Co-occurrence of acrocentric chromosome associations with dicentric chromosomes in irradiated human lymphocytes.

PURPOSE: The occurrence of acrocentric chromosome association (ACA) after radiation exposure is an interesting cytogenetic endpoint, known to show a dose-dependent increase in irradiated lymphocytes suggesting its potential use in radiation biodosimetry. Here, an attempt was made to study the complexity and correlation of the occurrence of ACA with dicentric chromosomes (DC) in lymphocytes exposed to gamma radiation. METHODS: Ninety metaphases each with DC and without DC were chosen randomly from lymphocytes irradiated with different doses (0, 1, 2, 3, 4 and 5 Gy) of gamma radiation. ACA along with chromosomal types of aberrations were scored and analyzed for complexity and co-occurrence, retrospectively. RESULTS: The number of associations between 2 and ≥ 3 acrocentric chromosomes showed an increase with each irradiation dose. Concomitantly, the total number of chromosomal type of aberrations showed an increase in number at each radiation dose studied. The number of DC showed an increase, however, metaphases containing 1DC decreased while ≥ 2DC increased as the radiation dose increased. The number of tricentric chromosomes increased at doses higher than 2 Gy. Importantly, the association of DC with an acrocentric chromosome was noticed at doses 2 Gy and above. A significant (p < 0.05) increase was noticed in ACA frequency in 1DC and ≥ 2DC metaphases at 1 and 2 Gy, in 1DC at 3 Gy, and in ≥ 2DC 4 and 5 Gy compared to the frequency in no DC metaphases. When average ACA frequency was plotted against DC frequency, a significant (p = 0.0009) correlation was observed, producing regression equation y = 0.9025x + 0.1283; R2 = 0.9522. CONCLUSION: The present analysis showed increasing ACA complexity with increasing radiation dose. Furthermore, a higher frequency of ACA in cells with 1DC or ≥ 2DC compared to the ACA in cells without DC from the same sample of irradiated lymphocytes demonstrated the co-occurrence of ACA and DC in the same cells.

Linear dose response of acrocentric chromosome associations to gamma irradiation in human lymphocytes.

PURPOSE: The frequency of acrocentric chromosome associations (ACA) was studied to determine the possible dose-response relationship of gamma irradiation in human lymphocytes. METHODS: Peripheral blood collected from three healthy donors was irradiated with 0, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 Gy of gamma radiation. Chromosomal preparations were made after 48 h of culture as per standard guidelines. The experiment was repeated three times, with a different donor each time. RESULTS: The ACA frequency in irradiated lymphocytes increased with radiation dose. The D-G type of association was most prominent and showed a significant dose-dependent increase in frequency. The dose response of ACA frequency to radiation was found to be linear: ACA frequency = 0.2923 (±0.0276) + 0.1846 (±0.0307) × D (correlation coefficient r = 0.9442). As expected, dicentric chromosome (DC) frequencies followed the linear quadratic fit model, with DC frequency = 0.0015 (±0.0013) + 0.0220 (±0.0059) × D + 0.0215 (±0.0018) × D^2 (correlation coefficient r = 0.9982). A correlation curve was prepared for ACA frequency versus DC frequency, resulting in the regression equation y = 1.130x + 0.4051 (R2 = 0.7408; p = 0.0014). CONCLUSION: Our results showed an increase in ACA frequency in irradiated lymphocytes with an increase in radiation dose; thus, ACA may serve as a candidate cytogenetic biomarker for radiation biodosimetry.

Radiomitigation by Melatonin in C57BL/6 Mice: Possible Implications as Adjuvant in Radiotherapy and Chemotherapy.

