14. Radioactivity

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Radioactivity Decays laws Production of isotopic tracer method Assay using radioactive substrate Biological metabolic and physiological tracer studies dose response relationship Labeling of molecules with radioactive substances Artificial production of radioactivity and their uses in medicine Effects of radiation on biomolecules and molecular structure Radiation effect on cell and organisms Radiation hazard and its protections. Mitesh Shrestha

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Science Park HS -- Honors Chemistry Early Pioneers in Radioactivity Roentgen: Discoverer of X- rays 1895 Becquerel: Discoverer of Radioactivity 1896 The Curies: Discoverers of Radium and Polonium 1900- 1908 Rutherford: Discoverer Alpha and Beta rays 1897

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Radioactivity • Radioactive decay is the process by which the nucleus of an unstable atom loses energy by emitting radiation including alpha particles beta particles gamma rays and conversion electrons. • A material that spontaneously emits such radiation is considered radioactive. • It results in a decrease of the original amount radioactive material over time -the unstable nuclei become stable by releasing the particles waves

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Geiger Counter • Used to measure radiation. • The more intense the radiation the more “clicks”.

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The Nuclear Stability Belt

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All the elements heavier than Bismuth At 83

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Kinds of Radioactivity The three main decays are Alpha Beta and Gamma

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Science Park HS -- Honors Chemistry

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Three Common Types of Radioactive Emissions Alpha Beta Gamma

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RADIATION ITS ORIGIN • ALPHA • BETA- • BETA+ • GAMMA • X-RAYS • NUCLEUS • NUCLEUS • NUCLEUS • NUCLEUS • ELECTRON CLOUD SPACE CHARGE RADIATION TYPE ORIGIN

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RADIATION AND ITS CHARGE --

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RADIATION AND ITS CHARGE • ALPHA • BETA – • BETA+ • GAMMA • X-RAYS • +2 • -1 • +1 • 0 • 0

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• Alpha particles can usually be stopped by a very thin barrier. Radioisotopes emitting alpha particles are usually not hazardous outside the body but they can cause damage if ingested. • Betas streams of electrons can pass through a hand but are usually stopped by a modest barrier such as a few millimeters of aluminum or even a layer of clothing. As with alphas beta particles are more hazardous if inhaled or ingested. • Gammas can be very penetrating and can pass through thick barriers. Several feet of concrete would be needed to stop some of the more energetic gammas. A natural gamma source found in the environment and in the human body is 40 K an isotope of potassium. • Neutrons are also very penetrating. Some elements like hydrogen capture and scatter neutrons. Water is commonly used as a neutron radiation shield.

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Radioactive Decay Law • Radioactive decay is the spontaneous release of energy in the form of radioactive particles or waves. • It results in a decrease of the original amount radioactive material over time -the unstable nuclei become stable by releasing the particles waves

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Radioactive Decay Law • Any radioactive isotope consists of a vast number of radioactive nuclei. • Nuclei does not decay all at once. • Decay over a period of time. • We can not predict when it will decay its a random process but...

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... We can determine based on probability approximately how many nuclei in a sample will decay over a given time period by asuming that each nucleus has the same probability of decaying in each second it exists. Radioactive Decay Law

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• Nt the number of nuclei present at a give time t • N0 the number of nuclei present at time t 0 • e is the natural expoentional logarithms • t time years days hours or seconds • λ half life a RATE of decay amount/time

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• Half-Life λ: The amount of time required for one-half or 50 of the radioactive atoms to undergo a radioactive decay. • Every radioactive element isotope/nuclide has a specific half-life associated with it. • Is a measure of how stable the nuclei is. • Half-Life ranges from fractions of a second to billions of years. • No operation or process of any kind i.e. chemical or physical has ever been shown to change the rate at which a radionuclide decays.

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Another Contribution from Rutherford: Half-life of Radioactive Atoms The half-life of a radioactive substance is the time required for one half of it to decay.

