M. Memita Devi

Nuclear magnetic resonance spectroscopy-based metabonomic investigation of biochemical effects in serum of γ-irradiated mice

Purpose: Radiation exposure induces change in many biological compounds. It is important to assess the physiological and biochemical response to an absorbed dose of ionising radiation due to intentional or accidental event and to predict medical consequences for medical management. In the present study, nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling was used in mice serum for identification of radiation-induced changes at metabolite level. Materials and methods: Mice were irradiated with 3, 5 and 8 Gray of γ-radiation dose and serum samples collected at day 1, 3 and 5 post irradiation were analysed by proton nuclear magnetic resonance (1H NMR) spectroscopy. 1H NMR spectra of serum were analysed by pattern recognition using principal component analysis. Results: Irradiated mice serum showed distinct metabonomic phenotypes and revealed dose- and time-dependent clustering of irradiated groups. 1H NMR spectral analysis exhibited increased lactate, amino acids, choline and lipid signals as well as decreased glucose signals. These findings indicate radiation-induced disturbed energy, lipid and protein metabolism. Conclusions: The information obtained from this study reflects multiple physiological dysfunctions. The study promises the application of NMR-based metabonomics in the field of radiobiology, for development of metabolic-based markers for screening of risk populations and medical management in these cases.

Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury

The majority of human mild traumatic brain injuries (mTBI; 70%) lack radiological evidence of injury, yet may present long term cognitive, and behavioral dysfunctions. With the hypothesis of evident damaged neural tissue and immunological consequences during acute phase of mTBI, we used closed skull weight-drop TBI model to address human mTBI condition. Serum cytokines (TNF-α, IL-10) and glial fibrillary acidic protein (GFAP) expression were examined at day 0 (control, pre-injury), 4h, day 1, day 3 and day 5 post injury (PI). Diffusion tensor imaging (DTI) was performed at similar timepoints to identify neuroinflammation translation into imaging abnormalities and monitor injury progression. DTI indices including mean diffusivity (MD), radial diffusivity (RD), fractional anisotropy and axial diffusivity values were quantified from cortex (CTX), hippocampus and corpus callosum regions. One way ANOVA showed significant increase in TNF-α at 4h and IL-10 at day 1 PI as compared to control. GFAP(+) cells were significantly increased at day 3 and day 5 as compared to control in CTX. Repeated measures ANOVA revealed significant decreases in MD, RD values in CTX at day 3 and day 5 as compared to day 0. A significant, inverse correlation was observed between cortical MD (r=-0.74, p=0.01), AD (r=-0.60, p=0.03) and RD (r=-0.72, p=0.01) values with mean GFAP(+) cells in the cortical region. These findings suggest that mTBI leads to elevated cytokine expression and subsequent hypertrophy of astrocytic processes. The increased numbers of reactive glial cells contribute diffusion restrictions in the CNS leading to reduced MD and RD values. These findings are in line with the deficits and pathologies associated with clinical mTBI, and support the use of mTBI model to address pathology and therapeutic options.

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.

Blood biomarkers in metal scrap workers accidentally exposed to ionizing radiation A case study

The detrimental effect of nuclear accidents due to localized or whole body radiation exposure results in severe cellular damage. The current study was carried out to evaluate radiation-mediated variability in blood components of metal scrap workers exposed accidently to cobalt-60 source. Blood samples collected initially from five hospitalized patients, coded P1–P5, were processed for total leukocyte counts (TLC), platelet (PLT) counts, haemoglobin, estimation of DNA double strand breaks by measuring phosphorylated form of H2AX (γ-H2AX) and chromosomal aberrations (dicentrics). Blood cells count (TLC), in all the patients except P2, was found decreased. Dicentrics increased in all the five patients. γ-H2AX was found significantly elevated in patients P2 and P4. After 3 days, 21 subjects working in close vicinity of accident site were evaluated for the above-mentioned markers to confirm their possibility of radiation exposure; however, all the parameters in these subjects were found within normal limits. Blood from patients P1–P5 was collected again after 11 days. Studies revealed exorbitant increase in γ-H2AX in lymphocytes and monocytes of patients P1, P4 and P5. TLC and PLT count in these patients had fallen further. Dicentrics declined with time in all the five patients. Based on the studied blood biomarkers, we conclude that the five subjects showed signs of radiation exposure. Measurement on radiation dose could not be performed in the current study; however, the generated data particularly on dicentrics provide ample evidence of radiation exposure.

Effect of vitamin D supplementation on muscle energy phospho-metabolites: a 31P magnetic resonance spectroscopy-based pilot study

Abstract There are several published reports on the prevalence of low vitamin D levels in otherwise healthy Indian population. Vitamin D deficiency has shown variable effect on muscle performance and strength but there is paucity of data on the effect of vitamin D deficiency on muscle energy metabolism. The present study was proposed to investigate the influence of severe vitamin D deficiency on high-energy metabolite levels in resting skeletal muscle and thereafter, monitor the response after vitamin D supplementation using 31P magnetic resonance spectroscopy (MRS). Study was conducted on 19 otherwise healthy subjects but with low serum 25(OH)D levels (<5 ng/ml). Subjects were supplemented with cholecalciferol at a dose of 60 000 IU/week for 12 weeks. MRS measurements of inorganic phosphate (Pi), phosphocreatine (PCr), phosphodiester (PDE) and ATP of the calf muscle were taken pre- and post-vitamin D supplementation. The study revealed significantly increased PCr/Pi ratio and decreased [Pi] and PDE/ATP ratio with raised serum 25(OH)D levels after 12 weeks of supplementation. The study indicates that serum 25(OH)D level plays an important role in improving the skeletal muscle energy metabolism and vitamin D deficiency might be one of the primary reasons for prevalence of low PCr/Pi ratio and high PDE values in normal Indian population as reported earlier. The findings of this preliminary study are highly encouraging and warrant further in-depth research, involving larger number of subjects of different age groups, regions and socio-economic sections of the society to further strengthen a correlation between vitamin D levels and muscle energy metabolism.

