368
Organic Free radicals
CHIMIA 2012, 66, No. 6
doi:10.2533/chimia.2012.368
Chimia 66 (2012) 368–371
© Schweizerische Chemische Gesellschaft
Biomimetic Models of Radical Stress and
Related Biomarkers
Chryssostomos Chatgilialoglu*, Carla Ferreri, Annalisa Masi, Michele Melchiorre, Anna Sansone,
Michael A. Terzidis, and Armida Torreggiani
Abstract: The biological consequences of free radical production is the central subject of a very lively scientific
debate, focusing on the estimation of the type and extent of damage, as well as the efficiency of the protective
and repair systems. When studying free radical based chemical mechanisms, it is very important to establish
biomimetic models, which allow the experiments to be performed in a simplified environment, but suitably
designed to be in strict connection with cellular conditions. The biomimetic modeling approach has been coupled
with physical organic chemistry methodologies and knowledge of free radical reactivity. Molecular basis of
important processes have been identified, building up molecular libraries of products concerning unsaturated
lipids, sulfur-containing proteins and nucleic acids, to be developed as biomarkers. Ongoing projects in our
group deal with lipidomics, genomics and proteomics of free radical stress and some examples will be described.
Keywords: Biomarkers · Cyclonucleosides · Free radicals · Tandem protein–lipid damage · Trans lipids
1. Biologically Relevant Small
Radicals
Superoxide dismutase (SOD) and nitric oxide synthase (NOS) are two classes
of enzymes that control the production of
superoxide radical anion (O2•–) and induce
the formation of nitric oxide (•NO), respectively, as shown in reactions (1) and (2).[1,2]
Under physiological conditions, concentrations of ~0.1 nM O2•– and ~10 nM
•
NO play a role in regulating the activation
of transcription factors, cell proliferation
and apoptosis. During inflammatory response, their concentration can increase
up to 100-fold excess. These two radicals
are the progenitors of endogenous reactive
oxygen species (ROS) and reactive nitrogen species (RNS).
The ROS/RNS network includes molecules such as hydrogen peroxide (H2O2),
hypochlorous acid (HOCl) and peroxynitrite (ONOO–), as well as radicals such as
hydroxyl radical (HO•), nitrogen dioxide
(•NO2) and carbonate radical anion (CO3•–).
The ROS/RNS network also functions as
an efficient cellular defense mechanism, by
playing an important role for eliminating
viral and microbial infections. The overproduction of ROS/RNS has been linked
with the etiology of various diseases. The
main processes that generate HO• radicals
are depicted in reactions (3)–(5) and are
the Fenton reaction of H2O2, the reduction
of HOCl by superoxide radical anion and
the spontaneous decomposition of protonated ONOO–, respectively.[1,2]
In our laboratory the effect of radical production has been studied in aqueous systems using a biomimetic modeling
approach, coupled with physical organic
chemistry methodologies. Molecular basis
of biologically relevant radical processes,
as well as molecular libraries of products
concerning the transformations of unsaturated lipids, sulfur-containing proteins and
nucleic acids have been obtained, showing
promising applications in biomarker discovery and disease prevention.
2O2
+
2H+
SOD
NOS
Arginine
H2O2 + Fe2+
HOCl + O2
ONOO– + H+
*Correspondence: Dr. C. Chatgilialoglu
Consiglio Nazionale delle Ricerche
ISOF – BioFreeRadicals
Via Piero Gobetti 101
I-40129 Bologna
Tel.: +39 051 639 8309
Fax: +39 051 639 8349
E-mail: chrys@isof.cnr.it
H2O
γ
2. Biomimetic Model Based on
Radiation Chemistry
Radiolysis of neutral water leads to reactive species, including solvated electrons
(eaq–), hydrogen atoms (H•) and hydroxyl
radicals (HO•), together with H+ and H2O2,
as shown in reaction (6). The values in parentheses represent the radiation chemical
yields (G) in units of µmol J–1.[3]
In a N2O-saturated solution (~0.02 M
of N2O), eaq– are transformed into HO• (reaction (7), k7 = 9.1×109 M–1 s–1), affording
a G(HO•) = 0.55 µmol J–1, i.e. HO• radicals
and H• atoms account for 90% and 10%,
respectively, of the reactive species.
