1 / 75

Illinois Institute of Technology

Illinois Institute of Technology. Physics 561 Radiation Biophysics Lecture 9: Cancer, Genetics, and High LET radiation 30 June 2014 Andrew Howard. Cancer, concluded Mutagenesis Animals & humans Dose-response Latency Genetics Chromosomal damage Recombination Frameshifts

seth-padilla
Download Presentation

Illinois Institute of Technology

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Illinois Institute of Technology Physics 561 Radiation Biophysics Lecture 9:Cancer, Genetics, andHigh LET radiation30 June 2014 Andrew Howard Stochastic Damage; High-LET

  2. Cancer, concluded Mutagenesis Animals & humans Dose-response Latency Genetics Chromosomal damage Recombination Frameshifts Genes and Introns Structural components Genetics, concluded RNA vs. DNA The genetic code Relative sensitivities Organismal differences High-LET radiation Energy deposition Impact on DNA Resistance to repair The Ullrich experiment Damage to chromosomes Cataracts Plan for this lecture Stochastic Damage; High-LET

  3. Is an Ames Test a Good Substitute for These Complex Systems? • No! • 1,8-dinitropyrene is the most mutagenic substance known in the Ames test; yet it is only weakly tumorigenic in rats. Stochastic Damage; High-LET

  4. Why might we care about dinitropyrene? • Most mutagenic substance known in Salmonella strain TA98: 72900 revertants/nanomole • Nitroarenes like this one were found to be present in used toner, i.e., combustion waste from Xerox toner • When this appeared, Xerox chemists reformulated their toner to drastically reduce the nitroarene content in the used toner. • Mermelstein (1981) Mutation Research89:187-196. • Löfroth et al(1980) Science209:1037-1039 andMermelstein et al (1980) Science209:1039-1043. • So: all’s well that ends well! Stochastic Damage; High-LET

  5. This is also a story about enzyme induction • Nitroarenes like dinitropyrene and other polynuclear aromatic hydrocarbons, (e.g. benzo(a)pyrene) are known to be inducers of enzyme activities • Some of these enzyme activities actually activate toxicants rather than detoxifying them • Most of the activity of these enzymes will detoxify; • But if 1% makes things worse, we want to understand that 1% activation • So we found that pretreatment with these compounds could induce subsequent binding of other compounds to mouse DNA:Howard et al (1986), Biochem. Pharm.35: 2129-2134. Stochastic Damage; High-LET

  6. Animal Cell-Line Cancer Studies • How similar are these rodent cell systems (CHO, mouse) to human cells? • Answer: Human cells: • Are more resistant to spontaneous immortalization • Tend to give more nearly linear responses to dose • Radical scavengers and cold don’t protect as much:That suggests that direct mechanisms prevail in humans and indirect mechanisms are more important in rodents Stochastic Damage; High-LET

  7. More on humans vs. rodents • High-LET studies indicate that repair is less effective in human cell systems than in animal cell systems • Is the timescale a factor in that? Humans live a lot longer than rodents. • Promotion can be studied in animal cells, along with initiation Stochastic Damage; High-LET

  8. Radiation Carcinogenesisin Human Populations • Occupational: radiologists, miners, dial painters • Medical exposures: • Ankylosing spondylitis • Nonmalignant disease in pelvis and breast • Multiple fluoroscopies in to chest (e.g. in TB patients) • Infants & children with enlarged thymus and ringworm • Children exposed in utero to diagnostic X-rays • Nuclear accidents and weapon detonations • Environmental background (see last chapter) Stochastic Damage; High-LET

  9. Dose-Incidence in Cancer Studies • We seek a relationship relating post-exposure incidence ID to dose D and normal incidence In • Model might be: • Linear: ID = In + 1D • Quadratic: ID = In + 2D2 • LQ: ID = In + 1D + 2D2 • Corrected for loss of clonogenic potential:ID = (In + 1D + 2D2)exp(-1D+2D2) Stochastic Damage; High-LET

  10. Linear, Quadratic, LQ Models • We try to devise low-dose models based on high-dose data, where the three models are close together. It’s often difficult: Stochastic Damage; High-LET

