Yasir Hasan Siddique

ESTIMATION OF LIPID PEROXIDATION INDUCED BY HYDROGEN PEROXIDE IN CULTURED HUMAN LYMPHOCYTES

Malondialdehyde (MDA) is used for the estimation of damage by reactive oxygen species. MDA is a major reactive aldehyde resulting from the peroxidation of biological membranes. The most common method used to assess MDA production is the thiobarbituric acid (TBARS) assay. However, the value of this method is curbed by low specificity and has been criticized for its use in human studies. In the present study we have used an alternative method for the estimation of MDA production i.e. reaction of MDA with a chromogenic agent 1-methyl-2-phenylindole at 45°C. The paper describes the method of preparing standards for the estimation of MDA (lipid peroxidation) after the treatment with an oxidative stress inducing agent hydrogen peroxide (H2O2). In the present study, the treatments of 1, 5, 10, 20, 50, 100, 150 and 200 μM of H2O2 induced significant increase in lipid peroxidation as compared to the untreated ones. The results suggest that the present method can be used to measure the lipid peroxidation in cultured human peripheral blood lymphocytes and is specific for MDA estimation.

Antigenotoxic effects of ascorbic acid against megestrol acetate-induced genotoxicity in mice

The genotoxicity of megestrol acetate was studied in mouse bone marrow cells using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) as parameters. Megestrol acetate (8.12, 16.25 and 32.50 mg/ kg of body weight) was injected intraperitoneally separately in different groups of animals. Both CAs and SCEs were statistically increased at 16.25 and 32.50 mg/kg of body weight. Our earlier in vitro studies show the generation of free oxygen radicals, by synthetic progestins responsible for the genotoxic damage. As the genotoxic effects of steroids can be reduced by natural products having antioxidant properties, and ascorbic acid possesses antioxidant activity, ascorbic acid (20, 40 or 60 mg/kg of body weight) administered together with megestrol acetate (32.50 mg/kg of body weight) significantly decreased CAs and SCEs, suggesting an antigenotoxic role of ascorbic acid against megestrol acetate induced genotoxic damage in mice bone marrow cells. The antigenotoxic effect was clearly dose dependent. The highest protective effect was observed at 60 mg/kg body weight of ascorbic acid treated with 32.50 mg/kg body weight of megestrol acetate.

Assessment of cell viability, lipid peroxidation and quantification of DNA fragmentation after the treatment of anticancerous drug mitomycin C and curcumin in cultured human blood lymphocytes.

Mitomycin C (MMC) is an antineoplastic agent used to fight a number of different cancers including cancer of the stomach, colon, rectum, pancreas, breast, lung, uterus, cervix, bladder, head, neck, eye and oesophagus. It is a potent DNA cross-linker. The prolonged use of the drug may result in permanent bone marrow damage and other various types of secondary tumors in normal cells. The toxic effect of anticancerous drugs may be reduced if supplemented with natural antioxidants/plant products. With this view, the effect of 5, 10 and 15 microM of curcumin was studied against the genotoxic doses of MMC, i.e. 10 and 20 microM, in cultured human lymphocytes using cell viability, lipid peroxidation and DNA damage quantification as parameters. The treatment of curcumin with MMC results in a significant dose-dependent increase in cell viability and decrease in lipid peroxidation and DNA damage suggesting a protective role of curcumin against the anticancerous drug mitomycin C.

Antigenotoxic effect of apigenin against anti-cancerous drugs

Mitomycin C and cyclophosphamide are well known anti-tumor drugs. Their genotoxic effects are well established in various test systems. The genotoxic effects in non-tumor cell are of special significance due to the possibility that they may induce secondary tumors in cancer patients. Apigenin is a well known anti-oxidant and possess number of properties that are beneficial in some way to humans. With this view, the present study deals with the effect of apigenin against the genotoxic doses of mitomycin C and cyclophosphamide using chromosomal aberrations, sister chromatid exchanges and cell cycle kinetics as a parameters. The treatment of apigenin results in a significant, dose dependent decrease in the genotoxic damage, induced by mitomycin C and cyclophosphamide. It is concluded that the apigenin is potent in reducing the genotoxic damage, induced by anti-cancerous drugs, thereby reducing the chances of developing secondary tumors during the therapy.

