Means ± SE with different letters superscripts in the same
P < 0.05 using one way ANOVA test. n= 5 rats/group. Unit of GR= mmol of GSH utilized/min/mg protein. Rats injected with Nitrosodiethyamine (NDEA) caused significant (P < 0.05) decreases in renal tissue levels of glutathione reeducates (GR), superoxide dismutase (SOD) and catalase (CAT) antioxidant enzymes when compared to those fed on basal diet. Oral feeding of ginger powder at doses 0.5, 1 and 2 % from basal diet to hepatocellular carcinoma rats for 16 weeks significantly (P < 0.05) increased tissue levels of SOD, GR and CAT enzymes when compared with the positive control group, in a dose dependent manner, as recorded in Table (3). The obtained result is agreement with Manju and Nalini (2005) which induced colon carcinogenesis in rats by injection dimethylhydrazine (DMH) and his result showed that decreased significantly in enzymic (GR, SOD and CAT), but animals received ginger by intragastric intubation daily at a dose of 50 mg/kg body weight increased antioxidant enzymes measured. GR play a significant role in protecting cells against cytotoxic and carcinogenic chemicals by scavenging reactive oxygen species (Michiels et al., 1994). In addition, it involved in the detoxification of xenobiotics, carcinogens, free radicals and peroxides by conjugating these toxic substances with GSH, ultimately protecting cells and organs against carcinogeninduced toxicity. Since the reactive ultimate carcinogenic form of NDEA is an electrophilic diazonium ion, glutathione-dependent enzymes may play an important role in carcinogen detoxification. These enzyme can also serve as anticarcinogens and also as inhibitors at initiation and promotion/transformation stage of carcinogenesis. Moreover, antioxidant enzymes such as SOD and CAT are widely distributed in all cells and are present in high amounts in erythrocytes (Speranza et al., 1993). These enzymes protect the red cells against O2º- and H2O2 mediated lipid peroxidation Scott et al., 1991). The increases in circulating lipid peroxides of carcinogenesis -treated animals as descripted in the previous studies (Manju and Nalini 2005), correlates with the decline in circulatory antioxidants such as GR, SOD and CAT as show in the present study. This may be due to their overutilization to scavenge the products of peroxidation as well as sequestration by tumor cells. To be more specific essential antioxidants from circulation such as the GR have been reported to be sequestered by the tumor cells, to meet the demands of the growing tumor (Buzby et al., 1980). In the circulation, the enhancement of the activities of the detoxifying and antioxidant enzymes by ginger may be a major mechanism by which it ameliorates the deleterious effects of NDEA. In this context, we had previously shown that ginger ameliorates high fat diet induced oxidative stress by increasing antioxidant levels (Jeyakumar et al., 1999). Recent studies have also shown the chemopreventive effect of ginger during 7,12-dimethylbenz a anthraceneinduced skin tumors (Katiyar et al., 1996). Figure (1): Effect of ginger powder (GP) on serum concentrations of Alfa fetoprotein (AFP) of hepatocellular carcinoma rats. Results in figure (1) showed that rats with (NDEA) had significant (p<0.05) increase in Alfa fetoprotein cancer biomark (AFP) levels (1.034), as compared to the normal rats. The administration of ginger powder at doses 0.5, 1 and 2 % produced decrease in (AFP) levels (9.8±0.35b, 8.0±0.44c and 5.0±0.48d (ng/mL), respectively), as compared to the positive control group. Obtained result agreement with Manju and Nalini (2005) how observed that the tumor incidence was 100% in group injected with (DMH) dimethylhydrazine, but there were no tumors in control rats and group treated with DMH+ and ginger extract by intragastric intubation at dose 50 mg/kg body weight everyday for 30 weeks. Suppressed tumor formation as shown in figure (1) and depletion antioxidant mentioned above associated with Enhanced concentrations of circulating lipid peroxidation in NDEA induced liver tumor bearing animals. (Manju and Nalini 2005). DiethyNitrosoamine DENA, a procarcinogen undergoes metabolism in the liver, resulting in the production of active carcinogenic electrophile (diazonium ion), which is capable of producing toxic effects at sites far from tumor (Bobek et al., 2000). The production of reactive oxygen metabolites (ROMs) during the hepatic metabolism of DENA and/or during the process of carcinogenesis is well documented. Early reports also suggest that tumor cells produce substantial amount of H2O2 that are released into the circulation (Fiala, 1977) In conditions of severe oxidative stress such as carcinogenesis, reactive oxidant species such as superoxide and hydroxyl radical are released into circulation resulting in increased susceptibility of the plasma and erythrocytes to lipid peroxidation in DENA treated rats. Ginger and its active principles such as gingerols and shagoals are known to possess antioxidant property, and scavenge-free radicals like superoxide and hydroxyl radicals (Cao et al., 1993; Reddy and Lokesh 1992). Results observed decreased concentrations of Alfa fetoprotein cancer biomark on ginger supplementation. Ginger inhibits lipid peroxidation and scavenges reactive oxygen species (Jeyakumar et al., 1999). The results of histopathological observations showed that In negative control group, the liver sections showed normal architecture, characterized by polyhedral shaped hepatocytes and cytoplasm granulated with small uniform nuclei. Hepatocytes were arranged in well-organized hepatic cords and separated by narrow blood sinusoids (Fig. 2-a). In contrast, the liver sections of rats injected with NDEA showed loss of lobular architecture, inflammatory cell infiltration (Figure 2- b), hepatic cell necrosis and vacuolations, hemorrhage in blood sinusoid (Figure 2-c). Also, the nuclei of many hepatocytes appeared malignant or have features of degenerating and dividing process (Figure 2-c). The pretreatment of rats received NDEA with ginger preserved the normal architecture of liver caused by NDEA and restored the altered histopathological changes in a dose-dependant manner (Figs. 2- d, e and f). Figure 2 . Effect of ginger powder (GP) on Histopathological examination of hepatocellular carcinoma rats. Paraffin sections of liver stained by haematoxylin and eosin for histopathological changes. Liver section of group (1, Negative control) showing normal architecture of liver histology and cells with granulated cytoplasm and small uniform nuclei (a; X400). Hepatotoxic liver after treatment of NDEA (group 2, Hepatocarcinogenesis control) showing loss of architecture of the hepatic lobules, inflammatory cell infiltration (star)+ (b; X400), hepatic cell necrosis (arrow head) and vacuolations (long arrow), hemorrhage in blood sinusoid (short arrow) (c; X400) and malignant nuclei (b and c; X400). Liver section of group (3 treated with NDEA + GP 0.5% )minimal pleomorphism, inflammatory cell infiltration (long arrow), hemorrhage in blood sinusoid (short arrow) and karyolysis (arrow head) (d; X400). Liver section of group (4 treated with NDEA + GP 1% )restoring to normalcy with little hepatic damage; hemorrhage in blood sinusoid (short arrow) and karyolysis (arrow head) (e; X400). Liver section of group (5 treated with NDEA + GP 2%) preserving almost the normal structure of the hepatocytes (f; X400). 4. Conclusion Our results strongly suggest that consumed of ginger during the initiation of carcinogenesis (group 5) significantly inhibited cancer incidence, decreased liver enzyme and enhanced enzymic antioxidant concentrations and total protein. The study has revealed that ginger has no toxic effects and has emphasized its protective role against DENA induced liver carcinogenesis. Ginger is a promising agent for the prevention of chemicals-induced toxicity through enhancing the antioxidative.