illinois institute of technology physics 561

04/19/23 Survival Curves; Modifiers p. 1 of 96

Illinois Institute of TechnologyPHYSICS 561

Radiation BiophysicsLecture 5:

Survival Curves and Modifiers of Response

Andrew Howard17 June 2014


Looking forward Be alert for changes in the posted

assignments: I may add a few things Midterm will cover chapters 1-7 and the bit of

extra material on free radicals that we discussed last week; therefore, it will not include the material from today’s lecture


Survival Curves

We discussed models for cell survival last time We looked at various ln(S/S0) vs. dose models

and the logic behind them Today we’ll focus on the graphical implications

and how we can look at the numbers Then we’ll talk about cell cycles and other good

solid cell-biology topics. (Warning: I’m more of a biochemist than a cell

biologist, so don’t expect high expertise in this later section!)


Errata in Chapter 8

Page 169, Paragraph 2, 1st sentence:Until the later 1950’s it was not possible to use …

Two sentences later:Bacteroides Bacillus

Fig. 8.1, p. 173:The label that says Dq is pointing at the wrong thing:it should be pointing at the place where the dashed line crosses the (Surviving faction = 1.0) value.

What Fig. 8.1 should have said


Dose, Gy

Su

rviving

Fra

ction A B

Slope = k = 1/D0

Dq = D0lnn = quasi-thresholdn


Shoulder of the Survival Curve

We recognize that with MTSH dose-response we have a region where the slope is close to zero. We describe that region as a shoulder:


Slopes in the MTSH model Remember that the MTSH model says

ln(S/S0) = ln(1-(1-exp(-D/D0))n) Because S/S0 = 1-(1-exp(-D/D0))n

So what is the slope of the S/S0 vs. D curve? … and … what is the slope of the ln(S/S0) vs. D curve? In particular, what is the slope’s behavior at low dose? Answer: calculate dS/S0/ dD and d(ln(S/S0))/dD and

investigate their behavior at or near D = 0. Note: we’re looking here at the n>1 case.


Slope investigation, part I

For S/S0 itself,d(S/S0)/dD = d/dD(1-(1-exp(-D/D0))n)

The 1 out front doesn’t affect the derivative: d(S/S0)/dD = -d/dD(1-exp(-D/D0))n)

We’ll do the rest of this calculation now based on the general formulas

– dun/dx = nun-1du/dx– deu/dx = eudu/dx


Arithmetic & Calculusof Survival Models

MTSH says S/S0 = 1 - (1-e -D/D0)n

What I want to investigate is the slope at low dose, I.e. for D << D0,

And at high dose, I.e. for D >> D0. But are we interested in the slope of S/S0 vs. D

or ln(S/S0) vs. D? Both! Slope = derivative with respect to D. So Slope = d/dD(1 - (1-e -D/D0)n) = -d/dD(1-e -D/D0)n


MTSH slope, continued Recalling that dun/dx = nun-1du/dx, for n>1, Slope = -n(1-e -D/D0)n-1 d/dD (1-e -D/D0)

= -n(1-e -D/D0)n-1 [-d/dD(e -D/D0 )] But we know deu/dx = eu du/dx,

so d/dD(e -D/D0 ) = e -D/D0 (-1/D0) = -1/D0 e -D/D0

Therefore Slope = -n(1-e -D/D0)n-1 (-)(-1/D0) e -D/D0

i.e. Slope = (-n/D0)(1-e -D/D0)n-1e -D/D0


Slope at D << D0

This formula for the slope is valid for all values of D,including D << D0 and D >> D0

For small D, i.e. for D << D0,Slope is -n/D0 (1-e -0/D0)n-1e -0/D0

= (-n/D0) (1-e0)n-1e-0; for n > 1 this is= (-n/D0)(1-1)n-11 = 0. Shazam.


Slope of ln(S/S0) vs D

The behavior of the slope of the ln(S/S0) vs D curve is not much harder to determine.

Recall d lnu /dx = (1/u)du/dx. We apply this here: d ln(S/S0) / dD = (1/(S/S0)) d(S/S0)/dD.

For very small D, S/S0 = 1,so d ln(S/S0) / dD = (1/1) * d(S/S0)/dD = d(S/S0)/dD .

