NOTE The report of the Committee without its annexes appears as Official Records of the General Assembly, Sixty-third Session, Supplement No. 46. The designations employed and the presentation of material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the United Nations concerning the legal status of any country, territory, city or area, or of its authorities, or concerning the delimitation of its frontiers or boundaries. The country names used in this document are, in most cases, those that were in use at the time the data were collected or the text prepared. In other cases, however, the names have been updated, where this was possible and appropriate, to reflect political changes. Scientific Annexes Annex A. Medical radiation exposures Annex B. Exposures of the public and workers from various sources of radiation INTROdUCTION 1. In the course of the research and development for and the application of atomic energy and nuclear technologies, a number of radiation accidents have occurred. Some of these accidents have resulted in significant health effects and occasionally in fatal outcomes. The application of technologies that make use of radiation is increasingly widespread around the world. Millions of people have occupations related to the use of radiation, and hundreds of millions of individuals benefit from these uses. Facilities using intense radiation sources for energy production and for purposes such as radiotherapy, sterilization of products, preservation of foodstuffs and gamma radiography require special care in the design and operation of equipment to avoid radiation injury to workers or to the public. Experience has shown that such technology is generally used safely, but on occasion controls have been circumvented and serious radiation accidents have ensued. 2. Reviews of radiation exposures from accidents have been presented in previous UNSCEAR reports. The last report containing an exclusive chapter on exposures from accidents was the UNSCEAR 1993 Report [U6]. 3. This annex is aimed at providing a sound basis for conclusions regarding the number of significant radiation accidents that have occurred, the corresponding levels of radiation exposures and numbers of deaths and injuries, and the general trends for various practices. Its conclusions are to be seen in the context of the Committee's overall evaluations of the levels and effects of exposure to ionizing radiation. 4. The Committee's evaluations of public, occupational and medical diagnostic exposures are mostly concerned with chronic exposures of …

sources and effects of ionizing radiation

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.

https://zenodo.org/record/975797/files/article.pdf

The Comet Assay for DNA Damage and Repair: Principles, Applications, and Limitations

The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells. Its use has increased significantly in the past few years. This paper is a review of the studies published to date that have made use of the comet assay. The principles of strand break detection using both the alkaline and neutral versions of the technique are discussed, and a basic methodology with currently used variations is presented. Applications in different fields are reviewed and possible future directions of the technique are briefly explored.

The comet assay: a comprehensive review

Evidence from many sources shows that the control of gene expression in higher organisms is related to the methylation of cytosine in DNA, and that the pattern of methylation is inherited. Loss of methylation, which can result from DNA damage, will lead to heritable abnormalities in gene expression, and these may be important in oncogenesis and aging. Transformed permanent lines often lose gene activity through de novo methylation. It is proposed that epigenetic defects in germline cells due to loss of methylation can be repaired by recombination at meiosis but that some are transmitted to offspring.

https://science.sciencemag.org/content/sci/238/4824/163.full.pdf

The inheritance of epigenetic defects.

The single cell gel electrophoresis (SCGE) assay is a rapid, simple, visual and sensitive technique for measuring DNA breakage in individual mammalian cells. Here we review the development of the SCGE assay (with particular reference to the alkaline version), existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA, and the potential applications of the technique.

The single cell gel electrophoresis assay (comet assay): A European review

Working paper no. 3 Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo

We have studied incision-break formation in unstimulated and stimulated populations of human T-lymphocytes using the comet (single-cell microgel electrophoresis) assay. The frequency of strand breaks 1 h after UV-irradiation appears to be far greater in unstimulated than in stimulated lymphocytes from normal donors and the excess of strand breaks was observed for a far longer time after irradiation. This result corroborates the greater sensitivity to UV-C irradiation observed in a colony-forming assay but suggests that the defect may relate to a defect in strand rejoining rather than a defect in incision. Few strand breaks were seen in either unstimulated or stimulated lymphocytes of four xeroderma pigmentosum donors, suggesting that the method may offer a rapid diagnostic assay for XP.

