Alpha particles: experimental work

The realization that radon is a significant dose com ponent for t h e general population led to a greatly increased inte rest in the radiation biology of alpha particles.

Experimental diffic u lties:

 Short range

 Changing energy and LET within target.

Lim ited early studies with alpha particle s led to the “conventi onal wisdom” that even a single particle traversal of the nucleus would kill the cell.

Exponenti a l survival curves interpreted as “single-hi t, single target.” (Barendsen, 1960).

Indirect mechanism s were invoked to explai n the survival (and m u tation) of cells exposed to alpha particles.

Munro (1970) plutonium n eedle experime nt showed nucleus m u ch m o re sensitive than the cytoplasm.

One of the first studi e s to show that cells could survive a large num ber of alpha particle nuclear traversals was [Lloyd, et al., Int. J. Radiat. Biol., 35, 23-31, 1979]

 Alpha particles derived fro m a Tandem accelerator, 5.6 MeV, 85 keV/µm at cell surface.

 Track detectors used to count particle fluence.

 Cell nuclei measured.

**The Lloyd paper contains one of the fi rst observat i ons that cells flatten out considerably when at tached , an d that the nuclear cr o ss section changes between spherical and attached cells. Serious im p lications for radiation effects with lim ited penetration alpha particles

 Pelleted and fixed, nuclear cross section ~ 100 µm 2

 Attached and flattened, nuc lear cross section ~ 300 µm 2 , thickness ~2 µm.

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[Lloyd, 1979]

D 0 = 4.4 x 10 6 alphas/cm 2

Conclusion was that ~ 14 alpha particles pass through a 313 µm 2 nucleus, at a dose = D 0 .

Relation to other work:

40 µm 2 1-2 traversals = mean lethal dose (Barendsen, 1960) 150 µm 2 ~5 traversals = mean lethal dose (Datta, 1976)

313 µm 2 ~14 traversals = mean lethal dose (Lloyd, 1979)

Inactivation cross section = 1/D 0 = 22 µ m 2 /track (“very sim ilar” to Barendsen, Datta).

The authors concluded that total track length deposited in nucleus is the critical factor.

 Flatter cells can withst and mor e traversal s .

Speculation: cells flattened agai nst bone surfaces may survive alpha particle traversals (from bone-seekers: 226 Ra, 239 Pu) but transform into osteosarcomas.

[Radiation Research, 123, 304-310, 1990]

Earlier studies used wide beams from isotope sources or accelerators.

Objective: development of a model system for alpha particle irradiations of cells under m o re controlled conditions.

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The charge on the al pha particle decreases as the velo city decreases.

Early m odels did not take this into account.

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 Changing the thickness of the m y lar filte r produces different energy spectra reaching the cells.

 Spectra becom e quite br oad at lower ener gies.

Track etch m e asurements in CR-39 indicate ~1900 tracks/mm 2 / s ec.

[ R ad iatio n Resear ch, 1 2 8 , 204 -20 9 , 1 991 ]

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Collimated 3.5 MeV alpha particles

Careful estimation of nuclear cross section, nuclear thickness, together with keV/ µm , allows calculation of dose to the nucleus.

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N

0 calculat e d at the center (5 th layer) of the nucleus.

N

 is the calculated energy deposited in th e nucleus by the traversal of a single

alpha particle.

N

 is the absorbed dose to the nucle us from a single alpha particle.

Nu mber of alpha part icle traver sals per nucleus was calcu lated three ways:

1. num ber of traversals = N T

alpha particle fluence = 1900/mm 2 . s ec ( m easured in CR-39 track detector) dose rate = 3.8 cGy/sec (measu red with ionization cham ber)

A = nuclear cross section (µm 2 )

N  D 0  1900  A

T 3 . 8

N

2. N 0



where

N

 = the nuclear dose from a single alpha particle,

and D N = the dose to the nucleus at D .

0 0

3. By sim p ly dividi ng t h e num ber of tracks/µm 2 , (as r ead fro m CR-39 track detector) at a dose equal to D 0 , by the nuclear area.

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Cross section: the relative probabilities of inducing lethal events at a given fluence.

 (  m 2 )  k L  

 L ( ke V /  m )

 0 . 065  D N

( cGy )

L = mean stopping power of alpha particle traversing the nucleus

ρ = the density of tissue

α = 1/ D N (cGy)

k = a constant to m a tch units

The inactivation cross sections are fairly constant, with the exception of the (very flat) AG1522 cells.