  11. Latency • Definition (in the cancer context):Time between the mutational events that began cellular transformation and the appearance of a medically observable malignancy • How long in humans? • A few years (blood or lymphatic cancers) • 15-30 years for solid tumors • Animals: scale these numbers to animal’s lifespan • These numbers are minima:leukemia can take > 15 yrs Stochastic Damage; High-LET

  12. Macroscopic Damage to Chromosomes • First half of chapter 13 • Structural changes in chromosomes • Inversions of fragments • Multiple hits • Isochromatid breaks • Dicentrics • Minutes • Cross over Stochastic Damage; High-LET

  13. Generalizations about Genetics • Main focus here is on ways that ionizing radiation can damage the physical and replicative properties of DNA • We’ll look at three length scales: • The individual base-pair (~0.5 nm) • The gene (1000 bp - many nm) • The entire chromosome (200 nm or more) • Focusing only on single-base mutations (substitutions, deletions, insertions) does not tell the whole story Rad Bio: Genetics

  14. Early studies • T.H. Morgan studiedchromosomes in Drosophila • Tradescantia (spiderwort)microspores: Sax, 1938-1950 • More Tradescantia: Lea et al, 1946:Provided quantitative data on chromosomal aberrations during first mitotic cycle Rad Bio: Genetics

  15. 1960’s-1970’s:chromosomes • Metaphase chromosomes readily studied • Human lymphocytes extensively studied that way • “Premature chromosome condensation” used to study interphase chromosomal organization Courtesy HelmholtzZentrum München Rad Bio: Genetics

  16. Chromosome Breakage • Quite common result of radiation exposure • Can happen before replication:then the structural defect will be replicated(if the cell survives) • Can happen afterward:then one of the two chromatidswill differ from the other one. • Types of damage:Subchromatid, Chromatid, Chromosome • Repair tends to be rapid Wheat chromosomesCourtesy USDA Rad Bio: Genetics

  17. Single-Hit Breaks • Cartoons show * as centromere and ^ as break • Single-Hit:A B C * D E F^ G H IA B C * D E F + G H I • This can recombine in a number of ways:A B C * D E F I H GI H G A B C * D E FG H I A B C * D E F • … or a circle plus a fragment without a centromere, which won’t be able to use the spindle machinery to get sorted. Rad Bio: Genetics

  18. Double Hits • A B C * D^ E F^ G H IA B C * D + E F + G H I • Numerous ugly combinations can arise, e.g.E F A B C * D I H G • Acentrics can readily arise Rad Bio: Genetics

  19. Multiple Hits in Replicated Chromosomes • See figs. 13.1 and 13.2 • Major losses of genetic information possible • Balanced translocations: no harm done! • Dicentrics--two centromeres in one pair of chromosomes. Rad Bio: Genetics

  20. SCE: another form of multiple-hit damage • Sister chromatid exchange: exchange of DNA fragments from one chromatid to another between the two chromatids of a single chromosome • Important for chemical mutagens • Not common with radiation Cartoon courtesy madsci.org Rad Bio: Genetics

  21. Double-stranded breaks Rad Bio: Genetics

  22. Recombination + Rad Bio: Genetics

  23. Breakage and Exchange Hypotheses • Breakage hypothesis (Sax, 1940’s onward):Types of aberrations that require one break follow linear dose-response; those requiring two or three follow quadratic or cubic dose-response. • Exchange hypothesis (Revell, 1950’s onward):emphasizes reciprocal exchanges among chromosomal materials: rate  D1.7 • Alpen says the data don’t support this one much. Rad Bio: Genetics

  24. Using FISH to sort this out • Fluorescence in situ hybridization: available before modern genetic tools • Probe segments bind to DNA regions of high homology • Used to sort out differences between breakage hypothesis and exchange hypothesis From Wikipedia Rad Bio: Genetics

  25. Gene Mutations • We’ve discussed these in detail previously • Types: • Deletions (frameshift) • Additions (slightly less likely) (frameshift) • Substitutions (e.g. C for T; no frameshift) • Remember that three bases code for an amino acid! • If we skip a single base, we can throw off every single amino acid that is coded for downstream of the error. • Mutation frequencies are often linear with dose Rad Bio: Genetics

