Scholarly article on topic 'Age-Related Impairment of Pancreatic Beta-Cell Function: Pathophysiological and Cellular Mechanisms'

Age-Related Impairment of Pancreatic Beta-Cell Function: Pathophysiological and Cellular Mechanisms Academic research paper on "Nano-technology"

Share paper
Academic journal
Frontiers in Endocrinology
OECD Field of science

Academic research paper on topic "Age-Related Impairment of Pancreatic Beta-Cell Function: Pathophysiological and Cellular Mechanisms"

Radiation Protection Dosimetry Vol. 99, Nos 1-4, pp. 57-62 (2002) Nuclear Technology Publishing


L. Sanche

Groupe des Instituts de Recherche en Sante du Canada en Sciences des Radiations Faculté de Medecine, Universite de Sherbrooke Sherbrooke, Quebec, Canada, J1H 5N4


Abstract — It has recently been shown that 3-20 eV electron impact on vacuum-dry samples of plasmid DNA induced substantial yields of single and double strand breaks (SSBs and DSBs). These results are summarised in the present article along with those obtained from the fragmentation of elementary components (i.e. condensed H2O, bases and sugar analogues) of DNA induced by low energy electron impact under ultra high vacuum conditions. By comparing the results from these experiments, it is possible to determine fundamental mechanisms by which low energy electrons damage DNA. The decay of transient anions formed on the DNA's basic components is found to play a crucial role in producing SSBs and DSBs. Since a large portion of the energy deposited by ionising radiation first leads to the production of low energy secondary electrons, these findings provide basic knowledge necessary to understand the genotoxic effects of high energy radiation and eventually modify these effects at the molecular level.


Many investigations during the past century have been devoted to the understanding of the alterations induced by high energy radiation in biological systems, more particularly within living cells and the DNA molecule. The biological effects of such radiation are not produced by the mere impact of the primary quanta, but rather, by the secondary species generated along the radiation track(1). As these species further react within irradiated cells, they can cause mutagenic, genotoxic and other potentially lethal DNA lesions1-2-4-1, such as single and double strand breaks (SSBs and DSBs).

Secondary electrons with energies below 20 eV are the most abundant of the secondary species produced by the primary interaction(5-7). For example, a 1 MeV primary photon or electron generates about 4 X 104 secondary electrons when its energy is deposited in biological matter1-5'6'8-1. It is therefore crucial to determine the action of secondary electrons within cells, particularly in DNA where they could induce genotoxic damage. To investigate such damage induced to vital cellular components, experimental efforts have been devoted to the isolation of biomolecules as thin multilayer films in ultra high vacuum (UHV), where they could be bombarded with a beam of low energy electrons (LEEs)(9-12). Such environmental conditions are necessary to avoid LEE interaction with small surface impurities, which could modify the probed damage. In these experiments, fragments of biomolecules induced by LEE bombardment can be either analysed in situ or outside UHV. In the former, the masses of ions and neu-

Contact author E-mail:

tral species desorbing from a multilayer film target are analysed during bombardment1-11-12-1 or the products remaining in the film are characterised by X ray photo-electron spectroscopy after bombardment1-13-1. If sufficient degraded material is produced, the film target can be extracted from the UHV chamber and the products analysed by standard methods of chemical identification1-9-1. In this article, some of these methods are briefly described and recent results obtained from experiments performed with these techniques on the DNA molecule and some of its elementary constituents are reviewed.


Damage to pure dry samples of supercoiled DNA is induced by bombardment with 3-20 eV electrons under a hydrocarbon-free 10-9 torr residual atmosphere1-9-1. Plasmid DNA [pGEM 32^--, 3199 base pairs] is first extracted from Escherichia coli DH5a, purified, and resuspended in nanopure water into a clean N2-filled glove box, where the following manipulations are performed. An aliquot of this pure aqueous DNA solution is deposited onto chemically clean tantalum substrates held at liquid nitrogen temperatures, lyophilised with a hydrocarbon-free sorption pump at 0.005 torr and transferred directly in a UHV chamber without exposure to air. After evacuation for ~24 h, room-temperature DNA solid films of 5 monolayer average thickness and 6 mm diameter are irradiated with a monochromatic LEE beam of the same diameter. Each target is bombarded for a specific time at a fixed beam current density -2.2 X 1012 and incident electron energy. After 24 h under UHV, the DNA still contains

