Direct comparison between protons and alpha particles of the same LET

Back ground:

High LET m o re effective than low LET…well established in…

 Cell survival

 Mutagenesis

 Chromosome aberrat i ons

Systematic studies of the same particle type with a wide range of LETs are difficult.

 Ranges are short.

 High particle energies require d to reach lo wer LET v a lues.

 Im portant for m e chanistic studi es of biological effects.

RBE vs LET

Barendsen ….cl assic graph

 Widely used to interpret high-LET effects.

 Has influenced selection of radia tion prot ection weighting factors.

 Barendsen used a mi xtur e of x-rays, deuterons and alpha particles.

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[B are ndse n , 1 9 68]

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Other workers have partially re produced the Barendsen data.

 Blakel ey used very high LET p a rticles

 None used protons > 11 keV/µm

 All used a m i x of particles.

e.g. , Bird et al., “Inactivation of Sync hronized Chinese Hamster V79 Cells with Charged Particle Track Seg m ents”, Rad. Res., 82, 277-289, 1980.

It was commonly assum e d that prot ons and alphas of the same LET lie on the same smooth curve .

Yet, track structure differences were predicted (“subt le differences”).

 These comm on assum p tions were challenged by a report from Belli et al.,

“RBE-LET relationship for the survival of V79 cell s irradiated with low energy protons”. Int. J. Radiat. Biol., 55, 93-104, 1989.

 Cells on mylar film

 Protons with LETs ranging from 10.6 – 34.5 keV/µm

 Survival curves showed increasing “linearity”

Dose calculated as D (Gy) = 0.16 (F)(L ) Where F= proton fluence ( µ m -2 ) And L = LET k e V/ µ m

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[Belli, 1989]

Cal culated RBEs rel a tive to x rays were l a rger than RBEs rep o rted in the literature

for alphas of the same LET.

Complications:

Survival curves had “tails” at the highest LET values, the two most critical points . Attributed to cells that received no dose (or a very reduced dose) due to:

 Rounding up, or actual detaching, of the cell from the mem b rane (?)

 Elevat ed cells (?)

 Partial shielding of cells at the edge of the dish (?)

Tested by washing and not harvesting cells near the edge of the dish. Seemed to im prove the data, but not show n….

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Implications:

 Protons have a higher RBE th an alphas at the same LET

 On the RBE vs LET plot, the protons “peak” at ~ 20 keV/µm whereas, the alphas (and everythi ng else) pe aks at about 100-200 keV/µm.

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[Belli, 1989]

This was s u rprising . The differ e nces in tr ack structure are “subtle”. Protons and alpha art i cles in these en ergy ranges are of particul ar interest:

 Neutrons, which are of great concern in radiation protection, produce low energy recoil protons in tissue: in the 10-90 keV/µm range.

 Radionuclide sources used in ther apy and radon produ ce “slow” alpha particles: in the 60-250 keV/µm range.

 Protons are used in t h erapy at t h e Br agg peak. A significant proportion of the dose is delivered by low energy prot ons.

The series of papers by Goodhead et al ., working with Belli, was a thorough study of both proton and alphas by t h e same gr oup usi ng the same endpoint and sam e irradiation facilities. Th e experiment was extended to additional endpoints, m u tation, DSB, to investigate the extent of this phenom enon.

What ARE the differences between 1.2 Me V protons and 30.5 MeV alpha particles?

	1.2 M e V pr oton	30.5 MeV al pha
LET	23 keV/µm	23 keV/µm
Velocity	1.5 x 10 7 m / s	3.8 x 10 7 m/ s
Range in water	30 µm	730 µm
Max E trans. to e -	2.6 keV	16.6 keV
Core radius	0.0006 µm (6 Å)	0.0015 µm (15 Å)
Penum bra radius	0.070 µm	1.76 µm

Objective: direct com p arison of prot ons and alphas in the LET range reported by Belli to have different RBEs

 Variable energy cyclotron

 4 different beam s: protons a nd alpha particles at 20 keV/ µ m and 23 keV/

µ m.

 This is the range in the Belli re port where the LET differences are substantial,

 The particle ranges are s till large enough to not cause serious experim e ntal difficulties.

 Irradiation of cells in “track segment” mode.

Beam path after leaving the vacuum of the accelerator beam line:

 21.1 mm air

 2.5 µ m Hostaphan foil (0.36 mg / c m 2 )

 cells (V7 9 cells ~ 4 µ m - thick)

Aim is to match LET at 3 µ m depth in the cells.

Dosimetry used

 Thin window ionization cham ber

 CR39 track etch det e ctors

 Detectors in exact po sition as the cells

Fairly good agreement between the two dosim etry methods.

(Proton dose at low energy lower in CR39 possible due to poor penetration of the protons into the track et ch detector.)

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Cell Survival Curves

Cells irradiated in a horizontal beam..”dry” briefly.

Cells were cut out on the Hostaphan film , leaving a 2mm edge strip unused .