In the presence of thiols like the amphiphilic 2-mercaptoethanol (RSH), hy-
(1)
H2O2 + O2
(2)
Citrulline + •NO
Fe3+ + HO– + HO•
O2 + Cl– + HO•
ONOOH
•NO
2
(4)
+ HO•
eaq– (0.27), HO• (0.28), H• (0.06), H+ (0.27), H2O2 (0.07)
eaq– + N2O + H2O
HO•/H• + RSH
HO• + N2 + HO–
RS• + H2O/H2
(3)
(5)
(6)
(7)
(8)
Organic Free radicals
drogen abstraction by HO• and H• directly
produces thiyl radicals (reaction (8), k8 =
6.8×109 M–1 s–1 for HO• and k8 = 1.7×109
M–1 s–1 for H•).[3]
To investigate the reactivity of reductive species (eaq– and H•), HO• radicals have
to be scavenged. To achieve this, the most
efficient procedure is based on the addition
of an alcohol, the most commonly used being t-BuOH (0.2–0.5 m), which efficiently
scavenges HO• radicals but not H• atoms
(reaction (9), k9 = 6.0×108 M–1 s–1 for HO•
and k9 = 1.0×105 M–1 s–1 for H•).[3,4]
Thiyl radicals can be also generated by
means of direct photolysis of 2-mercaptoethanol in the presence of i-PrOH (>0.2
M). In this condition, the generation of thiyl radicals and H• atoms occurs as shown
in reaction (10) and the H• atoms are efficiently quenched by the alcohol (reaction
(11), k11 = 7.4×107 m–1 s–1). The (CH3)2C(•)
OH radical in turn reacts with the thiol to
give the thiyl radical (reaction (12), k12 =
1.2×108 m–1 s–1).[3]
3. Vesicles as Model Membranes
The natural structure of cell membranes is a double layer of phospholipids,
which are amphiphilic molecules capable
of self-organization.[5] The hydrophobic
part mostly consists of fatty acid residues,
that are carboxylic acids with a long hydrocarbon chain (up to 26 carbon atoms), saturated or unsaturated with up to six double
bonds. A specific structural feature of naturally occurring mono- and polyunsaturated
fatty acid (MUFA and PUFA) residues is
the cis double bond geometry, whereas
PUFA have the characteristic methyleneinterrupted motif of the unsaturated chain.
Liposomes are the universally accepted models for cell membranes as they
can closely simulate the bilayer structure.
Liposomes can be represented as shown in
Fig. 1, i.e. a double layer formed by spontaneous organization of the phospholipid
components in water, delimiting an aqueous cavity. The fatty acid tails can be saturated or unsaturated, and the disposition of
the double bonds in the vesicle depends on
the supramolecular arrangement of the bilayer. Monolamellar vesicles are the closest models to membranes and they can be
formed by different techniques, such as the
extrusion[6] and the injection[7] methodologies.
4. Trans-Fatty Acid Residues as
Biomarkers of Radical Stress
Scheme 1 shows the reaction mechanism of cis–trans isomerization that consists of a reversible addition of radical RS•
to the double bond. Indeed, the reconsti-
CHIMIA 2012, 66, No. 6
(CH3)2C(OH)CH2• + H2O/H2
HO•/H• + t-BuOH
RSH
hν
(9)
(10)
RS• + H•
(CH3)2COH + H2
H• + i-PrOH
(CH3)2COH + RSH
369
(11)
i-PrOH + RS•
(12)
Fig. 1. Large unilamellar vesicles in two
different representations.
tution of the double bond is obtained by
β-elimination of RS• and the result is in
favor of trans geometry, the most thermodynamically favorable disposition.[8] It is
worth noting that i) the radical RS• acts as
a catalyst for cis–trans isomerization, and
ii) positional isomers cannot be formed as
reaction products because the mechanism
does not allow a double bond shift.[9,10]
Considering polyunsaturated substrates,
the isomerization mechanism occurs as
a step-by-step process, i.e. each isolated
double bond behaves independently as
discussed above.[9,10]
Vesicles formed by unsaturated lipid
were found to be a good biomimetic model
for the double-bond isomerization. From
the early studies,[9,10] arachidonic acid residues in membrane phospholipids emerged
as relevant markers to be investigated, in
order to distinguish endogenous trans isomers, formed by radical processes, from
the exogenous trans isomers, known to derive from partially hydrogenated fats in the
diet. Considering the biosynthetic paths of
omega-6 fatty acids, the double bonds 5
and 8 of arachidonic acid can evidently be
formed by desaturase enzymes and therefore have only a cis configuration, unless
these positions are involved in a radicalbased isomerization process in the membranes.