2.5 H2O molecules per base pair as structural water(14). After bombardment, the DNA is analysed by agarose gel electrophoresis and quantified as supercoiled (undamaged), nicked circle (SSB), full-length linear (DSB), and short linear forms(15). The measured yields reported in Figure 1 were found to be linear with electron exposure at each energy(9).

The incident electron energy dependence of these yields (Figure 1) shows that LEEs induce SSBs and DSBs, even at electron energies below the ionisation limit of DNA (-8 eV)(16). Also, the damage is highly dependent on the initial kinetic energy of the incident electron, particularly below 14 to 15 eV, where thresholds near 3 to 5 eV and intense peaks near 10 eV are observed. The results shown in Figure 1 can be understood by investigating the fragmentation induced by LEEs to the various sub-units of the DNA molecule, including its structural water. DNA is composed of two strands of repeated sugar-phosphate units hydrogen-bonded together by the bases which are chemically linked to the sugar moiety of the backbone. A short-chain segment shown in Figure 2(b) exhibits the repeated sugar-phosphate sub-units of the deoxyribose backbone to which are chemically bonded the bases

£ £ ■Q ©

o o < '

1 1 1 1 -DNA double j 1 i 1

- strand breaks /

_(DSBs) / \ -J ~

•- i , i (a)- i

-DNA single F^S

-strand breaks /

(SSBs) \ ■

i i (b)- 1

-Loss of

-super-coiled /


i , i (c): 1

adenine, cytosine and thymine. Results obtained from electron bombardment of solid films of DNA constituents are presented in the following sections. These include H2O, the bases and the backbone sugar-like analogues, tetrahydrofuran (I), 3-hydroxytetrahydrofuran (II), and a-tetrahydrofuryl alcohol (III) whose molecular structures are shown in Figure 2(a).


Figure 3 shows the H" ion yields desorbed by the impact of 1 -20 eV electrons on 10 ML films of I, II and III. The curves in Figure 3 are characterised by an onset at 6.0, 5.8, and 6.0 eV and a yield maximum centred at 10.4, 10.2, and 10.0 eV for I, II, and III, respectively. A second feature is also observed in the H" yield function for II; it appears as shoulder on the low energy side of the 10 eV peak and is characterised by a sharp onset and a peak maximum near 7.3 eV. These results were generated by measuring with a mass spectrometer the H" anions, desorbed by a collimated 4 nA electron beam of 80 MeV full width at half maximum, incident at an angle of 70° from the surface normal of the sample film(17). The multilayer films of I, II and III were grown in UHV on an electrically isolated polycrystalline platinum ribbon attached to the tip of a closed-cycle helium-refrigerated cryostat(17).

O = ^O-

O O ¿f

5 10 15

Incident electron energy (eV)

Figure 1. Measured yields, per incident electron, for the induction of DSBs (a), SSBs (b), and loss of the supercoiled DNA form (c), in DNA solids by electrons of 3-20 eV. The error bars correspond to one standard deviation of the measurement.

Figure 2. Schematic drawing of (a) the DNA backbone sugarlike analogues tetrahydrofuran (I), 3-hydroxytetrahydrofuran (II), and a-tetrahydrofuryl alcohol (III), and (b) a short-chain segment of a single-stranded deoxyribose backbone of DNA.


