Illinois Institute of Technology

04/20/23 Stochastic Damage; High-LET p. 1 of 75

Illinois Institute of Technology

Physics 561 Radiation Biophysics

Lecture 9:Cancer, Genetics, and

High LET radiation30 June 2014

Andrew Howard


Plan for this lecture 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

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.


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 Research 89:187-196. Löfroth et al(1980) Science 209:1037-1039 and

Mermelstein et al (1980) Science 209:1039-1043. So: all’s well that ends well!


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.


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


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


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)


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)


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:


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



Macroscopic Damage to Chromosomes

First half of chapter 13 Structural changes in chromosomes

– Inversions of fragments– Multiple hits– Isochromatid breaks– Dicentrics– Minutes– Cross over

04/20/23 Rad Bio: Genetics p. 13 of 75

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


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


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


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


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.


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


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.


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


Double-stranded breaks


Recombination

+


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.


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


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


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.


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


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


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


How Introns are Removed 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)

2000 base-pairs of DNA

transcription2000 bases of RNA

exon exon exon exon exonintron intron intron intron

960 bases

320-amino-acidprotein

translation

splicing


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

G T


Review of DNA backbone

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.

ON

OCH2

PO O-

O

PO O-

O

ONext sugar

(Base)

2’


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, U

rather 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


Types of RNA

Our focus on central dogma sometimes makes us forget about RNA types other than mRNA

Type # bases % of synthesis

% of RNA

DS? Role in translation

mRNA 150-104 25 3 no Protein template

tRNA 55-91 21 15 part Amino acid activation

rRNA 100-104 50 80 part Scaffolding, catalysis

sRNA 15-1000 4 2 hybrid various


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

U

A

A


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


Genetic code: mRNA version

Determined by hard work in ~1958-1965

Slight variations in a few organisms


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)


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


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


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.


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


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


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– /2 = condensed medium correction– = v/c for particle– I = <ionization potential for absorbing medium>

-dE/dx = [0.307z*2Z/(A2)]•{[ln((2m0c22)/(1-2)I)] - 2 - C/Z - /2}


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


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


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

Rel

ativ

e io

niza

tion

Range, cm0 10

1

4

425 MeVa neutrons in water


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 = [∫a

b f(x)dx ] / (b-a)

f(x)

xa b

f(x)

a b

Area under curve =

∫ab f(x)dx

<f(x)> = (f(a)+f(b))/2

(a+b)/2


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.


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


Average LET, keV µm-1

Table 14.1:

Radiation type (LETav)TS (LETav)E

60Co -rays 0.27 19.6250 kVp x-rays 2.6 25.83 MeV neutrons 31 44Radon rays 118 8314 MeV neutrons 11.8 125Recoil protons 8.5 25Heavy recoils 142 362•Looks like the (LETav)TS is more in accord with our intuitive notion of what constitutes LET!


Direct & Indirect Effects

High LET means that many ionizations occur in a small neighborhood LET Spur energy Events/µm Spacing KeV/µm eV nm

60Co 0.25 60 4 250Radon 118 60 2000 0.5

This fact by itself accounts for much of the difference in biological consequence of high LET radiation


Biological Effects of High-LET Radiation

RBE = relative biological effectiveness analogous to OER in its definitional form:

RBE = (dose for given end point for reference radiation) /(dose for given end point for test radiation)

Problem with this formulation:– assumes that dose-response curves are described

by identical functions, i.e.– RBE is independent of the response level at which

it is estimated. Often not true!


MTSH comparisons Cf. fig. 14.3: Xrays and fast neutrons

Results from hamster fibroblasts


Can we define a single RBE?

Not really, unless our survival curves are really just rescalings of one another (“dose-modifying effect”)

For MTSH data RBE will be close to constant if the extrapolation numbers aren’t too high

For LQ we’re definitely going to have to pick a survival ratio


Survival Level Matters!


LQ case

RBE varies significantly between S=0.1 and S=0.001:


Relationship between RBE and LET

RBE vs LET: Generally higher LET radiations have higher RBE, up to a certain

point. With some systems the curve turns over.

LET, keV/µm

RB

E

100 101 102 103

Two hamster cell lines (from fig.14.5)

1

23

45


Cell-Cycle Dependence for RBE

Low LET radiations exert much more effect in late G2 & M than elsewhere

High LET radiation exerts approximately equal effects throughout the cell cycle

Reason: irreparable damage early in the cycle will persist through mitosis, so it’s just as bad!


OER vs LET

OER for High-LET Radiation is generally smaller than for low-LET radiation because the damage is less dependent on oxygen fixation of radical species.

This plot shows OER going not to 1 but to ~1.5--because water-derived radicals are still produced at high LET; cf. Fig. 14.6.

