Hatano Y., Katsumura Y., Mozumder A. (Eds.) Charged Particle and Photon Interactions with Matter - Recent Advances, Applications, and Interfaces

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590 Charged Particle and Photon Interactions with Matter

21.8.2 non-Staining iMaging

Of great interest for the new generation of microbeam facilities is the use of more sophisticated

optical devices to both locate the biological targets of interest and, following the radiation exposure,

determine the damage induced at a single-cell level. Current biological targets used for the micro-

beam experiments are considerably larger than the radiation probe. For example, the cell nucleus

and cytoplasm are several microns in diameter against the submicron resolution achieved by some

facilities. In order to take full advantage of the microbeam targeting properties, it is crucial to be

able to visualize subcellular compartments other than the nucleus with high precision. For the

microbeam technique to be capable of detecting very small cellular responses, the potential con-

founding insult of external agents such as UV illumination and dye staining has to be eliminated/

minimized. A few laboratories have already started to employ non uorescent techniques (see

Section 21.5.4), and a very promising approach is represented by the use of quantitative phase

microscopy (QPM). Using QPM, high resolution images can be obtained by analyzing three images

acquired with conventional light microscopy. Commercial software that performs such analysis is

already available and only requires one in focus image and two slightly defocused (positively and

negatively) images. The considerable benet of being able to use conventional light microscopy on

line for microbeam facilities has however been judged against the resolution of the images obtained

and the processing speed.

Alongside the biological demands of performing microbeam experiments in tissues and 3D cell

systems, there is a need to equip the microbeam facilities with deep imaging techniques such as

multiphoton microscopy. Multiphoton uorescence microscopy is an imaging technique that allows

imaging of living tissue samples up to a few millimeters thick. It is based on the principle that two

long-wavelength photons (700–1000nm) simultaneously excite the same uorophore into a higher

energy state than they would individually. The uorophores then decay, emitting a uorescent sig-

nal relative to its energetic state (400–500nm). The main differences between multiphoton and

conventional uorescent microscopy are the longer excitation wavelength, which requires use of

lasers, required to produce a uorescent signal and the localized nature of the emission signal that

is only emitted from the uorophores where two exiting photons are focused. As a consequence, the

insult induced by multiphoton imaging is considerably lower, and high resolution images at depth

of several hundred microns can be obtained. Moreover, the use of tunable lasers allows multiphoton

microscopes to observe post-irradiation cellular dynamics by acquiring time-lapse images (Bigelow

etal., 2009).

21.8.3 hard x-ray opticS

As illustrated in Section 21.6.2, the nest x-ray focusing is currently achieved with diffraction ele-

ments (i.e., zone plates) that are wavelength dependent and characterized by low efciencies that

severely limit their application with conventional lab bench x-ray sources. The use of reecting

optics overcomes such problems, although available devices described earlier suffer from com-

plex alignment routines, considerable length, and limited focusing ability. However, new grazing

incident reecting optic devices for x-rays (i.e., polycapillary systems based on commercial micro-

channel plates originally designed as electron multipliers) are being designed and manufactured

offering promising alternatives for x-ray microbeams. In particular, the MOXI device (microfabri-

cated optical array for x-ray imaging) consists of two symmetrical arrays of microchannels etched

into silicon wafers and arranged in a circular pattern (see Figure 21.4). By precisely aligning the

two micro-channel arrays, a focusing action can be achieved with considerable efciency and mini-

mum aberrations when one or both arrays are appropriately bent (Prewett and Michette, 2001).

The benets offered by the MOXI devices lie in having a much larger numerical aperture com-

pared to zone plates (i.e., they can collect a larger fraction of the produced x-ray beam), a greater

focusing efciency, and the possibility to use higher energy x-rays. Moreover, the focal length of

Application of Microbeams to the Study of the Biological Effects of Low Dose Irradiation 591

such micro-optical devices can be varied providing zoom lens capability. Realization of MOXIs,

however, presents some technical challenges due to the required smoothness of the micro-channels

arrays

and the ability to control their individual bending.