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27

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How long is the half- life of Sodium-24

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30 Half-Life Calculation • You have 400 mg of a radioisotope with a half- life of 5 minutes. How much will be left after 30 minutes 5 min 400 200 100 50 12.5 25 6.25 3.125 1.5625 .78125 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min

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31 Medical Applications of Half-Life Nuclide Half-Life Area of Body I –131 8.1 days Thyroid Fe –59 45.1 days Red Blood Cells Sr –87 2.8 hours Bones Tc –99 6.0 hours Heart Na –24 14.8 hours Circulatory System

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Radioactive Half-life

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Radioactive Half-Life • The half-life of an element is the time it takes for half of the material you started with to decay Remember it doesn’t matter how much you start with. After 1 half-life half of it will have decayed. • Each element decays into a new element • C14 decays into N14 while U238 decays into Pb206 lead etc • The half-life of each element is constant. It’s like a clock keeping perfect time

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A decay chain Uranium-238 has a half life of 4½ million years and emits alpha- particles. It decays to produce thorium-234 which has a half life of 24 days and produces beta-particles to protactinium-234 with a half life of 7 hours emitting beta-particles to uranium-234 with a half life of a quarter of a million years which decays by emitting alpha-particles... and so on until it finally creates the stable isotope lead-206.

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DECAY CHAIN

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Sources of Radioactivity • Primordial - from before the creation of the Earth • Cosmogenic - formed as a result of cosmic ray interactions • Human produced - enhanced or formed due to human actions minor amounts compared to natural

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Where are the Sources of Radioactivity • Naturally Occurring Sources: – Radon from the decay of Uranium and Thorium – Potassium -40 – found in minerals and in plants – Carbon 14 – Found in Plants and Animal tissue • Manmade Sources: – Medical use of Radioactive Isotopes – Certain Consumer products –eg Smoke detectors – Fallout from nuclear testing – Emissions from Nuclear Power plants

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Production of isotopic tracer method • A chemical compound in which one or more atoms have been replaced by a radioisotope so by virtue of its radioactive decay it can be used to explore the mechanism of chemical reactions by tracing the path that the radioisotope follows from reactants to products. • Radiolabeling is thus the radioactive form of isotopic labeling. • Radioisotopes of hydrogen carbon phosphorus sulphur and iodine have been used extensively to trace the path of biochemical reactions. • All the isotopes of a given element will behave the same chemically. • An ideal tracer has the same physical or chemical or biological properties of interest as the tracee but it presents some peculiar characteristic that enables its detection in the system where the tracee is also present.

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Production of isotopic tracer method • The commonly used radioisotopes have short half lives and so do not occur in nature. • They are produced by nuclear reactions. • One of the most important processes is absorption of a neutron by an atomic nucleus in which the mass number of the element concerned increases by 1 for each neutron absorbed. For example – 13 C + n → 14 C • In this case the atomic mass increases but the element is unchanged. • In other cases the product nucleus is unstable and decays typically emitting protons electrons beta particle or alpha particles. When a nucleus loses a proton the atomic number decreases by 1. For example – 32 S + n → 32 P + p • Neutron irradiation is performed in a nuclear reactor. The other main method used to synthesize radioisotopes is proton bombardment. The proton are accelerated to high energy either in a cyclotron or a linear accelerator.

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Two main ways in which radioactive tracers are used • When a labeled chemical compound undergoes chemical reactions one or more of the products will contain the radioactive label. Analysis of what happens to the radioactive isotope provides detailed information on the mechanism of the chemical reaction. • A radioactive compound is introduced into a living organism and the radio-isotope provides a means to construct an image showing the way in which that compound and its reaction products are distributed around the organism.

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Assay using radioactive substrate • Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates. • The radioactive isotopes most frequently used in these assays are 14 C 32 P 35 S and 125 I. • Since radioactive isotopes can allow the specific labelling of a single atom of a substrate these assays are both extremely sensitive and specific. • They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts the complex mixtures of enzymes produced when you lyse cells. • Radioactivity is usually measured in these procedures using a scintillation counter.

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Biological metabolic and physiological tracer studies • Tracer-based metabolomics is a systems biology tool that combines advances in tracer methodology for physiological studies high throughput “-omics” technologies and constraint based modeling of metabolic networks. • It is different from the commonly known metabolomics or metabonomics in that it is a targeted approach based on a metabolic network model in cells.

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Biological metabolic and physiological tracer studies • Incorporation of radioactive thymidine in determining DNA synthesis. • The appearance of radioactivity in the product or the disappearance of radioactivity in the precursors over time is used to determine the rate of reaction of enzymes.

