Manju Lata Gupta

Modification of radiation-induced DNA double strand break repair pathways by chemicals extracted from Podophyllum hexandrum: an in vitro study in human blood leukocytes.

Radiation exposure is a serious threat to biomolecules, particularly DNA, proteins and lipids. Various exogenous substances have been reported to protect these biomolecules. In this study we explored the effect of pre‐treatment with G‐002M, a mixture of three active derivatives isolated from the rhizomes of Podophyllum hexandrum, on DNA damage response in irradiated human blood leukocytes. Blood was collected from healthy male volunteers, preincubated with G‐002M and then irradiated with various doses of radiation. Samples were analyzed using flow cytometry to quantify DNA double strand break (DSB) biomarkers including γ‐H2AX, P53BP1 and levels of ligase IV. Blood samples were irradiated in vitro and processed to determine time and dose‐dependent kinetics. Semiquantitative RT‐PCR was performed at various time points to measure gene expression of DNA‐PKcs, Ku80, ATM, and 53BP1; each of these genes is involved in DNA repair signaling. Pre‐treatment of blood with G‐002M resulted in reduction of γ‐H2AX and P53BP1 biomarkers levels and elevated ligase IV levels relative to non‐G‐002M‐treated irradiated cells. These results confirm suppression in radiation‐induced DNA DSBs. Samples pre‐treated with G‐002M and then irradiated also showed significant up‐regulation of DNA‐PKcs and Ku80 and downregulation of ATM and 53BP1 gene expressions, suggesting that G‐002M plays a protective role against DNA damage. The protective effect of G‐002M may be due to its ability to scavange radiation‐induced free radicals or assist in DNA repair. Further studies are needed to decipher the role of G‐002M on signaling molecules involved in radiation‐induced DNA damage repair pathways. Environ. Mol. Mutagen. 55:436–448, 2014.

Podophyllum hexandrum-Mediated Survival Protection and Restoration of Other Cellular Injuries in Lethally Irradiated Mice

This study aims at the development of a safe and effective formulation to counter the effects of lethal irradiation. The sub-fraction (G-001M), prepared from Podophyllum hexandrum has rendered high degree of survival (>90%) at a dose of 6 mg kg−1 body weight (intramuscular) in lethally irradiated mice. Therapeutic dose of G-001M, at about 20 times lower concentration than its LD100, has revealed a DRF of 1.62. Comet assay studies in peripheral blood leukocytes have reflected that, treatment of G-001M before irradiation has significantly reduced DNA tail length (P < .001) and DNA damage score (P < .001), as compared to radiation-only group. Spleen cell counts in irradiated animals had declined drastically at the very first day of exposure, and the fall continued till the 5th day (P < .001). In the treated irradiated groups, there was a steep reduction in the counts initially, but this phase did not prolong. More than 60% decline in thymocytes of irradiated group animals was registered at 5 h of irradiation when compared with controls, and the fall progressed further downwards with the similar pace till 5th day of exposure (P < .001). At later intervals, thymus was found fully regressed. In G-001M pre-treated irradiated groups also, thymocytes decreased till the 5th day but thereafter rejuvenated and within 30 days of treatment the values were close to normal. Current studies have explicitly indicated that, G-001M in very small doses has not only rendered high survivability in lethally irradiated mice, but also protected their cellular DNA, besides supporting fast replenishment of the immune system.

Modulation of ionizing radiation induced oxidative imbalance by semi-fractionated extract of Piper betle An in vitro and in vivo assessment

The study was planned to evaluate modulatory effect of aqueous extract of Piper betle leaf (PBL) on ionizing radiation mediated oxidative stress leading to normal tissues damage during radiotherapy and other radiation exposures. The total polyphenols and flavonoids known as free radical scavenger (chelators) were measured in the extract. To ascertain antioxidant potential of PBL extract, we studied free radical scavenging, metal chelation, reducing power, lipid peroxidation inhibition and ferric reducing antioxidant properties (FRAP ) using in vitro assays. Mice were exposed to varied radiation doses administered with the same extract prior to irradiation to confirm its oxidative stress minimizing efficacy by evaluating ferric reducing ability of plasma, reduced glutathione, lipid peroxidation and micro-nuclei frequency. PBL extract was effective in scavenging DPPH (up to 92% at 100 µg/ml) and superoxide radicals (up to 95% at 80 µg/ml), chelated metal ions (up to 83% at 50 µg/ml) and inhibited lipid peroxidation (up to 45.65% at 500 µg/ml) in a dose dependant manner using in vitro model. Oral administration of PBL extract (225 mg/kg body weight) 1 hr before irradiation in mice significantly enhanced (p < 0.01) radiation abated antioxidant potential of plasma and GSH level in all the observed organs. The treatment with extract effectively lowered the radiation induced lipid peroxidation at 24 hrs in all the selected organs with maximum inhibition in thymus (p < 0.01). After 48 hrs, lipid peroxidation was maximally inhibited in the group treated with the extract. Frequency of radiation induced micronucleated cells declined significantly (34.78%, p < 0.01) at 24 hrs post-irradiation interval by PBL extract administration. The results suggest that PBL extract has high antioxidant potential and relatively non-toxic and thus could be assertively used to mitigate radiotherapy inflicted normal tissues damage and also injuries caused by moderate doses of radiation during unplanned exposures.