Survival curves fit with the linear quadratic equation

-ln S = α D + β D 2

Dose = (LET)(track density)

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All cells irradiated as exponentially grow ing m onolayers.

RBE calculation

 B defined as the rati o of the effectiv eness of prot ons/alphas…at the same LET.

 At low doses B = r a tio of the α linear dose coefficien ts.

 B=1.69 and 1.43 for the V79 experim e nts

o Significantly greater than 1

o In agreement with Belli, 1989 (also used V79 cells)

o Belli calcu lated the RBEs th e same way (ratio of α linear dose coefficients)

 RBE also significantly > 1 for HeLa cells, but not the C3H 10T½ cells.

 Cell thick n ess did vary somewhat, but the range of the highest LET proton (1.22 MeV) was still 33 µ m.

Conclusions:

 Protons are more eff ec tive than alpha particle s at the same LET.

 The effect was l a rgest for V79 cells.

 Results support the conclusions of Belli, 1989.

Sugggesti o n…

 Lim ited range m a kes e xperim e nts difficul t.

 Use deuter ons to increase range?

 At the same velocity, deuterons have the s a me LET and track s t ructure as protons, but t w ice t h e range .

Objective: RBE differences observed in cell surviva l experim e nts by these investigat ors between alphas and prot ons of the same LET. Extend these observations to addit ional endpoints.

HPRT Gene Mutation assay: HPRT is hypoxanthine- guanosine phosphoribosyl transferase. This enzyme is respons i b le for putt i ng purine bases on the phospho- ribose structure. A key enzyme in DNA synthesis .

Methods:

 Doses used produce a survival of >10%

 Cells plated in the presence of 6-thi o -guanine, a purine analog.

 6-thi o -guanine is toxic to ce lls if incorporated into DNA.

 HPRT will accept the 6-thioguanine as a substrate and transfer to phosphori bose.

 Only cells that have an inactive HPRT will survive this “selection pressure”.

Mutation is a rare ev ent.

Need an assay to select m u tants from very large num bers of cells. Requires a hit in a specific DNA sequence, the HPRT gene.

Requires the cell to survive.

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Data plotted as #colonies/10 5 cells vs dose.

M = α D

where M = m u tation frequency, α is the slope, D is t h e dose.

The ratio of the slopes us ed to cal culate t h e RBE between alphas and protons directly (no x-rays involved.)

Mutation cross section:

Interpreted as the g e ometrical area of the target tim e s the probability that a track will cause a m u tation.

  0 . 16 L 



σ is the cross section in µ m 2

α is the slope (Gy -1 ) L is LET (keV/ µ m)

 Cross sections are lar g er for protons co mp ared to alp h as by a factor of ~2!!

 I.e., for each “hit” the proton is 2 tim es m o re likely to cause a mut a tion.

 No variation with LET (not surprising, since the dif f erence is only 20-23!!)

RBE for HPRT mutation

 Calculated as the ratio of the slope s in the m u tation induction graphs

 RBEs ar e ~2

 No variation with LET

Mutation frequency vs cell inactivation.

Protons are m o re m u tagenic than alpha particles, at the sam e level of survi v al.

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Relation to previous studies:

 Alpha particles at 90-130 keV/ µ m have 6-9 tim es higher m u tation freque ncy than the current study.

 Protons in the 90-130 keV/ µ m range not studied yet.

 Belli states “underw ay” in 1992. Not in the literature…

 Difficult to get protons to this range of LET

Objective: Extend the study to another im por tant cellular endpoint, DNA damage.

Methods:

 Cells lab e led with [ 3 H]Thd

 Irradiations with x-rays at 4 ˚ C, dose rate 3.8 Gy/m in

 Irradiations with protons or alphas , horizontal beam, cells at room tem p briefly, dose rate = 80-160 Gy/m in.

DSB assays

Sucrose gradient sedi mentation assay .

 Cells lysed at neutral pH.

 Transferred to top of sucrose gradient.

 Centrifuge 3000 rpm / 65 hrs/20 ˚ C.

 Draw fractions from tube botto m through filters that bind DNA.

 Count the filters.

Precipitation assay

 Non-denaturing conditions.

 High salt concentration and cold tem p causes high m o lecular weight DNA to precipitate.

 Centrifuged to separate pe llet from supern atant.

 Am ount of radioactivity remaining in the supernatant proportional to DSBs.

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Results:

 Slopes used to co mpare the eff ectiveness of alphas and prot ons.

 Protons slightly less effective b u t not statistically significant.

 RBEs compared to X rays are ~ 1 for both protons and alphas in sedimentation assay.

 RBEs for protons and alphas are both less than 1 in the precipitation assay

 Neither assay can di s tinguish SSB from DSB

These results are at odds with the results for survival and mutation

Co mplicat ions?

 Very high doses used.

 Dose rates and irradiation times were significantly different.

Perspective 1992:

 Literature reports on RBE values for DSB induction and yields of DSBs/Gy vary consi d erably.

 Neither assay can distinguish com p lex damage or small fragments.