BACKGROUND/AIM: Melatonin (N-acetyl-5-methoxytryptamine), a chief secretory molecule of the pineal gland, has multiple properties, and numerous clinical investigations regarding its actions are in progress. This study investigated the radiomitigative role of melatonin in C57BL/6 mice. MATERIALS AND METHODS: Melatonin (100 mg/kg) was orally administered once daily starting at 1 h on day 1 and subsequently every 24 h until day 7 after whole-body irradiation (WBI) and survival was monitored for 30 days. The bone marrow, spleen, and intestine were studied to evaluate the mitigative potential of melatonin after radiation-induced damage. RESULTS: Melatonin significantly improved the survival upto 60% and 90% after 9 Gy (lethal) and 7.5 Gy (sub-lethal) WBI, respectively. Melatonin alleviated WBI-induced myelosuppression and pancytopenia, and increased white blood cell, red blood cell, platelet, and lymphocyte (CD4+ and CD8+) counts in peripheral blood. Bone marrow and spleen cellularity were restored through enhanced haematopoiesis. Melatonin ameliorated the damage in the small intestine, and promoted recovery of villi length, crypts number, and goblet cell count. CONCLUSION: Melatonin mitigates the radiation-induced injury in the gastrointestinal and haematopoietic systems. The observed radiomitigative properties of melatonin can also be useful in the context of adjuvant therapy for cancer and radiotherapy.

PK-PD based optimal dose and time for orally administered supra-pharmacological dose of melatonin to prevent radiation induced mortality in mice.

AIMS: The study reports preclinical pharmacokinetics (PK) and correlation with pharmacological effect at suprapharmacological dose of orally administered melatonin along with time and dose optimization, which have been lacking in earlier reports of radioprotection using melatonin. METHODS: PK of melatonin in C57BL/6 mice was evaluated after dose of 250 mg/kg using HPLC. Tissue distribution study was conducted in vital organs following oral administration. Plasma total antioxidant capacity (TAC) was determined by ABTS+ radical assay and was correlated to plasma concentrations of melatonin. Using the outcomes of PK and Pharmacodynamics (PD), survival study was conducted for optimization of 'drug radiation gap period' (DRGP). Optimal oral dose for radioprotection was determined using survival as an end point. KEY FINDINGS: PK analysis of melatonin revealed Tmax at 5 min with closely spaced another distinct concentration peak at 20 min. Plasma TAC of melatonin showed similar peaks at 5 min and 45 min, with the highest TAC at 45 min. Survival following a lethal (9 Gy) radiation dose was 20% and 40% after 5 and 45 min of melatonin administration, respectively. DRGP for melatonin was thus 45 min, while optimal oral dose ranged from 125 to 250 mg/kg. PK parameters at 250 mg/kg dose were qualitatively similar to low dose of melatonin, thus preventing chances of unexpected toxicity. SIGNIFICANCE: Survival enhancement at 45 min suggested as probable interval required as 'DRGP'. The optimum oral therapeutic window appears large with no substantial toxicity. The outcomes will be useful in development of radioprotectors as well as other therapeutic applications.

Melatonin attenuates 60 Co γ-ray-induced hematopoietic, immunological and gastrointestinal injuries in C57BL/6 male mice.

