Questi on : “What is the biological eff e ct of a sin g le alpha particl e ?” Microbeams

Delivery of single or precise num ber of charged particles to individual cells. Accuracy allows tar g eting of nucleus versus cytoplas m.

Avoid the Poisson di stribution statistical problem .

Only a few m i crobeam facilities in the world

 Colum b ia University

 Gray Laboratory, U.K.

 Pacific Northwest National Laborator y (now at Tex a s A & M)

 MIT..(in p r ogress)

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Basic design:

 Microscope stage for targeting

 Particle counting either by a scintillator under the cells together with a photom ultiplier or an ioniza tion cham ber over the cells.

Clearly the m i ners exposed to high con centrations of radon show an increased incidence of lung cancer (up to 10 times the normal rate).

At the low levels of exposure for the general populati on, the use of epidemiological data used for risk a ssessment has very large uncertainties.

EPA estimate: 21,000 lung cancer deaths in th e U.S. per year directly attributable to radon exposure (confidence lim its: 7,000 to 30,000).

Nu m b er of traversals of bronchial cell nuclei

 96% in a m i ner > 1 particle per year

 1 in 10 7 cells > 1 particle per year in a household environm ent

Objectives:

What are t h e effects of a single alpha pa rticle on survival a nd m u tation frequency?

Methods:

Irradiation is automated, ~ 2 seconds per cell, up to 10, 000 cells per day. Spatial precision is ± 4 µm .

Modeling indicates that the beam would hit the nucleus 98.4% of the time.

Energy of the alpha particles is 5.5 MeV, range ~40 µm , LET = 90 keV/µm .

All cells on a plate irradiated then rem oved for standard colony form ing assay or m u tation analysis.

Mutation assay

 Mutation assay when cells are irradiat ed one at a time (even 10000/ day) requires a sensitive endpoint.

 A L cells have a standard set of ha m s ter chrom o somes, plus one hum a n chromosome

 The hu man chromos o me codes for cell surface mark ers that make the cells susceptible to killing by specifi c antibodies and com p lement.

 Cells plated in dish, exposed to anti body, only those with a mutation in the hum an g e ne will not express the su rface antigen and thus survive.

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Cells stained briefly with a non-toxic concen tration of Hoechst dye for targeting

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 Cell survival as a function of t h e num ber of alpha particles is sim ilar to other reports.

 D 0 is approxim a tely 4 particles (3.7).

 80% of cells survive passage of a single alpha particle.

 10% of cells survi v e 8 alpha particles!!

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 Traversal of the nucleus by a single al pha particle in creased the m u tation frequency by a factor of 2.

 At 1 particle per cell, the m i crobeam results agree with broad beam alpha particle irradiations where a m ean of 1 particle was irradiated.

 At 8 particles per cell, the m i crobeam m u tation frequency is higher than that seen with a broad beam delivering a m ean of 8 part icles per cell. (Are cells that received lower doses “overgrowing” t h e populat ion?)

Conclusions and Significance

 Nuclear traversal by a singl e alpha particle was only slightly cytotoxic , but

highly mutagenic.

 First dem o nstration t h at a single alpha particle traversing a nucleus will have a high probabil i t y of causing a m u tation.

 Highlights the need for radiation protection at low doses.

Prevailing dogm a: the nucleus is the targ et for genot oxic and cytotoxic effects.

Reports on the “Bystander Effect” indicat e that DNA damage can be caused in cells not directly traversed.

Objective: use mi crobeam to selectively i rradiate cytoplasm to determ ine if there are effects on survival or m u tation frequency.

The Columbi a mi crobeam pr oduces alpha particles of 5.5 MeV, range ~40 µm , LET = 90 keV/µm.

Irradiation of each cell takes about 6 seconds.

All cells on the dish irradiated, then tr ypsi n ized, removed and plated for either survi v al or m u tation analysis.

Mutation assay is the A L ham s ter line with one human chrom o some.

Hum a n chrom o some expresses cell surface antigens that m a ke cells susceptible to co m p lement inactivation. Wild-type cells are killed in the presence of antibody and com p lement

Cells with m u tations in this gene can survive.

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 Nucleus is green, cytoplasm is red.

 Irradiate 8 µm from the end of the nucleus on each end of the cell.

 Do not irradiate if o n e cell’s cyt oplasm overlaps another cell nucleus.

 Accuracy estimate: 75% had both ends hit , 15% one end h it, 10% m i ssed, no accidental nuclear hits.

Approach:

 Irradiate cytoplasm with 1, 2, 4, 8, or 16 alpha particles.

 Determ in e survival

 Determ in e m u tati on frequency

 Add scavengers of free radicals to dete r m i n e if the ef fect is mediated by reactive oxygen species (ROS).

 Imm uno-staining for specific o x idative DNA d a mage product 8-hydroxy- deoxygua nosine (8-OHdG).

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Lethality is m i nimal co m p ar ed to nuclear traversals:

 4 particles in cytoplasm, > 90% surviv al; (35% if 4 particles are in the nucleus)

 32 particles in cytopl asm , >70% surviv al; (<< 1% if 32 particles are in the nucleus)

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Mutation frequency:

 Background level is 43 ± 15 mutants/10 5 cells.

 The effect saturates afte r 4 particles per site.

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Mutation spectra:

Most of the m u tants from 8 particles th rough the cytoplasm were sim ilar to the spontaneous m u tants: small alte rations only in the CD59 gene.

80% of the m u tations seen after 8 particle s through the nucleus were m u lti-locus. Different m u tagenic m e chanism s involved?