But we’ve just shown that that derivative is zero, so d ln(S/S0) / dD = 0.


High-dose case

We’ve covered the low-dose case. What happens at high dose, i.e. D >> D0? What we’d like to show is that the slope of

lnS/S0 vs. D is -1/D0. Let’s see if we can do that. Slope = d ln(S/S0) / dD = (1/(S/S0) d/dD(S/S0) Thus slope =

(1 - (1-e -D/D0)n-1)d/dD(1 - (1-e -D/D0)n) For D >> D0, D/D0 is large and

-D/D0 is a large negative number;therefore e-D/D0 is close to zero.


High-dose case, continued

Slope = limD∞

{(1 - (1-e -D/D0)n)-1(-n/D0)(1-0)n-1e-D/D0)} That’s messy because the denominator and the

numerator both go to zero. There are ways to do that using L’Hôpital’s rule, but there are simpler ways that don’t involve limits.

The trick is to recognize that we can do a binomial expansion

I’ve done that in the HTML notes The result will be slope = -1/D0 and ln n is the Y

intercept of the extrapolated curve


What constitutes a high dose here?

The only scaling of the dose that occurs in the formula is the value of D0, so we would expect that we are in that high-dose regime provided that D >> D0.

In practice the approximation that the slope is -1/D0 is valid if D > 5 D0.


Linear-Quadratic Model This is simpler. ln(S/S0) = D + D2

Therefore slope = d/dD (ln(S/S0)) = d/dD(D + D2) Thus slope = + 2D. That’s a pretty simple form. At low dose, || >> 2||D, so slope = . At high dose (what does that mean?) || << 2||D,

so slope = 2D. What constitutes a high dose?

Well, it’s a dose at which || << 2||D, so D >> | / (2| Thus if dose >> | / 2|, then slope = 2D.


Implications of this model

At low dose slope = is independent of dose but is nonzero; thus ln(S/S0) is roughly linear with dose.

At high dose slope = 2D,i.e. it’s roughly quadratic.

How can we represent this easily?We discussed this last time:ln(S/S0) / D = + D,

so by plotting ln(S/S0) / D versus D, we can get a simple linear relationship.


Y-intercept =

Slope =

ln(S/S0)/DD

LQ: Plot of ln(S/S0) / D versus D

By observation, both and are negative.< 0 tells us that radiation harms cells; < 0 is an observed fact. Thus the intercept is below the axis and the slope is negative.


LQ graphical analysis: one step further

We can read -/ directly off an extrapolation of the plot: at D = Dz,ln(S/S0)/D = 0, + Dz = 0, = -Dz,

Dz = - / .Note < 0, < 0,so -/ < 0.

Y-intercept = Slope =

ln(S/S0)/DD

Dz


Where do linear and quadratic responses become equal?

At what dose does the linear response equal the quadratic response?

At that dose, D = D2, D = / So the value we read off the X-intercept of

the previous curve is simply the opposite of the dose value at which the two influences are equal.

We mentioned this last time, but we’re reminding you now


How plausible is all this?

Model studies suggest reasons to think that ln(S/S0) = D + D2 is a good approach.

Much experimental data are consistent with the model

Some of these LQ approaches allow for time-dependence to be built in.


LQ vs MTSH and thresholds

How does the question of comparing the LQ model to the MTSH model relate to the question of threshold doses?

A typical real-world question is a dose-response relationship for which the only reliable experimental results are obtained at high doses; at lower doses the confounding variables render the experiments uninformative.

Dose

Response


Dose-Response in Epidemiology

#2 is the linear non-threshold (LNT) model

Is #3 more realistic? This has regulatory

consequences!

Human health effect

Baseline

Dose

Experimental data

#3

#2

#1

Threshold

Tolerable dose #3

Tolerable dose #2Tolerable

dose #1

Limit of reliable measurementExtrapolations


Studying repair

We’ve been suggesting that LQ models and even some MTSH models are dependent on the idea that some DNA damage can be repaired accurately.

Let’s look for approaches to studying DNA damage that might provide a fuller understanding of the effects of repair.


