Cellular Responses to Radiation PDF

Radiobiology, Cellular Responses to Radiation and Radiosensitivity Dr Kholood Baron Recap to cell biology  Human cells are either somatic cells or germ cells.  Cells propagate through division: • Division of somatic cells is called mitosis and results in two genetically identical daughter ce...

Radiobiology, Cellular Responses to Radiation and Radiosensitivity Dr Kholood Baron Recap to cell biology  Human cells are either somatic cells or germ cells.  Cells propagate through division: • Division of somatic cells is called mitosis and results in two genetically identical daughter cells. each carrying a chromosome identical to that of the original cell. • Division of germ cells is called meiosis and involves two fissions of nucleus giving rise to four sex cells, each possessing half the number of chromosomes of the original germ cell (not identical to origin) .  Chromosome carries hereditary information in the form of genes. Cell Life Cycle  Cell proliferation cycle is defined by two time periods: • Mitosis M, where division takes place. • The period of DNA synthesis S.  S and M portions of the cell cycle are separated by two periods (gaps) G1 and G2 when, respectively • DNA has not yet been synthesized. • Has been synthesized but other metabolic processes are taking place.  Time between successive divisions (mitoses) is called cell cycle time. (This varies to each cell (tissue) type) Cell cycle time for stem cells in certain tissues is up to 10 days. cells are most radio-sensitive in the M and G2 phases, and most radio-resistant in the late S phase. Cell cycle time of malignant cells is shorter than that of some normal tissue cells, but during regeneration after injury normal cells can proliferate faster. q Cell death for stem cells and other cells capable of many divisions is defined as the loss of reproductive integrity (reproductive death). Purpose of Apoptosis • An organism induces cells that might poses a threat to commit suicide. • Apoptosis can be induced by radiation. This is generally a good thing because dead cells don’t become cancerous. Failure of Apoptosis • If radiation damages the genes that induce apoptosis, there is an increased possibility that any malfunctioning cancerous cells will proliferate. • As a “survival mechanism ” the cancer cells produce chemicals that suppress apoptosis. THIS a characteristic of all cancers. CLASSIFICATION OF RADIATIONS Radiation is classified into two main categories: • Non-ionizing radiation (cannot ionize matter). • Ionizing radiation (can ionize matter). Ionizing radiation contains two major categories • Directly ionizing radiation (charged particles). electrons, protons, alpha particles, heavy ions. • Indirectly ionizing radiation (neutral particles). photons (x rays, gamma rays), neutrons. Penetrability and Energy  All radiation has the ability to penetrate and transfer its energy to the material it is penetrating.  The term “Linear Energy Transfer”, or L E T, is used to describe the amount of energy imparted locally in a target. the unit for the LET is keV/ m .  The higher the value of LET, the greater the amount of energy being transferred per interaction, and the lower its penetrating ability. This also means the greater the risk of damage to the material absorbing the energy.  Alpha particles have a high LET. Their ability to penetrate anything is very low. Alpha particles can be shielded by a piece of paper.  Beta particles have a low LET and can only penetrate material of low density. They can be shielded with Plexiglas.  X ray and gamma rays also have a low L E T, they have the highest ability to penetrate material. High density materials are needed to shield against these waves. LET and Penetrability • Alpha particles impart a large amount of energy in a short distance. • Beta particles impart less energy than alphas, but are more penetrating. X and Gamma rays impart only a fraction of their total energy each time they interact with the target material. Interaction of Radiation with Cellular Constituents • X-rays interact with atoms by the photoelectric effect, Compton scattering, or pair production. (Compton scattering is the most likely interaction). • In these interactions the X-ray transfers energy to an electron which then travels through the cell. This electron then interacts directly or indirectly with the Cell DNA.( nucleus) • The result is that these molecules are excited , ionized, or broken. • In some cases ( indirect interaction) free radicals are produced. Ionizing radiation interaction with the cell Direct Effects  Involve a transfer of energy from the radiation directly to the target molecule (typically DNA).  Direct effects are the most important type of effect for high LET radiation (e.g., neutrons and alpha particles). Indirect Effects - Occur when radiation produces free radicals which react with the target molecule (typically DNA). - The free radicals must be produced very close to the target since they have such short lifetimes. - Indirect effects is most important for low LET radiation (e.g., x-rays and betas). - Indirect action can be modified by chemical sensitizers or radiation protectors In direct interaction causes Radiolysis of Water (splitting the water molecules by radiation)  The Water is the major constituent of the cell. Most radiation energy absorbed by the body is absorbed by water.  