21.8.4 biological advanceS

Alongside the advances in imaging mentioned earlier, a major development is the move from cell

targeting studies to tissue and ultimately in vivo approaches. A range of studies have now been

reported in 3-D model systems using microbeams. These have been relatively simple approaches

where a single region of a tissue reconstruct is irradiated and then measurements made at later times

using conventional approaches of xing these tissues, sectioning these into multiple slices, and

looking at distance dependence of various biological responses (Belyakov etal., 2005; Sedelnikova

etal., 2007). The coupling of multiphoton imaging approaches to microbeam exposure will allow

sophisticated real time imaging of responses at depth to delineate multicellular signaling pathways.

With higher energy microbeams, the opportunity to do in vivo exposures, especially in mouse mod-

els, will be possible. This is particularly timely as recent data has suggested long range bystander

responses can be observed using in vivo models, using relatively crude shielding opportunities

and there is an urgent need to test the responses using targeted microbeam approaches (Mancuso

etal.,

2008). Live cell imaging for cellular studies will also develop rapidly alongside multiple new

uorescent markers of response. A key requirement will be to utilize time-lapse approaches for fol-

lowing changes after microbeam irradiation. For high throughput, these will have to be done ofine

from the microbeam irradiation station. This requires good coordinate transfer approaches so that

irradiated

cells can be revisited and followed ofine.

21.9 summary

A range of approaches have been developed for producing microbeams for radiationbiology research.

These include the following: charged particles, x-rays, and electron microbeams. Signicant tech-

nological development in radiation delivery, beam detection, and positioning, alongside advances

in imaging has made microbeams powerful tools for assessing the effects of radiation exposure in

a range of biological models. Much of this has concentrated on the effects of low- dose exposures

of relevance to environmental and occupational exposures received by human populations. With

advances in both technology and biology, microbeam approaches will continue to make a signi-

cant

contribution to our knowledge of radiation effects in the future.

X-ray focus

MOXI device

X-ray source

Figure 21.4 Schematic illustration of the working principle of a MOXI lens.

592 Charged Particle and Photon Interactions with Matter

aCknowledgments

The authors are grateful to Cancer Research UK [CUK] grant number C1513/A7047, the European

Union NOTE project (FI6R 036465), and the U.S. National Institutes of Health (5P01CA095227-02)

for

funding their work.

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595

Redox Reactions of

Antioxidants:

Contributions

from

Radiation Chemistry

Aqueous Solutions

K. Indira Priyadarsini

Bhabha Atomic Research Centre

Mumbai,

India

22.1 Free radiCals: intraCellular generation and reaCtions

The general term free radical represents a molecule or atom having an unpaired electron, and

reactive free radicals are those in which the electron is readily available to react with other mol-

ecules. Oxygen-derived free radicals that have drawn the initial attention in biology are superoxide

(

i−

), hydroxyl (

•

OH), peroxyl (ROO

•

), oxyl (RO

•

), singlet oxygen (

), and hydroperoxyl (HOO

•

)

radicals. The term reactive oxygen species (ROS) was meant to denote these radicals collectively

(Halliwell, 1990; Halliwell and Gutteridge, 1993). With the discovery of nitric oxide (NO) and its

Contents

22.1 Free Radicals: Intracellular Generation and Reactions........................................................ 595

22.2 Oxidative

Stress and Antioxidants .......................................................................................597

22.3

Classication

of Antioxidants............................................................................................... 597

22.4

Redox

Reactions Involving Free Radicals............................................................................ 598

22.5

Radiation

Chemistry in the Study of Free Radicals and Antioxidants ................................ 598

22.6

Redox Studies of Antioxidants by Pulse Radiolysis............................................................. 601

22.6.1 Phenolic

Antioxidants............................................................................................... 601

22.6.1.1

Vitamin

C and Vitamin E..........................................................................603

