Lycopene modulates initiation of N-nitrosodiethylamine induced hepatocarcinogenesis: Studies on chromosomal abnormalities,membrane fluidity and antioxidant defense system

Oxidative damage due to free radicals generated during nitrosamine metabolism has been suggested as one of the major cause for the initiation of hepatocarcinogenesis. Lycopene, is a well known antioxidant and have promising preventive potentials, however the mechanism of action remain hypothetical and unclear. To investigate the involvement of lycopene extracted from tomatoes (LycT) against oxidative stress induced deleterious effect of N-nitrosodiethylamine (NDEA) on cellular macromolecules, female Balb/c mice were divided in four groups: Control, NDEA (cumulative dose of 200 mg NDEA/kg body weight injected intraperitoneally in 8 weeks), LycT (5 mg/kg body weight given orally on alternate days, throughout the study) and LycT + NDEA (co-administration of LycT and NDEA). NDEA treatment commenced after 2 weeks of LycT administration. At the end of NDEA exposure i.e., at 10th week, enhanced activities of hepatic phase I enzymes, levels of reactive oxygen species (ROS), lipid peroxidation (LPO) was observed in NDEA group which may have contributed in chromosomal aberrations, enhanced micronucleated cell score, membrane fluidity and serum liver marker enzymes. A significant decrease in enzymatic and non-enzymatic antioxidant system could delineate the mechanism behind such NDEA insults. LycT pre-treatment to NDEA challenged group showed lower chromosomal abnormalities, micronucleated cells score, ROS, LPO levels and liver enzymes. Lycopene aids in normalizing the membrane fluidity and enhancing the activity of antioxidant enzymes and reduced glutathione which could account for the reduced oxidative damage in LycT + NDEA group. It seemed that lycopene supplementation target multiple dys-regulated pathways during initiation of carcinogenesis. Thus, dietary supplementation with lycopene can serve as an alternate measure to intervene the initiation of carcinogenesis.     

Cancare-a herbal formulation inhibits chemically induced tumours in experimental animals

Cancer chemopreventive potential of Cancare, a multi-herbal formulation on chemically induced tumours was studied by N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in rats and 20-methylcholanthrene (20-MC) induced sarcoma development in mice. Oral administration of Cancare was found to inhibit the liver tumour development induced by N-nitrosodiethylamine. Animals administered with NDEA had visible liver tumours by the end of 30th weeks and the liver weight was raised to 6.1 +/- 1.4 g/ 100 g body wt. None of the animals treated with Cancare (150 mg/ kg) developed any visible liver tumours by this period and the liver weight was 3.0 +/- 0.6 g/ 100 g body wt. Gamma-Glutamyl transpeptidase, a marker of hepatocellularcarcinoma, which was raised to 83.7 +/- 8. 9 U/l in serum of NDEA treated group was reduced to 35.2 +/- 6.1 U/l by simultaneous administration of Cancare. Elevated levels of serum alkaline phosphatase, glutamate pyruvate transaminase, bilirubin, liver glutathione S-transferase, glutathione and gamma-Glutamyl transpeptidase in the NDEA administered group was significantly reduced by Cancare administration. Cancare administration inhibited the sarcoma development and increased the life span of mice administered with 20-MC dose dependently. All animals in the control group developed sarcomas by 150th day and dead by 174th day after 20-MC administration. Cancare administration (30 mg and 150 mg/kg) inhibited the sarcoma development (46.7 and 60%) as well as increased the life span (53.3 and 66.7%) as estimated on 240th day after 20-MC administration. The results are indicative of the chemopreventive potential of Cancare against chemically induced neoplasmas.     