Reactions happens due to transfer of energy from (electrons) to the water molecule. This will create Free Radicals: The unpaired electron makes the free radical extremely reactive. Within a nanosecond of its formation, a free radical will react with nearby molecule in the cell and damages it. Indirect Effect - When X-rays interact with water, two types of free radicals are formed: The presence of an excess of oxygen during irradiation of cells allows the formation of additional free radicals : The presence of an excess oxygen during irradiation of cells allows the formation of free radicals: It is worth noting that hydroxyperoxy free radical combination into the highly unstable hydrogen peroxide. The free radicals might react with molecules (RH) and produce a new radical species that proceeds to damage other molecules. • A hydrogen free radical might combine with an oxygen molecule to produce a hydroperoxyl radical. then combine with another hydroxyl free radical to produce hydrogen peroxide. H O ÷ HO + O2 HO2 • Other types of free radicals can combine with oxygen to produce organic peroxy free radicals: R + O2 ÷ RO2 • Much of the damage brought by radiation is believed to be due to oxygen containing radicals, ions and peroxides collectively known as reactive oxygen species (ROS). • The hydroxyl radicals free are believed to be particularly important.  Steps involved in producing biological damage by the indirect action of x rays are as follows: • Primary photon interaction (photoelectric effect, Compton effect, pair production) produces a high energy electron or positron. 2. • High energy light charged particle is moving through tissue to produce free radicals in water. 3. • Free radicals ( highly reactive) -- may produce chemical changes in DNA from the breakage of chemical bonds. 4. • Changes in chemical bonds result in biological effects. 1. Radiation leads to tissue damage. Its estimated that - Two thirds of biological damage caused by low (LET) radiation is due to indirect action - Biological damage by high LET is primarily by direct ionization action. - If the radiation directly affects somatic cells, the effects on the DNA (and hence the chromosomes) could result in a radiation-induced malignancy. - If the damage is to reproductive stem cells, the result could be a radiation-induced congenital abnormality. Subcellular Targets  Everything in the cell is subject to direct and indirect damage, but some targets are considered to be more important than others:  DNA   membranes (especially the nuclear membrane) The DNA molecule is considered to be the most important site of damage because it serves as the master blue print for the cell. DNA Damage  With low LET radiation, the damage tends to be isolated along the DNA molecule.  With high LET radiation, clusters of damage occur on a given chromosome.  Such clustered damage is more complex and difficult to repair than the isolated damage caused by low LET radiation.  Clustered damage are unique to radiation, The following table (UNSCEAR 2000) indicates the estimated DNA damage per gray (100 rads). Base damage 500 per cell Single strand breaks 1,000 per cell Double strand breaks 40 per cell DNA-protein crosslinks 150 per cell Type of DNA Damage 1- DNA Base Damage  Involves alterations to individual nitrogenous bases.  These alterations are primarily due to the formation of free radicals in the water that is closely bound to the chromosomes.  Example: the free radicals might induce the deamination of the nitrogenous base cytosine and convert it to uracil. It is even possible that the base might be completely removed. DNA Base Damage and Repair  DNA base damage is often (not always) is meant by the term mutation. And is referred to as a genetic (vs. chromosomal) mutation.  Large stretches of the chromosome consist of “nonsense” repetitive DNA. Base damage to these regions is inconsequential  Base damage is usually repaired. The classical base-excision repair mechanism operates as follows: • First, the damaged section is excised by a DNA glycosylase enzyme. Various glycosylases damage recognize different types of base damage. • Then a DNA endonuclease cuts the DNA “backbone” • Next, a phosphodiesterase removes the sugar and phosphate remnants. DNA Base Damage and Repair • After this, the excised segment is resynthesized by a polymerase using the undamaged strand as a template. • Finally, ligases attach the newly synthesized segment in place. Sometimes the damage necessitates the removal of a large segment of one side of the DNA molecule, not just one base and the associated sugar and phosphate groups. • Production by single particle crossing both strands? • Production by two independent events? • DSBs are more likely to result in the death of the cell than base damage. Even so, only a small percentage of DSBs are lethal. • Most DSBs are repaired and do not result in visible chromosomal aberrations  ** X-ray dose of ~1 Gy produces about 1000 single strand breaks and about 50-100 double strand breaks in a typical mammalian cell.  This dose causes about 50% cell death.  DSBs are not necessarily lethal. What actually happens in the cell depends on several factors, including: • The type and number of nucleic acid bonds that are broken • The intensity and type of radiation • The time between exposures • The ability of the cell to repair the damage • The stage of the cell’s reproductive cycle when irradiated. Chromosomal aberrations (Mutations)  Unrepaired DSBs can lead to chromosomal aberrations (mutation)  The broken ends of the chromosome at the site of the DSB getting attached to the ends of other broken, or unbroken, chromosomes.  