22.6.1.2

Water-Soluble

Analogues of Vitamin E.....................................................605

22.6.1.3

Flavonoids..................................................................................................605

22.6.1.4 Resveratrol .................................................................................................608

22.6.1.5 Curcumin ...................................................................................................608

22.6.1.6 Folic

Acid...................................................................................................609

22.6.2

Non-Phenolic

Antioxidants........................................................................................609

22.6.2.1

Melatonin ...................................................................................................609

22.6.2.2 β-Carotene ................................................................................................. 610

22.6.2.3 Cyclic

Nitroxides ....................................................................................... 611

22.6.3

Sulfur

and Selenium Antioxidants............................................................................ 611

22.7

i−

Radical Reactions and Development of SOD Mimics .................................................. 613

22.8 Conclusions........................................................................................................................... 615

Acknowledgments.......................................................................................................................... 616

References...................................................................................................................................... 616

596 Charged Particle and Photon Interactions with Matter

role in essential physiology, radicals centered on nitrogen, like nitric oxide (NO), nitrogen dioxide

(NO

), and peroxynitrite (ONOO

−

), are also included in the term ROS (Fang etal., 2002; Finkel and

Holbrook, 2000; Kohen and Nyska, 2002; Winterbourn, 2008). Some authors prefer to use the term

reactive nitrogen species to denote radicals derived from nitric oxide, etc. Other important radicals

that are also crucial in biology are those derived from sulfur, halogens, and carbon. In this chapter,

all

these radicals are collectively termed as ROS.

The

intracellular production of free radicals is mediated through oxygen-centered

i−

radi-

cals and the NO

•

radical. The

i−

radical is produced through multiple biological processes like

mitochondrial respiration, phagocytosis, etc. During mitochondrial respiration,

i−

is generated

by single-electron transfer to oxygen during the process of ATP synthesis (Sies, 1997; Cadenas and

Davies, 2000; Finkel and Holbrook, 2000; Fang et al., 2002).

i−

radicals are also generated by

enzymatic reactions like NADPH oxidation by NADPH oxidase, oxidation of hypoxanthine, and

xanthine by xanthine oxidase, at times by chemical reactions like autoxidation of monoamines, etc.

(Kohen and Nyska. 2002). Under normal physiological conditions, nearly 2% of the oxygen con-

sumed by the body is converted into

i−

and other radicals. Its percentage increases during condi-

tions such as in competitive sports, activation of immune system, and exposure to environmental

factors

like pollutants, radiation, UV light, etc.

Nitric

oxide (NO

•

) is produced from l-arginine by the three isoforms of nitric oxide synthase

(NOS) enzymes (Fukuto and Ignarro, 1997; Koppenol, 1998; Priyadarsini, 2000). Both NO

•

and

i−

radicals are not very reactive, but they are converted to powerful oxidizing free radicals

•

OH, ROO

•

, RO

•

, etc., by complex transformation reactions. Some of the radical species

produce high amounts of molecular oxidants like H

, peroxynitrite (ONOO

−

), hypochlorous

acid (HOCl), etc. (Finkel and Holbrook, 2000; Winterbourn, 2008). H

is produced from many

different processes, including dismutation of

i−

radicals, the two-electron reduction of oxygen

by cytochrome P-450, during glycine metabolism pathway, etc. HOCl is produced by the reac-

tion of H

and Cl

−

by myeloperoxidase (MPO) in immunologically activated macrophages or

phagocytes. ONOO

−

is generated by the diffusion-controlled reaction of NO

•

with

i−

. Under

favorable conditions, these molecular species act as sources of free radicals. H

is converted

•

OH radicals by the presence of trace metals and HOCl produces singlet oxygen. ONOO