Standardization of conditions for the metabolic activation of N-nitrosodiethylamine in mutagenicity tests

Like all nitrosamines, N-nitrosodiethylamine (NDEA) requires metabolic activation in order to exert its carcinogenic effects. This activation involves cytochrome P450s (CYP), which generates unstable metabolites that react with the DNA of cells in the immediate vicinity of metabolite formation. Although NDEA is carcinogenic, it has been considered a weak mutagen in classic genotoxicity assays. We used optimized Salmonella/mammalian microsome genotoxicity assays to assess the mutagenicity and toxicity of low concentrations of NDEA. Using a fixed concentration of NDEA (36.5 mg/ml), we varied the length of preincubation in the presence of different concentrations of an S9 metabolic activation mixture. Salmonella typhimurium strains TA97 and TA102 were resistant to NDEA-induced mutagenesis, even after a preincubation of up to 120 min and the use of different concentrations of the S9 mix. Strain TA98 was susceptible to mutagenesis by NDEA in the absence of the S9 mix and after preincubation with NDEA for 90 min. When bacteria of this strain were preincubated with NDEA for 60 min, mutagenesis was detected at an S9 mix concentration >9.55 mg/ml. NDEA also induced mutagenesis in strain TA100 after preincubation for 90 or 120 min, and this effect was dependent on the S9 concentration. E. coli strain BH990 also showed a concentration-dependent response, with only 60% of the cells surviving after a 120-min preincubation with NDEA in the presence of 19.1 mg S9 mix/ml.     

Lutein presents suppressing but not blocking chemopreventive activity during diethylnitrosamine-induced hepatocarcinogenesis and this involves inhibition of DNA damage

Cancer chemopreventive agents are classified as blocking or suppressing agents if they inhibit initiation or promotion/progression phase of carcinogenesis, respectively. Two experiments were conducted in order to classify lutein as a blocking and/or suppressing agent during rat hepatocarcinogenesis. Inhibitory effects of lutein on hepatic preneoplastic lesions (PNL) and DNA strand breakage induced in Wistar rats by the resistant hepatocyte model of hepatocarcinogenesis (initiation with diethylnitrosamine and promotion with 2-acetylaminofluorene coupled with partial hepatectomy) were investigated when the carotenoid was administered specifically during initiation (experiment 1) or promotion (experiment 2) phase. Animals received by gavage during 2 (experiment 1) or six (experiment 2) consecutive weeks on alternate days 70 mg/kg body weight of lutein. Rats treated with only corn oil during these same periods and submitted to this model were used as controls. Treatment with lutein during initiation did not inhibit nor induced (P>0.05) hepatic preneoplastic lesions and DNA damage. On the other hand, treatment during promotion inhibited (P<0.05) the size of hepatic macroscopic nodules and DNA damage and increased (P<0.05) lutein hepatic levels that reached levels seen in human liver samples. Lutein presented inhibitory actions during promotion but not initiation of hepatocarcinogenesis, being classified as a suppressing agent. This reinforces lutein as a potential agent for liver cancer chemoprevention.     

Suppression of N-nitrosodiethylamine induced hepatocarcinogenesis by silymarin in rats

Antioxidants are one of the key players in tumorigenesis, several natural and synthetic antioxidants were shown to have anticancer effects. In the present investigation the efficacy of silymarin on the antioxidant status of N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in Wistar albino male rats were assessed. The animals were divided into five groups. The animals in the groups 1 and 3 were normal control and silymarin control, respectively. Groups 2, 4 and 5 were administered with 0.01% NDEA in drinking water for 15 weeks to induce hepatocellular carcinoma (HCC). Starting 1 week prior to NDEA administration group 4 animals were treated with silymarin in diet for 16 weeks, 10 weeks after NDEA administration group 5 animals were treated with silymarin and continued till the end of the experiment period (16 weeks). After the experimental period the body weight, relative liver weight, number of nodules, size of nodules, the levels of lipid peroxidation, glutathione (GSH), and the activities of antioxidant enzymes were assessed in both haemolysate and liver tissue. In group 2 hepatocellular carcinoma induced animals there was an increase in the number of nodules, relative liver weight. The levels of lipid peroxides were elevated with subsequent decrease in the body weight, (glutathione) GSH, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD). In contrast, silymarin + NDEA treated groups 4 and 5 animals showed a significant decrease in the number of nodules with concomitant decrease in the lipid peroxidation status. The levels of GSH and the activities of antioxidant enzymes in both haemolysate and liver were improved when compared with hepatocellular carcinoma induced group 2 animals. The electron microscopy studies were also carried out which supports the chemopreventive action of the silymarin against NDEA administration during liver cancer progression. These findings suggest that silymarin suppresses NDEA induced hepatocarcinogenesis by modulating the antioxidant defense status of the animals.     