All features below 15 eV in Figure 3 are characteristic of dissociative electron attachment (DEA) to I, II and III (i.e. the formation at a specific energy of a transient anion of the parent molecule which dissociates into H" and the corresponding neutral radical). The steep rise in the H" signal with an energetic threshold near 14.5 eV is characteristic of non-resonant dipolar dissociation (DD) of C-H bonds in I, II, and III; it could also partially arise from DD of the O-H bond in II and III. DD results from the dissociation of an electronic excited state into a positive and a negative ion fragment. The formation of H" via DEA from I, II and III has been discussed in detail by Antic et al(17). These authors considered the possibility of H" arising from dissociation of the tetrahydrofuran ring, the OH and the -CH2OH group. Other decay channels of the transient anions may result in the formation of larger anion fragments, such as the OH", CH2OH", and the (M -1)" ion fragment and compete with H" production, but these heavier ions were not observed to desorb. Typically, large mass fragments do not possess sufficient kinetic energy to escape induced polarisation and thus remain trapped within the film(18). Owing to the strong simi-

Electron energy (eV)

Figure 3. H- desorption yields stimulated by the impact of 1-20 eV electrons on a 10 monolayer (ML) thick film of I, II, and III. The small arrows indicate the positions of the two Feshbach resonances associated with dissociative electron attachment to the hydroxyl group in II and III, respectively. The smooth solid lines serve as guides to the eye, and the curve baselines have been shifted vertically for clarity.

larity of the H- desorption profiles for I, II and III, these authors concluded that 'the majority of the anion yield for all three systems arises from at least one transient anion associated with electron attachment to the furan ring and located near 10 eV'. Considering the largely Rydberg character of the excited states in I near the energy range of the observed resonance, they further suggested that this resonant state is of the core-excited type, possibly with dissociative valence a* con-figurational mixing. Lepage et al(19) have observed a broad resonance at similar electron energies in the excitation function of several vibrational modes in I, where they found good correlation with the gas-phase resonance observed in cyclopentane; they have tentatively assigned it as a core-excited shape resonance. This assignment is consistent with that made for the resonance observed near 10 eV for the (M - 1)- fragment (as well as all the lower mass fragments) produced by DEA to gaseous furan(20).


Electron impact dissociation of DNA bases has been investigated in the gas(21) and the condensed phase(10,11,13). In both phases, a large variety of stable anions is produced via DEA to the bases for electron energies below 30 eV. The anions H-, O-, CN-, CH2-, OCN- and OCNH2- were observed from gaseous thy-mine, whereas electron impact on gaseous cytosine resulted in the formation of the anions H-, C-, O-and/or NH2-, CN-, OCN-, C4H5N3- and/or CH4N3O-and C3H3N2- (11). All four bases were investigated in the form of thin multilayer films held at room temperature, but fewer anions of different masses were measured than in the gas phase. The difference is principally due to the inability of the heavier anions to overcome the polarisation potential they induce in the film, causing them to remain undetected in the target(18). In fact, only the light anions H-, O-, OH-, CN-, OCN- and CH2-were found to desorb by the impact of 5-30 eV electrons on the physisorbed DNA bases adenine, thymine, guanine and cytosine via either single or complex multibond dissociation(11). As an example, the CN- yield functions produced by 3 -20 eV electron impact on thin films of each of the four bases are shown in Figure 4. All yield functions exhibit maxima which are characteristic of DEA to the bases. Whereas anions such as H-can be induced via a simple bond cleavage from the bases, the production of CN- occurs under more complex ring dissociation. From the structure of the DNA bases, it can be readily seen that extraction of a CN-anion must involve more than a single bond cleavage. In other words, during the lifetime of the negative ion, not only exocyclic single bond occurs, but also complex endocyclic multibond cleavages are involved via most likely either stepwise(22) or concerted reactions(23). The similarity of the CN-, OH- and O- ion yield functions with resonant peaks at 9.0 eV and 10 eV from guanine

or cytosine and thymine films, respectively, observed by Abdoul-Carime et al(11), suggests that they arise from the formation of a same excited isocyanic anion intermediate (OCNH)*", via closely lying but distinct resonances; i.e. (G", C", or T") ^ R + (OCNH)*", where R represents the remaining radical; then (OCNH)*" further undergoes fragmentation into different possible dissociative channels: CN" + OH, OH" + CN or O" + CNH.