We extended the biomimetic investigation to biological systems, in order to prove
the ‘endogenous’ trans lipid formation under strictly physiological conditions. It was
important to deal with ‘trans-free’ conditions, which means that the presence of any
external source of trans fatty acid isomers
is carefully checked. Cell membrane lipid
composition was monitored during incubation in the absence and presence of thiol
compounds, ensuring that no contribution
of trans compounds could come from the
medium. The trans-arachidonate content
determined in membrane phospholipids of
human leukemia cell lines (THP-1) provided the first indication of the occurrence of
an endogenous isomerization process.[11]
This opened new perspectives for the role
of trans lipids in the lipidome of eukaryotic cells[12] and was followed by several
other investigations in living systems.[13]
More recently peroxidation and isomerization processes, both supported by the
free radical reactivity of thiol compounds,
were evaluated in a biomimetic model of
linoleic acid micelles. Indeed, the catalytic
katrans = 2.9 x 105 M–1 s–1
kacis = 1.6 x 105 M–1 s–1
RS•
+ R
1
R2
kacis
RS
kfcis
H
R1
kfcis = 1.7 x 107 s–1
H
R2
katrans
kftrans
RS•
+
R2
R1
kftrans = 1.6 x 108 s–1
Scheme 1. Reaction mechanism for the cis–trans isomerization catalyzed by thiyl radicals; Rate
constants of addition (ka) and fragmentation (kf) at 25 °C.
370
Organic Free radicals
CHIMIA 2012, 66, No. 6
tyric acid and alanine, respectively (Fig.
2).[18] Concomitant formation of diffusible
sulfur-centered radicals (MeS• or S–•), able
to migrate into the lipid bilayer, can induce
cis–trans isomerization of unsaturated
fatty acid residues.[19] Reductive radical
stress, causing a desulfurization process,
is therefore coupled with the formation of
S-centered radicals that in turn can diffuse
apart and become responsible for the damage transfer from proteins to lipids.
These reductive modifications assayed
in different peptide/protein sequences (e.g.
Met-enkephalin,[20] β-amyloid,[21] RNase
A,[22] Human serum albumin[23]) constitute
an integration of the molecular inventories
that up to now take into account only oxidative transformations. They can be useful
to achieve an integrated vision of the free
radical reactivity in a multifunctional system and, overall, for wider applications in
the redox proteomics field.
Fig. 2. Tandem protein–lipid damage under reductive radical stress.
process of cis–trans isomerization can occur parallel with radical chain peroxidation, producing in the same experiment
trans fatty acids and hydroperoxides.[14]
Mono-trans isomers of cholesteryl linoleate and arachidonate have been synthesized by thiyl radical-catalyzed cis–
trans isomerization and fully characterized. This small trans library allowed the
identification to be carried out in human
plasma by GC, Raman, and IR analyses,
demonstrating for the first time the presence of specific regioisomers connected to
free radical stress.[15]
Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid present in fish oils
used for omega-3 enriched foods and diets.
The natural cis double bond geometry can
be transformed to the trans configuration
during the deodorization process utilized
in the food industry. The analytical discrimination of the five possible mono-trans
regioisomers represents a limiting step for
the recognition and structure−activity relationship in connection with the harmful
effects of trans-fatty acids in health. We
carried out a dual synthetic strategy, providing new access to mono-trans EPA isomers and valuable information on GC and
NMR characteristics for further applications in metabolomics and lipidomics. This
library was used as an analytical reference
for isomer determination in deodorized
fish oils and the follow-up of rats fed fish
oil diets, evidencing for the first time that
mono-trans EPA isomers are incorporated
in liver mitochondria membranes after dietary intake.[16]
compounds in the cell, and iii) the evidence
of trans-lipid increase in cultured cells in
the presence of thiols ensuring that no contribution of trans compounds could come
from the medium.[17]
Possible scenarios for the production
of diffusible thiyl radicals were expanded
by the discovery of free-radical damage
to sulfur-containing proteins resulting in
consequent isomerization of membrane
lipids. The complex scenario of radical
stress reactions affecting peptides/proteins
can be better elucidated through the design of biomimetic studies simulating the
consequences of the different free radicals
attacking amino acids. In this context, ionizing radiations allowed the examination
of the specific damage caused by H-atoms
and electrons coupled with protons, thus
establishing the molecular basis of reductive radical stress. This is an innovative
concept that complements the well-known
oxidative stress, also in view of a complete
understanding of the global consequences
of radical species reactivities on living
systems. A recent review summarizes the
knowledge of the chemical changes present in sulfur-containing amino acids occurring in polypeptides under reductive radical
conditions, in particular the transformation
of Met and Cys residues into α-amino bu-
5’,8-Cyclo-2’-deoxyadenosine
and
5’,8-cyclo-2’-deoxyguanosine in their 5’R
and 5’S diastereomeric forms are tandemtype lesions observed among the DNA
modifications and identified in mammalian cellular DNA in vivo (Fig. 3).[24] These
lesions result from the chemistry of the
C(5)’ radicals generated by the attack of
HO• radicals to 2-deoxyribose units.
