Primary hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Accumulating evidence has suggested that several mechanisms contribute to the carcinogenesis of HCC. Recent efforts to control the incidence of HCC have focused on developing effective new chemoprevention strategies. HCC induced by diethylnitrosamine (DEN) in Wistar rats that shows similarities to human HCC is an ideal model for investigating the effect of intervention by chemopreventive agent. DEN, a hepatocarcinogen, is known to induce perturbations in the nuclear enzymes involved in DNA repair/replication. Investigations have provided evidence that DEN causes a wide range of tumors in all animal species, and these compounds are considered to be effective health hazards to man.
Man is exposed to DEN through diet, in certain occupational settings, and through the use of tobacco products, cosmetics, pharmaceutical products, and agricultural chemicals. It has been reported that DEN, after its metabolic biotransformation, produces the promutagenic adducts, O6-ethyl deoxyguanosine and O4- and O6-ethyl deoxythymidine that can produce DNA chain damage, depurination or binding to DNA, and often generates a miscoding gene sequence, paving a way for the initiation of liver carcinogenesis. It has also been reported to produce reactive oxygen species (ROS), a potentially dangerous by-product of cellular metabolism that may directly affect cellular development, growth, and survival. Oxidative stress caused by ROS has been reported in membrane lipid peroxidation, DNA damage, and mutation associated with the initiation of various stages of the tumor formation process.
Polyphenolic compounds have the most promising pharmaceutical properties and have received greater attention than any other class of natural products to counter the ill effects of oxygen radicals. Umbelliferone (UMB), otherwise known as 7-hydroxycoumarin, is a coumarin derivative of benzopyrone that is naturally present in edible fruits, such as Bengal quince (Aegle marmelos Correa) and bitter orange (Citrus aurantium). UMB is known to have a wide spectrum of pharmaceutical effects including antioxidant, antidiabetic, antihyperglycemic, and anti-inflammatory properties. UMB acts as a fluorescent probe and is used in the synthesis of drugs, especially anticancer drugs, and in the treatment of monensin and allergic disorders. The ultraviolet activity of UMB is used as a sunscreen agent and an optical brightener for textiles. It has also been used as a gain medium for dye lasers. UMB can be used as a fluorescence indicator for metal ions such as copper and calcium. It acts as a pH indicator in the 6.5–8.9 range. It is acutely toxic to laboratory animals in chronic oral gavage administration at doses ≥ 200 mg/kg and exposure of UMB induces irritation of the eyes, respiratory system, and skin.
Materials and methods
Microscopic observations of UMB in DEN-treated rat liver are given in Fig. 2. Fig. 2 shows the histopathological examination of liver section. Control (Group 1) rats revealed normal liver parenchyma cells with granulated cytoplasm, small uniform nuclei, and central vein surrounded by cords of hepatocytes. Group 2 DEN-treated rats showed loss of architecture and lobules of neoplastic hepatocytes with a fecal area of fatty change. Group 3 rats exhibited normal architecture, indicating the non-toxic nature of UMB. Groups 4 and 5 rats co-treated with UMB and DEN showed moderate cancerous change, fatty change, and hydropic degeneration. Group 6 rats showed fewer neoplastically-transformed cells and the hepatocytes maintained near-normal architecture.
In recent years, there has been a growing interest in dietary substances obtained from natural products having chemoprotective properties against chemical carcinogens. HCC is a common cancer and is the third leading cause of death worldwide. DEN is known to induce the reproducible and complete carcinogenic biochemical changes involved in the progression of HCC. ROS are potentially dangerous byproducts of cellular metabolism that have directly affected cellular growth, development, and survival. Lipid peroxidation is one of the major mechanisms of cellular injury caused by free radicals and acts as an important causative factor in carcinogenesis. DEN intoxication has been reported to generate lipid peroxidation byproducts that may interact with various biomolecules that lead to oxidative stress. This may be due to the uncontrolled generation of free radicals that overwhelms the antioxidant defense system. DEN-induced rats showed increased lipid peroxidation levels (thiobarbituric acid reactive substances, malondialdehyde and conjugated dienes) in both plasma and liver tissue. UMB administration to DEN-treated rats at three different doses 10 mg/kg, 20 mg/kg, and 30 mg/kg body weight every day led to significantly deceased levels of lipid peroxidation both in the plasma and liver when compared with animals induced with DEN alone. This shows the anti-lipid peroxidative role of UMB and is probably mediated by UMB’s ability to inhibit free radical generation. These results also correlate with the previous findings from our laboratory. The strong inhibitory effect of UMB at a dose of 30 mg/kg body weight/day was noticed.