Hari N. Bhilwade

Schisandrin B exhibits anti-inflammatory activity through modulation of the redox-sensitive transcription factors Nrf2 and NF-κB

Schisandrin B (SB), a dibenzocyclooctadiene derivative isolated from Schisandra chinensis and used commonly in traditional Chinese medicine for the treatment of hepatitis and myocardial disorders, has been recently shown to modulate cellular redox balance. Since we have shown that cellular redox plays an important role in the modulation of immune responses, the present studies were undertaken to study the effects of SB on activation and effector functions of lymphocytes. SB altered the redox status of lymphocytes by enhancing the basal reactive oxygen species levels and altering the GSH/GSSG ratio in lymphocytes. It also induced nuclear translocation of redox sensitive transcription factor Nrf2 and increased the transcription of its dependent genes. SB inhibited mitogen-induced proliferation and cytokine secretion by lymphocytes. SB also significantly inhibited mitogen-induced upregulation of T cell costimulatory molecules and activation markers. It was observed that SB inhibited mitogen-induced phosphorylation of c-Raf, MEK, ERK, JNK, and p38. It suppressed IκBα degradation and nuclear translocation of NF-κB in activated lymphocytes. Anti-inflammatory effects of SB were significantly abrogated by the inhibitors of Nrf2 and HO-1, suggesting the involvement of this pathway. Similar anti-inflammatory effects of SB on lymphocyte proliferation and cytokine secretion were also observed in vivo. To our knowledge, this is the first report showing that the anti-inflammatory effects of SB are mediated via modulation of Nrf2 and NF-κB in lymphocytes.

Gamma ray induced DNA damage in human and mouse leucocytes measured by SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay.

The studies reported in this communication had two major objectives: first to validate the in-house developed SCGE-Pro: a software developed for automated image analysis and data processing for Comet assay using human peripheral blood leucocytes exposed to radiation doses, viz. 2, 4 and 8 Gy, which are known to produce DNA/chromosome damage using alkaline Comet assay. The second objective was to investigate the effect of gamma radiation on DNA damage in mouse peripheral blood leucocytes using identical doses and experimental conditions, e.g. lyses, electrophoretic conditions and duration of electrophoresis which are known to affect tail moment (TM) and tail length (TL) of comets. Human and mouse whole blood samples were irradiated with different doses of gamma rays, e.g. 2, 4 and 8 Gy at a dose rate of 0.668Gy/min between 0 and 4 degrees C in air. After lyses, cells were electrophorased under alkaline conditions at pH 13, washed and stained with propidium iodide. Images of the cells were acquired and analyzed using in-house developed imaging software, SCGE-Pro, for Comet assay. For each comet, total fluorescence, tail fluorescence and tail length were measured. Increase in TM and TL was considered as the criteria of DNA damage. Analysis of data revealed heterogeneity in the response of leucocytes to gamma ray induced DNA damage both in human as well as in mouse. A wide variation in TM and TL was observed in control and irradiated groups of all the three donors. Data were analyzed for statistical significance using one-way ANOVA. Though a small variation in basal level of TM and TL was observed amongst human and mouse controls, the differences were not statistically significant. A dose-dependent increase in TM (P<0.001) and TL (P<0.001) was obtained at all the radiation doses (2-8 Gy) both in human and mouse leucocytes. However, there was a difference in the nature of dose response curves for human and mouse leucocytes. In human leucocytes, a linear increase in TM and TL was observed up to the highest radiation dose of 8 Gy. However, in case of mouse leucocytes, a sharp increase in TM and TL was observed only up to 4 Gy, and there after saturation ensued. In human samples, the dose response of both TM and TL showed best fits with linear model (r(TM)=0.999 and r(TL)=0.999), where as in mouse, the best fit was obtained with Sigmoid (Boltzman) model. From the present data on leucocytes with increase in TM and TL as the criteria of DNA damage, it appears that mouse is relatively more sensitive to radiation damage than humans.

Schisandrin B, attenuates cisplatin-induced oxidative stress, genotoxicity and neurotoxicity through modulating NF-κB pathway in mice.

Abstract This study aimed to investigate the potential beneficial effect of an antioxidant lignan, Schisandrin B (Sch B), against cisplatin (cDDP) induced oxidative stress mediated geno- and neuro-toxicities. A dose of 10 mg/kg cDDP induced considerable genotoxicity in mice, and Sch B treatment attenuated the cDDP-induced DNA damage as assessed by the comet assay in the brain. The frequency of micro-nucleated erythrocyte production in bone marrow was also significantly reduced by Sch B treatment in cDDP-treated mice. In neurobehavioral studies, Sch B significantly prevented the memory deficits induced by cDDP, and had an anxiolytic effect in the elevated plus maze task. Sch B treatment significantly attenuated lipid peroxidation, acetylcholinesterase activity and nitrite levels induced by cDDP. Furthermore, Sch B effectively inhibited NF-κB and p53 activation, and cleaved caspase-3 expression in cDDP-treated mice. Hence, Sch B with potent antioxidant and neuro-protective property with no mutagenic activity would be beneficial complementary food factor against cDDP induced oxidative stress.