Protection of hematopoietic, immunological, and gastrointestinal injuries from deleterious effects of ionizing radiation is prime rational for developing radioprotector. The objective of this study, therefore, was to evaluate the radioprotective potential of melatonin against damaging effects of radiation-induced hematopoietic, immunological, and gastrointestinal injuries in mice. C57BL/6 male mice were intraperitoneally administered with melatonin (50-150 mg/kg) 30 min prior to whole-body radiation exposure of 5 and 7.5 Gy using 60 Co-teletherapy unit. Thirty-day survival against 7.5 Gy was monitored. Melatonin (100 mg/kg) pretreatment showed 100% survival against 7.5 Gy radiation dose. Melatonin pretreatment expanded femoral HPSCs, and inhibited spleenocyte DNA strands breaks and apoptosis in irradiated mice. At this time, it also protected radiation-induced loss of T cell sub-populations in spleen. In addition, melatonin pretreatment enhanced crypts regeneration and increased villi number and length in irradiated mice. Translocation of gut bacteria to spleen, liver and kidney were controlled in irradiated mice pretreated with melatonin. Radiation-induced gastrointestinal DNA strand breaks, lipid peroxidation, and expression of proapoptotic-p53, Bax, and antiapoptotic-Bcl-xL proteins were reversed in melatonin pretreated mice. This increase of Bcl-xL was associated with the decrease of Bax/Bcl-xL ratio. ABTS and DPPH radical assays revealed that melatonin treatment alleviated total antioxidant capacity in hematopoietic and gastrointestinal tissues. Present study demonstrated that melatonin pretreatment was able to prevent hematopoietic, immunological, and gastrointestinal radiation-induced injury, therefore, overcoming lethality in mice. These results suggest potential of melatonin in developing radioprotector for protection of bone marrow, spleen, and gastrointestine in planned radiation exposure scenarios including radiotherapy. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 501-518, 2017.

Frequency of gamma H2AX foci in healthy volunteers and health workers occupationally exposed to X-irradiation and its relevance in biological dosimetry.

Gamma-H2AX (γ-H2AX) assay is a marker to measure double-strand breaks in the deoxyribonucleic acid. Variables such as age, oxidative stress, temperature, genetic factors and inter-individual variation have been reported to influence the baseline γ-H2AX focus levels. Therefore, knowledge on baseline frequency of γ-H2AX foci in a targeted population would facilitate reliable radiation triage and dose estimation. The objective of the present study was to establish the baseline data using blood samples from healthy volunteers (n = 130) differing in age, occupation and lifestyle as well as from occupationally exposed health workers (n = 20). The γ-H2AX focus assay was performed using epifluorescence microscopy. In vitro dose-response curve for γ-H2AX foci was constructed in blood samples (n = 3) exposed to X-rays (30 min post-exposure). The mean γ-H2AX focus frequency obtained in healthy volunteers was 0.042 ± 0.001 and showed an age-related increase (p < 0.001). Significantly higher (p < 0.005) focus frequencies were observed in health workers (0.066 ± 0.005) than in healthy volunteers. A sub-group analysis did not show a significant (p > 0.1) difference in γ-H2AX focus frequency among sexes. Blood exposed in vitro to X-rays showed dose-dependent increase in γ-H2AX foci frequency (Y = 0.1902 ± 0.1363 + 2.9020 ± 0.3240 * D). Baseline frequency of γ-H2AX foci obtained from different age groups showed a significant (p < 0.01) influence on the dose-response coefficients. The overall results demonstrated that the γ-H2AX assay can be used as a reliable biomarker for radiation triage and estimating the radiation absorbed dose by considering variables such as age, occupation and lifestyle factors.

Evaluation of sesamol-induced histopathological, biochemical, haematological and genomic alteration after acute oral toxicity in female C57BL/6 mice.

The objective of this study was to evaluate organ-wise toxicological effects of sesamol and determine the LD50 cut-off value and GHS category following acute oral toxicity method OECD 423. An acute oral toxicity study was carried out in female C57BL/6 mice. Observations for physical behaviour and measurements on haematology, biochemistry, histology of vital organs were performed. In addition, genotoxicity assessment using comet and micronuclei assays was also performed. Acute toxicological effects were observed at 2000 mg/kg, while no adverse effects observed at 300 mg/kg. The effects of 2000 mg/kg were manifested as severe histopathological changes in all organs (femur, spleen, gastrointestine, lungs, heart, kidney, liver, stomach and brain) and excessive DNA strands breaks occurred in femoral bone marrow cells and splenocytes. A single dose of sesamol (2000 mg/kg, body weight) caused the death of two mice (out of three) within 2 h. Hence, sesamol is in GHS category 4 (>300-2000) with LD50 cut-off value of 500 mg/kg body weight. In contrast, this study is correlated with the obtained GHS category 4 and LD50 cut-off value 580 mg/kg body weight by ProTox. In conclusions, the present study has classified sesamol toxicity and assessed organ-wise acute oral toxicity of sesamol in female C57BL/6 mice. Therefore, these findings may be useful for the selection of dosages for further pre-clinical evaluation and potential drug developmental of sesamol.