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Dose response relationship • The dose–response relationship or exposure– response relationship describes the change in effect on an organism caused by differing levels of exposure or doses to a stressor usually a chemical after a certain exposure time. • This may apply to individuals e.g.: a small amount has no significant effect a large amount is fatal or to populations e.g.: how many people or organisms are affected at different levels of exposure.

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Biological Effects of Radiation

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Acute Exposure Large Doses Received in a Short Time Period  Accidents  Nuclear War  Cancer Therapy Short Term Effects Acute Radiation Syndrome 150 to 350 rad Whole Body Anorexia Nausea Erythema Fatigue Vomiting Hemorrhage Epilation Diarrhea Mortality

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Effects of Acute Whole Body Exposure on Man Absorbed Dose Rads Effect 10000 1200 600 450 100 50 25 5 Death in a few hours Death within days Death within weeks LD 50/30 Probable Recovery No observable effect Blood changes definite 1st Blood change obs

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Chronic Exposure Doses Received over Long Periods  Background Radiation Exposure  Occupational Radiation Exposure 50 rem acute vs 50 rem chronic  acute: no time for cell repair  chronic: time for cell repair Average US will receive 20 - 30 rem lifetime Long Term Effects  Increased Risk of Cancer  0.07 per rem lifetime exposure  Normal Risk: 30 cancer incidence

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• Ionization within body tissues: similar to water • Ionization causes many derivatives to be formed: Peroxides Free Radicals Oxides • These compounds are unstable and are damaging to the chemical balance of the cell. Various effects on cell enzymes and and structures occur. • Radiation is not the only insult responsible Pollutants Vitamin imbalance poor diet Sickness and Disease Cellular Effects

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Cellular Effects cont Cells often recover from damage Repeated Insults may cause damage to be permanent  Cell Death  Cell Dysfunction - tumors cancer cataracts blood disorders  Mitosis Cell Division Delayed or Stopped  Chromosomal breaks  Organ Dysfunction at High Acute Doses

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Variations in Sensitivity Wide variation in the radiosensitivity of various species  Plants/microrganisms vs. mammals Wide variation among cell types  Cells which divide are more sensitive  Non-differentiated cells are more sensitive  Highly differentiated cells like nerve cells are less sensitive

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Effects on the Fetus The fetus consists of rapidly dividing cells Dividing cells are more sensitive to radiation effects than nondividing cells Effects of low level radiation are difficult to measure A lower dose limit is used for the fetus

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Genetic Effects It is possible to damage the hereditary material in a cell nucleus by external influences like Ionizing radiation chemicals etc. Effects that occur as a result of exposure to a hazard while in-utero are called teratogenic effects Teratogenic effects are thought to be more severe during weeks 8-17 of pregnancy - the period of formation of the body’s organs A higher incidence of mental retardation was found among children irradiated in-utero during the bombings of Hiroshima and Nagasaki

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Maternal Factors Pregnancy Statistically a radiation exposure of 1 rem poses much lower risks for a woman than smoking tobacco or drinking alcohol during pregnancy Smoking General Babies weigh 5-9 oz. Less than average 1 pack/day Infant Death 1 in 5 1 pack/day Infant Death 1 in 3 Alcohol 2 drinks/day Babies weigh 2-6 oz. Less than average 1 in 10 2-4 drinks/day Fetal alcohol syndrome 1 in 3 4 drinks/day Fetal alcohol syndrome 1 in 3 to 1 in 2 Radiation 1 rem Childhood leukemia deaths before 12 years 1 in 3333 1 rem Other childhood cancer deaths 1 in 3571

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Dose Response Curves Dose Dose Effects occur after a threshold Effects occur at any level stochastic Acute effects Chronic effects Biological effects The stochastic model is more conservative and is used to establish dose limits for occupational exposure

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Rate of Absorption Most important factor in determining when effects will occur Recovery is less likely with higher dose rates than lower dose rates for an equivalent amount of dose more permanent damage More recovery occurs between intermittent exposures less permanent damage

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Area Exposed The larger the portion - the more damage if all other factors are the same Blood forming organs are more sensitive A whole body dose causes more damage than a localized dose such as in medical therapy. Dose limits take this into consideration

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High-dose effects include: • nausea fatigue • erythema diffuse redness over an area of skin after irradiation • epilation loss of hair • blood disorders • intestinal disorders • fever • dry and moist desquamation shedding of the outer layer of skin • depressed sperm count in the male • temporary or permanent sterility in the male and female • injury to the central nervous system at extremely high radiation doses.