Squalene as Novel Food Factor

Currently, health beneficial roles of natural products attract much attention and diverse functional ingredients have been extensively studied their preventive effect in many diseases such as cardiovascular diseases and cancer. Squalene is one of those examples. It distributes in nature from plant to animal but extraordinarily concentrated in the liver of certain species of shark (Squalidae family) as it was first identified as a healing substance in the shark liver oil. It is now well-known that squalene is the physiological substance functioning in animal as the precursor of cholesterol biosynthesis. On the other hand, it has long history of using as an attractive resource for functional food, supplement or even pharmaceutics because it has unique physical property and wide variety of physiological functions such as anticancer and anti-hyper cholesterolemia. The antioxidant and oxygen carrying properties of squalene predicts its potential in preventing cardiovascular disease. We reviewed recent progress in functional studies of squalene both in vitro and in vivo models.

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.

Age-dependent changes in spontaneous frequency of micronucleated erythrocytes in bone marrow and DNA damage in peripheral blood of Swiss mice.

Age-dependent changes in chromosomal damage in bone marrow - a self-proliferating tissue - in the form of spontaneously occurring micronucleated erythrocytes, and DNA damage in peripheral blood were examined in male and female Swiss mice. In the erythrocyte population in the bone marrow, polychromatic (immature) erythrocytes showed a significant increase in the frequency of micronuclei as a function of age of the mice (1-20 months). The increase in micronucleus frequency was less in normochromatic (mature) erythrocytes. The female mice showed a higher frequency of micronuclei than the male mice in all the age groups examined. However, the female to male ratio of micronucleus frequencies in total erythrocytes as well as in polychromatic erythrocytes decreased with age. DNA damage, measured as tail moment in the single-cell gel electrophoresis in peripheral blood of different age groups of mice (1, 6, 12 and 18 months) showed a gradual increase with age. Female mice showed more DNA damage than 1-month and 18-month-old male mice. In conclusion, these results show that there is an accumulation of genetic damage in bone marrow and DNA damage in peripheral blood of mice during ageing, and that females show more alterations than males.

Magnitude of radiation-induced DNA damage in peripheral blood leukocytes and its correlation with aggressiveness of thymic lymphoma in Swiss mice

Abstract Purpose: The present study is aimed to investigate the magnitude and kinetics of DNA damage in peripheral blood leukocytes of mice exposed to whole body gamma irradiation (WBI; 3 Gy) and its correlation with aggressiveness of thymic lymphoma (TL). Materials and methods: DNA damage was monitored in peripheral blood cells of individual mice by comet assay at different intervals of post-irradiation, which were correlated with weight of TL in respective mice at 120th day. To further study genomic radiosensitivity in TL development, peripheral blood samples collected at the 15th and 90th day of post-irradiation from control and WBI animals were irradiated (0.5 Gy) ex vivo followed by assessment of DNA damage by comet assay. Results: The maximum DNA damage (tail moment) was observed at 5 min after WBI, which decreased at longer period, and was minimum at the 7th day after WBI. However, residual damage was observed in comparison to control and it persisted up to 90 days of irradiation. Tail moment values observed at an early time (5 min) of post-irradiation was better correlated (correlation coefficient, r = 0.84) with weight of TL than at longer time period (60 days; r = 0.21). Our results showed that in ex vivo irradiated (0.5 Gy) peripheral blood, the magnitude of DNA damage was higher in samples obtained from WBI mice than sham-irradiated controls suggesting enhanced genomic radiosensitivity in WBI mice. Genomic susceptibility to radiation observed in peripheral blood from WBI animals showed better correlation with weight of TL at the 15th day (r = 0.9) post-irradiation period than at the 90th day (r = 0.44). Conclusion: These results suggest that the magnitude of radiation-induced initial DNA damage in peripheral blood leukocytes and genomic radiosensitivity could be an indicator of TL aggressiveness in mice.

Dihydroxyselenolane (DHS) supplementation improves survival following whole-body irradiation (WBI) by suppressing tissue-specific inflammatory responses.

Dihydroxyselenolane (DHS), a simple water-soluble organoselenium compound, was evaluated for radioprotection in BALB/c mice after whole-body irradiation (WBI) (8Gy (60)Co, 1Gy/min), by monitoring 30-d post-irradiation survival and biochemical/histological changes in radiosensitive organs. Intraperitoneal administration of DHS at 2mg/kg for five consecutive days before irradiation and three times per week during the post-irradiation period showed maximum benefit (40% improvement in 30 d post-irradiation survival). DHS treatment, despite inducing expression of glutathione peroxidases (GPx1, GPx2, and GPx4) in spleen and intestine, did not protect against radiation-induced acute (10-day) haematopoietic and gastrointestinal toxicities. DHS treatment significantly reduced radiation-induced DNA damage in peripheral leukocytes and inflammatory responses in intestine, lung, and circulation. The anti-inflammatory effect of DHS was associated with reductions in lipid peroxidation, expression of pro-inflammatory genes such as Icam-1, Ccl-2, and iNos-2, and subsequent infiltration of inflammatory cells. Irradiated mice treated with DHS survived until day 30 post-irradiation and showed restoration of spleen cellularity and intestinal villi, but had moderately increased systemic and tissue-specific inflammatory responses. Another organoselenium compound, selenomethionine, evaluated in parallel with DHS at the same dose and treatment schedule, showed comparable radioprotective effects. The mechanism of radioprotection by DHS is mainly via suppression of inflammatory responses.