The Elkind-Sutton Experiment

Provides a way of probing repair functions in cells Procedure:

– Irradiate and establish survival curve(“conditioning dose”)

– Take cells surviving at S=0.1 and subject them to further irradiation at varying time intervals after reaching S=0.1

If repair is taking place, then the appearance of a curve similar to the original shoulder is indicative of full recovery


Results


Interpretation

If slope and implied n value are equivalent to the original curve, then repair is complete

Smaller n values indicate insufficient time has elapsed

n=1 implies repair has not begun


Elkind-Sutton and LET

We might expect more complicated results if we vary the LET for the two dosing regimens

Low-LET first, high-LET second gives two lines of different slope, independent of the time interval

High-LET first, low-LET second gives line followed by usual Elkind-Sutton distribution


Low-LET followed by High-LET


High-LET, then Low-LET


The Cell Cycle Cells have a definite cycle over which specific activities occur. Particular activities are limited to specific parts of the cycle Howard and Pelc (1953) characterized four specific phases:

– M (mitosis, i.e cell division)– G1 (growth prior to DNA replication)– S (synthesis, i.e DNA replication)– G2 (preparation for mitosis)

Mitosis(M)

Presynthetic(G1)

Synthesis(S)

Post-synthetic(G2)


What happens in S phase?

DNA is replicated; thus, we have twice as much DNA at the end of S as at the beginning.

During mitosis the two duplexes of DNA can separate One goes to one daughter cell, the other to the other


How much time do these segments take?

Depends on the overall mitotic rate and the type of cell:Cell Cycle Times in hours:Segment CHO HeLaM 1 1G1 1 11S 6 8G2 3 4------------------------------------------TOTAL 11 24


Pie charts of cycle percentages

The point is that different kinds of cells spend differing amounts of time in the various phases


Phase Sensitivity

Many cells are much more sensitive to radiation in some parts of the cell cycle than they are in others.

Why?– Repair is more vigorous in some stages– Unrepaired damage has more opportunity to

manifest itself as clonal alteration close to mitosis– Access of repair enzymes to damaged DNA is

sometimes influenced by how organized the DNA is.


What phases are sensitive? In general, cells are radioresistant when they are

synthesizing DNA. Cells that are synthesizing DNA are taking up label;

the time that they’re doing that is correlated with survival:

% o

f la

bele

d ce

lls

Sur

vivi

ng F

ract

ion% of labeled cells

Surviving Fraction

0.490

Time in Hours0 24


Survival Curves in Various Phases

See fig. 8.8:– Late S is least radiosensitive– Early S next least– G-1 somewhat sensitive– G-2 and M most radiosensitive

M and G2 curves are essentially straight lines (log-linear dose-response), suggesting that repair is unavailable or of little influence


Figure 8.8, reimagined with LQ model


Radiation-InducedCell Progression Delay

Note that various biochemical signals regulate progression from one phase of the cycle to another.

To study this, you need synchronized cells . . . Sample study (Leeper, 1973):

– CHO cells exposed to 1.5 Gy in mid-G1 experienced a delay of 0.5 h in cell division

– 1.5 Gy in late S or early G2 caused a delay of 2-3 h– Dose-dependent: (4h for 3Gy, 6-7h for 6 Gy)


Shape of the culture matters! How the cells grow influences how much the cells’

progression is altered by radiation Monolayers’ progression is altered less than cells

in a multicellular spheroid geometry

0.22 hours per Gray

0.53 hours per Gray


Is that such a big deal?

Probably not: The cells in the spheroidal mass divide

half as fast even in the absence of radiation, possibly due to contact inhibition.

Therefore it may simply be that the whole mitotic clock has been slowed down, including the clock as it’s been influenced by radiation.


Causes for these effects

Why are cells more radiosensitive in M and G2?– Reduced availability of repair enzymes– Repackaged DNA is hard to repair

How is cell progression influenced by radiation?– Damage to protein kinases and cyclins involved

in cellular checkpoints– Premature degradation of p21, maybe…– Sample 1994 study:

Edgar et al, Genes Dev. 440: 52 (1994)


Effectors of Radiation Sensitivity

Biological– Cells go through life cycles & are

much more sensitive to radiation damage at some stages than at others

Chemical Physical


Assignment related to amino acids

1. There are exactly twenty amino acids that serve as the building blocks for proteins in almost all organisms. The general formula for 19 of these 20 amino acids is +NH3-CHR-COO-, where R is any of 19 different side-chain groups. The simplest of these R groups is H, for which the amino acid is called glycine; the most complicated is a moiety known as indole, for which the amino acid is called tryptophan.