Previous results from electron-bombarded amorphous H2O and D2O films are also of relevance to DNA damage, since on average 2.5 H2O molecules were estimated to be present for each base pair in the DNA samples used(9) to produce the data shown in Figure 1. The damage induced by low energy electron impact on amorphous ice films has been measured by recording


a ; ■

f "„V

'■ . ■ r*




■ ■

-Í■X* • ■■

> .Hi? ■:

r: ■ ■


■ 'H


■ ■ ■ ■ o

m * mB mf

■ ■ ■


10 15 20 25 30 35 40 45 5 10 15 Incident electron energy (eV)

Figure 4. Incident electron energy dependence of CN ion yields desorbed under 500 nA electron bombardment of 8 ML thick films of (a) adenine, (b) thymine, (c) guanine, and (d) cytosine.


H-(24), h2<25-26», D(2S), O(3P) and O0D2)(27) desorption yield functions in the range 5-30 eV. Most of these functions exhibit resonance structures below 15 eV which are characteristic of transient anion formation. From anion yields, DEA to condensed H2O was shown to result principally into the formation of H~ and the OH radical from dissociation of the 2B1 state of H2O~ located around 8 eV with smaller contributions from the 2A1 and 2B2 anionic states formed near 9 and 11 eV, respectively1-24-1. At higher energies non-resonance processes such as dipolar dissociation, lead to H2O fragmentation with the assistance of a broad resonance extending from 20 to 30 eV(24-26).


In previous sections, it has been shown that the incident electron energy dependence of the damage to elementary constituents of DNA, probed in the form of desorbed anions, exhibits strong variations due to DEA. From comparison of these maxima in the anion desorption yield functions of these constituents, to the DNA results, it becomes quite obvious that the strong energy dependence of the DNA strand breaks below 15 eV in Figure 1 can be attributed to the initial formation of transient anions, decaying into the DEA and/or dissociative electronic excitation channels. However, since the basic DNA components (i.e. the sugar and base units and structural H2O) can all be fragmented via DEA between 5 and 13 eV, it is not possible to attribute unambiguously SSBs and DSBs to the initial dissociation of a specific component. For example, the maximum in the SSB yield lying near 8 eV is very close to the maximum in H- + OH production from H2O, but the DNA bases also produce H- and the corresponding radical with high efficiency near 9 eV(11). What appears to be more convincing is the coincidence in the 10 eV peak in H- production in Figure 3 from tetrahydrofuran and its analogues, with that in the DSB yield seen in Figure 1. This is not surprising, since two events are necessary to create a DSB, and the probability for such breaks is likely to be larger when the first hit results in a SSB (i.e. in a break on the sugar-phosphate backbone). Another interesting aspect of the DSB yield curve is the absence of damage induced by direct (non-resonant) electronic transitions. Indeed, the two points in Figure 1 around 14 and 15 eV lie at the zero base line, indicat-

ing that below ~16 eV a DSB occurs exclusively via the decay of transient anions. These observations suggest that below 16 eV, a DSB occurs via molecular dissociation on one strand initiated by the decay of a transient anion, followed by reaction of at least one of the fragmentation products on the opposite strand. This hypothesis is further supported by the observation of electron-initiated fragment reactions (such as hydrogen abstraction, dissociative charge transfer, atom and functional group exchange, and reactive scattering) occurring over distances comparable to the DNA's doublestrand diameter (~2 nm) in condensed films containing water or small linear and cyclic hydrocarbons(28-30).


Results on the damage induced by low energy electrons to DNA and some of its basic constituents have been reviewed in this article. The incident electron energy dependence of the yield of anions desorbed from solid H2O, sugar-phosphate backbone sub-units and DNA bases exhibits strong variations typical of dissociative electron attachment to these molecules. These results were compared to the measured yields per incident electron for the induction of SSBs and DSBs of supercoiled DNA measured between 3 and 20 eV. Such a comparison shows that below 15 eV, the DNA damage results principally from the formation of transient molecular anions localised on the DNA's basic components. These transitory states arise from the fundamental elec-trodynamic and exchange forces acting between the electron and a specific sub-unit of the DNA. Because of the universality of these fundamental interactions, they are expected to also be operative in living cells. Thus, from a radiobiological perspective, the results summarised in this article suggest that the abundant low energy (0-20 eV) secondary electrons, and possibly their ionic and radical reaction products, play a crucial role in the nascent stages of cellular DNA radiolysis and may already induce substantial damage long before their thermalisation along ionising radiation tracks.


