alterations induced by DENA on the liver tissues macroscopically and microscopically. In addition, tiopronin and hesperidin protected against DENA-induced elevation of serum α-fetoprotein level. Furthermore, tiopronin maintained the normal activity of ASK1/P38/P53 signaling cascade while hesperidin prevented DENA-induced overexpression of PI3K/Akt/CDK2 pathway.
In conclusion, this study demonstrated that tiopronin is an antioxidant hepatoprotective agent with a novel mechanistic pathway to inhibit DENA-induced HCC in rats. The mechanism by which tiopronin exerts its action is via maintaining the normal activity of ASK1/p38/p53 signaling cascade. In addition, hesperidin downregulated PI3K/Akt/CDK2 pathway to protect against DENA-induced HCC.
Study limitations:
The current study exhibited at least three limitations:
1. First, the experimental design examined only one dose of tiopronin (60 mg/Kg b.wt.) and one dose of hesperidin (200 mg/Kg b.wt.).
2. Second, the possible toxicity of tiopronin or hesperidin themselves was not described due to the lack of a control group receiving their treatment only without DENA.
3. Third, the total protein level of ASK1/P38/P53 and PI3K/Akt signaling cascades have not been measured and correlated to the active form of protein.
Future work:
This study has thrown up many questions that are in need of further investigations. Future work needs to be done to establish whether a pharmacologic treatment with ASK1 or P38 activators can cancel the anti-tumor activity of tiopronin. The effect of tiopronin on DENA-induced expression of CYP2E1 should be studied as a possible mechanism for tiopronin chemoprevention. Furthermore, a study is needed to investigate the possible downstream targets of P53 such as cell cycle arrest, repair of DNA damage and apoptosis. In addition, dose response curve of tiopronin with different doses and times of exposure are required to determine the drug safety margin as well as investigating the possible protective effects of tiopronin against other cancer models.