Antioxidant and anticarcinogenic activity of Lycovin--an indigenous herbal preparation

Aqueous extract of Lycovin has been found to be a potent inhibitor of lipid peroxide formation, (IC50 = 500 micrograms/ml) and scavenger of hydroxyl radical (IC50 = 44 micrograms/ml) and superoxide radical (IC50 = 30 micrograms/ml) in vitro. Lycovin syrup 1.5 ml and 7.5 ml/kg body wt administered orally, reduced the development of sarcoma induced by 20 MC by 35% and 70% respectively. Lycovin syrup was also found to inhibit the hepatocarcinogenesis induced by NDEA. The tumour incidence was 100% in the control group, while none of the drug treated animals developed tumour. Liver weight, gamma-glutamyl transpeptidase (GGT), GSH-S-transferase (GST), reduced glutathione, (GSH) and aniline-4-hydroxylase in liver were elevated in NDEA alone treated animals. The serum parameters indicative of liver injury such as bilirubin, lipid peroxides, alkaline phosphatase and glutamate pyruvate transaminase were also elevated by NDEA administration. These elevated parameters were significantly reduced in animals treated with Lycovin syrup along with NDEA in a dose dependent manner. Even though the exact mechanism of action is not known at present, the observed anticarcinogenic activity may be due to the inhibition of P.450 enzyme activity and subsequent inhibition of the production of the ultimate carcinogen as well as scavenging of oxygen free radicals during promotion of the transformed cell.     

Modelling of the parkinsonian syndrome by the administration of kainic acid into the caudate nucleus

The experiments on rats have shown that bilateral administration of kainic acid (0.1-0.15 microgram) into the rostral parts of caudate nuclei led to the development of hypokinesia and rigidity. An increase in the electrical activity--the formation of the generator of pathologically increased excitement (GPIE) was noted in a zone of kainic acid injection. Rigidity and hypokinesia were attenuated and the GPIE activity was depressed after cyclodol (1-10 mg/kg) or L-DOPA (100-200 mg/kg) administration. Combined administration of cyclodol (2 mg/kg) and L-DOPA (50 mg/kg) induced potentiated antiparkinsonian effect. Dopamine microinjections into the GPIE area depressed its activity and abolished rigidity and hypokinesia. These data suggest that the Parkinson syndrome develops under the influence of GPIE induced by kainic acid administration into caudate nuclei.     

The chemopreventive effect of Ginkgo biloba and Silybum marianum extracts on hepatocarcinogenesis in rats

Background/objective

This study was designed to evaluate the potential chemopreventive activities of Ginkgo biloba extract (EGb) and Silybum marianum extract (silymarin) against hepatocarcinogenesis induced by N-nitrosodiethylamine (NDEA) in rats.

Methods

Rats were divided into 6 groups. Group 1 served as normal control rats. Group 2 animals were intragastrically administrated NDEA at a dose of 10 mg/kg five times a week for 12 weeks to induce hepatocellular carcinoma (HCC). Groups 3 and 4 animals were pretreated with silymarin and EGb respectively. Groups 5 and 6 animals were posttreated with silymarin and EGb respectively. The investigated parameters in serum are alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT) and vascular endothelial growth factor (VEGF). The investigated parameters in liver tissue are malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and comet assay parameters.

Results

In NDEA group, MDA level was elevated with subsequent decrease in GSH level and SOD, GPx and GR activities. In addition, NDEA group revealed a significant increase in serum ALT, AST and GGT activities and VEGF level. Furthermore, NDEA administrated animals showed a marked increase in comet assay parameters. These biochemical alterations induced by NDEA were confirmed by the histopathological examination of rat livers intoxicated with NDEA that showed an obvious cellular damage and well differentiated HCC. In contrast, silymarin+NDEA treated groups (3&;5) and EGb+NDEA treated groups (4&;6) showed a significant decrease in MDA level and a significant increase in GSH content and SOD, GPx and GR activities compared to NDEA group. Silymarin and EGb also beneficially down-regulated the increase in serum ALT, AST, GGT activities and VEGF level induced by NDEA. In addition, silymarin and EGb significantly decreased comet assay parameters. Histopathological examination of rat livers treated with either silymarin or EGb exhibited an improvement in the liver architecture compared to NDEA group.