Inactivation cross sections agree with literature values.

Total track length: the num ber of traversals x nuclear thickness.

The total track length for inactivation is nearly constant, with the exception of the AG1522 cells.

[RBEs cal c ulated using “dose at cell surface”.] Observations:

 The sensitivity to 60 Co varies with cell line.

 RBEs ar e nearly constant.

 No correlation between N T and RBE.

Conclusions:

 Average num ber of alpha particles leading to a lethal lesion is greater than 1 (range 2-6) and dependent on t h e cell line used.

 Total track length in the nucleus is a bett er measure t o co mpare the sensitivities of different cell lin es.

 Non-lethal lesions may be responsible for the greater carcinogenicity of alpha particles.

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Environm ental exposure to radon produces a low dose, where very few cells are hit at all. Ho wever the few cells t h at are hit receive a substantial dose (on the order of 0.5 Gy).

Irradiation with a wide field of alpha par ticles from an accelera tor or an isotope source m e ans that Poisson statistics m u st be used in the interpretation of dose.

P ( n ) 

( e  n 

n !

n n )

where n = th e av erage number of hits/target, and n = the specific nu m b er of hits/ target

For an average dose of one alpha particle per cell, the actual d i stribution is predicted to be:

0 particles	37%
1 particle	37%
2 particles	18%
3 particles	6%
4 particles	1.5%
5 particles	0.3%

Clearly this co mplicates the interpretation of the results.

Experimental approaches to d e liver exac tly defined num bers of alpha particles include

 Microbeam s

 Retrospect ive i m aging of cells grown on a track detector

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 Cell culture dish has LR115 tr ack detector as a base.

 Cells plated, locations recorded.

 Irradiation from below with alphas from a 210 Po source

 Allow cells to form colonies (6 days).

 Re-scan dish, re-visit original lo cations, colony criteria = 50 cells.

 Etch LR115 track detector by floa ting on alkaline etching solution.

 Re-scan dish, record track locat ions.

 Spatial resolution of the relo cation hit det e rm ination = 0.9 µ m.

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Survival is sim i lar to othe r reports using m i crobeam s.

No dependence of survival on mem b rane or cytoplasm hits observed, at t h e dose level used.

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Com p arison of the predicted Poisson dist ribut ion of tracks per nucleus to the actual num ber of tracks observed per nucleus.

Determ in ation of penetration depth using m y lar thickness.

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Track diameter increases at “track ends”.

Conventionally assumed ch aracteristics of alpha particle irradiation.

 No matter how low the dose, any cell tr aversed receives a relatively high dose (~0.5 Gy).

 Cells not traversed are unaffect ed.

Previous work:

 Chrom o somal instab ility in progeny.

 Nu m b er of cells showing instability was greater than predicted based on Poisson statistics.

Possible problem s with interpreting these results.

 There is always a Poisson distri bution of cells hit and non-hit.

 Is the instability observed in the progeny of a hit cell or a non-hit cell (i.e., is there a “bystander effect ”?

Objective:

Create experim e ntal conditions where the surviving population was largely non- hit, but was present (“bystanders”) during the irradiation.

Approach:

 Interpose a grid between the cells and the alpha source.

 This will shield cells from irradiation.

 CR-39 track etch t e chniques to verify grid effect.

 Harvest cells

o determ ine survival

o determ ine genetic effect (insta bility: non-clonal aberrations in decendants in a colony derived fro m one of the original cells)

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The maxim u m expected propor tion of cells exhibiting instability was calculated.

 The particle fluence is known,

 Using Poisson statisti cs the proporti on of cells surviving a particle traversal is estimated from the cell survival curve.

With a dose of 1 Gy, the m a jority of cells irradiated are inactivated.

 With no grid: 20% of survivors were irradiated

 With grid present: 3% of survi vors were irradiated.

Results:

Adding a grid reduc ed the num ber of irradiated cells present

The mean num ber of aberrations was th e same with or without the grid.

Implications:

 Mechanism not understood (re active oxygen species invol ved?)

 Risk from exposure t o alpha particles not lim ited to cells actually hit.

 Potential role in carcinogenesis.

 Does risk estimation at low doses underestimate the effect?