Stress-Mediated Adaptive Response Leading to Genetic Diversity and Instability in Metabolite Contents of High Medicinal Value: An Overview on Podophyllum hexandrum

Podophyllum hexandrum, known for its diversified clinical importance particularly for antineoplastic activity and valuable source for biological protection against high doses of radiation, has its unique position in the plant kingdom. Detailed understanding of mechanism and opportunity of chemical manipulations has amplified the scope of its bioactivity. Podophyllotoxin, the major active principle of this plant, has passed through various structural deviations with the basic aim of making the end product clinically more effective with minimal toxicity. However, over exploitation and limited growth has categorized this plant under endangered species. Depending upon the geographical variations, different species and subspecies of this plant have been explored. Morphological variations and quantitative differences in active principles are the major concern of its unstable medicinal value in whole and semifractionated preparations. The current review has addressed the issues related to the genetic diversity of P. hexandrum, extrinsic and intrinsic stresses responsible for its diversified nature, chemical modifications to enhance its multitasking bioactivity, and efforts for its cultivation and production of important metabolites to avoid collection of wild species due to its critically endangered nature.

A Combination of Podophyllotoxin and Rutin Attenuates Radiation Induced Gastrointestinal Injury by Negatively Regulating NF-κB/p53 Signaling in Lethally Irradiated Mice.

Development of an effective radio protector to minimise radiation-inflicted damages have largely failed owing to inherent toxicity of most of the agents examined so far. This study is centred towards delivering protection to lethally irradiated mice by pre-administration of a safe formulation G-003M (combination of podophyllotoxin and rutin) majorly through regulation of inflammatory and cell death pathways in mice. Single intramuscular dose of G-003M injected 60 min prior to 9 Gy exposure rescued 89% of whole body lethally irradiated C57BL/6J mice. Studies have revealed reduction in radiation induced reactive oxygen species (ROS), nitric oxide (NO) generation, prostaglandin E2 (PGE2) levels and intestinal apoptosis in G-003M pre-treated mice intestine. Restricted nuclear translocation of redox-sensitive Nuclear factor-κB (NF-κB) and subsequent downregulation of cyclo-oxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS; EC 1.14.13.39) and tumor necrosis factor (TNF-α) levels demonstrated the anti-inflammatory effect that G-003M exerts. Support to early hematopoietic recovery was exhibited through G-003M mediated induction of granulocyte colony stimulating factor (G-CSF) and interleukin (IL-6) levels in lethally irradiated mice. Considerable attenuation in radiation induced morphological damage to the intestinal villi, crypts and mucosal layers was observed in G-003M pre-treated mice. Additionally, our formulation did not reduce the sensitivity of tumor tissue to radiation. Altogether, these results suggest that G-003M ameliorates the deleterious effects of radiation exposure by minimising ROS and NO generation and effectively regulating inflammatory and cell death pathways. Mechanism of protection elucidated in the current study demonstrates that G-003M can be used as a safe and effective radio protective agent in radiotherapy for human application.

