Nagarajan Rajendra Prasad

Neuroprotective Effects of Hesperidin, a Plant Flavanone, on Rotenone-Induced Oxidative Stress and Apoptosis in a Cellular Model for Parkinson’s Disease

Rotenone a widely used pesticide that inhibits mitochondrial complex I has been used to investigate the pathobiology of PD both in vitro and in vivo. Studies have shown that the neurotoxicity of rotenone may be related to its ability to generate reactive oxygen species (ROS), leading to neuronal apoptosis. The current study was carried out to investigate the neuroprotective effects of hesperidin, a citrus fruit flavanol, against rotenone-induced apoptosis in human neuroblastoma SK-N-SH cells. We assessed cell death, mitochondrial membrane potential, ROS generation, ATP levels, thiobarbituric acid reactive substances, reduced glutathione (GSH) levels, and the activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) using well established assays. Apoptosis was determined in normal, rotenone, and hesperidin treated cells, by measuring the protein expression of cytochrome c (cyt c), caspases 3 and 9, Bax, and Bcl-2 using the standard western blotting technique. The apoptosis in rotenone-induced SK-N-SH cells was accompanied by the loss of mitochondrial membrane potential, increased ROS generation, the depletion of GSH, enhanced activities of enzymatic antioxidants, upregulation of Bax, cyt c, and caspases 3 and 9, and downregulation of Bcl-2, which were attenuated in the presence of hesperidin. Our data suggests that hesperidin exerts its neuroprotective effect against rotenone due to its antioxidant, maintenance of mitochondrial function, and antiapoptotic properties in a neuroblastoma cell line.

Caffeic acid modulates ultraviolet radiation-B induced oxidative damage in human blood lymphocytes

Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppressin in the human skin cells. These biological changes are responsible for photocarcinogenesis and photoaging. Normal lymphocytes are highly sensitive to the damaging effect of UV-radiation and undergo cell death. In the present study, the photoprotective effect of caffeic acid (3,4-dihydroxy cinnamic acid), a dietary phenolic compound, has been examined in the UVB (280-320) irradiated human blood lymphocytes. Lymphocytes pretreated with increasing concentration of caffeic acid (l, 5 and 10 microg/mL) for 30min were irradiated and lipid peroxidation, antioxidant defence status, cell viability (by MTT assay) and DNA damage (by comet assay) were examined. UVB-irradiation causes increased levels of lipid peroxidation, DNA damage and decreased antioxidant status, cell viability in human lymphocytes. Caffeic acid pretreatment significantly reduced the levels of lipid peroxidation markers i.e. thiobarbituric acid reactive substance (TBARS), lipid hydroperoxide (LPH), conjugated diene (CD) and decreased DNA damage (tail length and % tail DNA) in UVB-irradiated lymphocytes. Further, caffeic acid pretreatment significantly maintains antioxidant status and decreased UVB-induced cytotoxicity. The maximum dose of caffeic acid (l0 microg/mL) normalized the UVB induced cellular changes indicating the photoprotective effect of caffeic acid in irradiated lymphocytes.

Radiosensitizing effect of ferulic acid on human cervical carcinoma cells in vitro

Radiotherapy may be effectively combined with plant derived radiosensitizers. Ferulic acid, a naturally occurring phenolic acid, has been reported to have free radical producing properties. In the present study, the radiosensitisation potential of ferulic acid has been tested in two cervical cancer cell lines (HeLa and ME-180) in vitro. Percentage of growth inhibition (MTT assay), colony survival, levels of lipid peroxidation (TBARS, CD and LHP), antioxidant status (SOD, CAT, GPx and GSH), oxidative DNA damage (% tail DNA, tail length, tail moment and Olive tail moment), apoptotic morphological changes (AO/EtBr staining) and intracellular ROS levels (DCFH-DA) were estimated. The present results show that ferulic acid (FA) enhances radiation effects by increasing lipid peroxidative markers in HeLa and ME-180 cells. We observed significant enhancement of ROS levels during ferulic acid plus radiation treatment. FA treatment alone increased intracellular ROS levels indicate its prooxidant nature. Similarly, we observed enhanced oxidative DNA damage and apoptotic morphological changes in FA plus radiation treated cells. The present data suggest radiation sensitizing property of FA in cervical cancer cells. Further investigations warrants to substantiate the present findings.

Antioxidant potential of sesamol and its role on radiation-induced DNA damage in whole-body irradiated Swiss albino mice

Sesamol (SM) is a dietary phytochemical present in the processed sesame oil. In this present study we have evaluated the antioxidant potential of SM and its role in the protection of radiation-induced DNA damage in γ-irradiated mice. The antioxidant properties of SM were evaluated by using different in vitro antioxidant assays. SM shows scavenging effect against hydroxyl (OH), superoxide anion (O(2)(-)), nitric oxide, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS(+)) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. Our results demonstrate that SM exhibits strong antioxidant property in all the in vitro assays. When mice were exposed to 7 Gy γ-radiations there was an increase in % tail DNA, tail length, tail moment and Olive tail moment in blood lymphocytes. SM (100mg/kg b.wt) pretreatment significantly decreased the % tail DNA, tail length, tail moment and Olive tail moment in irradiated mice lymphocytes. These results suggest that SM protects γ-radiation-induced DNA damage in mice lymphocytes, which may be attributed to its antioxidant property.