−

physiological pH is in equilibrium with the protonated form peroxynitrous acid (ONOOH), which

reacts with substrates through the

•

OH radical and the nitrogen dioxide (

) radical (Goldstein

and Czapski, 1995; Goldstein et al., 1996; Koppenol, 1998), and at physiological concentrations

of carbon dioxide, ONOO

−

is converted to another powerful oxidant, the carbonate radical anion

(

i−

) (Augusto et al., 2002; Bartesaghi et al., 2004; Goldstein and Merenyi, 2008). Free radi-

cals like

•

OH radicals, on reacting with cellular organelles, produce different types of secondary

radicals, and sometimes these radicals react with oxygen to produce peroxyl radicals. All these

secondary and primary reactions lead to the production of several different types of peroxyl

radicals inside cells, such as those obtained from lipids, DNA bases, amino acid radicals, thiyl

radicals, etc. Peroxyl radicals often induce chain reactions (Sonntag, 1987; Cadenas and Davies,

2000; O’Neill and Wardman, 2009). In Scheme 22.1, different primary and secondary pathways

leading to intracellular generation of ROS are given.

Free-radical induced chemical reactions inside cells are quite complex and the outcome of such

free-radical initiated cellular changes depend on several factors like site of generation, nature, and

type of cell, expression of redox proteins, activation of repair mechanisms, and many other fac-

tors (Halliwell and Gutteridge, 1993; Finkel and Holbrook, 2000; Valko et al., 2006; Winterbourn,

2008). For example, free-radical reactions with membrane lipids cause lipid peroxidation, which

leads to changes in membrane uidity and structure. Free-radical reactions with proteins can lead

to their denaturation and loss of vital enzyme activity. Free-radical reactions with DNA cause base

modication, single and double strand breaks, and mutations. Under normal conditions, most of the

free-radical mediated changes in biomolecules are reversed by the repair mechanisms, but under

pathological

conditions, the damage may become irreversible and lead to a disease.

Redox Reactions of Antioxidants 597

22.2 oxidative stress and antioxidants

In living cells, the redox environment is strictly controlled. The word “redox state” is used to describe

the cellular redox potential, which uctuates signicantly during the cellular functions (Schafer and

Buettner, 2001; Kohen and Nyska. 2002; Mats et al., 2008). When less reactive free radicals (less

powerful oxidants) are converted to more reactive species inside cells, there is a shift from reduc-

ing status to oxidative status. This shift toward an oxidative environment leads to oxidative stress.

Change in the cellular redox environment also affects signal transduction pathways, DNA, RNA

and protein synthesis, and regulation of cell cycle. Oxidative stress has now been implicated in

many diseases such as atherosclerosis, Parkinson’s disease, Alzheimer’s disease, cancer, etc., in

humans (Halliwell and Gutteridge, 1993; Finkel and Holbrook, 2000; Fang et al., 2002; Valko et al.,

2006; Winterbourn, 2008). Cells can overcome small perturbations in oxidative status and regain

their original status by endogenous antioxidant systems involving superoxide dismutase (SOD),

catalase, glutathione peroxidase, and low molecular weight antioxidants like ascorbate, tocopher-

ols, reduced coenzyme, urate, glutathione, etc. These systems either prevent the production of free

radicals or inhibit free-radical mediated damage to biomolecules, or they repair the damaged bio-

molecule. When the endogenous defense fails, cells need to be supplemented with exogenous agents

known as antioxidants. An antioxidant is therefore a reducing agent that is capable of preventing

the pro-oxidation process, or oxidative damage in cells. The recent denition of an antioxidant is

“acompound that when present in low concentrations signicantly prevents or delays oxidation of

the

bulk oxidizable substrate.”