Conclusions

The obtained findings suggested that silymarin and EGb may have beneficial chemopreventive roles against hepatocarcinogenesis through their antioxidant, antiangiogenic and antigenotoxic activities.     

Chemo-preventive effect of Star anise in N-nitrosodiethylamine initiated and phenobarbital promoted hepato-carcinogenesis

The generation of free radicals is a cause of many pathological conditions like diabetes mellitus, cancer, stroke, etc. Free radicals cause damage to cellular DNA and initiate carcinogenesis. Free radicals also bring about proliferation of cells via cell signaling. An inverse relationship between the consumption of vegetable diets and the risk of cancer has been established. In the present study, Star anise (Illicium verum), which is a commonly used condiment in Indian cuisine, was assessed for its anti-carcinogenic potential in N-nitrosodiethylamine (NDEA) initiated and phenobarbital (PB) promoted hepato-carcinogenesis. Rats were randomly selected for eight experimental groups. The carcinogenesis was induced by injecting the rats, with a single dose of NDEA (200 mg/kg body weight) intraperitoneally as initiator, followed by promotion with PB (0.05%) in drinking water for 14 consecutive weeks. The treatment with NDEA increased liver weight, while Star anise (Star) treatment reduced the liver weight of rats. The treatment with Star throughout for 20 weeks or during the promotion stage (6-20 weeks) significantly reduced the nodule incidence and nodule multiplicity in the rats, while the treatment with Star at the initiation phase (first 4 weeks) only could not reduce these parameters. The treatment with Star for 20 consecutive weeks significantly reduced the nodule size and nodule volume. The treatment with Star throughout as well as at the promotion stage lowered the lipid peroxidation (LPO) in liver and erythrocytes, while the LPO was not lowered, when Star was administered during initiation stage only. The treatment with Star restored the liver and erythrocyte super-oxide dismutase (SOD) activities to normal in the carcinogenesis-induced rats. The liver catalase (CAT) activity increased in all the treated groups. The erythrocyte CAT activity increased in the rats treated with Star during initiation and promotion stage only. The liver glutathione (GSH) level increased significantly in the groups treated with Star. The erythrocyte GSH level was lowered in the rats treated with NDEA and PB, however, Star treatment helped in increasing the erythrocyte GSH level to some extent. The liver and erythrocyte glutathione-S-transferase (GST) activity increased in all the groups treated with NDEA and PB. The treatment with Star decreased GST level significantly. These results indicate that the treatment with Star reduces the tumor burden, lowers oxidative stress and increases the level of phase II enzymes, which may contribute to its anti-carcinogenic potential.     

Facts and theories concerning the mechanisms of carcinogenesis

Carcinogenesis can be induced experimentally by exposure to exogenous agents or it can occur spontaneously without intentional or active intervention. Carcinogenesis can be actively induced by chemicals, radiation, infectious biological agents, transgenesis, or selective breeding. In the human and occasionally when testing potential carcinogens in animals, cancer may result from passive exposure to carcinogens encountered in the ambient environment or from changes in the internal milieu of the animal. Many carcinogens alter the structure of DNA resulting in carcinogenesis, but a significant number of carcinogens do not appear to act through this mechanism. When the action of specific carcinogenic agents is considered in relation to the stages of cancer development, initiation, promotion, and progression, the mechanism of the induction of carcinogenesis by DNA-reactive agents that alter genomic structure can be reconciled with those agents that do not act in this manner. As some cells are fortuitously initiated by uncontrolled variables such as irradiation and through changes in normal processes, the stimulation of growth and altered genetic expression by nongenotoxic agents may result indirectly in cancer development. The final stage of carcinogenesis, progression, can occur spontaneously, enhanced by formation and propagation of genetic errors due to increased cellular proliferation associated with the promotion stage. In addition, chemical and viral agents that lack the capacity for initiation and promotion may actively convert cells in the stage of promotion to the stage of progression. Therefore, the diverse mechanisms of action of carcinogenic agents in relation to their effects on specific stages in the natural history of cancer development allow for greater congruence of many of the theories of carcinogenesis. The influence of the roles of nongenotoxic carcinogenic agents and the potential role of progressor agents on the carcinogenesis process allow a more accurate identification of the potential risk that specific carcinogenic agents pose for increasing human cancer.     

