Evaluation of Luteolin in the Prevention of N-nitrosodiethylamine-induced Hepatocellular Carcinoma Using Animal Model System

Hepatocellular carcinoma (HCC) is one of the commonest tumors worldwide. The treatment of HCC is vital for disease diagnosis and prognosis, as the liver is the most important organ controlling metabolic functions. Now-a-days, western folklore medicines are largely dependent on the phyto compounds which are highly effective in therapy and with low side effects. Luteolin is a flavonoid (3,4,5,7-Tetrahydro flavones) possess anti-inflammatory, anticancer and anti allergic property. The present study evaluates the efficacy of luteolin against N-nitrosodiethylamine (DEN) induced HCC in albino rats. In the highlight of the above, luteolin was evaluated for its efficacy against DEN induced HCC in male Wistar albino rats. The Biochemical parameters such as tissue damaging enzymes viz., AST, ALP, LDH and γ-GT, enzymatic antioxidants viz., SOD, CAT, GSH and GPx and histopathological changes have been estimated. The tissue damaging enzymes were found to be high in DEN alone treated group whereas the enzymatic antioxidants decreased destructively. Severe lesions and cirrhosis were observed in the toxin (DEN alone) treated group. The luteolin treated DEN group altered the tissue damaging enzymes and the enzymatic antioxidants. The damaged lesion in the histoarchitecture of DEN treated rat liver was almost completely restored. Finally this study strongly demonstrates that luteolin has potent curative property against HCC in albino rats.     

Protective influence of vitamin E on the antioxidant defence system in the whole blood and liver of normal and alloxan-induced diabetic rats

The effect of oral administration of vitamin E for twenty-eight consecutive days on blood glucose, reduced glutathione levels, antioxidant enzymes (superoxide dismutase and catalase activities), and levels of malondialdehyde (as an index of free radical-mediated lipid peroxidation) was observed in the whole blood and liver of normal and alloxan-induced diabetic rats. It was found that oral administration of vitamin E significantly (p<0.05) lowered the blood glucose level and increased the body weight of the diabetic rats. The activities of superoxide dismutase and levels of reduced glutathione increased significantly (p<0.05) while the level of lipid peroxidation decreased.     

Protective Effect of Emblica officinalis Against Alcohol-Induced Hepatic Injury by Ameliorating Oxidative Stress in Rats

The effect of Emblica officinalis fruit extract (EFE) against alcohol-induced hepatic damage in rats was investigated in the present study. In vitro studies showed that EFE possesses antioxidant as well nitric oxide (NO) scavenging activity. In vivo administration of alcohol (5 g/kg b.wt/day) for 60 days resulted increased liver lipid peroxidation, protein carbonyls, nitrite plus nitrate levels. Alcohol administration also significantly lowers the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and reduced glutathione as compared with control rats. Administration of EFE (250 mg/kg body weight) to alcoholic rats significantly brought the plasma enzymes towards near normal level and also significantly reduced the levels of lipid peroxidation, protein carbonyls and restored the enzymic and non-enzymatic antioxidants level. This observation was supplemented by histopathological examination in liver. Our data indicate that the tannoid, flavonoid and NO scavenging compounds present in EFE may offer protection against free radical mediated oxidative stress in rat hepatocytes of animals with alcohol-induced liver injury.     

Hypoglycaemic, hypolipidemic and antioxidant properties of tulsi (Ocimum sanctum linn) on streptozotocin induced diabetes in rats

Effect of oral administration of 200 mg/Kg body weight of the aqueous extract ofOcimum sanctum (Tulsi) mixed with diet for eight weeks to diabetic (streptozotocin induced) rats was studied. There was significant reduction in fasting blood glucose, serum lipid profile, lipid peroxidation products, (LPO) and improvement in glucose tolerance. The aqueous extract also decreased LPO formation (thiobarbituric acid reactive substances TBARS) and increased antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione transferase (GT) and one antioxidant reduced glutathione (GSH) in plasma and rat liver, lung, kidney and brain. The decrease in TBARS and increase in GSH, SOD, CAT, GPX, and GT clearly shows the antioxidant property ofOcimum sanctum.     