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Erythema - radiodermatitis

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Desquamation These early somatic effects are called acute radiation syndrome ARS.

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Low-dose chronic irradiation does not impair fertility. The health effects analysis of 150000 American radiologic technologists has revealed no effect on fertility. The number of births that occurred during a 12-year sampling period equaled the number expected. Animal data in this area are lacking. Those that are available indicate that even when radiation is delivered at the rate of 100 rad per year no noticeable depression in fertility is noted

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Irradiation in utero Irradiation in utero concerns the following two types of exposures: • that of the radiation worker • that of the patient Substantial animal data are available to describe fairly completely the effects of relatively high doses of radiation delivered during various periods of gestation. Because the embryo is a rapidly developing cell system it is particularly sensitive to radiation. With age the embryo and then the fetus becomes less sensitive to the effects of radiation and this pattern continues into adulthood.

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After maturity has been reached radiosensitivity increases with age.

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All observations point to the first trimester during pregnancy as the most radiosensitive period.

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Within 2 weeks of fertilization the most pronounced effect of a high radiation dose is prenatal death which manifests as a spontaneous abortion. Observations in radiation therapy patients have confirmed this effect but only after very high doses. Fortunately this response is of the all-or-none variety: Either a radiation-induced abortion occurs or the pregnancy is carried to term with no ill effect.

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During the period of major organogenesis from the 2nd through the 10th week two effects may occur. Early in this period skeletal and organ abnormalities can be induced. As major organogenesis continues congenital abnormalities of the central nervous system may be observed if the pregnancy is carried to term. If radiation-induced congenital abnormalities are severe enough the result will be neonatal death. After a dose of 200 rad 2 Gy t to the mouse nearly 100 of fetuses suffered significant abnormalities. In 80 this was sufficient to cause neonatal death.

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The incidence of childhood leukemia in the population at large is approximately 9 cases per 100000 live births. According to the Oxford Survey if all 100000 had been irradiated in utero perhaps 14 cases of leukemia would have resulted. Although these findings have been substantiated in several American populations no consensus has been reached among radiobiologists that this effect after such low doses is indeed real.

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Time of X-Ray Examination Relative Risk First trimester 8.3 Second trimester 1.5 Third trimester 1.4 Total 1.5 Relative Risk of Childhood Leukemia After Irradiation In Utero by Trimester

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Time of Exposure Type of Response Natural Occurrence Radiation Response 0-2 wk Spontaneous abortion 25 0.1 2-10 wk Congenital abnormalities 5 1 2-15 wk Mental retardation 6 0.5 0-9 mo Malignant disease 8/10000 12/10000 0-9 mo Impaired growth and development 1 Nil 0-9 mo Genetic mutation 10 Nil Summary of Effects After 10 Rad In Utero

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Uses of Radioactive Material

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Consumer Products  Building materials  Tobacco Po-210  Smoke detectors Am-241  Welding rods Th-222  Television low levels of X-rays  watches other luminescent products tritium or radium  Gas lantern mantles  Fiesta ware Ur-235  Jewelry

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Smoke Detectors Alpha particles from americium-241 red lines ionize the air molecules pink and blue spheres. The ions carry a small current between two electrodes. Smoke particles brown spheres attach to ions reducing current and initiate alarm.

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Medical Diagnostic  X-rays  Nuclear Medicine Tc-99m Tl-201 I-123  Positron Emission Tomography PET Therapeutic  X-rays Linear Accelerators  Radioisotopes Brachytherapy Cs-137 Ir-192 Ra-226 Teletherapy Co-60 Radiopharmaceuticals I-131 Sr-89 Sm-153

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Industrial Radiography Use of high activity sealed sources to examine structural components such as beams or pipes

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Radioisotopes in Medicine • Nuclear medicine uses radiation to provide diagnostic information about the functioning of a persons specific organs or to treat them. Diagnostic procedures using radioisotopes are now routine. • Radiotherapy can be used to treat some medical conditions especially cancer using radiation to weaken or destroy particular targeted cells. • Over 40 million nuclear medicine procedures are performed each year and demand for radioisotopes is increasing at up to 5 annually. • Sterilisation of medical equipment is also an important use of radioisotopes.