Early stages of hepatic carcinogenesis after vagotomy

Initial stages of hepatocarcinogenesis have been studied in nonoperated and vagotomized animals. As a carcinogenic substance diethylnitrosamine (DENA) has been used. In order to estimate manifestation of the changes, the histochemical method for revealing glucoso-6-phosphatase activity, adenosine triphosphatase and gamma-glutamyltranspeptidase in the liver has been applied. The disturbance of vagus innervation is stated to delay the course of early stages of hepatocarcinogenesis, induced with DENA.     

Vanadium inhibits the development of 2-acetylaminofluorene-induced premalignant phenotype in a two-stage chemical rat hepatocarcinogenesis model

In recent years, research on the biological influence of micronutrients in cancer has grown enormously. Among these, vanadium, a dietary micronutrient present in mammalian tissues has received considerable attention as a limiting agent. In the present study, attempts have been made to investigate the in vivo antitumour potentials of this micronutrient at the 0.5 ppm dosage in drinking water in a defined model of a two-stage experimental rat hepatocarcinogenesis. The chemopreventive effect of vanadium was assessed by studying certain biomarkers, such as development of gamma-glutamyltranspeptidase (GGT)-positive foci, levels of some essential trace elements, in situ expression of proliferating cell nuclear antigen (PCNA) and chromosomal aberrations. Hepatocarcinogenesis was induced in male Sprague-Dawley rats by chronic feeding of 2-acetylaminofluorene (0.05% in basal diet) on and from week 4. Vanadium administration throughout the experiment reduced the relative liver weight, nodular incidence (66.70%), total number and multiplicity (79.93%) and restored hepatic levels of selenium (Se) and iron (Fe) (P < 0.001) when compared to the carcinogen control. Moreover, long-term vanadium treatment significantly abated the expressions of GGT (P < 0.001) and PCNA with concomitant reduction in PCNA immunolabeling index (P < 0.001; 36.62%). Finally, the anticlastogenic potential of vanadium was reflected through its ability to inhibit early chromosomal aberrations (P < 0.001; 45.17%) in 2-AAF-challenged rat hepatocytes. Our results suggest that supplementary vanadium at a dose of 0.5 ppm, when administered continuously throughout the study, than administered either in the initiation or promotion phase alone, is very much effective in suppressing neoplastic transformation in vivo. We conclude the significant role of vanadium in limiting cell proliferation and chromosomal aberrations during the preneoplastic stages of hepatocarcinogenesis in rats.     

Potential chemoprevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by polyphenolics from Acacia nilotica bark

Chemopreventive potential of Acacia nilotica bark extract (ANBE) against single intraperitoneal injection of N-nitrosodiethylamine (NDEA, 200 mg/kg) followed by weekly subcutaneous injections of carbon tetrachloride (CCl₄, 3 ml/kg) for 6 weeks induced hepatocellular carcinoma (HCC) in rats was studied. At 45 day after administration of NDEA, 100 and 200 mg/kg of ANBE were administered orally once daily for 10 weeks. The levels of liver injury and liver cancer markers such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), γ-glutamyl transferase (γ-GT), total bilirubin level (TBL), α-feto protein (AFP) and carcinoembryonic antigen (CEA) were substantially increased following NDEA treatment. However, ANBE treatment reduced liver injury and restored liver cancer markers. ANBE also significantly prevented hepatic malondialdehyde (MDA) formation and reduced glutathione (GSH) in NDEA-treated rats which was dose dependent. Additionally, ANBE also increased the activities of antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) in the liver of NDEA-administered rats. Eventually, ANBE also significantly improved body weight and prevented increase of relative liver weight due to NDEA treatment. Histological observations of liver tissues too correlated with the biochemical observations. HPLC analysis of ANBE showed the presence of gallic, protocatechuic, caffeic and ellagic acids, and also quercetin in ANBE. The results strongly support that A. nilotica bark prevents lipid peroxidation (LPO) and promote the enzymatic and non-enzymatic antioxidant defense system during NDEA-induced hepatocarcinogenesis which might be due to activities like scavenging of oxy radicals by the phytomolecules in ANBE.     