Effect of Curcumin on Lifespan, Activity Pattern, Oxidative Stress, and Apoptosis in the Brains of Transgenic Drosophila Model of Parkinson's Disease

Background. A time dependent loss of dopaminergic neurons and the formation of intracellular aggregates of alpha synuclein have been reported in PD model flies. Methods. The progeny (PD flies) expressing human alpha synuclein was exposed to 25, 50, and 100 µM of curcumin mixed in the diet for 24 days. The effect of curcumin was studied on lifespan, activity pattern, oxidative stress, and apoptosis in the brains of PD model flies. The activity of PD model flies was monitored by using Drosophila activity monitors (DAMs). For the estimation of oxidative stress, lipid peroxidation and protein carbonyl content were estimated in the flies brains of each treated groups. The cell death in Drosophila brain was analyzed by isolating brains in Ringers solution placing them in 70% ethanol and stained in acridine orange to calculate the gray scale values. Results. The exposure of flies to 25, 50, and 100 µM of curcumin showed a dose dependent significant delay in the loss of activity pattern, reduction in the oxidative stress and apoptosis, and increase in the life span of PD model flies. Conclusion. Curcumin is potent in reducing PD symptoms.

Evaluation of the Toxic Potential of Graphene Copper Nanocomposite (GCNC) in the Third Instar Larvae of Transgenic Drosophila melanogaster (hsp70-lacZ)Bg9

Graphene, a two-dimensional carbon sheet with single-atom thickness, have attracted the scientific world for its potential applications in various field including the biomedical areas. In the present study the graphene copper nanocomposite (GCNC) was synthesized, characterized and evaluated for its toxic potential on third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. The synthesized GCNC was analyzed by X-ray diffraction (XRD), scanning/transmission electron microscopy (SEM/TEM), atomic force microscopy (AFM), and fourier transform infrared spectroscopy (FTIR). The GCNC in 0.1% DMSO was sonicated for 10 min and the final concentration of 0.033, 0.099, 0.199 and 3.996 µg/µl of diet were established. The third instar larvae were allowed to feed on it separately for 24 and 48 hrs. The hsp70 expression was measured by O-nitrophenyl-β-D-galactopyranoside assay, tissue damage by trypan blue exclusion test and β-galactosidase activity was monitored by in situ histochemical β-galactosidase staining. Oxidative stress was monitored by performing lipid peroxidation assay and total protein estimation. Ethidium bromide/acridine orange staining was performed on midgut cells for apoptotic index and the comet assay was performed for the DNA damage. The results of the present study showed that the exposure of 0.199 and 3.996 µg/µl of GCNC were toxic for 24 hr of exposure and for 48 hr of exposure: 0.099, 0.199 and 3.996 µg/µl of GCNC was toxic. The dose of 0.033 µg/µl of GCNC showed no toxic effects on its exposure to the third instar larvae for 24 hr as well as 48 hrs. This dose can be considered as No Observed Adverse Effect Level (NOAEL).

Antigenotoxic effect of apigenin against mitomycin C induced genotoxic damage in mice bone marrow cells

The antigenotoxic effect of apigenin was studied against the genotoxic damage induced by mitomycin C on mouse bone marrow cells using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) as parameters. Apigenin was studied at three different doses i.e. 10, 20 and 40 mg/kg b.w. and was found to be non-genotoxic at all the above three doses. Mitomycin C at 2 mg/kg b.w. was given along with the 10, 20 and 40 mg/kg bw of apigenin. A significant decrease in SCEs and CAs was observed, suggesting a protective role of apigenin against the genotoxicity of mitomycin C on mice bone marrow cells.