Antioxidant activity ofcurculigo orchioides in carbon tetrachloride—induced hepatopathy in rats

In this study antioxidant activity of methanol extract of rhizomes ofCurculigo orchioides (MEC) was investigated using carbon tetrachloride (CCl₄)-intoxicated rat liver as the experimental model. The hepatotoxic rats were administered MEC for 90 days (daily, orally at the dose of 70 mg per kg body weight). Lipid peroxidation (LPO) in CCl₄-intoxicated rats was evidenced by a marked increment in the levels of thiobarbituric acid reactive substances (TBARS) and diene conjugates (CD), and also a distinct diminution in glutathione (GSH) content in the liver. In CCl₄+MEC—treated rats these biochemical parameters attained an almost normal level. The decreased activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GRD) in CCl₄—intoxicated rats, and its retrieval towards near normalcy in CCl₄+MEC—administered rats revealed the efficacy of MEC in combating oxidative stress due to hepatic damage. Elevated level of glutathione transferase(GTS) observed in hepatotoxic rats too showed signs of retuming towards normalcy in MEC co-administered animals, thus corroborating the antioxidant efficacy of MEC. The findings provide a rationale for further studies on isolation of active principles and its pharmacological evaluation.     

Evaluation of the radioprotective effect of turmeric extract and vitamin E in mice exposed to therapeutic dose of radioiodine

The aim of this study was to evaluate the radioprotective effect of turmeric extract (40 mg/kg body weight) and vitamin E (α- tocopherol acetate, 400 IU/kg body weight) supplementation on lipid peroxidation, reduced glutathione and antioxidant defense enzymes in various organs like liver, kidney and salivary glands at 24 h in adult Swiss mice. ¹³¹Iodine exposure significantly increased lipid peroxidation in kidney and salivary glands in comparison to control animals. Pre supplementation with turmeric extract for 15 days showed significant lowering of lipid peroxidation in kidney. On the other hand vitamin E pre supplementation showed marked reduction in lipid peroxidation in salivary glands. Reduced glutathione levels decreased significantly in liver after radiation exposure. However, pre supplementation with turmeric extract and vitamin E did not improve glutathione levels in liver. In conclusion, we have observed differential radioprotective effect of turmeric extract and vitamin E in kidney and salivary glands. However, Vitamin E seems to offer better radioprotection for salivary glands which is known to be the major site of cellular destruction after radioiodine therapy in patients.     

Free radical injury and antioxidant status in patients with benign prostate hyperplasia and prostate cancer

Reactive oxygen species and other free radicals are known to be the mediators of phenotypic and genotypic changes that lead from mutation to neoplasia. There are some primary antioxidants such as glutathione peroxidase (GPx), glutathione S-transferases (GSTs) and reduced glutathione, which protect against callular and molecular damage caused by the reactive oxygen metabolites (ROMs). The present study was conducted to determine the level of malondialdehyde (MDA), as an index of lipid peroxidation, along with the GPx, GSTs activities and level of reduced glutathione in 45 prostate cancer (PC) patients, 55 benign prostate hyperplasia (BPH) patients as compared to the controls. Significant higher levels of MDA and GSTs activities in the serum, (P<0.005) and significant lower levels of reduced GSH concentration and GPx activity in blood haemolysates (P<0.05) of PC and BPH patients were observed as compared to the controls. The relatively higher GSTs activity and low level of reduced GSH may be due to the response of increased reactive oxygen metabolites production in the blood. The higher MDA and lower GPx activities may be inadequate to detoxify high levels of H₂O₂ into H₂O leading to the formation of the^*OH radical followed by MDA. This result hypothesizes that oxidant-antioxidant imbalance may be one of the major factor responsible for the development of prostate cancer and benign prostate hyperplasia.     