 Exposure t o high levels of radon has b een linked to an in creased incidence of lung cancer in uranium m i ners.

 Epidem iological studies estimate that 16,000 cases of lung cancer per year in the U.S. are caused by radon.

 Uncertainties exist in the conversion of exposure, measured in working le vel m onths (WLM) to a dose (Gy).

 The biological stages of carcinoge nesis fol l owing radon exposure and leading to a carcinoma of the lung are not known.

 Objective: to generate a relations hip betw een exposure (WLM) and dose (mGy) to target cells in the lung: deep-lung fibroblasts.

Approach:

 The rat is used as a m odel system .

 Deep lung-fibroblasts are the target cell.

 Exposures in vitro use cells isolated from the rat, then exposed to radon gas and proge ny in vitro . [Rn C l 2 source as a generator. 222 Rn equilibrated with cell culture mediu m , then the cells are ad ded.]

 Exposure in vivo to radon aerosol (uranium ore dust 5.3 m g /m 3 ).

 Endpoint is m i cronucleus formation in cells duri ng first di visi on after irradiation.

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In vivo ex posures:

 In vivo exposures of 0, 115, 213, and 323 WLM.

 Concentration was 1015 WL!!

 Exposure over 1, 2 or 3 days at ~ 100 WLM/day.

 Rats sacrificed 4 hrs after end of exposure ,

 lung fi broblasts isol ated, grown in cytochalasin B for 66-72 hrs

 score m i cronuclei/1000 binucleated cells

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In vitro exposures:

 Harvest rat lung fi broblasts

 Grow in culture for 16 hrs or 96 hours; irradiate

 16 hours cells still in G 0 /G 1 , i.e., not dividi ng

 96 hours, > 50% have entered S or G 2 /M

 after irradiation, grow in cytochalasin B for an additional 66-72 hours

 score m i cronuclei/1000 binucleated cells.

The dose response to 222 Rn in vitro is not different for dividi ng or non-divi ding cells.

Significance:

 This study is the first to use deep lung fi broblasts (sl o w turnover in vivo, can be stim ulated to divi de in vitro).

 In vivo dose-response was linear despite the long ti me of the irradiations 1,2, 3 days; i.e., no loss of dama ged cells during t h e experim e nt.

 This study produced a m odel for a “bio logical indicator of dose”: 1 WLM produced dam a ge equivalent to 0.79 mGy.

Previous work has been done with alveolar m a crophages.

 Can isolat e by “lavage”

 Much higher exposures used (1000 WLM!!)

 Cytochalasin B or BrdUrd not used in the m a crophage study

 Induction of m i cronuclei was 5 x lower in macrophages 0.012 MN/100 cells/WLM com p ared to the l ung fibroblast study: 0.058 MN/100 cells/WLM.

 Macrophages m i grate: may not have been present for the whole irradiation.

Previous work looked at the cells of the upper respiratory tra c t.

This group and others looki ng at diffe rent cell types, different endpoint s

1.7 – 2.5 mGy/WLM in rat tracheal ep ithelial cells-chrom osome aberrations

2.6 mGy/WLM in deep lung fibroblast s —colony form ing assay.

The current study use d the same cell type, endpoint, non-divi di ng cells, both in vivo and in vitro .

 Radon produces 55% of the effective dose equivalent to the general population.

 Most of this dose is delivered to bronchia l epithelial cells.

 Estimation of risk from radon exposure uses the epidem iological approach.

 Data ar e n o t sufficient for a biologi cal approach using RBEs and specific biological endpoint s.

Alpha particles penetrating through lung ti ssue have a va riety of different LETs. Co mplicat es the cal culation of dose to a particular site.

Objective: investiga tion of the early steps in the chain of events leading from radon exposure to tum o r form ation

Approach:

 The rat is used as a m odel system .

 Deep lung-fibroblasts are the target cell.

 Also used a Chines e Ham s ter cell line, CHO, al so a fibroblast cell line.

 Exposures in vitro use cells isolated from the rat, then exposed to radon gas and proge ny in vitro . [Rn C l 2 source as a generator. 222 Rn equilibrated with cell culture mediu m , then the cells are ad ded.]

 Exposure in vivo to radon aerosol (uranium ore dust 5.3 m g /m 3 ).

 Endpoint is m i cronucleus formation in cells duri ng first di visi on after irradiation.