Effect of L-ascorbic Acid on the climbing ability and protein levels in the brain of Drosophila model of Parkinson's disease.

ABSTRACT In the present study, the effect of l-ascorbic acid (AA) was studied on the climbing ability of the Parkinsons disease (PD) model Drosophila expressing normal human alpha synuclein (h-αs) in the neurons. These flies show locomotor dysfunction as the age progresses. AA at final concentration of 11.35 × 10−5 M, 22.71 × 10−5 M, 45.42 × 10−5 M, and 68.13 × 10−5 M was added to the diet, and the flies were allowed to feed for 21 days. AA at 11.35 × 10−5 M did not show any significant delay in the loss of climbing ability of PD model flies. However, AA at 22.71 × 10−5 M, 45.42 × 10−5 M, and 68.13 × 10−5 M showed a dose dependent significant (p < .05) delay in the loss of climbing ability of PD model flies as compared to the untreated PD flies. The total protein concentration in brain homogenate was measured in treated as well as control groups after 21 days, no significant difference was obtained between treated as well as control (PD flies and l-dopa) groups. The results suggest that AA is potent in delaying the climbing disability of the PD model flies expressing h-αs in the neurons.

Protective effect of apigenin against N-nitrosodiethylamine (NDEA)-induced hepatotoxicity in albino rats.

A number of pharmacological properties have been attributed to apigenin. In the present study the effect of apigenin was investigated with respect to hepatotoxicity induced by N-nitrosodiethylamine (NDEA), a compound that is present in many food stuffs and has been reported to be a hepatocarcinogen. Male rats were exposed to NDEA (0.1mg/ml) dissolved in drinking-water separately, and with 10, 20, or 40mg/ml of apigenin for 21 days. The activity of glutamic-oxaloacetic transaminase (SGOT), glutamic-pyruvic transaminase (SGPT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) was measured in blood serum. Lipid peroxidation, protein carbonyl content and micronucleus frequency were determined in hepatocytes. To assess the effect on DNA damage, the comet assay was performed on hepatocytes, blood lymphocytes and bone-marrow cells of the exposed rats. The results of the study reveal that the treatment of NDEA together with apigenin showed a significant dose-dependent decrease in the serum concentration of the enzymes SGOT, SGPT, ALP and LDH (p<0.05). Histological sections of the liver also showed a protective effect of apigenin. A significant dose-dependent reduction in lipid peroxidation and protein carbonyl content was observed in rats exposed to NDEA (0.1mg/ml) together with apigenin (p<0.05). The results obtained for the comet assay in rat hepatocytes, blood lymphocytes and bone-marrow cells showed a significant dose-dependent decrease in the mean tail length (p<0.05). The present study supports the role of apigenin as an anti-genotoxic and hepatoprotective agent.

GC-MS analysis of Eucalyptus citriodora leaf extract and its role on the dietary supplementation in transgenic Drosophila model of Parkinson's disease.

The role of Eucalyptus citriodora L. leaf extract was studied on the transgenic Drosophila model of flies expressing normal human alpha synuclein (h-αS) in the neurons. These flies exhibit locomotor dysfunction as the age progresses. The leaf extract was prepared in acetone and was subjected to GC-MS analysis. The GC-MS analysis revealed the presence of nine major compounds. E. citriodora extract at final concentration of 0.25, 0.50 and 1.0μl/ml was supplemented with the diet and the flies were allowed to feed for 21days. The effect of extract was studied on the climbing ability and the oxidative stress on the PD model Drosophila expressing normal human alpha synuclein (h-αS) in the neurons. The supplementation of 0.25, 0.50 and 1.0μl/ml of E. citriodora extract showed a dose dependent significant delay in the loss of climbing ability and reduction in the oxidative stress in the brain of PD model flies. The results also support the utility of this model in studying PD symptoms.