Development of hematopoietic syndrome mice model for localized radiation exposure.

Current models to study the hematopoietic syndrome largely rely on the uniform whole-body exposures. However, in the radio-nuclear accidents or terrorist events, exposure can be non-uniform. The data available on the non-uniform exposures is limited. Thus, we have developed a mice model for studying the hematopoietic syndrome in the non-uniform or partial body exposure scenarios using the localized cobalt60 gamma radiation exposure. Femur region of Strain 'A' male mice was exposed to doses ranging from 7 to 20 Gy. The 30 day survival assay showed 19 Gy as LD100 and 17 Gy as LD50. We measured an array of cytokines and important stem cell markers such as IFN-γ, IL-3, IL-6, GM-CSF, TNF-α, G-CSF, IL-1α, IL-1ß, CD 34 and Sca 1. We found significant changes in IL-6, GM-CSF, TNF-α, G-CSF, and IL-1ß levels compared to untreated groups and amplified levels of CD 34 and Sca 1 positive population in the irradiated mice compared to the untreated controls. Overall, we have developed a mouse model of the hematopoietic acute radiation syndrome that might be useful for understanding of the non-uniform body exposure scenarios. This may also be helpful in the screening of drugs intended for individuals suffering from radiation induced hematopoietic syndrome.

Combination of podophyllotoxin and rutin modulate radiation-induced alterations of jejunal proteome in mice.

Purpose: Gastrointestinal (GI) injuries post ionizing radiation (IR) becomes a crucial factor in survival. Thus, the current study was aimed to explore the molecular mechanisms behind IR produced GI proteome alterations and their amelioration by a safe radioprotective formulation candidate, G-003M (podophyllotoxin+rutin).Materials and method: C57BL/6 mice were administered with G-003M 1 h before 9 Gy whole body γ irradiation. 2DE-MS analysis was conducted to identify differential expression of jejunum proteins with fold change >1.5 (p < .05) at various time-points. Results: G-003M pre-administration decreased total number of differential proteins. It mediated protection to cytoskeleton, modulated stress, apoptosis and inflammatory proteins. Direct effect on eukaryotic translation initiation factor 4H (Eif4h), thioredoxin domain-containing protein 17 (Txndc17) and interferon-induced protein 35 (Ifi35) was observed. Bioinformatics depicted transcription factor-MYC, was also positively modulated by G-003M. Further, it also enhanced level of citrulline (ELISA analysis), and restored crypts and villi lengths (histological analysis) against severe damage caused by lethal irradiation.Conclusion: Current findings reveal that G-003M may be an efficient candidate in protecting key proteins of metabolic and biochemical pathways assisting in the rapid recovery of GI proteome. This fairly improved the chances of animal survival exposed to lethal doses of whole body radiation.

A Combination of Podophyllotoxin and Rutin Alleviates Radiation-Induced Pneumonitis and Fibrosis through Modulation of Lung Inflammation in Mice.

Pneumonitis and pulmonary fibrosis are predominant consequences of radiation exposure, whether planned or accidental. The present study, demonstrates radioprotective potential of a formulation, prepared by combining podophyllotoxin and rutin (G-003M), in mice exposed to 11 Gy thoracic gamma radiation (TGR). Treated mice were observed for survival and other symptomatic features. Formation of reactive oxygen species (ROS)/nitric oxide (NO) was measured in bronchoalveolar lavage cells. DNA damage and cell death were assessed in alveolar cells by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Total protein (TP), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were measured in bronchoalveolar lavage fluid (BALF)/serum of mice to assess lung vascular permeability. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), cluster of differentiation 45, inducible nitric oxide synthase (iNOS), and nitrotyrosine were also estimated in lungs/BALF of differentially treated mice. Our observations revealed 100% survival in G-003M-pretreated mice against 66.50% in 11 Gy TGR exposed. Other symptoms like reduction in graying of hair, weight loss, and breathing rate were also observed in pretreated groups. Significant decline in ROS/NO and cell death in formulation pretreated mice were also observed. Decreased level of TP, LDH, and ALP in BALF/serum samples revealed G-003M-induced inhibition in lung permeability. Level of IL-6, TNF-α, and TGF-ß1 in the lungs of these mice was found corresponding to control group at 8 weeks posttreatment. On the contrary, these cytokines raised significantly in 11 Gy TGR-exposed mice. Lung pneumonitis and fibrosis were found significantly countered in these mice. The observations revealed that G-003M could regulate immune system by curtailing radiation-induced oxidative and inflammatory stress, which has helped in minimizing radiation-inflicted pneumonitis and fibrosis.

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.

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.

γH2AX formation kinetics in PBMCs of rabbits exposed to acute and fractionated radiation and attenuation of focus frequency through preadministration of a combination of podophyllotoxin and rutin hydrate.

DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G-003M on foci generation and persistence. G-003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co(60) gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G-003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G-003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger (P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11-15 in terminally ill animals. G-003M pretreatment significantly (P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re-appearance of foci is a strong indicator of imminent death in animals, and that G-003M provides protection against radiation. Environ. Mol. Mutagen. 57:455-468, 2016. © 2016 Wiley Periodicals, Inc.

Countering effects of a combination of podophyllotoxin, podophyllotoxin ß-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.