Synthetic procedures of compounds
1–4 were developed starting from 8-bromopurine derivatives under continuous
radiolysis or photolysis.[25,26] These procedures involve a radical cascade reaction
that mimics the DNA damage causing the
formation of 5’,8-cdAdo and 5’,8-cdGuo
lesions. The reaction of HO• radicals with
2’-deoxyadenosine and 2’-deoxyguano-
NH2
N
HO
O
N
N
N
N
HO
O
N
N
N
OH
3 (5'S)
N
2 (5'R)
O
O
N
OH
1 (5'S)
HO
Fig. 3. The four purine
5’,8-cyclo-2’-deoxynucleosides.
NH2
OH
5. Reductive Radical Stress and
Tandem Lipid-Protein Damage
There are strong experimental supports that the origin of endogenous formation of trans lipids is correlated with radical stress. However, up to now it has not
been demonstrated what the real culprits
are for the cis–trans isomerization. Thiyl
radicals are in the pole position on the basis of i) the efficiency of the thiyl radicalcatalyzed cis–trans isomerization in vitro,
ii) the presence of many sulfur-containing
6. Purine 5’,8-Cyclonucleoside
Lesions
O
N
NH
N
NH2
HO
O
N
OH
4 (5'R)
NH
N
NH2
Organic Free radicals
CHIMIA 2012, 66, No. 6
NH2
N
HO
O
N
N
N
N
HO
kc = 1.6 x105 s–1
OH
N
OH
6
O
N
O
N
N
O
5
HO
Scheme 2. Radical
cyclization of C(5)’
radicals in purine
2-deoxyribo derivatives.
NH2
N
O
N
NH
N
NH2 k = 6.9 x105 s–1
c
HO
OH
7
O
N
NH
N
NH2
OH
8
Fig. 4. Radiationinduced formation
of 5’,8-cdAdo and
5’,8-cdGuo in N2/O2
saturated aqueous
solutions of DNA.
sine under normoxic conditions was also
investigated.[27] The minor path of C(5)’
radical formation was found to be ca. 10%
by quantifying the hydrated 5’-aldehyde in
both experiments. Pulse radiolysis studies
focused on the rate constants of cyclization
at nucleoside level. Scheme 2 shows these
cyclizations as well as the corresponding
rate constants at room temperature. Both
cyclizations are in the range 105–106 s–1,
radical 7 being four times faster that radical
5.[25,28] Quantitative determination of these
lesions in biological samples as biomarkers of free radical damage is a challenge.
Results reported for irradiated samples of
calf thymus DNA have been critically reviewed,[24] underlining the need for further
research for the potential involvement of
these lesions in human health. Fig. 4 illustrates the purine 5’,8-cyclonucleosides
measured as the sum of diastereomers by
HPLC-MS/MS in enzymatically digested
DNA samples: the diastereomeric ratio
5’R/5’S being ~4 and ~3 for 5’,8-cdAdo
and 5’,8-cdGuo, respectively.[29]
7. Conclusions and Future
Directions
This mini-review offers an overview of
the main processes studied in our laboratory. Ongoing projects also deal with the application of the molecular libraries as biomarkers of free radical stress in lipidomics,
genomics and proteomics. Our biomimetic
approach is a convenient tool for the examination of free radical reactivity, particularly for a chemical biology approach
where the behavior of free radical species
in the liposomal or oligonucleotide models can satisfactorily be transferred to the
transformations occurring in the biological context. In this view an improvement
of interdisciplinarity is needed, among the
fields of chemistry, biochemistry, biology
and medicine, in order to create a common
territory where the achievements of free
radical mechanisms can be straightforwardly transferred to a better comprehension of the biological pathways in health
and diseases.
Acknowledgements
Financial support from the Ministero
dell’Istruzione,
dell’Università
della
Ricerca
(PRIN-2009K3RH7N_002)
and
Marie Curie Intra-European Fellowship
(CYCLOGUO–298555) as well as the sponsorship of COSTAction CM0603 on ‘Free Radicals
in Chemical Biology (CHEMBIORADICAL)’
are gratefully acknowledged.
Received: March 6, 2012
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