Gene expression and apoptosis response in hepatocellular carcinoma cells induced by biocompatible polymer/magnetic nanoparticles containing 5-fluorouracil

BackgroundSuper-paramagnetic iron oxide nanoparticles (SPION) contain a chemotherapeutic drug and are regarded as a promising technique for improving targeted delivery into cancer cells.ResultsIn this study, the fabrication of 5-fluorouracil (5-FU) was investigated with loaded Dextran (DEX-SPION) using the co-precipitation technique and conjugated by folate (FA). These nanoparticles (NPs) were employed as carriers and anticancer compounds against liver cancer cells in vitro. Structural, magnetic, morphological characterization, size, and drug loading activities of the obtained FA-DEX-5-FU-SPION NPs were checked using FTIR, VSM, FESEM, TEM, DLS, and zeta potential techniques. The cellular toxicity effect of FA-DEX-5-FU-SPION NPs was evaluated using the MTT test on liver cancer (SNU-423) and healthy cells (LO2). Furthermore, the apoptosis measurement and the expression levels of NF-1, Her-2/neu, c-Raf-1, and Wnt-1 genes were evaluated post-treatment using flow cytometry and RT-PCR, respectively. The obtained NPs were spherical with a suitable dispersity without noticeable aggregation. The size of the NPs, polydispersity, and zeta were 74 ± 13 nm, 0.080 and −45 mV, respectively. The results of the encapsulation efficiency of the nano-compound showed highly colloidal stability and proper drug maintenance. The results indicated that FA-DEX-5-FU-SPION demonstrated a sustained release profile of 5-FU in both phosphate and citrate buffer solutions separately, with higher cytotoxicity against SNU-423 cells than against other cells types. These findings suggest that FA-DEX-SPION NPs exert synergistic effects for targeting intracellular delivery of 5-FU, apoptosis induction, and gene expression stimulation.ConclusionsThe findings proved that FA-DEX-5-FU-SPION presented remarkable antitumor properties; no adverse subsequences were revealed against normal cells.How to cite: Mahdia SA, Kadhimb AA, Albukhaty S, et al. Gene expression and apoptosis response in hepatocellular carcinoma cells induced by biocompatible polymer/magnetic nanoparticles containing 5-fluorouracil. Electron J Biotechnol 2021;52. https://doi.org/10.1016/j.ejbt.2021.04.001     

An opium alkaloid-papaverine ameliorates ethanol-induced hepatotoxicity: Diminution of oxidative stress

In this communication, we show the modulatory potential of papaverine, an opium alkaloid and a well known vasodilator agent on the ethanol-induced hepatic oxidative stress in male Wistar rats. Ethanol treatment (50% v/v) enhanced lipid peroxidation significantly accompanied by a decline in the activities of glutathione peroxidase (G-Px), glutathione reductase (GR) and depletion in levels of hepatic glutathione (GSH). Ethanol administration increased hepatic glutathione-s-transferases (GST). Enhanced lipid peroxidation induced by ethanol was significantly reduced when papverine was coadministered (P<0.05). In addition, the depleted levels of glutathione and inhibited activities of G-Px and GR recovered significantly (P<0.05) levelling off to control values on co-exposure. Papaverine (200 mg/kg bw) effectively antagonised the ethanol-induced lipid peroxidation and impaired glutathione levels and glutathione dependent enzyme systems. Our results suggest that papaverine is an effective chemopreventive agent in the liver and may suppress the ethanol-induced hepatotoxicity.     

