High LET Alpha Particles - Research Proposal Example

Complex chromosome aberrations are expected to be induced in human Peripheral Blood lymphocytes (PBLs) on exposure to ‘high LET alpha particles’. The objective of the study is to assess the efficiency of detecting chromosomal aberrations by three-color FISH and mFISH and also to evaluate the role of non-transmissible complexes as biomarkers in case of chronic exposure to high-LET alpha particles under lab conditions. The population in the study involved was retired male workers from Russian Nuclear Industry who were classified into three subject groups. Customised software and standardised methodology developed at State Research Centre Institute of Biophysics, Moscow was used in the study to compare and analyse the stain pattern. PLAIN LANGUAGE SUMMARY Human chromosomes can undergo structural changes on exposure to high LET alpha particles. The study tries to evaluate the efficacy of three-color FISH and mFISH methodology to assess and demarcate these changes. Retired male workers from Russian Nuclear Industry who were expected to have high exposure to high LET alpha particles during work conditions were involved in the study and standard methodologies were used for the study. Aims of the project To assess the efficiency of detecting chromosomal aberrations by three-color FISH and mFISH To assess the role of non-transmissible complexes as biomarkers in case of chronic exposure to high-LET alpha particles under lab conditions. Works that led to the project A number of experiments have been reviewed by the author to support the background of the study. Seven studies have been referenced in the paper to support the view that complex aberrations are induced on exposure to high-LET radiation. The definition of insertion which is the characteristic feature of aberrations also has been included in the review. A number of studies involving the FISH technique have profoundly emphasised the importance and relevance of complexity of radiation- induced chromosome aberrations. The relevance of the fact that the rearranged chromosomes may comprise of parts of multiple chromosomes in the study also has been reviewed. mFISH methodology demonstrated in two different studies one by R. M. Anderson, S. J. Marsden, S. J. Paice, A. E. Bristow, M. A. Kadhim, C. S. Griffin and D. T. Goodhead and another by R. M. Anderson, D. L. Stevens and D. T. Goodhead has been supportive to this observation. The induction of complex chromosome aberrations in human peripheral blood lymphocytes (PBLs) when exposed to a low dose of high-LET under laboratory conditions has been supported by different studies and it has been found that the insertions which were visible were parts of other complex aberrations. The references made have supported the background information that the complexity of the aberration induced is much related to the alpha particle exposure. The study by R. M. Anderson, S. J. Marsden, S. J. Paice, A. E. Bristow, M. A. Kadhim, C. S. Griffin and D. T. Goodhead has related the in vivo experiment with relevance to the exposure to high-LET particles. It has been opined that long after the exposure, the possibility of efficient detection of stable insertions could be unlikely .The role of Radon, which is a natural emitter of alpha particle and which is present in the environment has been confirmed by the background information as a consistent source of high LET alpha particle exposure. This upon inhalation gets into the blood stream and then transported into various organs resultantly causing complex chromosome aberrations in peripheral blood lymphocytes. These effects are found to be very common as with the case of in vitro exposures. This relation with the experiments done in vitro and the high probability of human getting affected by LET alpha particle exposure leading to complex chromosome aberrations have contributed to the scope of the study. Experimental method & Design The population involved in the study was retired male workers from Russian Nuclear Industry and was classified into three subject groups. The first subject group (subject 3-6) comprised of workers in the age group 55 to 63. The other two groups were the control population which were aged 38 years (subject 1) and the third one with age 40 (subject 2). The blood samples were collected adhering to the guidelines of Russian Health Ministry and the Russian Atomic Ministry. The collection process was done by trained medical staff of State Research Centre Institute of Biophysics, Moscow (SRC-IB) during annual health examinations. Fixed chromosome preparations from anonymized samples were made available for the study. The plutonium body burden and the particulars of the dose was determined at the same institute with reference to the results suggested by the biophysics studies on urine conducted on outpatients and inpatients. The complex changes that happen after radionuclides enter into ones body through chronic inhalation of plutonium aerosols during the work conditions with probable conditions of contact with radionuclides has been confirmed by reference to results of previous studies. It was confirmed that plutonium and americium would be assigned biokinetic parameters according to ICRP 67 after radionuclides gets dissolved into the blood compartments. With this knowledge, the software developed at State Research Centre Institute of Biophysics was used to assess the plutonium body burden and the doses. The Lymphocyte culturing and Chromosome preparation was done as the collected whole blood was cultured in phytohemagglutinin-supplemented medium containing 20% fetal calf serum for fourty eight hours. Later in the final two and a half hours of the culture, 0.2 mg/ml of Demecolcine (0.2 mg/ml) was added and by utilising standard cytogenetic techniques the metaphase cells were collected. For the Three-Color Fluorescence In Situ Hybridization (FISH), these metaphase cells were hardened and pretreated with RNase A. The pretreated cells were then treated with pepsin after washing in 23 SSC and PBS. It was later washed with PBS,50 mM MgCl2/PBS, 50 mM MgCl2/1% formaldehyde/PBS twice before finally dehydrating it in ethanol. The nalysis was carried out using an Olympus fluorescence microscope with for DAPI, FITC, FITC/DAPI/Texas Red and FITC/DAPI/TRITC filters. In the case of Multiplex FISH (mFISH) the metaphase chromosomes isolated from whole blood were hardened and pretreated similarly as done in the case of Three-Color Fluorescence In Situ Hybridization. The cells were later denatured using 70% formamide/23 SSC and dehydrated using ethanol for hybridization. Meanwhile the commercially available probe was denatured. Later the cells and the probe were hybridized and then washed in 0.43 SSC/0.3% Igepal. The cells were stained and stored in 2208 C. The same software as in previous cases was used to analyse the chromosome aberrations. The microscope used to view the chromosomes was Olympus BX51 fluorescence microscope with individual filters. The staining pattern in the case of Three-colour FISH was classified using Savage and Simpson scheme where as in the case of mFISH, the only difference in the aberration classification was that the each minute and complex aberrations were observed and assigned in this case. The statistical comparison of the data was done using Fisher’s exact test or its multi sample equivalent in terms of abnormal cell proportion. Exact binomial test or its multi sample equivalent was used for the data analysis with regard to the mean number of aberrations per cell PROJECT JUSTIFICATION The cost involved for the study has been justified by the relevance of the study in human health. The population involved in the study is scientifically known to have deposits of radio active plutonium. Though the metabolic effect would differ in the case of Radon, to which human are commonly exposed in natural environment, the results of the study can be a generic background to the effects of these exposures. The results of the study are also expected to be more precise as it has involved standardised methodologies. The expertise involved in the sampling methodology also would justify the cost efficacy of the study. A number of linked studies has been utilised which aided in the cost reduction. As the study is of a pilot-nature, the equipments, the results and other consumables would be resourceful for further follow up studies. Works cited Anderson,R M. Viktoria, Tsepenko, V. Gasteva, G N. Molokanov, A A. Sevan’kaev, V A & Goodhead, D T, “mFISH Analysis Reveals Complexity of Chromosome Aberrations in Individuals Occupationally Exposed to Internal Plutonium: A Pilot Study to Assess the Relevance of Complex Aberrations as Biomarkers of Exposure to High-LET ά Particles”.Radiation Research 163 (2005).26–35 Read More