Countering effects of a combination of podophyllotoxin, podophyllotoxin β-D-glucoside and rutin hydrate in minimizing radiation induced chromosomal damage, ROS and apoptosis in human blood lymphocytes

The present study was conceptualized with the aim of developing a safe radioprotector for human application against radiation induced toxicity. For this study, a formulation (G-002M) prepared by combining three active principles isolated from rhizomes of Podophyllum hexandrum, was evaluated for its potential to protect genomic DNA of human blood cells exposed to different doses of radiation (5,7&10Gy). Blood samples were pretreated (-1hr to exposure) with G-002M. Parameters of Premature Chromosome Condensation (PCC) assay like PCC-index, PCC-rings and PCC-fragments were used to estimate radiation induced chromosomal aberrations. Radiation (7Gy) induce ROS generation and its modulation by G-002M was determined by flow-cytometry and fluorescent microscopy while apoptosis (0,2,24&48 hr) was analyzed using TUNEL assay. Effect on spindle organization in G2/M arrested cells by all the three compounds individually was studied using immunofluorescence microscopy. Irradiation caused dose dependent linear increase in PCC-rings and fragments, while decline in PCC index. G-002M pretreatment significantly decreased these chromosomal aberrations at all the radiation doses and assisted cell survival as indicated by increased PCC index compared with radiation only group. Significant decrease in radiation induced intracellular ROS (45 ± 3%) and apoptosis (49.9%) was also exhibited by the formulation. On podophyllotoxin treatment, most of the cells have shown blocked spindles however, depicted normal arrangement. G-002M also demonstrated a highly significant Dose Modifying Factor or DMF (PCC-rings: 2.27 and PCC-fragments: 1.60). Present study based on many parameters along with DMF study, strongly suggests that G-002M is an effective formulation with a potential to minimize chromosomal damage even at very high radiation doses.

Podophyllotoxin and Rutin Modulates Ionizing Radiation-Induced Oxidative Stress and Apoptotic Cell Death in Mice Bone Marrow and Spleen

The present study is aimed to investigate the radioprotective efficacy of G-003M (combination of podophyllotoxin and rutin) against gamma radiation-induced oxidative stress and subsequent cell death in mice bone marrow and spleen. Prophylactic administration of G-003M (−1 h) rendered more than 85% survival in mice exposed to 9 Gy (lethal dose) with dose reduction factor of 1.26. G-003M pretreated mice demonstrated significantly reduced level of reactive oxygen species, membrane lipid peroxidation, and retained glutathione level. In the same group, we obtained increased expression of master redox regulator, nuclear factor erythroid-derived like-2 factor (Nrf-2), and its downstream targets (heme oxygenase-1, Nqo-1, glutathione S-transferase, and thioredoxin reductase-1). In addition, G-003M preadministration has also shown a significant reduction in Keap-1 level (Nrf-2 inhibitor). Radiation-induced lethality was significantly amended in combination-treated (G-003M) mice as demonstrated by reduced 8-OHdG, annexin V FITC+ cells, and restored mitochondrial membrane potential. Expression of antiapoptotic protein Bcl-2 and Bcl-xL was restored in G-003M pretreated group. However, proapoptotic proteins (Puma, Bax, Bak, Caspase-3, and Caspase-7) were significantly declined in this group. Further analysis of immune cells revealed G-003M-mediated restoration of CD3 and CD19 receptor, which was found decreased to significant level following irradiation. Similarly, Gr-1, a marker of granulocytes, was also retained by G-003M administration prior to radiation. Modulatory potential of this formulation (G-003M) can be exploited as a safe and effective countermeasure against radiation-induced lymphohemopoietic injury.

Proteomic analysis of irradiated lung tissue of mice using gel-based proteomic approach

Abstract Purpose: Exposure to radiation causes severe alterations of protein expression level inside the cell, thus it may influence the biological events and stress response. In the present investigation, we have demonstrated the effect of radiation on mice lung tissues. Materials and methods: Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF was used to check the expression changes in lung proteome profile of strain ‘A’ female mice after exposure to lethal doses of gamma irradiation at different time periods (24 and 48 h). Identified proteins were analysed for their altered expression and were further validated by Western blotting and enzyme-linked immunosorbent assay (ELISA). Results: Nine significant differentially expressed proteins were identified from irradiated lungs tissues. The expression level of zinc finger protein was found to be up regulated at 24 h irradiation in comparison to 48 h irradiation. Conclusions: Zinc finger protein may be considered as a radiation responsive protein. Alteration in its expression pattern may primarily affect binding specificity of the protein that can further result in the interference in transcriptional control of multiple stress responsive genes.

Giant Unruptured Calcified Aneurysm of Non-coronary Sinus of Valsalva

A tion. Chest X-ray showed an ill-defined opacity in right lower chest. Echocardiography, CECT thorax, CT and coronary angiography showed a large unruptured aneurysm with organised thrombus, arising from the non-coronary sinus of Valsalva with calcification around the communication and surrounding aorta without any atrial or ventricular communication and a normal aortic valve, which was compressing the right lung (Fig. 1 a i t V w i a a e a t w a