Ameliorating reactive oxygen species-induced <Emphasis Type="Italic">in vitro</Emphasis> lipid peroxidation in brain,liver, mitochondria and DNA damage by <Emphasis Type="Italic">Zingiber officinale</Emphasis> Roscoe

Iron is an essential nutrient for a number of cellular activities. However, excess cellular iron can be toxic by producing reactive oxygen species (ROS) such as superoxide anion (O₂⁻) and hydroxyl radical (HO^·) that damage proteins, lipids and DNA. Mutagenic and genotoxic end products of lipid peroxidation can induce the decline of mitochondrial respiration and are associated with various human ailments including aging, neurodegenerative disorders, cancer etc. Zingiber officinale Roscoe (ginger) is a widely used spice around the world. The protective effect of aqueous ethanol extract of Z. officinale against ROS-induced in vitro lipid peroxidation and DNA damage was evaluated in this study. The lipid peroxidation was induced by hydroxyl radical generated from Fenton’s reaction in rat liver and brain homogenates and mitochondrial fraction (isolated from rat liver). The DNA protection was evaluated using H₂O₂-induced changes in pBR-322 plasmid and Fenton reaction-induced DNA fragmentation in rat liver. The results indicated that Z. officinale significantly (P<0.001) protected the lipid peroxidation in all the tissue homogenate/mitochondria. The extract at 2 and 0.5 mg/ml could protect 92 % of the lipid peroxidation in brain homogenate and liver mitochondria respectively. The percent inhibition of lipid peroxidation at 1mg/ml of Z. officinale in the liver homogenate was 94 %. However, the extract could partially alleviate the DNA damage. The protective mechanism can be correlated to the radical scavenging property of Z. officinale. The results of the study suggest the possible nutraceutical role of Z. officinale against the oxidative stress induced human ailments.     

Ethanolic leaves extract of <Emphasis Type="Italic">Trianthema portulacastrum</Emphasis> L. ameliorates aflatoxin B<Subscript>1</Subscript> induced hepatic damage in rats

Aflatoxins are potent hepatotoxic and hepatocarcinogenic agents. Reactive oxygen species and consequent peroxidative damage caused by aflatoxin are considered to be the main mechanisms leading to hepatotoxicity. The present investigation aims at assessing the hepatoprotective effect of ethanolic leaves extract of Trianthema portulacastrum on aflatoxin B₁ (AFB₁)-induced hepatotoxicity in a rat model. The hepatoprotection of T. portulacastrum is compared with silymarin, a well known standard hepatoprotectant. Lactate dehydrogenase, alkaline phosphatase, alanine and aspartate aminotransferases were found to be significantly increased in the serum and decreased in the liver of AFB₁ administered (1 mg/kg bw, orally) rats, suggesting hepatic damage. Marked increase in the lipid peroxide levels and a concomitant decrease in the enzymic (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione-S-transferase) and nonenzymic (reduced glutathione, vitamin C and vitamin E) antioxidants in the hepatic tissue were observed in AFB1 administered rats. Pretreatment with T. portulacastrum (100 mg/kg/p.o) and silymarin (100 mg/kg /p.o) for 7 days reverted the condition to near normal. The results of this study indicate that the ethanolic leaves extract of T. portulacastrum is a potent hepatoprotectant as silymarin.     

Toxicology and free radicals scavenging property of Tamra bhasma

Free radicals are implicated in various chronic diseases. There has always been a search for new antioxidants. In this paper we have investigated Tamra bhasma, a metallic ayurvedic preparation. It is a time-tested medicine in Ayurveda and is in clinical use for various ailments specifically the free radical mediated diseases. Our results show that Tamra bhasma inhibits lipid peroxidation (LPO), prevents the rate of aerial oxidation of reduced glutathione (GSH) content and induces the activity of superoxide dismutase (SOD) in rat liver homogenate in the bi-phasic manner. The drug was orally given for 7, 15 and 30 days in different doses. Best protective response was found at the dose of 0.5mg/100g body weight in albino rats, although it showed some histopathological changes at the dose of 20mg/100g body weight. The results suggest that this Ayurvedic preparation is not merely a source of copper metal, but it is a strong anti-oxidant with no detectable adverse effect in lower doses of therapeutic range.     