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Effects of radiation on biomolecules and molecular structure • There are many kinds of radiations that can increase mutations. • Most adverse health effects of radiation exposure may be grouped in two general categories: – Deterministic effects harmful tissue reactions due in large part to the killing/ malfunction of cells following high doses and – Stochastic effects i.e. cancer and heritable effects involving either cancer development in exposed individuals owing to mutation of somatic cells or heritable disease in their offspring owing to mutation of reproductive germ cells. • Radiation is often classified as ionizing or non-ionizing depending on whether ions are emitted in the penetrated tissues or not. • The ability of radiation to cause human cancer especially leukemia was dramatically shown by the increased rates of leukemia among survivors of the atomic bombs dropped in World War II and more recently by the increase in skin cancer in individuals exposed to too much sunlight UV radiation.

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Effects of radiation on biomolecules and molecular structure • Accumulated evidence in radiobiological studies has suggested DNA as the principle target for the biologic effects of radiation. It is now well established that radiation produces a wide spectrum of DNA lesions which include damages to nucleotide bases base damages DNA single-strand breaks SSBs and double-strand breaks DSBs.

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Effects of radiation on biomolecules and molecular structure

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Ionizing radiation as physical mutagen • Ionizing radiation is a high-energy kind of radiation that causes ions and free radicals to form. • Ionizing radiation is made up of energetic subatomic particles ions or atoms moving at high speeds usually greater than 1 of the speed of light and electromagnetic waves on the high-energy end of the electromagnetic spectrum. • X rays gamma rays γ beta particle radiation and alpha particle radiation also known as alpha rays are ionizing form of radiation. • Ionization usually occurs because the radiation source has very large energy For example radiation from radioactive substances uranium radium cobalt X-ray and cosmic ray If DNA molecules are hit by the radiation the DNA chain will loose. In consequence the DNA chain cannot function in protein synthesis.

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Ionizing radiation as physical mutagen • Aside from hydrogen bonds covalent bonds also hold DNA together. The backbones of the two DNA strands are made of nucleotides linked together by covalent bonds. So if ionizing radiation comes along and breaks these bonds the DNA will be chopped up into tiny little pieces The cell will try to repair these DNA breaks but it is very difficult for the cell to correctly put all those DNA pieces back together again. • Inevitably some mistakes may be made and these mistakes are mutations because they change the DNA sequence. This means that ionizing radiation can cause mutations in cells that are deep inside not just the cells on the surface of body. These mutations can eventually lead to cancer ionizing radiation is known to cause leukemia and thyroid cancer. • DSBs caused by ionizing radiation or other carcinogenic chemicals are considered the most relevant lesion for mutations and carcinogenesis. Unrepaired and misrepaired DSBs are serious threats to the genomic integrity. DSBs lead to chromosomal aberrations which simultaneously affect many genes to cause malfunction and death in cells.

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Ionizing radiation as physical mutagen • When cells are exposed to ionizing radiation radiochemical damage can occur either by direct action or indirect action. • Direct action occurs when alpha particles beta particles or x- rays create ions which physically break one or both of the sugar phosphate backbones or break the base pairs of the DNA. • Ionizing radiation can also impair or damage cells indirectly by creating free radicals. Free radicals are molecules that are highly reactive due to the presence of unpaired electrons on the molecule. Free radicals may form compounds such as hydrogen peroxide which could initiate harmful chemical reactions within the cells. As a result of these chemical changes cells may undergo a variety of structural changes which lead to altered function or cell death.

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Ionizing radiation as physical mutagen Direct action of ionizing radiation on DNA. Single strand and double strand breaks

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Ionizing radiation as physical mutagen Indirect action of ionizing radiation on DNA.

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Ionizing radiation as physical mutagen

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Ionizing radiation as physical mutagen

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Non – Ionizing radiation as physical mutagen • Non-ionizing radiation refers to any type of electromagnetic radiation that does not carry enough energy per quantum photon energy to ionize atoms or molecules—that is to completely remove an electron from an atom or molecule. • Causes molecular vibration electron raised to higher level and new bonds can be formed like thymine dimer.