The importance of distinct metabolites of N-nitrosodiethylamine for its in vivo mutagenic specificity

Although N-nitrosodiethylamine (NDEA) is a potent carcinogen in rodents and a probable human carcinogen, little attempts were made to characterize its mutation spectrum in higher eukaryotes. We have compared forward mutation frequencies at multiple (700) loci with the mutational spectrum induced at the vermilion gene of Drosophila, after exposure of post- and pre-meiotic male germ cells to NDEA. Among 30 vermilion mutants collected from post-meiotic stages were 12 G:C-->A:T transitions (40%), 8 A:T-->T:A transversions (27%), and 4 structural rearrangements (13%). The remainder were three A:T-->G:C transitions, two G:C-->C:G transversions and one G:C-->T:A transversion. The results show that although NDEA induces predominantly transitions (40% G:C-->A:T and 10% A:T-->G:C), the frequencies of transversions (37%, of which 27% of A:T-->T:A transversions) and especially of rearrangements (13%) are remarkably high. This mutation spectrum differs significantly from that produced by the direct-ethylating agent N-ethylnitrosourea (ENU), although the relative distribution of ethylated DNA adducts is similar for both carcinogens. These differences, in particular the occurrence of rearrangements, are most likely the result of the requirement of NDEA for bioactivation. Since all four rearrangements were collected from non-metabolizing spermatozoa (or late spermatids), it is hypothesized that they derived from acetaldehyde, a stable metabolite of NDEA. Due to its cytotoxicity, attempts to isolate vermilion mutants from NDEA-exposed pre-meiotic cells were largely unsuccessful, because only two mutants (one A:T-->G:C transition and one 1bp insertion) were collected from those stages. Our results show that NDEA is capable of generating carcinogenic lesions other than base pair substitutions.     

Protective role of Vitamin E pre-treatment on N-nitrosodiethylamine induced oxidative stress in rat liver

Nitrosamine compounds are known hepatic carcinogens. In the metabolism of nitrosamines, such as N-nitrosodiethylamine (NDEA), there is evidence of the formation of reactive oxygen species (ROS) resulting in oxidative stress, which may be one of the factors in the etiology of cancer. The formation of ROS may alter the antioxidant system, while the presence of Vitamin E may counteract NDEA induced oxidative stress. This study was planned to determine whether pre-treatment with Vitamin E (40 mg/kg body weight, i.p., twice a week for 4 weeks) to NDEA induced rats provides protection against oxidative stress in liver caused by the carcinogen. A single necrogenic dose of NDEA (200mg/kg body weight) was administered i.p. to the male albino rats with or without Vitamin E pre-treatment and the animals were sacrificed on Days 7, 14 or 21 after the administration of NDEA. The result showed enhanced levels of hepatic lipid peroxidation (LPO) and conjugated dienes of NDEA treated rats as the indices of oxidative stress, however, Vitamin E pre-treated rats administered NDEA showed decreased LPO and conjugated dienes (Day 21). Superoxide dismutase (SOD) activity in liver was not altered significantly in NDEA treated rats with or without Vitamin E pre-treatment. Catalase (CAT) activity was inhibited with NDEA treatment, however, Vitamin E pre-treatment showed recovery in hepatic CAT activity (Days 14 and 21). Total and Se-glutathione peroxidase (GSH-Px) activities and glutathione-S-transferase (GST) activity in liver increased in NDEA treated rats irrespective of Vitamin E pre-treatment. Glutathione reductase (GSH-R) activity as well as total glutathione (GSH) content in liver decreased in NDEA treated animals, both of which were recovered in Vitamin E pre-treated rats administered NDEA. Activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were increased significantly following NDEA treatment to rats with or without Vitamin E pre-treatment. The activities of AST and ALT enzymes were significantly reduced on Days 14 and 21 and ALP activity was reduced on Day 21 in NDEA+Vitamin E treated animals when compared to NDEA treated alone. LDH enzyme activity was normalized on Day 14 in Vitamin E pre-treated animals administered NDEA. However, the AST, ALT and ALP enzyme activities remained high in all treatment groups as compared to control group. Normal control and Vitamin E treated alone rats revealed normal histology of liver. On the other hand, NDEA treated animals showed alterations in normal hepatic histoarchitecture, which comprised of necrosis and vacuolization of the cells. However, the rats treated with Vitamin E+NDEA showed that the liver cells were normal, with very little necrosis (Day 21). This study concludes that the pre-treatment with Vitamin E prior to the administration of NDEA, reduced the degree of oxidative stress, although this vitamin produced only slight changes in the hepatic injury, in a time-dependent manner.     