Role of oxidative stress in ethanol induced germ cell apoptosis — An experimental study in rats

The study was undertaken to evaluate the possible involvement of oxidative stress in the pathogenesis of ethanol induced testicular atrophy in rats. Adult male rats were orally administered ethanol at a dose of 1.6 g/kg body weight/day for four weeks. Twenty-four hours after the last treatment the rats were sacrificed using anesthetic ether. Testes were removed and weighed. Apoptosis was studied by using the Feulgen reaction on 5 μ thin paraffin sections of testis. Testicular homogenate was prepared and centrifuged. The supernatant was used for the estimation of extent of lipid peroxidation and antioxidant defense status. There was significant reduction in body weight: and in testicular weight and diameter in ethanol treated rats. Extent of germ cell apoptosis was significantly high in ethanol treated rats. Ethanol treated rats showed significantly high tissue TBARS level and glutathione S-transferase activity; and low tissue ascorbic acid, reduced glutathione, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities. Chronic ethanol administration resulted in high oxidative stress in the testes either due to increased extent of lipid peroxidation or due to decreased antioxidant defenses, and thereby induces germ cell apoptosis leading to testicular atrophy.     

The response of liver lipid peroxidative and antioxidant defense systems of protein undernourished rats to liver regeneration

The response of liver lipid peroxidative and antioxidant defense system of protein undernourished rats to liver regeneration induced by partial hepatectomy was examined in rats. Animals were divided into four groups; A,B,C and D of four animals each. Animals in group A were maintained on 16% casein diet while those in groups B, C and D were placed on low-protein diet (5% casein) for fourteen weeks and fed ad libitum. 72 hours before sacrifice, partial hepatectomy was carried out on animals in group D while animals in group C were sham-operated. The results show that protein undernutrition induced an increase in lipid peroxidation but reduced catalase activity, glutathione level and superoxide dismutase activity when compared with well-nourished rats. Liver regeneration however, resulted in significant increases in lipid peroxidation and catalase activity but significant reductions in glutathione level and superoxide dismutase activity in protein undernutrition rats when compared with their sham-operated counterparts. These results suggest that liver regeneration induced by partial hepatectomy exacerbates lipid peroxidation in protein undernutrition rats and that Catalase plays a major role in the mopping up of reactive oxygen species generated following liver regeneration in partially hepatectomised protein undernutrition rats.     

Glutathione and some related enzymes in erythrocytes of patients with liver diseases

Red-cell glutathione (GSH) contents and enzyme activities of glutathione peroxidase (GSH-Px) as well as glutathione reductase (GSSG-R) were assayed in 30 normal subjects, 60 cirrhotic patients and 60 cancer patients. The mean GSH level and GSSG-R activity studied showed increased values in all diseased groups compared to normal values with significant elevations only in liver metastases group. Reduced enzyme activity of GSH-Px has been noted in both the cirrhotic groups but the decrease was significant only in alcoholic cirrhotic group. Decreased red-cell GSH-Px activities below the normal range were found in all the cancer patients with remarkable reduction in liver metastases than in hepatocellular carcinoma.     

Evaluation of antioxidant profile and activity of amalaki (Emblica officinalis), spirulina and wheat grass

Aqueous and alcoholic extracts of amalki (Emblica officinalis), spirulina and wheatgrass were prepared and analyzed for antioxidant vitamin content (vitamin C and E), total phenolic compounds. Antioxidant status, reducing power and effect on glutathione S-transferase (GST) activity were evaluated in vitro. Vitamin C content of crude amalaki powder was found to be 5.38 mg/g, while very less amount 0.22 mg/g was detected in wheat grass. Amalki was rich in vitamin E like activity, total phenolic content, reducing power and antioxidant activity. Total antioxidant activity of aqueous extract of amalki, spirulina and wheat grass at 1mg/ml concentration were 7.78, 1.33 and 0.278 mmol/l respectively. At similar concentrations the total antioxidant activity of alcoholic extract of amalaki, spirulina and wheat grass was 6.67, 1.73 and 0.380 mmol/l respectively. Amalki was also found to be rich source of phenolic compounds (241mg/g gallic acid equivalent). Alcoholic extract of wheat grass showed 50 % inhibition in FeCl₂- ascorbic acid induced lipid peroxidation of rat liver homogenates in vitro. Both aqueous and alcoholic extracts of amalaki inhibited activity of rat liver glutathione S-transferase (GST) in vitro in dose dependant manner. Since GST acts as powerful drug metabolizing enzyme its inhibition by amalaki offers possibility of its use for lowering therapeutic dose of herbal preparations. The aqueous extracts of both amalki and spirulina also showed protection against t-BOOH induced cytotoxicity and production of ROS in cultured C₆ glial cells.     