The author would like to thank Ms Francine Lussier for the preparation of this manuscript. This work was supported by the Canadian Institutes of Health Research.


1. von Sonntag, C. The Chemical Basis for Radiation Biology (London: Taylor and Francis) (1987).

2. Ward, J. F. In: Advances in Radiation Biology 5, Eds J. T. Lett and H. Adler. (New York: Academic Press) pp. 181-239 (1977).

3. Yamamoto, O. In: Aging, Carcinogenesis and Radiation Biology, Ed. K. Smith (New York: Plenum) pp. 165-192 (1976).

4. Fuciarelli, A. F. and Zimbrick, J. D. Eds. Radiation Damage in DNA: Structure/Function Relationships at Early Times (Columbus, OH: Batelle (1995).

5. International Commission on Radiation Units and Measurements. Average Energy Required to Produce an Ion Pair. Report 31. (Washington, DC: ICRU) (1979).

6. LaVerne, J. A. and Pimblott, S. M. Electron Energy-loss Distributions in Solid, Dry DNA. Radiat. Res. 141, 208-215 (1995).

7. Cobut, V., Frongillo, Y., Patau, J. P., Goulet, T., Fraser, M-J. and Jay-Gerin, J.-P. Monte Carlo Simulation of Fast Electron and Proton Tracks in Liquid Water—1. Physical and Physicochemical Aspects. Radiat. Phys. Chem. 51, 229 (1998).

8. Bartels, D. M., Cook A. R., Mudaliar M. and Jonah, C. D. Spur Decay of the Solvated Electron in Picosecond Radiolysis Measured with Time-correlated Absorption Spectroscopy, J. Phys. Chem. A 104, 1686-1691 (2000).

9. Boudaiffa, B., Cloutier, P., Hunting, D., Huels, M. A. and Sanche, L. Resonant Formation of DNA Strand Breaks by Low Energy (3-20 eV) Electrons. Science 287, 1658-1660 (2000).

10. Herve du Penhoat M. A., Huels, M. A., Cloutier, P., Jay-Gerin, J. P. and Sanche, L. Electron Stimulated Desorption of H-from Thin Films of Thymine and Uracil. J. Chem. Phys. 114, 5755-5764 (2001).

11. Abdoul-Carime, H., Cloutier, P. and Sanche, L. Low-energy (4-50 eV) Electron-stimulated Desorption of Anions from Physi-sorbed DNA Bases. Radiat Res. 155, 625-633 (2001).

12. Abdoul-Carime, H., Dugal, P. C. and Sanche, L. Damage Induced by 1-30 eV Electrons on Thymine and Bromouracil Substituted Oligonucleotides. Radiat. Res. 153, 23-28 (2000). ^

13. Klyachko, D. V., Huels, M. A. and Sanche, L. Halogen Anion Formation in 5-halouracil Film: X-rays Compared to Subionis- | ation Electrons. Radiat. Res. 151, 177-187 (1999). O

14. Swarts, S. G., Sevilla, M. D., Becker, D., Tokar J. C. and Wheeler K. T. Radiation-induced DNA Damage as a Function d of Hydration. I. Release of Unaltered Basis. Radiat Res. 129, 333-344 (1992). f

15. Boudaiffa, B., Hunting, D. J., Cloutier, P. Huels M. A. and Sanche, L. Induction of Single and Double Strand Breaks in o PlasmidDNA by 100 to 1500 eV Electrons. Int. J. Radiat. Biol. 76, 1209-1221 (2000). h

16. Colson, A. O., Besler, B. and Sevilla, M. D. Ab Initio Molecular-orbital Calculations on DNA Base Pair Radical Ion-effect : of Base Pairing on Proton-transfer Energies, Electron-affinities and Ionization-potentials. J. Phys. Chem. 96, 9787 (1992). p