Upcoming Problem 1, cont’d

Much of the chemistry that these R groups participate in in proteins is ionic in nature, involving charges or partial charges; but usually these ionic interactions involve pairs of electrons rather than unpaired electrons. An exception is the amino acid tyrosine, which can participate in free-radical (unpaired-electron) interactions. Draw a structure of the tyrosyl free radical and explain why it might have a reasonably long lifetime, as compared to a hydroxyl radical or some other short-lived free radical.


Second upcoming problem 2. Most biochemical oxidation-reduction reactions involve

transfers of pairs of electrons and therefore do not involve free radical mechanisms. A sizeable minority, however, do involve free radicals. Which of the following biochemical oxidizing or reducing agents are capable of participating in single-electron (free-radical) reactions, and which are not? Explain briefly. You may need to look up the structures of some of these compounds.

(a) ferric iron, Fe3+

(b) nicotinamide adenine dinucleotide, oxidized form (NAD) (c) flavonamide mononucleotide (FMN)

(also spelled flavin amide mononucleotide; it’s an instance of what are generally known as flavin prosthetic groups)


Errata

Page 205, in the EXAMPLE:4.0 µM l-1 should be 4.0 µM, or 4.0 µmol l-1

Page 206, last sentence:If the lesions produced by high LET radiation are predominantly of type II (irrepairable), then m-1 will be disappearingly small and no oxygen sensitization will be detectable.

Page 213, last paragraph: cysteine, not crysteine


Cellular Life Cycles (review)Phases

Mitotic M (short)– Sensitive

Presynthetic G1 (variable)– Radiation causes 1/2 h delay here

Synthetic S (4 - 8 h)– DNA synthesis– Least sensitive

Postsynthetic G2 (usually short 1 - 2h)– Radiation causes 3 - 4 h delay here– End of G2 sensitive

__________Overall Process 14 h


What happens in G1?

Routine cellular metabolism Both buildup of new cellular structures and gathering

energy to do so, i.e. both catabolism and anabolism:

Metabolism as a whole consists of: Catabolism Anabolism Energy-producing Energy-requiring Breakdown of complex Build-up of complex

molecules into simpler molecules from simplerones, producing ATP precursors, using ATP


A mitotic cell

Nucleus

(DNA location;

cellular organization)

Mitochondrion(energy metabolism)

Ribosomes(protein synthesis)

Mitotic Spindle

Other organelles


Timescales of damage (fig. 4.1)Physics (~10-16 s)

– Primary interaction event with biomolecule or H2O

– Excitations & ionizationsFast Chemistry (10-15 - 10-7 s)

– Water radicals and other quasi-stable species form

– Reactive species diffuse and cause damage

Slower chemistry(10-7 to 10-3 s)

– Further damage via diffusion

– Chemical restitution & repair

Biochemistry (msec-min):enzymatic repair of damage

Biology (hrs-days):biological repopulation from surviving cells


Physical & Chemical Effectors

Water free radicals

h + H2O •OH, •H

•O2-, etc

ionized species

Pure water


How does water matter?

In a dry setting, the damage must be direct In a wet environment, water-derived free

radicals and ions are the source of much of the chemical damage

Experiments that can eliminate secondary (radical-mediated) damage show much reduced radiosensitivity


An example from my field

Protein crystals must be irradiated wet because they fall apart when they’re dry.

Protein crystals irradiated at 300K are destroyed:

– In days on a conventional X-ray source– In minutes on a 2nd-generation synchrotron– In seconds on a 3rd-generation synchrotron

They’re essentially immortal at 110K except on a 3rd-generation synchrotron source, where they live for 5-100 minutes


Radiosensitivity of Enzymes Most enzymes are more

radiosensitive in the presence of substantial hydrations than when dried

Data can’t readily be measured below ~4% hydration

But those aren’t very physical anyway (except maybe with integral membrane proteins)


Why does this work this way?

