COST Action CM1201
Biomimetic Radical Chemistry
2nd MC meeting & 1st Annual Scientific Meeting
May 5th – 7th 2013
Bologna, Italy
Organizing Committee:
Chryssostomos Chatgilialoglu
Carla Ferreri
Annalisa Masi
Michele Melchiorre
Anna Sansone
Michael A. Terzidis
Armida Torreggiani
ISOF, Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, I-40129 Bologna
2
Main Programme
Sunday Evening, May 5th 2013: Palazzo GRASSI (Bologna, Italy)
18:30-20:30
nd
2 Management Committee Meeting
Agenda
1. Welcome to participants
2. Adoption of agenda
3. Approval of minutes and matters arising of last meeting
4. Update from the Action Chair
a. Status of Action, including participating countries
b. Action budget status
c. STSM status and new applications
5. Promotion of gender balance and of Early Stage Researchers (ESR)
6. Update from the Grant Holder
7. Update from the COST Office
8. Update from the DC Rapporteur
9. Annual Progress Conference (preparation and/or feedback from DC)
10. Follow-up of MoU objectives
a. Progress report of working groups
11. Scientific planning
a. Scientific strategy
b. Action Budget Planning
c. Long-term planning (including anticipated locations and dates of future activities)
d. Dissemination planning (Publications and outreach activities)
12. Requests for new members
13. Non-COST applications to the Actions
14. AOB
15. Location and date of next meeting
16. Summary of MC decisions
17. Closing
20.30-23:30
FESTIVE DINNER
3
Monday, May 6th 2013: Consiglio Nazionale delle Ricerche, Bologna
09:30-09:50
09:50-10:10
10:10-10:30
10:30-10:50
10:50-11:10
11:10-11:40
11:40-12:00
12:00-12:20
12:20-12:40
12:40-13:00
13:00-13:20
13:30-14:30
14:30-14:50
14:50-15:10
15:10-15:30
15:30-15:50
15:50-16:10
16:10-16:40
16:40-17:00
17:00-17:20
17:20-17:40
17:40-18:00
Registration and Welcome from the Action Chair (Chrys Chatgilialoglu)
WG2: Models of DNA Damage and Consequences. Session 1
Jean-Luc Ravanat
“Recent aspects of radiation-induced DNA lesions”
Martine E. Lomax
“Processing AP sites as single lesions and within clustered damaged sites when
present in mononucleosomes”
Tsvetan G. Gantchev
“The dynamic structure of cis-platinum (CPT) intra- and inter-strand cross-linked
DNA and the interactions with hydrated electrons”
Leif A. Eriksson
“Computational Modeling of ROS/Radical-induced DNA Damage Processes”
COFFEE BREAK
WG1: Radical Enzymes. Session 1
Bernard T. Golding
“How do microbes degrade hydrocarbons in the oxygen-free biosphere?”
Jerzy L. Gebicki
“Inhibitory effect of flavonoids on catalase: structure-activity relationship”
Aleksandra Jankovic
“Impacts of manganese (II) pentaazomacrocyclic superoxide dismutase mimic in
diabetes”
Matthias Boll
“Reduction of aromatic rings by dearomatizing arylcarbonyl-CoA reductases”
Myriam Seemann
“GcpE and LytB, two Fe/S enzymes involved in the biosynthesis of isoprenoids”
LUNCH – CNR cafeteria
WG3: Membrane Stress, Signalling and Defences. Session 1
Shlomo Sasson
“The impact of nutritional overload-induced phospholipid remodeling on
pancreatic β-cell function and dysfunction”
Elena E. Pohl
“Protein activation by reactive aldehydes”
Manar Aoun
“Novel biomarkers for radical stress attack in mitochondria and plasma: mtDNA
lesions, trans-PUFA in membrane phospholipids and isoprostanes”
Branka Mihaljevic
“Radical scavenging activity of trans-resveratrol: photophysical and
photochemical properties of resveratrol”
Michele Melchiorre
“Free radical stress and fatty acid-based lipidomics”
COFFEE BREAK
WG4: Bio-Inspired Synthetic Strategies. Session 1
Armido Studer
“Oxidative biomimetic carbene catalysis”
Lars Engman
“Catalytic Antioxidants”
Guillaume Povie
“Catechol derivatives in radical chain reactions”
Angeles Martin
“New synthetic strategies based on C–, O– and N–centered radicals”
FREE EVENING
4
Tuesday, May 7th 2013: Consiglio Nazionale delle Ricerche, Bologna
09:30-09:50
09:50-10:10
10:10-10:30
10:30-10:50
10:50-11:10
11:10-11:40
11:40-12:00
12:00-12:20
12:20-12:40
12:40-13:00
13:00-13:20
13:30-14:30
14:30-14:50
14:50-15:10
15:10-15:30
15:30-15:50
15:50-16:10
16:10-16:40
16:40-17:00
17:00-17:20
17:20-17:40
17:40-18:00
WG2: Models of DNA Damage and Consequences. Session 2
Michael A. Terzidis
“Purine 5’,8-cyclo-2’-deoxynucleoside lesions”
Krzysztof Bobrowski
“Pulse radiolysis: a tool for investigating radical processes in biological molecules”
Massimo Bietti
“Laser flash photolysis as a tool for the study of hydrogen atom transfer and
electron transfer reactions of oxygen centered radicals”
Dimitar Angelov
“Nucleosome remodeling and base excision repair”
Alexandros G. Georgakilas