22.3 ClassiFiCation oF antioxidants

Low molecular weight antioxidants are classied into many different types (Sies, 1997; Shi and

Noguchi, 2000; Fang et al., 2002). Depending on their solubility and polarity, they are either lipid-

soluble or water-soluble antioxidants. Vitamin E is an example of a lipid-soluble antioxidant, and

vitamin C or ascorbic acid is an example of a water-soluble antioxidant. They are also classied as

synthetic and natural antioxidants. Synthetic antioxidants are much less in number as compared

to the natural ones. Vitamins, avonoids, carotenoids, plant polyphenols, etc., are natural antioxi-

dants. Butylated hydroxy toluene (BHT) and tertiary butylhydroquinone (TBHQ) are examples of

synthetic antioxidants. Depending on their mechanism of action, they are classied as preventive

HOCI

ROO

MPO

Peroxidase

Xanthine oxidase

NADPH oxidase

L-Arginine

NO synthase

–

SOD

GSH

OH + RH

(Lipid/protein/DNA)

ONOOH

ONOO

–

+CO

ONOOCO

–

•

sCheme 22.1 Reactions leading to the intracellular formation of ROS.

598 Charged Particle and Photon Interactions with Matter

or chain-breaking antioxidants. Preventive antioxidants like desferrioximine are compounds that

form chelates with transition metals, thereby helping in the prevention of free-radical production.

Radical-scavenging antioxidants are the most important class of antioxidants. Ascorbic acid, being

a water-soluble compound, can scavenge

•

OH radicals that are produced in the cytosol, but lipid-

soluble compounds like vitamin-E, quercetin, etc., scavenge the lipid peroxyl radicals; therefore

these compounds are also termed as chain-breaking antioxidants. Vitamin E is one of the rst

reported lipid-soluble chain-breaking antioxidants, which is often considered as a standard to com-

pare the antioxidant capacity of new molecules (Pryor, 1989). Antioxidants like ascorbic acid

help in recycling chain-breaking antioxidants.

22.4 redox reaCtions involving Free radiCals

It is obvious that the function of an antioxidant is to minimize the oxidative stress, and an important

requirement for an antioxidant is to convert reactive free radicals and molecular oxidants to less

reactive species (Halliwell and Gutteridge, 1993). Due to the presence of an unpaired electron, a

reactive free radical in general participates in electron and hydrogen transfer reactions, causing

both oxidation and reduction. The oxidizing and reducing ability of a free radical not only depends

on its reduction potential, but also on the energy and electronic state of the molecule with which it

is reacting. Another parameter that decides the reactivity of a free radical is the rate constant for a

particular reaction. Therefore, knowledge of both thermodynamic and kinetic parameters is neces-

sary for diverting the course of a free-radical reaction (Wardman, 1989; Buettner, 1993; Kohen and

Nyska. 2002). The most powerful oxidizing radical is the

•

OH radical and the most powerful reduc-

ing radical is the hydrated electron (

−

), and the reactions with these radicals take place with rate

constants close to diffusion-controlled limits (Sonntag, 1987; Buxton et al., 1988). Peroxyl radicals

are of many different types and their reduction potentials vary from 0.4 to 1.3V vs. normal hydrogen

electrode (NHE) (Jonsson, 1996), and their rate constants with antioxidants can vary from 10

−1

(Neta et al., 1990).

i−

radicals can either act as an oxidant or a reductant, and their

reaction rate constants are much less than those with

•

OH radicals (Sonntag, 1987). The molecular

oxidants can induce both one- and two-electron oxidation reactions (Koppenol, 1994; Furtmuller

et al., 2005). H

is a powerful two-electron oxidant, but reacts slowly with many molecules and

does not directly react with several biomolecules. But HOCl, with a lower reduction potential, reacts

much faster with many biomolecules. NO is a poor oxidant, but reacts with

i−

to produce per-

oxynitrite, a powerful oxidant. Peroxynitrite reacts with biomolecules both by one-electron and

two-electron transfer. In addition to the thermodynamic and kinetic properties, another important

factor that is to be considered while extrapolating these results to real biological systems is the

diffusion coefcient of the radical.

i−

radicals have very low membrane permeability and react

in the same reaction region where it is generated. H

is cell permeable and therefore can travel

through different reaction zones. Other oxygen free radicals with short lifetimes like

•

OH radicals

have much restricted reaction zones (Finkel and Holbrook, 2000; Winterbourn, 2008). Table 22.1

lists different types of ROS important in biology along with their one-electron reduction potentials.