The nature of the aberration depends on the stage of the cell cycle at which the exposure occurred. In G1 each chromosome consists of one DNA molecule. In G2 each chromosome consists of two DNA molecules (one per chromatids).  The aberrations that result from DSBs in G1 are called chromosomal aberrations.  Those that result from DSBs in G2 are sometimes called chromatid aberrations.  Likely Results of Chromosomal Aberrations If the cell attempts to divide, it is likely that some of the chromosomal material will not be properly distributed between the two daughter cells.  Example, the spindle fibers cannot attach to and separate the acentric fragments (genetic material).  Dicentric chromosomes might even be pulled in both directions.  Due to the chromosomal aberrations, the cell might: • Survive but never attempt to divide. • Die at mitosis without dividing. • Divide successfully, but the daughter cells die because they lack a complete complement of DNA.  This referred to as “mitotic death” Radiation can induce cell death via apoptosis. Cytogenetic Dosimetry For low LET radiation, the relationship between dose and the number of double strand breaks -DSBs (and therefore dicentric chromosomes) seems to be linear-quadratic. Possible outcomes of cell irradiation: • No effect. • Division delay: The cell is delayed in going through division. • Apoptosis: The cell dies before it can divide. • Reproductive failure: The cell dies when attempting the mitosis. • Genomic instability: There is a delay in reproductive failure. • Mutation: The cell survives but contains a mutation. • Transformation: The mutation leads to a transformed phenotype and possibly carcinogenesis. • Bystander effects: An irradiated cell may send signals to neighboring unirradiated cells and induce genetic damage in them. • Adaptive responses: The irradiated cell becomes more radio-resistant. DNA damage has no effect if • The damage is completely repaired • The damage is unrepaired (or misrepaired) but it is limited to a non-functional (meaningless) segment of DNA • The damage is unrepaired (or misrepaired) and the damaged cells die or become non-functional. There is no significant effect if only a small number of cells die or become non-functional Effects of radiation can be classified as somatic or genetic : • Somatic effects are harm that exposed individuals suffer during their lifetime, such as radiation induced cancers (carcinogenesis), sterility, opacification of the eye lens and life shortening. • Genetic or hereditary effects are radiation induced mutations to an individual’s genes and DNA that can contribute to the birth of defective descendants. The effects can be classified into early or deterministic (have a threshold), or delayed or stochastic(no threshold.  Effects are also classified into somatic and hereditary. The somatic include early and delayed effects (cancer)  Stochastic effect : the probability of occurrence increases with increasing dose but the severity in affected individuals does not depend on the dose (e.g., induction of cancer and genetic effects).  Deterministic (non-stochastic) effects are highly predictable response to radiation. There is a threshold of radiation dose after which the response is dose related. Some of the known deterministic effects are radiation-induced lung fibrosis, organ dysfunction, fibrosis, lens opacification, blood changes, decrease in sperm count). Cell Survival Rate  While it is difficult to determine the number of nonlethal mutations induced by radiation, it is relatively easy to quantify cell death.  Effects of radiation on tissue as a function of dose are presented in the form of: • Cell survival curves. • Dose response curves. In radiation biology, cell death is defined as the inability of individual cells to reproduce. Cell Survival Curves Cell survival curve (surviving fraction against absorbed dose) describes the relationship between: • Surviving fraction of cells, i.e., the fraction of irradiated cells that maintain their reproductive integrity. • Absorbed dose. Cell survival against dose is graphically represented by plotting the surviving fraction S(D) on a logarithmic scale on the ordinate against dose D on a linear scale on the abscissa.  The mean lethal dose is the dose that kills 63% of the original population. An optimist would say that it is the dose at which 37% survive. CELL SURVIVAL CURVES Type of radiation influences the shape of the survival curve. • For (high LET) the cell survival curve is almost an exponential function of dose (shown by an almost straight line on a log-linear plot. • For (low LET) the survival curves show an initial slope followed by a shoulder region and then become nearly straight at high doses. 1- Q Repair.  One explanation for the shoulder on the survival curve is that it indicates the presence of a DNA repair mechanism (called Q repair).  Dq then becomes a measure of the effectiveness of this repair process  Do a measure of the cell's sensitivity to radiation induced death. 