Tissue Effects of Radiation at the Cellular Level Jeffrey Bryan, DVM, MS, PhD, DACVIM(Oncology) firstname.lastname@example.org
Cellular Radiation Effects • Cell membrane - Alteration in permeability • Cellular organelles - Functional Aberrations • Nuclear membrane - Altered permeability & Function • DNA - Chromosomes - Functional aberrations
DNA (Chromosomes) • The DNA makes up the chromosomes of the cell and carries all of the functional encoding information of the cell or organism • All of the chromosomes together make up the genome • The genome is composed of many genes (60,000 in humans) • The individual genes are composed of sequences of nitrogenous bases attached to the molecular backbone. These sequences encode for protein functions etc. which control all cell functions • Large areas of a DNA strand may not be expressed in individual cells
DNA Structure • Double stranded helix (twisted ladder millions of rungs long) with side rails of ladder composed of Sugar molecules bound together by a phosphate • Rungs are composed of the nitrogenous bases Adenine, Thymine, Guanine and Cytosine. • Adenine and Thymine combine to make up one type of rung and Guanine and Cytosine combine to make up another type. • A given base may be on either side of the helix
DNA Structure • DNA is a very large molecule. There are about 2 x 109 base pairs in the mammalian genome distributed across 15-100 chromosomes. • The stearic configuration (shape) of the molecule changes constantly and is important to function. • DNA is replicated at cell division
Mechanism of radiation Injury • Direct ionization of a portion of the DNA molecule. • Indirect injury by free radicals in the DNA environment. • H+, 0H-, H202-, etc.
DNA Radiation Injuries • Base pair deletion • Cross-linking injuries • Single Strand Break • Double Strand Break • Multiple (complex) lesions
Radiation Induced Chromosomal Aberations • Chromatid exchanges. • Sister Unions • Acentric Fragments • Rings • Dicentric Unions
Radiation Induced Chromosomal Aberrations http://www.geneticarchaeology.com/research/DNA_Damage_To_Nuclear_Test_Vets_Prompts_Call_For_Study_Of_Children.asp
Radiation Induced Chromosomal Aberrations Chromosome 5 pair
DNA Replication • DNA is replicated during S Phase prior to the onset of mitosis • The original DNA is used as a template for the building of the new DNA. • Quite rapid process, requires less than 15 hours.
Cell Division • Mitosis • Multistep process • DNA organizes into identifiable chromosomes (Prophase ) • DNA aligns with centromeres on equatorial plate (Metaphase) • DNA Separates and moves to opposite ends of cell (Anaphase) • Cell cytoplasm divides at equatorial plate (Telophase)
Mitosis • Cell resumes normal functional operations (interphase) • Only requires about one hour • Through this process radiation induced aberrations in the DNA may result in significant loss of DNA to one or both of the daughter cells.
Cell Cycle • Tissues grow and are maintained through cell replication (regeneration) • Some cells never divide once adulthood is reached. • There are a specific set of steps involved • G1 (G0) Gap Phase 1 Functional cell • S Synthesis DNA synthesis • G2 Gap phase 2 Rest • M Mitosis Cell Division
Repair of Radiation Injury • Cellular mechanisms are in place which can repair most if not all types of radiation injury to the DNA. • Repair is a time sensitive process • Repair is a cell cycle dependent process • Repair is a dose rate dependent process • Repair is dose dependent • Repair is radiation type dependent
Cellular Mechanisms of Repair • Base Excision Repair • Damaged bases must be repaired • The complementary base on the opposite strand serves as a template. • This type of repair is quite efficient • Loss of this repair mechanism increases the incidence of mutations.
Cellular Mechanisms of Repair • Nucleotide Excision Repair (NER) Repairs DNA damage due to pyrimidine dimer adducts added to the DNA by injury. - Enzymatic removal of lesion and associated backbone. - Lesion is then sealed by DNA polyemerase and ligase. - Defective mechanism increases sensitivity to UV light
Cellular Mechanisms of Repair • Double Strand Break Repair • Non-homologous End Joining • Occurs primarily in G1 phase when no sister chromatid is present. • In some instances the base pair sequence is filled in by repair processes without a template. • Complex process with multiple pathways • Because it is an error prone process it tends to promote development of mutations.
Cellular Mechanisms of Repair • Double Strand Break repair • Homologous Recombination repair • Uses sister chromatid as a template to faithfully recreate the damage section and join the ends together properly • Occurs in S phase when sister chromatids present • Error free process • Loss of ability increases radiation sensitivity and mutation rate.
Cellular Mechanisms of Repair • Single strand break repair • Occurs via similar pathway to Base Excision Repair. • Efficiently done and vast majority of lesions are repaired. • Predominately error free process
Cellular Mechanisms of Repair • Because of the efficiency of repair mechanisms for all but double strand breaks the majority of the cell killing occurring at low doses is due to double strand breaks which are not repaired. • At high doses accumulated DNA injury due to many single strand breaks and base pair deletions becomes more important.
Types of DNA Damage • Lethal Damage • Irreversible and irreparable – fatal to cell • Potentially Lethal Damage (PLD • Damage which is lethal unless modified by post irradiation events • Sublethal Damage (SLD) • Repairable injury to the DNA
Lethal Damage • Non repairable injury associated with double strand breaks • Increases with LET up to a point • Increases with higher doses
Potentially Lethal Damage • Not repaired and is lethal under normal circumstances. • Repair increased by conditions which are suboptimal to the division of the cell • Reduced temperature • Hypoxia • Low pH • Others • Increased capability = radioresistance
Sublethal Damage Repair (SLD) • Refers to DNA damage that is repaired • Splitting radiation dose increases survival • Occurs in 1-6 hours after irradiation • Affected by phase of cell cycle • Affected by cell cycle time • Long cycle usually increases repair • Indicated by shoulder on survival curve
Repair is a time sensitive process • Repair of DNA injury of all types is essentially complete by 6 hours post irradiation. • External factors that affect cellular metabolic rate may delay or accelerate it • Foundation of modern radiotherapy
Repair is a cell cycle dependent process • Different phases have different repair capabilities • Mitosis has the least repair capability • G2 • G1/G0 • S phase has the most repair capability • Capability varies in G1 and S
Next Time • Cell Cycle and Differentiation Effects