Radioprotective potential of melatonin against 6°Co γ-ray-induced testicular injury in male C57BL/6 mice.

BACKGROUND: Melatonin, the chief secretary product of pineal gland, is a strong free radical scavenger and antioxidant molecule. The radioprotective efficacy and underlying mechanisms refer to its antioxidant role in somatic cells. The purpose of this study, therefore, was to investigate the prophylactic implications of melatonin against γ-ray-induced injury in germinal cells (testes). C57BL/6 male mice were administered melatonin (100 mg/kg) intra-peritoneally 30 min prior to a single dose of whole-body γ-irradiation (5 Gy, 1 Gy/minute) using (60)Co teletherapy unit. Animals were sacrificed at 2h, 4h and 8h post-irradiation and their testes along with its spermatozoa taken out and used for total antioxidant capacity (TAC), lipid peroxidation, comet assay, western blotting and sperm motility and viability. In another set of experiment, animals were similarly treated were sacrificed on 1(st), 3(rd), 7(th), 15(th) and 30(th) day post-irradiation and evaluated for sperm abnormalities and histopathological analysis. RESULTS: Whole-body γ-radiation exposure (5 Gy) drastically depleted the populations of spermatogenic cells in seminiferous tubules on day three, which were significantly protected by melatonin. In addition, radiation-induced sperm abnormalities, motility and viability in cauda-epididymes were significantly reduced by melatonin. Melatonin pre-treatment significantly inhibited radiation-induced DNA strands breaks and lipid peroxidation. At this time, radiation-induces activation of ATM-dependent p53 apoptotic proteins-ATM, p53, p21, Bax, cytochrome C, active caspase-3 and caspases-9 expression, which were significantly reversed in melatonin pre-treated mice. This reduced apoptotic proteins by melatonin pre-treatment was associated with the increase of anti-apoptotic-Bcl-x and DNA repair-PCNA proteins in irradiated mice. Further, radiation-induced decline in the TAC was significantly reversed in melatonin pre-treated mice. CONCLUSIONS: The present results indicated that melatonin as prophylactic agent protected male reproductive system against radiation-induced injury in mice. The detailed study will benefit in understanding the role of melatonin in modulation of radiation-induced ATM-dependent p53-mediated pro-vs.-anti apoptotic proteins in testicular injury. These results can be further exploited for use of melatonin for protection of male reproductive system in radiotherapy applications involving hemibody abdominal exposures.

Hoechst 33342 induced reactive oxygen species and impaired expression of cytochrome c oxidase subunit 1 leading to cell death in irradiated human cancer cells.

Oxidative stress and mitochondrial dysfunction in cancer cells represent features that may be exploited therapeutically. We determined whether minor groove binding ligand Hoechst 33342, known to induce mitochondrial dysfunction via increase in reactive oxygen species (ROS), enhances killing of human head and neck cancer (KB) cells mediated by impaired expression of mitochondrial protein involved in electron transfer. Elevation in ROS generation, increase in ΔΨm, down regulation of cytochrome c oxidase (CO), alteration in expression of antioxidant enzymes viz. Mn-SOD and Catalase, and release of cytochrome c into the cytosol, were observed in time-dependent manner when cells were irradiated (5 Gy) in presence of Hoechst 33342. Persistent increase in ROS observed till 48 h following treatment decreased the clonogenic survival and viability to a large extent via increase in ΔΨm, release of cytochrome c and non-coordinated expression of antioxidant enzymes. Treatment with antioxidants PEG-MnSOD and PEG-catalase inhibited the increase in ROS and loss of cell survival, suggesting the involvement of ROS in the Hoechst 33342-induced cell death. The result demonstrated significant sensitization of cancer cells to radiation-induced toxicity in presence of Hoechst 33342 via increasing ROS to a toxic level and impairing CO expression and antioxidant enzymes. This understanding is expected to benefit both in elucidating the detailed mechanisms of actions of DNA interacting drug and designing better molecules for enhancing radiation-induced cell death among cancer cells.