Normal response– Arylesterase: less water means less damage– Dry conditions mean that radiosensitivity

depends entirely on direct, not indirect damage Abnormal response: Cholinesterase

– harder to explain– Augustinsson suggests: at low [H2O], free

radicals are detoxified by nonfunctional sulfhydryls in the protein

– At higher water concentrations the sulfhydryls can’t get to the damage in time


Do we care?

Not much:– We can’t really alter the hydration of most cell

systems without destroying them– We definitely can’t influence the hydration states

of whole organisms without doing damage that is probably much more significant than that of the radiation

But these studies may help us understand mechanisms of damage, and that could be relevant


Do we care, vol. II DNA and RNA are heavily hydrated because they’re

charged and because they tend to associate directly with solvent or with soluble proteins like histones

Proteins vary a lot in their hydration:– Soluble proteins are heavily hydrated– Integral membrane proteins have very little water

around them Is there a difference in radiosensitivity?

– Not much research that I could find– One instance: Takáts et al. (1993) J.Rad.Res. 34:141


Temperature-sensitivity

Direct damage will be essentially temperature-independent

Indirect damage should be temperature dependent because it relies on diffusion of radicals and ions from the site of their production to the macromolecule

Ionizations and excitations may display different temperature dependencies because once the molecule is excited, its chemistry may depend on thermal interactions


Arrhenius plots

We can examine temperature dependence via the Arrhenius plot, wherein we expect k = Qexp(-G‡/RT)

Thus ln k = ln[Qexp(-G‡/RT)] = lnQ - G‡/RT, so if we plot ln(k) as a function of 1/T then the relationship should be linear, and the slope will be -G‡/R, i.e it will be proportional to the activation energy G‡.

If multiple processes are involved we may get a non-log-linear response.

Over temperature ranges typical of the internals of homeothermic organisms, we’re not going to see much effect!


Physical meaning of G‡

Chemical reactions are characterized by an activation energy barrier, i.e., an energy that must be put into the system in order to get from reactant to product or vice versa.

G‡ is the height of that activation barrier.

Energy

Reaction coordinate

ReactantProduct

G‡


Radiation & Temperature

Kinetics

37oC = 310K27oC = 300K

p199 T < 100K 100 < T < 170 K 170 < T < 420 K


Radiation and Temperature Effects in various temperature ranges:

Tmin Tmax Effects

0K 100K temperature-insensitive;no charge migration, target must be

hit.

100K 170K excitation localized at site;exciton migration is crucial

170K 420K disruption of disulfides, ionization Does this matter much with biological systems (esp. in homeotherms), where T ~ 310K almost all the time?Probably not, other than for understanding mechanisms.


Oxygen and Radiation

FACT: O2 is a radiation-sensitive moleculee.g. O2 + e- O2

-• (superoxide) interactions with macromolecules

It’s tempting to think that all biology occurs in the presence of 19-21% O2, but it doesn’t!

H2O free-radical chemistry in the presence of O2 is different from H2O free radical chemistry in the absence of O2.(recall Fricke dosimetry story)

P(O2) in tissue varies widely– Hemoglobin transports O2

– Myoglobin stores O2


Damage Fixation by Oxygen

R• + O2 RO2•

semi stable10-7 - 10-3 sec?

________________________________________

Mitigators of O2 fixationradical presence

R• + R´SH R-H + R´-S•R´-S• + R´-S• R´-S-S-R´ (dimerization)


Minor syntactic point

Beware of the word fixation It doesn’t mean correction:

it refers to stabilization Stabilizing a radical doesn’t make it less

dangerous;it makes it more dangerous!

So if we say that oxygen is involved in fixation of damage, we mean that it makes the damage worse, not better!


Experiments on Oxygen Sensitivity

More cells are killed in air than in N2:

Survival in the absence of oxygen

Survival in the presence of oxygen


LQ: oxic vs. anoxic


Radiosensitivity as function of P(O2)

Shigella experiments show threshold in P(O2) below which few cells are killed

Alpen, fig. 9.3


Oxygen Enhancement Ratio

OER [dose in N2 for surviving fraction S/So] /

_____________________________

dose in O2 for surviving fraction S/So

if S = So/10,then OER = DN / DO

This definition issomewhatarbitrary,but it works!