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Scavenger experiments:

 Add DMSO to reduce ROS directly: DM SO can protect against indirect dam a ge

 Add BSO to deplete intracellular glutathi one (GSH). GSH is the cells natural scavenger of ROS.

DMSO added before and afte r irradiation reduced level of m u tants (4-5 fold). Pretreatment with BSO for 18 hrs reduces GSH lev e l s to 5% of controls.

Mutation frequency increased 4-5 fold.

DMSO an d BSO alone were n on-toxic and non-m utagenic.

DMSO treatment had no effect on the mu tation frequency following 4 particles through the nucleus.

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Detection of oxidiz e d nucleoside (8-OHdG).

 8 particles through t h e cytoplasm

 Fluorescence intensity increased by ~ 2-fold

 Tim e course indicated maxim u m signal intensity seen at 5 mi nutes after irradiation.

 Signal intensity decreased to co ntrol le vel s by 45 m i nutes after irradiation.

Conclusions:

 Nuclear traversals -- direct effect

 Cytoplasm traversals -- indir ect effect med i ated by ROS

 Mutation spectra of cytoplasm traver sals look like spontaneous m u tations.

 Cytoplasmic m u tations mediated by RO S? Scavenger experiments support this. Detection of 8-OHdG sup ports this.

 Mechanism? Hydroxyl radicals invol ved?

 Migration length of OH · is too short, ~ 4 nm , targeted site s were ~8 µm from the nucleus.

 Cascade to longer-li v ed free radicals?

 Mitochondria invol ved? Coul d explain saturation effect.

Significance:

 Nuclear traversals produced 3-4 fold m o re m u tations at equal num bers of particles.

 However, cytoplasmic trave rsal may be more important for carcinogenesis

 At equitoxic doses (e.g., 90% survi v al) cytoplasm i c irradiation induced 7- fold m o re m u tations than nuclear traversals.

 Cytoplasmic irradiation could actua lly be m o re of a health risk.

Estimation of risk from exposure to ra don is based on epidem iological data in m i ners, and extrapol ated to the lo w exposure levels present in hom es.

Bronchial epithelial cells in m i ners m a y ha ve been traversed by several particles in a short period.

For the individual in a domestic radon situ ation, it is highl y unlikely tha t any cell will be traversed by m o re than one al pha particle in an entire lifetime.

Objectives:

 Investigate the onco g enic effects of exactly one alpha particle.

 Co mpare the effects of exactly one to a Poisson mean of one alpha particle.

 This is a check on whether the cells receiving m o re than one particle dom inate the response.

 “Control” study: com p arison of exactly four with a Poisson mean of four particles.

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The mi crobeam facil ity at Colum b ia.

Continuous im provements in autom a tion ma ke it possible to irradiate ~ 3000 cells per hour.

“Broad beam” studies produce the statis tical Poisson distri buti on of parti c le traversals.

For a m ean of 1 part icle:

 37% will see 0 traversals

 37% will see 1 traversals

 26% will see 2 or m o re traversals

The average dose wi ll be the same as the group that all received exactly one

traversal.

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Cells are s t ained wit h a Ho ech st dye to visualize the nucleus for targeting. Broad beam groups are also stained as a control.

Transformation was judged by m i croscopic inspection of colonies aft e r 7 weeks of growth. Methods previously reported. Such transform e d cel ls produce tum o rs when injected into animals.

Transformed cells lose contact inhibition.

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[Hall, 2000]

Transformed cells lose anchorage dependence.

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[Hall, 2000]

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Transformation frequency/10 4 surviving cells.

 Control rates are sim i lar.

 Broad beam results are greater t h an m i crobeam results only at 1 particle.

 At 2, 4, and 8 particles the m i crobeam results are th e same (within the error bars) as the broadbeam results.

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 Cells traversed by exactly one alpha par ticle show a lower risk than those receiving a mean of one.

 Exactly one alpha p a rticle is not si gnificantly differe nt than control s receiving zero!!

 This im plies that the majority of tran sformed cells resulting from a mean of one traversal m u st come fro m the subpopu lation tha t received m o re than one traversal.

 If traversal by only one particle does not significantly raise the risk of oncogenic transform a tion, estimations by extrapolation from higher doses received b y min e rs will overestimat e the risk.

Previous work by this group with this cell line

 Exact numbers of alpha particles: survi v al and m u tation endpoi nts.

 Cytoplasmic irradiation.

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 Now carried out to 20 particles per nucleus.

 D 0 is approxim a tely 3.6 al pha particle traversals.

 Survi v al after 20 particles through the nucleus is ~ 1%.

A L cells: ham s ter with one human chro m o so me that expresses cell surface markers for com p lement inactivation.

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 ~500 cells plated in irradiation dish

 At random, 5, 10 or 20% of the cells in the dish irradi ated with 20 alpha particles each

 >99% of the survivi ng cells are unirradi ated, but the m u tation rate is 3 times higher tha n control.

 Bystander effect in cells not traversed by alpha particles.

 The effect appears to “saturate”.

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 For the population where 20% of the cells were exposed to 20 alpha particles, 99.8% of the progeny are cal culated to be from unirradiated cells.

 The mut a tional spectra ar e clearly different from those that occur spontaneously.

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 The bystander m u tagenicity was not affected by ad dition of DMSO.

 The effect is not due to a free radical ROS .

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 Lindane blocks gap junctions a nd reduces the bystander effect.

 Cell-cell contact plays an im portant role in this effect.