2.Target Theory (multitarget/multihit processes). - Survival curves are explained in terms of the number of targets in a cell that must be hit for cell death to occur. Cell death is assumed to be due to DSBs. The latter can be produced by a single event when both strands are broken at once, or by two separate events when each strand is broken individually DOSE RESPONSE CURVES Plot of a biological effect observed (e.g., tumour induction or tissue response) against the dose given is called a dose response curve. Generally, as the dose increases so does the effect. Dose response curves may or may not have a threshold dose. Factors Affecting Radiosensitivity 1- Radiation Quality  The amount of biological damage per unit dose increases with the linear energy transfer (LET) of the radiation.  The higher LET, the greater the radiation energy lost per unit distance, and the greater the density of ions and free radicals in the charged particle tracks.  As LET increases, more energy is deposited in the individual cells, but fewer cells are affected per unit dose.  Increasing the LET of the radiation increases the complexity of the damage to the DNA. This makes repair more difficult. 2. Dose Rate Effect  The dose delivery can be classified as a: • Chronic Exposure. The dose is delivered at a low rate over a long time, e.g., 0.1 rad/hr for 10,000 hours (total 1000 rad). • Fractionated Exposure. The dose is delivered in discrete quantities, e.g., 100 rad are delivered per week for 10 weeks (total dose: 1000 rad). • Acute Exposure. The total dose is delivered at once, (total 1000 rad). - For low LET radiation, the magnitude of the effect per unit dose is greatest following acute exposures and least with chronic exposures - Chronic exposures with high LET radiation may be more carcinogenic than acute exposures, i.e., an inverse dose rate effect. 3- Tissue Exposed  The most radiosensitive tissues possess cells are: • dividing at the time of exposure (mitosis is the most sensitive stage of the cell cycle) • of an undifferentiated type, i.e., unspecialized in structure and function Examples of Radiosensitive Tissues: • germinal cells of the ovary and testis (spermatogonia) • hematopoietic (blood forming) tissues: red bone marrow Spleen lymph nodes • epithelium of skin • epithelium of gastrointestinal tract • lymphocytes (exception to the “law”) • oocytes (exception to the “law”) Examples of Radioresistant Tissues: • bone • muscle • cartilage • nervous tissue During embryonic and fetal development, the cells of these tissues are reproducing and much more sensitive to radiation. 4- Exposure Time During the Cell Cycle  For low LET radiation, the most sensitive stages of the cell cycle, with respect to cell death, are mitosis and late G1 (at the G1 -S border).  Because the chromosomes are condensed during mitosis and the repair mechanisms have poor access to the DNA molecule.  For high LET radiation, all phases of the cell cycle appear equally sensitive.  In radiation therapy, the cancer cells most likely to be killed are those in the sensitive stage of the cell cycle. Cells in other stages of the cell cycle survive.  Radiation can slow down a cell’s passage through G1 and G2. this is to give the cell more time to repair damage prior to division. 5. Adaptive Response  Low doses of radiation appear capable of initiating changes in cells that reduce the consequences of subsequent exposures.  For example, This adaptive response results in a lower than expected number of chromosomal aberrations following a second “challenge” dose.  This adaptive response seems to involve the activation of certain genes that increase the production of enzymes involved in DNA repair. 6. Oxygen Tension  Cells with normal concentrations of oxygen tend to be 2-3 times as sensitive to low LET radiation as hypoxic (low in oxygen) cells.  Poorly vascularized tissue, i.e., tumors, tend to be hypoxic. While, tissues well supplied with blood tend to have normal oxygen tensions.  This effect of oxygen is probably due to several things, e.g.,: • Increasing the production of H2O2 and other reactive oxygen species. • increasing the stability and toxicity of free radicals. - Because oxygen can combine with the free electrons produced during the radiolysis of water. This can increase the biological. 7. Chemical Protective Agents  Certain chemicals, injected in substantial quantities 30 minutes or so prior to an acute exposure, can significantly reduce the effective dose of the radiation.  Examples of radioprotective drugs include cysteine, cystamine, glutathione and sulphydryl groups.  radioprotective drugs focuses on the protection from large acute exposures and may provide protection from late effects such as cancer. NOTE: RADIOPROTECTORS AND RADIOSENSITIZERS  Some chemical agents may alter the cell response to ionizing radiation, either reducing or enhancing the cell response: • Chemical agents that reduce cell response to radiation are called radioprotectors. They generally influence the indirect effects of radiation by scavenging the production of free radicals. • Chemical agents that enhance cell response to radiation are called radiosensitizers. They generally promote both the direct and indirect effects of radiation. 8- Sex  Females tend to be slightly more radioresistant than males. This can be related to differences in hormonal. Variability in female hormonal levels has been linked to variations in lymphocyte radiosensitivity  In term of cancer, males are more susceptible to radiation induced leukemia, while females are more susceptible to radiation-induced thyroid cancer. 9- Age: Radio sensitivity according to age in Descending order into : • Fetus, • Child • Teenage • Adults.