Photoprotective effect of sesamol on UVB-radiation induced oxidative stress in human blood lymphocytes in vitro

Normal lymphocytes are highly sensitive to the damaging effect of radiation and undergo cell death. In the present study, the photoprotective effect of sesamol, a constituent of sesame oil, has been examined in the UVB-(280-320nm) irradiated human blood lymphocytes. Lymphocytes pretreated with increasing concentrations of sesamol (1, 5 and 10μg/ml) for 30min, were irradiated and lipid peroxidation and antioxidant defense were examined. UVB-irradiated lymphocytes exhibited increased levels of lipid peroxidation and disturbances in antioxidant defense. Sesamol pretreatment resulted in significant reduction in lipid peroxidation marker, thiobarbituric acid reactive substances (TBARS). Further, antioxidants like reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) increased, in a dose-dependent manner, in sesamol pretreated and UVB-irradiated lymphocytes. The maximum dose of sesamol (10μg/ml) normalized the UVB induced lipid peroxidation, indicating the photoprotective effect of sesamol in irradiated lymphocytes.

Ferulic acid, a dietary phenolic acid, modulates radiation effects in Swiss albino mice.

The radioprotective efficacy of Ferulic acid (FA) against whole body gamma radiation was studied in Swiss albino mice. To study the radiation protection, mice were administered with ferulic acid intraperitoneally (i.p) (50 mg/kg body weight.), once daily for five consecutive days. One hour after the last administration of ferulic acid on the sixth day, animals were whole body exposed to 8 Gy gamma radiations. Effect of ferulic acid pretreatment on radiation-induced changes in antioxidant enzymes and lipid peroxidation status in spleen, liver and intestine was analyzed. A significant increase in the antioxidant enzymatic status and decreased lipid peroxidation marker levels were observed in ferulic acid pretreated group, when compared to the irradiated animals. Our study also shows increased % tail DNA, tail length, tail moment and Olive tail moment in irradiated mice blood lymphocytes. Ferulic acid (50 mg/kg body weight) pretreatment significantly decreased the % tail DNA, tail length, tail moment and Olive tail moment in irradiated mice lymphocytes. The histological observations indicated a decline in the villus height and crypt number with an increase in goblet and dead cell population in the irradiated group, which was normalized by ferulic acid pretreatment. In conclusion, present study indicated ferulic acid treatment prevents radiation-induced lipid peroxidation, DNA damage and restored antioxidant status and histopathological changes in experimental animals.

Umbelliferone modulates gamma-radiation induced reactive oxygen species generation and subsequent oxidative damage in human blood lymphocytes

The purpose of this study was to investigate the antioxidant potential of umbelliferone, 7-hydroxy coumarin, and its role in the protection against radiation-induced oxidative damage in cultured human blood lymphocytes. It was found that the antioxidant effect of umbelliferone was dose dependent in hydroxyl (OH(•)), superoxide anion (O(2)(•-)), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical cation (ABTS(•+)) and 1,1-diphenyl-2-picrylhydrazyl (DPPH(•)) radical scavenging assays. To explore the radioprotective effect of umbelliferone, freshly isolated human blood lymphocytes were treated with 124 μM umbelliferone (optimum dose-fixed by MTT assay) 30 min before 3Gy irradiation. It was found that umbelliferone pretreatment inhibited radiation-induced reactive oxygen species generation in 3Gy exposed lymphocytes. Microscopic observations showed that there was a significant apoptotic cells (ethidium bromide/acridine orange staining) and decreased mitochondrial membrane potential (Rhodamine 123 staining) in irradiated lymphocytes. On the other hand, 124 μM umbelliferone treatment significantly decreased % of apoptotic cells and prevented radiation induced mitochondrial depolarization in lymphocytes. Further, it was noticed that there was an increased DNA damage (comet assay), lipid peroxidation with decreased antioxidant enzymatic i.e., superoxide dismutase, catalase and, glutathione peroxidase activities in 3Gy irradiated lymphocytes. Conversely, umbelliferone (124 μM) treatment before irradiation decreased comet attributes and lipid peroxidative markers with improved antioxidant enzyme activities in irradiated lymphocytes. Taken together, the results of this study clearly suggest the radioprotective effect of umbelliferone in human lymphocytes by inhibiting reactive oxygen species generation and its subsequent toxicity.