Quantitative Oxygen Sensitivity

Paul Howard-Flanders and Tikvah Alper:Define “S/SN” to be the ratio of the 10% survival dose under experimental conditions to the 10% survival dose in N2, i.e. in the absence of oxygen

“S/SN” = (m[O2] + K) / ([O2] + K)

– for any concentration of oxygen.– m and K are separately determined for each

system– m is dimensionless, K is a concentration– m represents maximum relative sensitivity

so m 1.

04/19/23 Response Modifiers; Tumors p. 72 of 96

Extrema of “S/SN”

At [O2] = 0, S/SN = (m*0 + K) / (0 + K) = 1

For [O2] >> K,S/SN = (m[O2] + K) / ([O2] + K) = m[O2] / [O2] = m

Thus justifying description of m as maximum relative sensitivity


How to compute m and K

m is available from asymptotic behavior– It’s equal to S/SN for [O2] >> K.

– In practice most systems are equally radiosensitive from about 1% [O2] on up

To compute K, note that if [O2] = K, thenSK/SN = (mK + K) / (K+K) = (m+1)/2

Therefore if we know m from asymptotic behavior,we can examine a curve like 9.3 to find the point where S/SN = (m+1)/2 and read off K from the abscissa


Howard-Flanders Coefficients Coefficients m and K for various organisms:Organism m K, µMShigella flexneri Y6R 2.9 4.0

Escherichia coli B/r 3.1 4.7

Saccharomyces cerevisiae 2.4 5.8

Can be read off curves like fig. 9.3


Curve-fits for Organismal Data These are idealizations, of course! Note that 11.3 µM = 1%


Why does this happen?

Alper’s model:2 types of damage from primary radiation event

– Type I: lesion requires oxygen for lethality– Type II: always lethal, independent of oxygen

Thus: type I can be chemically restituted Restitution competes with oxygen fixation Posit: n1 type I lesions, n2 type II lesions,

krep = rate of repair, kfix = rate of O2 fixation, then:

m-1 = n1/n2 and K = krep/kfix


What happens with high-LET?

Essentially all damage is type 2 Nothing depends on restitution Therefore n1/n2 << 1, m-1=0, m=1

Thus S/SN = ([O2] + K) / ([O2] + K) = 1,independent of K.

The fact that high-LET produces this behavior is mildly supportive of the appropriateness of Alper’s model, although it’s far from conclusive


Time-Dependence Study the kinetics via short bursts of radiation We look to see whether providing O2 at a certain

time-point before or after irradiation sensitizes cells– Oxygen sensitizes the cell if present before

irradiation– If available till ~3 msec after irradiation, it still matters– If oxygen is made available later than a few msec

post-irradiation, it doesn’t sensitize the cell This suggests that the oxygen-dependent free

radicals have lifetimes shorter than 3 msec.


Michael’s results with Serratia

Time constant ~ 0.5 msec for sensitization

Dose: 280 Gy of electrons


What radicals are involved? The short time-span shown in this experiment suggests

that free radicals are involved:but which ones?

Michael suggests that superoxide (O2-•), hydroperoxyl

(H-O=O•), and eaq- have shorter lifetimes than is

consistent with 0.5 msec timing Other researchers continue to plug for superoxide and

hydroperoxyl as candidates OH• doesn’t interact with O2 that much except via CO2•-:

OH• + HCO2- -> CO2•- + H2O

CO2•- + O2 -> CO2 + O2-•


Superoxide We’re still not sure if superoxide is

a major actor in oxygen-mediated damage But this is how superoxide is detoxified

by superoxide dismutase (SOD):– 2 O2•- + 2H+ H2O2 + O2 (catalyzed by SOD)

– H2O2 H2O + (1/2)O2 (catalyzed by catalase)

Exogenous SOD doesn’t help alleviate damage(but maybe that’s because it isn’t tied to catalase, so peroxide can build up to toxic levels?)