 60 Co irradiations used as a phot on control both in vitro and in vivo .

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Dose-response for micronuclei form ation in rat lung fibroblasts is linear for 60 Co, both in vitro and in vivo.

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The dose-response for m i cronucleus form ation is sim ilar in vitro and in vivo , for both CHO (fibroblast) cells and for prim ary lung fi broblast s .

The dosimetry is accurate for the in vitro exposures RBEs are approxim a tely 10.

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Significance:

The isolation procedures did not a ffect the dose-response relationship

Two im portant physi cal f actors can influence RBE

 dose rate

 LET

Differences in dose rate between the 60 Co controls (0.5 Gy/m in) and the radon exposures (dose delivered over hours in vitro and over several days in vi vo) did not seem to ch ange the d o se response relationship.

The LET s p ectrum of the alpha pa rticles reaching the fibroblasts in vivo is unknown. There may be a spread of LET both above and below the 100 keV/µm “maxi m um”.

“The Bystander Eff ect”

Little and colleagues ha ve dev e loped a plutonium -based alpha particle irradiator, sim ilar to the one at Los Alamos described by Raju.

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The range of the alpha par ticles was calibrated using the track-etch approach with increasing thicknesses of m y lar.

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Results indicate the alpha particles are 3. 65 MeV at the cell surface, with an LET of 112 ke V/ µ m. The particle fluence is 0.0054 tracks/ µ m 3 , which produces a dose rate of 9.9 cGy/m i n. A camera shutter m e chanism allo ws exposures down to 0.067 sec.

With a dose rate of 0.366 tracks/nucleus/ m in , exposures of 2 sec or less produced, on average, very low num bers of alpha particle tracks/nuc leus (0.0004 – 0.0112).

 Very few nuclei (< 1%) were actually traversed by an alpha p a rticle at t h ese doses.

SCE

Cells irradiated, synchronized i n G 1 . Brom odeoxyuridine added, cells allowed to grow for 2 cell cycl es, chro mat i ds stain differently. SCE can be detected.

SCE is a crossing over event between the chromosomes attached at the centromer e .

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Sister chromatid exchange (SCE) versus exposure t i me (dose). Background rate has been subtracted.

The SCE r a te incr eas es rapidly and plateaus . At plateau, the rate is 1.4 t i mes the background rate.

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Scoring of SCE expressed as S C E p e r chr o moso me.

 At 0.31 mGy, 30% of cells showed increased SCE.

 At 2.45 mGy, 43% of cells showed increased SCE.

 13% of cells showed SCE per chrom o so me > 0.6, a level rarely seen in control cells.

 At these doses only 0.1 to 0.5% of cell nuclei were trav ersed by an alpha particle.

A com p arable level of exchange with x-rays requires 1-2 Gy (RBE >100!!). Significance of S C E in mammalian cells i s not clear.

The doses used here are too low to pr oduce d e tectable levels of cell killing.

Implications:

Cells not directly traversed show a chro mosomal ch ange.

The risk to individuals may not be eas ily extrapolated from ep idem iological data at high doses with low LET radiation.

Alpha particle dose in the range used in this study (0.31-4.9 m G y) are well within the range of exposure reported to occur from radon in hom es.

More indirect evidence fro m th e L ittle lab on the existence of a

“Bystander Effect” using a different endpoint : HPRT mutation.

Objective: measure effects of very low doses of alpha particles: doses relevant to environm ental exposures.

Requires a m o re sensitive endpoint.

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N.B. Dose = 0.174 Gy (17.4 cGy) fo r each track through the nucleus. Increasing the exposure results in m o re cells receiving the same dose.

At high doses, wh en there is more than one track per cell, the bystander effect is overshadowed by direct dose.

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 Dose response becomes non-linear at low doses.

 Unexpectedly higher response than pr edicted from extrapolation of the high dose result s down to the low dose region.

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Mutation frequency per al pha particle track increased 5 -fold at the lowes t fluence.

Conclusion is that the enhan c ed m u tation rate at lo w particle fluences was the result of mutations in non- irradi ated bystander cells.

“The Bystander Eff ect”

 probabl y a variety of different mechanism s

 some reports say gap junction, intr acellular comm unication is required,

 other reports say it is not

 does it occur in vivo?

 Im plications for radi ation prote c tion a nd the current debate over the “linear no-threshold’ hypothesis.