Osmolyte changes the binding affinity and mode of interaction of minor groove binder hoechst 33258 with calf thymus DNA.

The aim of this work was to investigate the effect of altered water activity on Hoechst 33258-calf thymus DNA (CtDNA) interaction by using osmotic stress approach. Water activity was changed by using osmolytes viz., sucrose and triethylene glycol (TEG). We have reported the results of thermal denaturation, absorption and fluorescence spectroscopy and binding affinity measurements as a function of osmolytes concentration. TEG dramatically lowered the thermal stability of CtDNA, ΔT(m)=-16 °C whereas sucrose induced very little decrease. Hoechst 33258 increases the stability of CtDNA, but in the presence of TEG, the ΔT(m) was -37 °C and a marginal decrease was observed with sucrose. Binding affinity of Hoechst 33258 with CtDNA was found to be reduced from 4.75×107 to 0.16×107 M⁻¹ in TEG and this was accompanied with the increased uptake of 74±2 water molecules. In the presence of sucrose this uptake of water molecules was found to be 30±1. Method of continuous variation suggests that the osmolytes lowered the stoichiometry of Hoechst 33258-CtDNA complex. On the contrary, van't Hoff plot revealed the hydrophobic interaction (ΔS=130.66 J mol⁻¹ K⁻¹) between the Hoechst 33258 and CtDNA. The detailed absorption and fluorescence spectral measurements including the fluorescence lifetime and anisotropy indicated bound state of Hoechst 33258 in osmotic stress condition. Fluorescence lifetime measurement revealed that the contribution from the planar conformer of Hoechst 33258 dominated the binding interaction with CtDNA in presence of TEG. These results can be useful for understanding of interaction of Hoechst 33258 with genomic DNA in a complex environment having altered water activity.

Interaction of DNA minor groove binder Hoechst 33258 with bovine serum albumin.

Hoechst 33258 belongs to bisbenzimidazole class of molecules having anticancer properties for their ability to inhibit topoisomerase and many other cellular processes. The aim of the present study is to understand the nature of Hoechst 33258-bovine serum albumin (BSA) binding interactions by using absorption, fluorescence and circular dichrorism (CD) measurements under simulative physiological conditions. The absorption spectra of BSA indicated the binding of Hoechst 33258 with BSA. The analysis of fluorescence data indicated the presence of both dynamic and static quenching mechanism in the binding. The associative binding constant and number of binding sites were found to be K=2.08=10(7) M(-1) and n=1.36 respectively. Biexponential fluorescence lifetime distribution of Hoechst 33258 in the presence of BSA has altered viz. tau(1) was increased significantly from 0.3 ns (60%) to 1.2 ns (13%) whereas a marginal increase in tau(2) from 3.6 ns (40%) to 4.0 ns (87%). Fluorescence anisotropy value of Hoechst 33258 has increased from 0.14 to 0.34 upon the addition of BSA. Thermodynamic parameters were also calculated using Van't Hoff plot by conducting fluorescence titration at four different temperatures, DeltaH=+102.785 kJ mol(-1), DeltaS=+490.18 kJ mol(-1), DeltaG=-491.708 kJ mol(-1). The CD spectrum of BSA revealed that the binding of Hoechst 33258 to BSA causes loss in the secondary structure but increases the thermal stability of the protein. The results indicated that hydrophobic interactions were the predominant intermolecular forces in stabilizing BSA-Hoechst 33258 complex. The possible implications of these results will be on designing better therapeutic minor groove binding drug molecules.