7-Hydroxycoumarin prevents UVB-induced activation of NF-κB and subsequent overexpression of matrix metalloproteinases and inflammatory markers in human dermal fibroblast cells

Ultraviolet B (UVB) irradiation alters multiple molecular pathways in the skin, thereby inducing skin damage. Human dermal fibroblasts (HDFa) were subjected to single UVB-irradiation (18mJ/cm(2)) resulting in reactive oxygen species (ROS) generation, oxidative DNA damage and upregulation of nuclear factor kappa B (NF-κB) expression. Further, it has been observed that there was a significant cytokine production (TNF-α and IL-6) in UVB irradiated HDFa cells. Our results show that 7-hydroxycoumarin (7-OHC) prevents UVB-induced activation of NF-κB thereby subsequently preventing the overexpression of TNF-α and IL-6 in HDFa cells. Further, 7-OHC prevents UVB-induced activation of cyclooxygenase-2 (COX-2) expression, an inflammatory mediator in skin cells. Moreover, 7-OHC inhibited mRNA expression pattern of matrix metalloproteinases (MMP-1 and MMP-9) in UVB irradiated skin cells. Furthermore, 7-OHC restored antioxidant status, thereby scavenging the excessively generated ROS; consequently preventing the oxidative DNA damage. It has also been noticed that 7-OHC prevents UVB mediated DNA damage through activation of DNA repair enzymes such as XRCC1 and HOGG1. In this study, we treated HDFa cells with 7-OHC before and after UVB irradiation and we found that pretreatment showed better results when compared to posttreatment. Further, 7-OHC showed 9.8416 sun protection factor (SPF) value and it absorbs photons in the UVB wavelength rage. Thus, it has been concluded that sunscreen property, free radical scavenging potential and prevention of NF-κB activation play a role for photoprotective property of 7-OHC.

Resveratrol loaded gelatin nanoparticles synergistically inhibits cell cycle progression and constitutive NF-kappaB activation, and induces apoptosis in non-small cell lung cancer cells.

PURPOSE Previously, we reported that the prepared resveratrol (RSV) loaded gelatin nanoparticles (GNPs) possessed enhanced anticancer effect than free RSV in non-small cell lung carcinoma cells and Swiss albino mice. The present study aims to explore the relevant mechanism of cell death induced by the combination of RSV-GNPs in NCI-H460 cells. METHODS AND RESULTS To increase its bioavailability and anticancer efficacy, we have encapsulated RSV-GNPs by Coacervation method. The detailed methods of preparation and characterization of RSV-GNPs were reported in our earlier publication. RSV-GNPs treated cells showed a further increased level of lipid peroxidative markers, i.e. TBARS and LHP in NCI-H460 cells. Activities of antioxidant enzymes SOD, CAT, GPx and GSH levels were decreased upon the treatment with RSV-GNPs in NCI-H460 cells. The nuclear fragmentation was evaluated by DAPI staining and data showed condensed apoptotic bodies upon treatment with the combination of RSV-GNPs compared to RSV alone treatment group. In addition, cell death induced by RSV-GNPs was mainly due to apoptosis which was characterized by a nuclear DNA fragmentation in a ladder-pattern was obtained from the genomic DNA analysis. Moreover, Western blotting analysis showed that apoptosis induced by RSV-GNPs is associated with the increased Bax, p53, p21, caspase-3 protein levels, and decreased Bcl-2 and NF-κB proteins expression, which indicates the involvement of mitochondria-dependent apoptosis in the anticancer efficacy of RSV-GNPs in NCI-H460 cells. It was also found that this enhanced anticancer efficacy of RSV-GNPs induced cell arrest in the G0/G1 phase of cell cycle. CONCLUSIONS Taken together, the results of our study clearly suggested that the cell death induced by the combination of RSV-GNPs would involve alteration in expression of p53, p21, caspase-3, Bax, Bcl-2 and NF-κB, indicating oxidative mechanism in NCI-H460 cells. Based on these results, it is concluded that GNPs is an ideal way to deliver RSV because of its high loading efficiency and superior efficacy in NCI-H460 cells.

Apigenin protects gamma-radiation induced oxidative stress, hematological changes and animal survival in whole body irradiated Swiss albino mice

Aim: The present study was undertaken to find out the possible radioprotective efficacy of apigenin against whole body gamma (γ)-irradiation, oxidative damage and hematological alterations in Swiss albino mice. Materials and Methods: The percentage of mice surviving 30 days, of exposure to radiation dose (7-11 Gy) was used to construct survival dose response curves. Apigenin (15 mg/kg body weight) was administered intraperitonially ( i . p ) for 7 consecutive days, once daily, and then the mice were exposed to single dose of 7 Gy of γ-radiation. Mice were sacrificed at 24 hours post irradiation, and liver and intestine were taken for various biochemical estimations viz. lipid peroxidation (LPO) markers [thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD) and lipid hydroperoxides (LOOH)], antioxidant status [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH)]. Further blood was collected for the hematological studies [Haemoglobin (Hb) content, red blood cell (RBC) and white blood cell (WBC) count]. Results: Apigenin 15 mg/kg body weight elevated radiation LD 50 from 8.2 to 10 Gy, indicating the dose modifying factor (DMF) of 1.21. It was found that there was an increased LPO level with decreased antioxidant status in 7 Gy irradiated Swiss albino mice. It has been also observed that Hb content, WBC and RBC count was decreased in irradiated Swiss albino mice. Conversely, significant decrease in LPO and restoration of antioxidant status and hematological changes was observed in apigenin pretreated group. Conclusion: Hence, apigenin pretreatment renders protection against 7 Gy radiation-induced biochemical and hematological alterations in Swiss albino mice.