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Non – Ionizing radiation as physical mutagen • UV is normally classified in terms of its wavelength: – UV-C 180-290 nm--"germicidal"--most energetic and lethal it is not found in sunlight because it is absorbed by the ozone layer – UV-B290-320 nm--major lethal/mutagenic fraction of sunlight – UV-A 320 nm--visible--"near UV"--also has deleterious effects primarily because it creates oxygen radicals but it produces very few pyrimidine dimers. Tanning beds will have UV-A and UV-B. • Ultraviolet ray generally do not cause ionization However the energy from ultraviolet ray will be absorbed by purine and pyrimidine so that the atom becomes more reactive the electron undergoes excitation. Consequently DNA double-helix becomes in disorder and inhibits replication One of the effects caused by ultraviolet ray is skin cancer. • The major lethal lesions are pyrimidine dimers in DNA produced by UV-B and UV-C--these are the result of a covalent attachment between adjacent pyrimidines in one strand

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When cells are exposed to UV light in the 240- to 300-nm range nucleic acid bases acquire excited energy states producing photochemical reactions between DNA bases. The principal products in DNA at biologically relevant doses of UV light are cyclobutane dimers formed between two adjacent pyrimidine bases in the DNA chain. Both thymine-thymine and thymine-cytosine dimers are formed. Non – Ionizing radiation as physical mutagen

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Cyclobutane dimer Non – Ionizing radiation as physical mutagen

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Handling of radio labeled materials

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Personal Protective Clothing • Required PPE: For any work with an open radioactive source wear: – disposable gloves latex or nitrile gloves are generally suitable – a full-length lab coat worn closed with sleeves rolled down – close-toed shoes. Never wear sandals or other open-toed shoes while working with radioactivity. • Safety Glasses: You should wear safety glasses for any radioisotope procedure but it is especially important whenever there is a potential for the build-up of pressure that could release a spray of material. • Protecting Your Wrists: Lab coat cuffs may hang down and drag across contaminated surfaces. To protect the skin of your wrists consider one of the following steps: • Wrap tape around your lab coat sleeve or put a rubber band around the sleeve to keep the cuff from dragging. • Wear long gloves and tuck your lab coat into the gloves. • Wear Tyvek sleeve protectors. • Survey the skin of your wrists frequently as you work. • Contaminated Lab Coats: See Spills Incidents for information about how to handle a contaminated lab coat. • Extra Clothing: Keep an extra set of clothing and shoes in the lab in case your clothing becomes contaminated. • Petroleum-Based Hand Creams: Avoid using petroleum-based hand creams when wearing gloves because petroleum-based hand creams may increase glove permeability.

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Food and Beverages • No Eating or Drinking: Do not eat or drink in any room labeled with a Caution: Radioactive Materials sign on the door. • When you see this sign on a door youll know that you are never permitted to eat or drink in that room. • No Storage: Do not store food beverages or medicines in refrigerators freezers or coldrooms where radioactive materials are used or stored. • Storing Food Items in Your Desk: You may store your food water bottles beverages medicines coffee mugs eating utensils etc. in your closed desk in a radioisotope use lab but you are not permitted to have these items out on top of your desk or any other surfaces.

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Security • Stock Vials: Lock radioactive stock materials and sealed sources in a secured container or a secured storage area when not in use. A stock material is radioactive material as provided by the vendor and does not include material withdrawn from the original stock for experimental use. • Tethered Lock Box: If you store your stock vials in a lockbox the lockbox must be tethered to a surface with a secure cable or the lock box must either be kept in a locked freezer or refrigerator. • Locking the Lab: Do not leave radioactive materials unsecured in an unattended lab even for a short time unless the lab is locked. • Supervising Visitors: Supervise your own visitors to the lab. • Greeting Visitors: When visitors who are not accompanied by authorized lab personnel enter the lab courteously find out who they are and why they are there. • Missing Materials: If you discover that radioactive material is missing or lost and cannot be accounted for notify authority no later than the next business day.

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Signs and Labels • Room Labeling: Label radioisotope use rooms with Caution Radioactive Material signs. If there are no signs on a room in which radioactive materials are used or stored contact EHS to request labeling for the room. • Container Equipment Labeling: Label any container of radioactive material or piece of equipment in which radioactive material is stored and any contaminated area or item regardless of the level of radioactivity with Radioactive tape. Labeling contaminated items and containers of radioactive material is an important tool for contamination control and is a courtesy to other laboratory personnel.