Study of acute biochemical effects of thallium toxicity in mouse urine by NMR spectroscopy.

Thallium (Tl) is a toxic heavy metal and its exposure to the human body causes physiological and biochemical changes due to its interference with potassium‐dependent biological reactions. A high‐resolution 1H NMR spectroscopy based metabonomic approach has been applied for investigating acute biochemical effects caused by thallium sulfate (Tl2SO4). Male strain A mice were divided in three groups and received three doses of Tl2SO4 (5, 10 and 20 mg kg−1 b.w., i.p.). Urine samples collected at 3, 24, 72 and 96 h post‐dose time points were analyzed by 1H NMR spectroscopy. NMR spectral data were processed and analyzed using principal components analysis to represent biochemical variations induced by Tl2SO4. Results showed Tl‐exposed mice urine to have distinct metabonomic phenotypes and revealed dose‐ and time‐dependent clustering of treated groups. The metabolic signature of urine analysis from Tl2SO4‐treated animals exhibited an increase in the levels of creatinine, taurine, hippurate and β‐hydroxybutyrate along with a decrease in energy metabolites trimethylamine and choline. These findings revealed Tl‐induced disturbed gut flora, membrane metabolite, energy and protein metabolism, representing physiological dysfunction of vital organs. The present study indicates the great potential of NMR‐based metabonomics in mapping metabolic response for toxicology, which could ultimately lead to identification of potential markers for Tl toxicity. Copyright

Phosphorous magnetic resonance spectroscopy-based skeletal muscle bioenergetic studies in subclinical hypothyroidism.

Background: Subclinical hypothyroidism (sHT) is considered to be a milder form of thyroid dysfunction. Few earlier studies have reported neuromuscular symptoms as well as impaired muscle metabolism in sHT patients. Aim/objective: In this study we report our findings on muscle bioenergetics in sHT patients using phosphorous magnetic resonance spectroscopy (31P MRS) and look upon the possibility to use 31P MRS technique as a clinical marker for monitoring muscle function in subclinical thyroid dysfunction. Subjects and methods: Seventeen normal subjects, 15 patients with sHT, and 9 patients with hypothyroidism performed plantar flexion exercise while lying supine in 1.5 T magnetic resonance scanner using custom built exercise device. MR Spectroscopy measurements of inorganic phosphate (Pi), phosphocreatine (PCr), and ATP of the calf muscle were taken during rest, at the end of exercise and in the recovery phase. PCr recovery rate constant (kPCr) and oxidative capacity were calculated by monoexponential fit of PCr vs time (t) at the beginning of recovery. Results: We observed that changes in some of the phosphometabolites (increased phosphodiester levels and Pi concentration) in sHT patients which were similar to those detected in patients with hypothyroidism. However, our results do not demonstrate impaired muscle oxidative metabolism in sHT patients based upon PCr dynamics as observed in hypothyroid patients. Conclusions:31P MRS-based PCr recovery rate could be used as a marker for monitoring muscle oxidative metabolism in sub clinical thyroid dysfunction.

NMR spectroscopy based metabolic profiling of urine and serum for investigation of physiological perturbations during radiation sickness

Radiation accidents are rare events that induce radiation syndrome, a complex pathology which is difficult to treat. In medical management of radiation victims, life threatening damage to different physiological systems should be taken into consideration. The present study was proposed to identify metabolic and physiological perturbations in biofluids of mice during different phases of radiation sickness using 1H nuclear magnetic resonance (1H NMR) spectroscopy and pattern recognition (PR) technique. The 1H NMR spectra of the biofluids collected from mice irradiated with 5 Gray (Gy) at different time points during radiation sickness were analysed visually and by principal components analysis. Urine and serum spectral profile clearly showed altered metabolic profiles during different phases of radiation sickness. Increased concentration of urine metabolites viz. citrate, α ketoglutarate, succinate, hippurate, and trimethylamine during prodromal and clinical manifestation phase of radiation sickness shows altered gut microflora and energy metabolism. On the other hand, serum nuclear magnetic resonance (NMR) spectra reflected changes associated with lipid, energy and membrane metabolism during radiation sickness. The metabonomic time trajectory based on PR analysis of 1H NMR spectra of urine illustrates clear separation of irradiated mice group at different time points from pre dose. The difference in NMR spectral profiles depicts the pathophysiological changes and metabolic disturbances observed during different phases of radiation sickness, that in turn, demonstrate involvement of multiple organ dysfunction. This could further be useful in development of multiparametric approach for better evaluation of radiation damage as well as for medical management during radiation sickness.