17. Antic, D., Parenteau, L., Lepage, M. and Sanche, L. Low-energy Electron Damage to Condensed Phase Deoxyribose Ana- O logues Investigated by Electron Stimulated Desorption of H- and Electron Energy Loss Spectroscopy. J. Phys. Chem. 103, f 6611-6619 (1999). d

18. Huels, M. A., Parenteau, L., Michaud, M. and Sanche, L. Kinetic-energy Distribution of O- Produced by Dissociative Electron Attachment to Physisorbed O2. Phys. Rev. A. 51, 337-349 (1995). 3

19. Lepage, M., Letarte, S., Michaud, M., Motte-Tollet, F., Hubin-Franskin, M.-J., Roy, D. and Sanche, L. Electron Spectroscopy . of Resonance-enhanced Vibrational Spectroscopy of Gaseous and Solid Tetrahydrofuran. J. Chem. Phys. 109, 5980 (1998). (¡3

20. Muftakhov, M., Asfandiarov, N. L. and Khvostenko, V. I. Resonant Dissociative Attachment of Electrons to Molecules of g 5-membered Heterocyclic Compounds and Lactams. J. Electron Spectrosc. Relat. Phenom. 69, 165 (1994). ¡2

21. Huels, M. A., Hahndorf, I., Illenberger, E. and Sanche, L. Resonant Dissociation of DNA Bases by Subionization Electrons. 3 ' J. Chem. Phys. 108, 1309-1312 (1997). b

22. Andrieux, C. P., LeGorande, A. and Saveant, J.-M. Electron Transfer and Bond Breaking: Examples of Passage from a r

Sequential to a Concerted Mechanism in the Electrochemical Reductive Cleavage of Arylmethyl Halides. J. Am. Chem. Soc. o

114, 6892-6904 (1992). G

23. Stepanovic, M., Pariat, Y. and Allan, M. Dissociative Electron Attachment in Cyclopentanone, Butyrolactone, Ethylene i

Carbonate, and Ethylene Carbonate-d: Role of Dipole Bound Resonances. J. Chem. Phys. 110, 11376-11382 (1999). d

24. Simpson, W. C., Orlando, T. M., Parenteau, L., Nagesha, K. and Sanche, L. Dissociative Electron Attachment in Nanoscale V Ice Films — Thickness and Charge Trapping Effects. J. Chem. Phys. 108, 5027 (1998). g

25. Kimmel, G. A., Orlando, T. M., Vezina, C. and Sanche, L. Low-energy Electron-stimulated Production of Molecular Hydro- 3 gen from Amorphous Water Ice. J. Chem. Phys. 101, 3282-3286 (1994). 3

26. Kimmel, G. A. and Orlando, T. M. Observation of Negative Ion Resonances in Amorphous Ice via Low-energy (5-40 eV) 3 Electron-stimulated Production of Molecular Hydrogen. Phys. Rev. Lett. 77, 3983 (1996). 4

27. Kimmel, G. A. and Orlando, T. M. Low-energy (5-120 eV) Electron-stimulated Dissociation of Amorphous D2O Ice — D(S- 0 2), O(P-3(2,1,0)), and O(D-1(2)) Yields and Velocity Distributions. Phys. Rev. Lett. 75, 2606 (1995). 5

28. Sieger, M. T., Simpson, W. C. and Orlando, T. M. Production of O2 on Icy Satellites by Electronic Excitation of Low-temperature Water Ice. Nature 394, 554 (1998).

29. Huels, M. A., Parenteau, L. and Sanche, L. Substrate Sensitivity of Dissociative Electron Attachment to Physisorbed Aniline. Chem. Phys. Lett. 279, 223-229 (1997).

30. Bass, A. D., Parenteau, L., Huels, M. A. and Sanche, L. Reactive Scattering of O- in Organic Films at Sub-ionization Collision Energies. J. Chem. Phys. 109, 8635-8643 (1998).