Manipulating constitutive levels of SOD by genetic means gives muddy results

Human Cu-Zn SOD


Thiol Mitigators

We expect that oxygen competes with thiols, such that the more thiol mitigation is involved, the less oxygen-dependent fixation of damage can occur:

1. [macro]-R• + R´SH [macro]-R-H + R´S•

2R´S• R´-S-S-R´

2. [small]• + R´SH [small]-H + R´S•

2R´S• R´-S-S-R´


Glutathione as mediator

Glutathione can readily dimerize under oxidizing conditions:R-SH + HS-R R-S-S-H

Reasonably prevalent in cells(millimolar: [glut] >> [cys])

In principle cysteine could also operate this way, but its cellular concentration is too low

Cysteine

glutathione


Are thiols really important?

Some argument about that Revesz’s results support the competition model Maybe only exogenous thiols really influence

radiosensitivity Introducing cysteine or synthetic thiols does definitely

mitigate damage:RSH + OH• RS• + H2ORS- + OH• RS• + OH-

. . . And then these RS• radicals recombine as disulfides


Dose reduction factors

We envision the effects of these exogenous thiols as involving dose reduction

The quantitation is equivalent to reducing the available dose to influence the biological system

Example, WR2721 or amifostine:– Works well in vitro– Limited utility in vivo– Fairly high toxicity


Sensitization by Nitroaromatics How do we make the cells in the rapidly growing tumor

as rad-sensitive as they would be if PO2 were higher?

Nitroaromatics react with radicals to “fix” (stabilize) the damage

metronidazole

misonidazole


Nitroaromatic action

Like oxygen, nitroaromatics react with short-lived radicals to produce longer-lived and therefore more reactive radicals

Concentrations required to enhance radiosensitivity are much higher than with O2

These compounds have other applications, but they can be used therapeutically as potentiators of radiation damage


Oxygen vs. Mizonidazole (fig. 9.5)

oxygen misonidazole


5-Halogen-substituted pyrimidines

These are molecules that resemble thymine The halogen at the 5- position looks like the

methyl group in thymine and can be incorporated in place of thymine in DNA

Most common: 5-bromodeoxyuridine Sensitization produced by ready reaction with

the aqueous electron


The 5-BrdU radical Semi-stable radical is actually resident in the DNA

and can influence chemistry in neighboring bases Other mechanisms are probably acting too.

N

HN

R

O

Br

+ eaq-

N

HN

R

O

Br

•-

N

HN

R

O

+ Br-

•


Cell Death Clonogenic cell death:

inability to produce several generations’ worth of progeny

Acute pathological cell death: necrosis– Cells typically swell, then lyse– Accompanied by inflammation

Apoptosis– Programmed cell death– Shrinkage, fragmentation, phagocytosis– p53 is activator of genes that regulate it


Gilbert & Lajtha’s cell types

A B C

D E F

See next slide for explanation!


Cell populations in TissueA. Simple transit population

Cells in, cells out Spermatozoa, blood cells

B. Decaying population(e.g. oocytes)C. Closed, static population (neurons?)

D. Dividing, transit population– Some cell division,

so more leave than enter– Differentiating blood cells

E. Stem cell population(many kinds)F. Closed,dividing population

– No cells in or out—just a lot of division

– Tumors, eye-lensepithelial cells


Cell population kinetics

Cell types that divide are the most sensitive. Cells are most sensitive during G2 and M,

so cells that spend a lot of timein G2 and M are more sensitive

If a cell population is exposed to radiation, the outcome depends on there being an adequate number of (clonogenically) surviving cells.


Growth Fraction Lajtha (1963): described “G0” phase in cell cycle:

cell is not engaged in proliferation but could later re-enter proliferative stage

Growth fraction is defined as fraction of total cellular population that is clonogenically competent and actually in the process of DNA replication and cell division.

Measurement: uses 3H-thymidine uptake Significance: cells in G0 have time to repair DNA damage

– Works even if [repair enzymes] is low during G0– This is suspected but not proven


The expanded cell cycle (Lajtha)

G0 is seen as an alternative to normal cycling Cells may re-enter the cycle after a change in

environmental conditions or upon receiving a signal

M

G1

S

G2

G0

illinois institute of technology physics 561

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