  26. Effect of Frameshift • Suppose the DNA duplex looks like this:5’- A - T - C - G - C - A - A - 3’ (template strand)3’- T - A - G - C - G - T - T - 5’ (coding strand) • Now we delete one base, so that the coding strand is3’- T - A - G - G - T - T - 5’ • Now the amino acids coded for will be different. Rad Bio: Genetics

  27. Frameshifts (continued) • Most common form of (local) DNA damage • Defined as any form of DNA damage that leads to a loss of one base during replication or transcription. • Chemistry • Deletion of bases • Fragmentation of sugar-phosphate backbone • Distortion of base-base hydrogen bonds and other 3-D elements Rad Bio: Genetics

  28. Consequences of frameshifts • Result in complete misreading of remainder of message • Obviously they’re less dangerous near the end of a gene! • Frameshift in an intron near the end of a gene might mean the mRNA will be correct Rad Bio: Genetics

  29. Genes • A gene is a unit within a chromosome (i.e., a stretch of DNA) that gets transcribed and thereby codes for a specific function. • Most genes ultimately code for proteins; but • Some code for transfer RNAs, rRNA, sRNA • Eukaryotic genes often have exons and introns: • Exons are segments of a gene that are ultimately turned into proteins • Introns are segments of a gene that are removed from the message before translation Rad Bio: Genetics

  30. How Introns are Removed 2000 base-pairs of DNA • Suppose we begin with a 2000-base-pair gene. • It will be transcribed to make a 2000-base messenger RNA molecule • That gets chopped up to make up a ~960 base ultimate message • This will produce a 320-amino acid protein (960/3 = 320) transcription 2000 bases of RNA exon exon exon exon exon intron intron intron intron splicing 960 bases translation 320-amino-acidprotein Rad Bio: Genetics

  31. Structural components of genes • Bases • Adenine • Cytosine • Guanine • Thymine • Phosphate-deoxyribose backbone • Double helix enables the replication and transcription processes and offers physical stabilization A C T G Rad Bio: Genetics

  32. Review of DNA backbone O • Sugar-phosphate backbone on each strand has a nitrogenous acid base attached at the 1’ position of the ribose ring • The lack of an -OH group on the 2’ position prevents formation of a reactive intermediate; this makes DNA more stable than RNA • DNA’s double-helical structure also helps its stability. (Base) O P O- N OCH2 O 2’ O O P O- O Next sugar Rad Bio: Genetics

  33. Differences between DNA and RNA • RNA has a hydroxyl at the 2’ position on the ribose ring • RNA is made up of A, C, G, Urather than dA, dC, dG, dT • DNA is replicated using a molecular machine called DNA polymerase • DNA codes for RNA (tRNA, mRNA, rRNA, or sRNA) using a molecular machine called RNA polymerase • DNA is more chemically stable than RNA Stochastic Damage; High-LET

  34. Types of RNA • Our focus on central dogma sometimes makes us forget about RNA types other than mRNA Stochastic Damage; High-LET

  35. RNA hairpins • DNA is almost always double helical;Watson-Crick base pairing contributes to stability • RNA sometimes has complementary bases that allow for bases to pair up around a (hairpin) loop 5’-A-U-G-G-C-C-G-A-A-U-G-C-C-A-U-3’ 5’-A-U-G-G-C-C-G3’-U-A-C-C-G A U A Stochastic Damage; High-LET

  36. Special features • Code is redundant • 43 = 64 codons • Only 20 amino acids plus a few control codes;most amino acids have more than one codon each associated with them(minimum=1, maximum=6). • Middle base is generally conserved; all the codons for any given amino acid generally have the same middle base. • First RNA base may be sloppy Stochastic Damage; High-LET

  37. Genetic code: mRNA version • Determined by hard work in ~1958-1965 • Slight variations in a few organisms Stochastic Damage; High-LET

  38. Errors in Fidelity and Their Consequences • How do chemicals & radiation affect fidelity(rate of mutation)? • Increase likelihood of replication error • Radiation: bond disruption in bases(or sugars) • Direct (radiation breaks bonds in DNA) • Indirect (radiation causes free radicals; free radicals break bonds in DNA) Stochastic Damage; High-LET