Cytotoxicity and Apoptosis Inducing Activities of 2-Amino-4H-chromene-3-carbonitrile Derivatives Loaded on Gold Nanoparticles Against Human Breast Cancer Cell Line T47D

Chemotherapy drugs, used for prevention of uncontrolled cell proliferation in certain tissues as well as inducing apoptosis in tumor cells, are important candidates for treatment of cancer. The synthesized 2-amino-4H-chromene-3-carbonitrile derivatives effective on cancerous cells resistant to other drugs such as Paclitaxel were used due to their ability in induction of apoptosis. The growth inhibitory and inducing apoptosis activities were determined. In order to make it target-oriented, the best compound was conjugated with gold nanoparticles (NPs) by aspartic acid with chemical reduction method. Cytotoxicity effect of 2-amino-4H-chromene-3-carbonitrile derivatives against the T47D breast cancer cell line was determined by MTT assay. The synthesis of gold NPs was confirmed by transmission electron microscopy, UV–Vis and dynamic light scattering. To assess the effects of compounds on the process of apoptosis, staining methods with acridine orange–ethidium bromide and Hoechst staining by fluorescence microscopy and DNA fragmentation by the diphenylamine method were used. The synthesized compounds containing two NH₂ groups on benzene rings, demonstrated more cytotoxicity effect. The effect of conjugation with gold NPs and the induction of apoptosis were studied with the best compound. The cytotoxicity effects of the synthesized 2-amino-4H-chromene-3-carbonitrile compounds were changed by replacement of NO₂ group on thiol ring with different chemical groups on the benzene ring. Analyses of treated cell lines by conjugated and non-conjugated forms of compounds verified their ability in inducing apoptosis while conjugated form demonstrated higher apoptosis.     

Chemoprotective Role of Triphala Against 1,2-Dimethylhydrazine Dihydrochloride Induced Carcinogenic Damage to Mouse Liver

The present study was carried out to investigate the protective role of Triphala (a combination in equal proportions by weight of fruit powder of Terminalia belerica, Terminalia chebula and Emblica officinalis) against 1,2-dimethylhydrazinedihydrochloride (DMH) induced Endoplasmic reticulum stress (ER stress) in mouse liver. An oral dose of 3 mg/kg body wt in drinking water for 5 weeks significantly (P < 0.001) increased the levels of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum Alkaline phosphatase (ALP) and total bilirubin thus suggesting damage to mouse liver and biliary dysfunction. The DMH administration invariably led to increase in the liver microsomal proteins of molecular weight of about 29 (ERp29) and 53 kDa (ERp53) and decrease in the protein of molecular weight of 36 kDa (ERp36) thereby suggesting the interference of DMH and its metabolites with normal protein biosynthesis and folding, in the reticular membranes of the liver cells thus developing ER stress. Histological studies show necrosis, large sized hepatocytes with increased N:C ratio, aberrant mitotic figures and prominent nucleoli in the liver of DMH treated mice. In animals fed 5% Triphala in diet (w/w) during DMH administration, there was significant decrease in the above changes in the liver suggesting the suppression of DMH induced ER stress in liver. Triphala significantly (P < 0.05) decreased lipid peroxidation and also the activity of lactate dehydrogenase (LDH) in mouse liver. It simultaneously increased the level of reduced glutathione (GSH) and the activity of glutathione-S-transferase (GST) thereby suggesting that it prevents peroxidative damage and also diverts the active metabolites (electrophiles) of DMH from their interactions with critical cellular bio-molecules which could be responsible for its protective action against DMH.