UV-C sensitivity of unstimulated and stimulated human lymphocytes from normal and xeroderma pigmentosum donors in the comet assay: a potential diagnostic technique.

We have used the T-Lymphocyte cloning technique as a method of monitoring the human population for somatic cell mutant frequency. We present a statistical analysis of the experimental factors which may influence the observed mutant frequency. We have obtained consistently high plating efficiencies of T-cells from the mononuclear cell fraction from donor blood samples (mean of 56%, based on 123 observations from 70 individuals). Nevertheless, an inverse correlation of mutant frequency with plating efficiency was observed, and some experimental factors (serum and interleukin-2 batch, and worker) may have a significant effect on the observed mutant frequency. We discuss the difficulties that these possible effects present in establishment of a reference database and design of long-term studies. No significant effect of donor sex on mutant frequency was observed, but age (1.3% increase per year for normal adults) and smoking (56% increase over normal non-smokers) both significantly increased the mutant frequency. We discuss the utility of the assay for the monitoring of populations for heritable DNA damage, and we compare the results to those obtained with lymphocytes using other endpoints, e.g. chromosome aberrations, micronuclei and sister-chromatid exchange.

A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating T-lymphocytes.

We have measured DNA strand breaks induced by ionising radiation in nucleated cells from freshly isolated whole blood from normal human subjects. Samples were taken after subjects had fasted overnight and again 1 h after they had eaten breakfast in combination with approximately 35 mg/kg vitamin C. Damage was measured by single cell gel electrophoresis (the 'comet' assay), in which DNA single strand breaks generate a comet tail streaming from the nucleus. In repeat experiments on 6 subjects a reduction in DNA damage, as indicated by a highly significant decrease in overall comet length, was observed following vitamin C ingestion, both in the unirradiated control blood samples and in the dose response to ionising radiation damage. In addition, consistent differences in dose response between individual subjects were found. The peak effect was 4 h after intake of food and vitamin C. An effect was also seen with vitamin C alone and after breakfast without additional vitamin C. Protection against strand breakage was also seen in Ficoll-separated mononuclear cells but evidence was not obtained for protection of separated, mitogen stimulated T-lymphocytes either against ionising radiation cell killing in a clonal assay, or against clastogenicity assessed by micronucleus formation following one cell division. Exposure of separated lymphocytes in vitro to vitamin C, at doses greater than 200 microM, did not offer protection but induced strand breakage. Our results raise the possibility that variation in normal diet may not only affect susceptibility to endogenous oxidative damage, but may affect some responses of the individual to radiation.

Effect of diet and vitamin C on DNA strand breakage in freshly-isolated human white blood cells

A cell-kinetic model for the application of the micronucleus test to polychromatic erythrocytes in mouse fetal liver, fetal blood, and maternal bone marrow after exposure to clastogenic agents is described. The time of expression and dose-response relationships obtained with γ-radiation, methyl methanesulphonate, procarbazine, mitomycin C and benzo[a]pyrene are analysed in terms of this model. The numbers of target cells damaged per unit dose has been calculated and the dose equivalents obtained. Maternal and fetal cells show similar sensitivity to γ-radiation, but fetal cells are markedly more sensitive to MMS and procarbazine. This probably due to differences in tissue distribution and metabolism. Maternal and fetal erythroid tissues can show linear and exponential dose-response relationships, which may not coincide (e.g. with MMS). It is concluded that risks from fetal exposure to genotoxic agents cannot be reliably predicted from in vivo tests restricted to adult animals. However, the micronucleus technique appled to fetal erythroid cells proveds a rapid and reliable short-term test, appropriate to minimising risks of genome damage during prenatal development.

Jane Cole

Papers

Working paper no. 3 Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo

UV-C sensitivity of unstimulated and stimulated human lymphocytes from normal and xeroderma pigmentosum donors in the comet assay: a potential diagnostic technique.

A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating T-lymphocytes.

Effect of diet and vitamin C on DNA strand breakage in freshly-isolated human white blood cells

Short-term tests for transplacentally active carcinogens. I. Micronucleus formation in fetal and maternal mouse erythroblasts.