OE

R

LET, keV/µm1 100010 100

1

2

1.5


LET vs Fractionation

Recall that repair-competent systems respond less to fractionated doses than to single doses whereas repair-deficient systems are fractionation-independent

For high-LET radiation, fractionation does not decrease biological effects


Contrarian results for fractionation Sometimes in tumors fractionation increases the

undesirable biological effect, i.e. more healthy cells die per 100 tumor cells killed!

– Why? - no explanation in Alpen– 2-stage model for cancer: (Ullrich)– Also: high dose rate (equivalent to fewer fractions)

is more likely to simply kill the cell rather than producing clonogenically competent but mutated cells(remember the concept of correcting for mortality in epidemiology)


Late Effects of High LET Radiation

You can induce cancer with neutrons Ignores fractionation or (!) damage is

more likely with fractionation Tumors also arise from heavy-ion

irradiation


LET vs. Cross Section Alpen looked at particle fluence as a dose parameter--

requires constant LET over volume Result: power-law relationship between cross section for

tumor induction and LET (fig.14.7)

Cro

ss S

ect

ion

, µm

2

LET, keV/µm0.1 1 10 100

.1

10

0.001


General note re linearity

Ordinary linear relationship:– Both axes on linear scales.– y = ax + b,– a = slope, b = Y intercept

Semi-log linear relationship:– X linear scale, Y logarithmic– ln y = ax + b– exp(ln y) = exp(ax+b)– y = exp(b) exp(ax) = keax

– for k = exp(b)

x

y

b

x

ln y

b


Third possibility: power law

Here we have linearityon a log-log plot:

ln(y) = alnx + b exp(ln(y)) = exp(alnx)exp(b) y = k exp(a ln x) for k=exp(b) But aln x = ln(xa), so y = kexp(ln(xa)) = k xa

So this is a power law.

ln x

ln y

b


Probability of TraversalsTable 14.2 shows that multiple traversals of the nucleus are very rare even at high dosesIt also shows a close correspondence between cross section for tumor production and cross section for traversalDose, <fluence/ Probability of traversalsGy cell> 0 1 2 >= 10.01 0.006 0.993 0.006 2.1*10-5 0.0060.02 0.013 0.987 0.013 6.8*10-5 0.0130.05 0.032 0.968 0.031 5.0*10-4 0.0320.15 0.097 0.907 0.088 4.0*10-3 0.0920.20 0.129 0.878 0.114 7.0*10-3 0.1210.30 0.193 0.823 0.159 1.5*10-2 0.1760.40 0.258 0.772 0.199 2.5*10-2 0.227


Number of Traversals


Cell Transformation in High-LET Irradiation

Assertion: non-local effects on DNA predominate over point mutation events

Certain assay systems suggest this assertion: Kronenberg et al, human TK6 lymphoblasts--

entire hprt gene is missing after irradiation This gene codes for hypoxanthine phosphoribosyl transferase,

an important enzyme in DNA synthesis “If large genomic changes are brought about by heavy ion

radiation, then a multistep process may be short- circuited to a single event.”


Cataracts

The vertebrate eye is a remarkable organ

All the living components must be completely transparent for maximum fidelity of light transmission.

Even the lens is made up of living cells!

A loss of transparency is a cataract,either evanescent or permanent.

A variety of insults can give rise to a loss of transparency.


Cataracts

Cataracts are common with high-LET radiation Reminder: cataracts involve loss of transparency in the

lens because problems with differentiation will lead to failure of alignment of the fibers

Cataracts happened with workers in early accelerator facilities

RBE values are high, and tend to be higher for lower doses (i.e. low doses cause almost as many cataracts as higher doses)


Cataracts from high-LET radiation

It’s long been known that high-LET radiation can give rise to cataracts, even at low doses.

Relative biological effectiveness of high-LET radiation (neutrons) is 30 or more for low doses

RBE goes down for higher doses Beams of ions (Z > 20) produce similar

effects, again including lowered RBE for higher doses.


Cataracts from specific types of radiation

Neutrons:– In animals: cause cataracts with RBE~2 to 100– But with humans: cataracts become rare up to 2 Gy,

almost universal at D > 11 Gy. Argon and iron ions: RBE ~12-40 for low doses (below

0.25 Gy), more like 2-5 for higher doses Why does the RBE depend on dose?

– Can’t kill a cell more than once?– Mechanistic explanations (see Ullrich discussion)?


The Ullrich Tumor Experiments

Ullrich found that with low doses of neutrons, fractionation never diminished incidence of tumors

With certain types (lung, mammary) there was an enhancement in tumor rate with fractionation

Dose-response was nonlinear Saturation and fall-off of incidence with high

doses


Ullrich Experiment: Why? Mechanisms of Genetic Damage Low vs High LET

– Low-LET radiation exerts many of its effects at the level of point mutations(single-base substitutions, deletions, or additions)

– High-LET exerts most of its effects on a more macroscopic scale

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