22.5 radiation Chemistry in the study oFFree

radiCals

and a

ntioxidants

Since the last half century, radiation chemists have contributed signicantly to the understanding of

fundamental biological processes, and thousands of research papers were published in the literature

in the interfacial research areas of chemistry and biology. Radiolysis of water was the common

interlink between these two branches of science, as living cells contain nearly 70% of water and

radiation induced chemical changes being non-specic, cause radiolysis of water (Bensasson et al., 1983;

Sonntag, 1987; Buxton et al., 1988; Richter, 1998). Decades of research by the radiation chemists

Redox Reactions of Antioxidants 599

have led to the complete understanding of the primary radiolytic processes in water. This also provided

a unique method to generate almost all the ROS exclusively and study their reactions with antioxi-

dants. Since several chapters in this book discuss the radiation chemistry of water in detail, it is

mentioned

only briey in this chapter.

Interaction

of ionizing radiation with dilute aqueous solutions causes initial excitation and ion-

ization of water molecules, followed by ion–molecule reactions, dissociation reactions, solvation

reactions, spur expansion reactions, etc., and at ∼10

−7

s after initial interaction of ionizing radiation,

the species present in a homogeneous distribution during the radiolysis of water can be represented

Equation 22.1 (Buxton et al., 1988; Richter, 1998):

H O e OH H H O H H O

aq2 2 2 2 3

− +

• •

, , , , , (22.1)

Out of these radical species, H

•

and

−

are reducing in nature, while

•

OH radicals are oxidizing in

nature. Employing suitable additives, such as inorganic salts and dissolved gases, it is possible to

convert these primary radicals of water radiolysis to new radicals (Buxton et al., 1988; Neta and Huie,

1988; Neta et al., 1990). The chemical reactions leading to the conversion of these primary species into

different oxidizing and reducing radicals are given in Scheme 22.2 and the relevant reactions and their

corresponding rate constants are listed in Table 22.2. With the development of the pulse radiolysis

technique, it was possible to monitor the free-radical reactions with antioxidants in real time scales.

The pulse radiolysis technique utilizes short pulses of charged particles and high-energy photons from

accelerators. These short pulses of electrons/charged particles or photons induce a non-equilibrium

situation in a small reaction zone in a very short time scale, such that a sufciently high concentration

of transient free-radical species is formed. These free-radical species are detected within their short

lifetimes, by following changes in spectroscopic properties, electrochemical properties, or changes

in spin density (Baxendale and Busi, 1981; Bensasson et al., 1983). Although modern pulse radiolysis

techniques are capable of producing much shorter pulses (<10

−12

s), most of the relevant studies with

antioxidants have been performed in the time scale of nanoseconds to a few seconds.

table 22.1

redox

otentials

of s

ome

mportant

eactive

xygen

pecies

name of the radical symbol E

(v vs. nhe) Couple

Hydroxyl

•

1.9

•

OH/OH

−

Superoxide

•−

−0.33

(

O /O

2 2

•−

)

1.80

(

•−

, 2H

O + O

)

Singlet

oxygen

0.65

O /O

•−

Alkoxyl

•

1.0–1.6

(RO

•

, H

/ROH)

Alkyl

peroxyl

ROO

•

∼0.43–1.26

ROO

•

, H

/ROOH)

Nitric

oxide

•

1.21 NO

/NO

0.39 NO/NO

−

Nitrogen dioxide

•

0.99

NO /NO

2 2

−

Carbonate

•−

1.78

CO /CO

•− −

Lipid peroxyl

LOO

•

1.0

LOO

•

/LOOH

Hydrogen

peroxide H

1.32 H

/2H

0.30

•

Hypochlorous

acid HOCl 1.08 HOCl/H

O, Cl

−

0.17

HOCl,

O, Cl

•

Peroxynitrite ONOO

−

0.2 ONOO

•

/ONOO

−

1.6

ONOO /NO

−

•