  39. Chromosomal Instability • The observation is that cell death continues for several cellular generations after the initial exposure • The explanation is that the chromosome becomes unstable, i.e. it loses some of its ability to remain structurally sound. • This instability is heritable and therefore accounts for some of these long time-scale effects • Little (1990): decreases in cloning efficiency lasts for 20-30 population doubling times in mammalian cells Stochastic Damage; High-LET

  40. Results in intact eukaryotes • Not as easy to study as in culture • Most work historically has been done in Drosophila • Rate of production of lethal mutations is linear with dose • Dose rate is irrelevant • Male gamete’s sensitivity varies greatly with the stage of development at which irradiation occurs • Rate of mutations ~ 1.5*10-8 - 8*10-8 per locus per cGy. • n.b.: a locus is a testable genetic element; it’s typically one gene, although it could be a group of genes controlled by a single promoter Stochastic Damage; High-LET

  41. Results in Mammals • Concern was raised in the 1960’s that we shouldn’t base mammalian exposure risks on Drosophila data • Mouse specific locus test: suggests • 2.2*10-7 mutations/locus/cGy (~5x higher). • Dose-rate matters: fewer observed mutations at lower dose rate. • Difficult to extrapolate these mouse results to humans • Rates around1 - 5*10-7 is typical. Stochastic Damage; High-LET

  42. Repair • Cell has mechanisms to recognize & replace faulty bases before they have a chance to be replicated • Some injury may disrupt a large enough segment of DNA that repair either fails or is error-prone Stochastic Damage; High-LET

  43. High LET Radiation • Review definition of LET:Rate of change of energy with distance along a track • Caveat I: Local Deposition, i.e.,depositions far from track don’t count • Caveat II: LET only associated with charged particles Stochastic Damage; High-LET

  44. LET Equation: Bethe-Block Formulation • If we let: • z* = Effective charge of projectile particle • Z = Atomic number of atoms in medium • A = Atomic weight of atoms in medium • C = sum of electron shell corrections • d/2 = condensed medium correction • b = v/c for particle • I = <ionization potential for absorbing medium> • -dE/dx = [0.307z*2Z/(Ab2)]•{[ln((2m0c2b2)/(1-b2)I)] - b2 - C/Z - d/2} Stochastic Damage; High-LET

  45. What does this mean? • It’s a messy equation, but we can derive several pieces of wisdom from it. • -dE/dx is inversely proportional to 2, so slow particles dump more energy per unit length • -dE/dx is proportional to z*2, so highly charged particles deposit more • Chemistry of target doesn’t matter too much, because Z/A is almost constant • The term inside the curly brackets is a correction Stochastic Damage; High-LET

  46. Where is Energy Deposited? • Most of the energy is deposited just before the particle stops moving • We’re interested in average LET over a region or track Stochastic Damage; High-LET

  47. Depositions with heavy particles • “Bragg peak”: most of the energy is deposited over a narrow linear track. • W.H. Bragg (father of W.L. Bragg) characterized this 4 425 MeVa neutrons in water Relative ionization 1 Range, cm 0 10 Stochastic Damage; High-LET

  48. Calculating Averages of Continuous Variables We wish to calculate the mean of a continuous function f(x) over a range of x from a to b,Then <f(x)>a,b = [∫ab f(x)dx ] / (b-a) f(x) f(x) <f(x)> = (f(a)+f(b))/2 Area under curve = ∫ab f(x)dx x b a b a (a+b)/2 Stochastic Damage; High-LET

  49. Applying this to LET • Average LET over energies:(LETav)S = { ∫E1E2[LET(E)]dE } /(E2-E1) • So for a slowly varying LET function we assume that it is close to linear, i.e.(LETav)S = [(LET)1 + (LET)2] / 2 • These are instances of track segment averages, (LETav)TS;they’re somewhat different from energy averages, for which we average over energy deposited. Stochastic Damage; High-LET

  50. Alternative averages • Energy-averaged LET(see previous slide; energy range from E1 to E2):<LET>E = { ∫E1E2 LET(E) dE } / (E2-E1)where E is the energy that we’re averaging over. • Track-averaged LET(track from position a to position b):<LET>TS = { ∫ab LET(x)dx } / (b-a)where x is the linear dimension through which the energy is being deposited a b Stochastic Damage; High-LET

More Related