Protective effects of garlic oil on hepatocarcinoma induced by N-nitrosodiethylamine in rats

To investigate the protective effects and the possible mechanisms of garlic oil (GO) against N-nitrosodiethylamine (NDEA)-induced hepatocarcinoma in rats, Wistar rats were gavaged with GO (20 or 40 mg/kg) for 1 week, and then were gavaged with GO and NDEA (10 mg/kg) for the next 20 weeks. The changes of morphology, histology, the biochemical indices of serum, and DNA oxidative damage of liver were examined to assess the protective effects. Lipid peroxidation (LPO), antioxidant defense system, and apoptosis-related proteins were measured to investigate potential mechanisms. At the end of the study (21 weeks), GO administration significantly inhibited the increase of the nodule incidence and average nodule number per nodule-bearing liver induced by NDEA, improved hepatocellular architecture, and dramatically inhibited NDEA-induced elevation of serum biochemical indices (alanine aminotransferase , aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase) and hepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in a dose-dependent manner. The mechanistic studies demonstrated that GO counteracted NDEA-induced oxidative stress in rats illustrated by the restoration of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) levels, and the reduction of the malondialdehyde (MDA) levels in liver. Furthermore, the mRNA and protein levels of Bcl-2, Bcl-xl, andβ-arrestin-2 were significantly decreased whereas those of Bax and caspase-3 were significantly increased. These data suggest that GO exhibited significant protection against NDEA-induced hepatocarcinogenesis, which might be related with the enhancement of the antioxidant activity and the induction of apoptosis.     

Garlic Oil Attenuated Nitrosodiethylamine-Induced Hepatocarcinogenesis by Modulating the Metabolic Activation and Detoxification Enzymes

Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes.     

Dietary influence of selenium on the incidence of N-nitrosodiethylamine-induced hepatoma with reference to drug and glutathione metabolizing enzymes

The dietary administration of selenium (sodium selenite; 4 p.p.m.) daily has been found to be highly effective in reducing the incidence of cancer induced by N-nitrosodiethylamine (DEN) in Wistar strain rats. Selenium treatment either before initiation, during initiation and selection/phenobarbital promotion phases of hepatocarcinogenesis has been found to be effective in elevating hepatic microsomal cytochrome b(5), NADPH-cytochrome C reductase and cytosolic aryl hydrocarbon hydroxylase activities to a statistically significant level measured either in the hyperplastic nodule or in the surrounding liver tissues compared to control animals. Moreover, selenium treatment throughout the study, decreases the cytosolic glutathione S-transferase and microsomal UDP-glucuronyl transferase activities by a significant degree when compared to control rats. Alterations in glutathione metabolizing enzyme activities (glutathione reductase, gamma-glutamyl transpeptidase, gamma-glutamylcysteine synthetase and glucose-6-phosphate dehydrogenase) were also observed in selenium-treated groups. Our results confirm the fact that selenium is particularly protective in limiting the action of DEN during the initiation phase of hepatocarcinogenesis.