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Setting Up a Radioactive Materials Work Area • Absorbent Paper: Cover the work surface with protective and absorbent bench paper to trap droplets of contamination. Its especially convenient to cover the entire work area and then to use smaller pieces on top of the large piece. Its easier to replace the small piece when it becomes contaminated than to replace the entire covering. • Dedicated Equipment: Your radioisotope work area should have a set of equipment that is only used for radioactive material work. Depending on your protocol this may include pipettors a microcentrifuge timers mixers a water bath etc. • An example of a good radioactive materials work area showing the use of absorbent paper shielding dedicated pipettors and labeling of equipment.

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Good Laboratory Practices • Familiarity with Radioisotope Properties: Be familiar with the properties of the radioisotope you plan to use and with any precautions and concerns specific to that radioisotope and material. For instance there are special precautions for working with 35 S-methionine because of its volatility. • Rehearsing Procedures: Rehearse unfamiliar radioisotope procedures before radioactive material is actually used. This helps you to see where all the necessary materials should be placed it helps you to work efficiently and it helps you to identify moments during the procedure when aerosols or contamination is most likely to occur. • Preoperational Survey: Are you sure your work area is free of contamination when you start You are encouraged to survey your work area carefully before you start in case someone else left the work area contaminated or in case you missed contamination the last time you worked. • Radiation Monitoring Badges: Wear radiation monitor badges when appropriate. Wear ring badges under gloves to prevent the ring from getting contaminated. Make sure you dont discard the ring when you remove your gloves. • Changing Gloves: Change your gloves frequently. Your radioactive solutions especially when aliquoting from the stock vial are likely to be highly concentrated. It is very easy to contaminate your gloves and to spread contamination.

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Good Laboratory Practices • Mouth Pipetting: Never pipette radioactive materials by mouth. • Surveys While You Work: Even though you are only required to do a postoperational survey it is very good practice to survey frequently and extensively as you work. Dont assume that contamination will only be found on the bench top. • O-Rings: if you are relying on tubes with o-rings to contain your radioactive material during hybridizations for instance be sure to check the condition of the tubes to be sure the o-rings arent dried out. • Volatile Radioactivity: Work in a hood during procedures using volatile materials such as I-125 or S-35 methionine/cysteine. • Waste: Cover radioactive waste cans at all times and store waste cans away from areas in which people spend substantial amounts of time. Provide shielding for waste cans with significant external radiation levels. • Postoperational Surveys: Survey yourself and your clothing when radioisotope work is finished and before leaving the lab.

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Microcentrifuge Use • It is difficult to keep a microcentrifuge used for radioactive material work free of contamination. Contaminated microcentrifuges must be cleaned up after use to prevent contamination from spreading to other tubes and to your gloves. The following steps may help reduce the incidence of contamination: • Wipe down the exterior of the tubes before placing them in the microfuge. • Dont fill tubes more than 2/3 full. • Use tubes with locking caps or with screwcaps the type with O-rings. • Consider using an aerosol-tight rotor so that only the interior of the rotor becomes contaminated.

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Fume Hoods and Biosafety Cabinets • Work with certain radioactive materials such as volatile I-125 or millicurie amounts of S-35 methionine/cysteine must be performed in a designated radioactive materials RAM fume hood. • Do not use biological safety cabinets or laminar flow hoods for work with volatile radioactive materials since the air from the cabinet may be exhausted back to the room.

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Biosafety regulations in the handling of recombinant DNA processes • In the context of the NIH Guidelines recombinant and synthetic nucleic acids are defined as: – molecules that a are constructed by joining nucleic acid molecules and b that can replicate in a living cell i.e. recombinant nucleic acids – nucleic acid molecules that are chemically or by other means synthesized or amplified including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules i.e. synthetic nucleic acids or – molecules that result from the replication of those described in i or ii above. • NIH GUIDELINES FOR RESEARCH INVOLVING RECOMBINANT OR SYNTHETIC NUCLEIC ACID MOLECULES NIH GUIDELINES April 2016 • http://osp.od.nih.gov/sites/default/files/NIH_Guidelines.ht ml_Toc446948317