“Radiation-induced clustered DNA lesions: induction and repair”
COFFEE BREAK
WG1: Radical Enzymes. Session 2
Frank Dekker
“Novel inhibitors and activators of lipoxygenases and their role in NF-κB signaling”
Athanassios Nicolaides
“High- and room-temperature isomerizations of dimers of highly pyradalized
alkenes”
Tamis Darbre
“Peptide dendrimers with bipyridine at the core as models of metaloenzymes: pHtuned metal coordination and peroxidase activity”
Radu Silaghi-Dumitrescu
“Computational approaches on two free radical generating systems: the peroxide
chemistry specific to bleomycin and P450, and the cobalt corrins”
Ioulia Smonou
“Biocatalysed reactions and one-pot synthesis of pharmaceutical precursors”
LUNCH – CNR cafeteria
WG4: Bio-Inspired Synthetic Strategies. Session 2
Ullrich Jahn
“New catalytic electron transfer- induced and thermal radical cyclizations”
Gabor Speier
“Radical chemistry of a flavin coenzyme mimic“
Ioannis N. Lykakis
“Green photocatalytic organic transformations using mesoporous-metal-oxidesupported gold nanoparticles and polyoxometalates”
Cyril Ollivier
“Bio-inspired electron transfer processes for fine chemical synthesis”
Jozsef Kaizer
“Iron complexes as functional models for the soluble methane monooxygenase
enzyme“
COFFEE BREAK
WG3: Membrane Stress, Signalling and Defences. Session 2
Tomris Ozben
“Bleomycin increases trans lipid isomers in human testicular cancer cell
membrane”
Ivana Tartaro Bujak
“The influence of natural occuring antioxidants on lipid peroxidation and
isomerization processes in model micellar system“
Naila Rabbani
“Glycation of HDL and LDL by methylglyoxal causes structural remodelling linked
to increased atherogenicity”
Kyriakos E. Kypreos
“Novel causative relationship between low HDL and diet-induced nonalcoholic
fatty liver disease”
FREE EVENING
5
List of Participants
Dimitar
ANGELOV
Ecole Normale Supérieure de
Lyon – Laboratoire de Biologie France
Moléculaire de la Cellule
[email protected]
Manar
AOUN
Université de Montpellier 1 –
Institut Universitaire de
Recherche Clinique
France
[email protected]
Massimo
BIETTI
Università di Roma "Tor
Vergata" – Dip. di Scienze e
Tecnologie Chimiche
Italy
[email protected]
Krzysztof
BOBROWSKI
Institute of Nuclear Chemistry
and Technology – Warsaw
Poland
[email protected]
Matthias
BOLL
University of Freiburg –
Institute of Biology II
Germany
[email protected]
Chryssostomos
CHATGILIALOGLU
Consiglio Nazionale delle
Ricerche – ISOF – Bologna
Italy
[email protected]
Tamis
DARBRE
University of Bern –
Department of Chemistry and
Biochemistry
Switzerland
[email protected]
Frank
DEKKER
University of Groningen –
Department of Pharmaceutical Netherlands [email protected]
Gene Modulation
Lars
ENGMAN
Uppsala University –
Department of Chemistry
Sweden
[email protected]
Leif A.
ERIKSSON
University of Gothenburg –
Department of Chemistry and
Molecular Biology
Sweden
[email protected]
Carla
FERRERI
Consiglio Nazionale delle
Ricerche – ISOF – Bologna
Italy
[email protected]
Tsvetan
GANTCHEV
Bulgarian Academy of Sciences
– Institute of Molecular Biology Bulgaria
– Sofia
Lodz University of Technology
– Institute of Applied Radiation Poland
Chemistry
Lodz University of Technology
– Institute of Applied Radiation Poland
Chemistry
National Technical University of
Athens – Department of
Greece
Physics
Lidia
GEBICKA
Jerzy L.
GEBICKI
Alexandros G.
GEORGAKILAS
Bernard
GOLDING
University of Newcastle upon
Tyne – School of Chemistry
Instituto de Productos
Antonio J.
Naturales y Agrobiología del
HERRERA GONZALEZ CSIC – Tenerife
Romanian Academy – Institute
E. Gabriela
of Physical Chemistry “Ilie
IONITA
Murgulescu” – Bucharest
Academy of Sciences –
Ullrich
Institute of Organic Chemistry
JAHN
and Biochemistry, Prague
6
[email protected]
[email protected]
[email protected]
[email protected]
U.K.
[email protected]
Spain
[email protected]
Romania
[email protected]
Czech
Republic
[email protected]
Aleksandra
JANKOVIC
University of Belgrade –
Department of Physiology
Serbia
aleksandra.jankovic@ibiss.
bg.ac.rs
József
KAIZER
University of Pannonia –
Department of Chemistry –
Veszprem
Hungary
[email protected]
Kyriakos E.
KYPREOS
University of Patras –
Department of Medicine –
Pharmacology laboratory
Greece
[email protected]
Martine
LOMAX
University of Oxford – Gray
Institute for Radiation Oncology U.K.
and Biology
[email protected]
x.ac.uk
Ioannis N.
LYKAKIS
Aristotle University of
Thessaloniki – Department of
Chemistry
Greece
[email protected]
Annalisa
MASI
Consiglio Nazionale delle
Ricerche – ISOF – Bologna
Italy
[email protected]
Angeles
MARTIN HERNANDEZ
Instituto de Productos
Naturales y Agrobiología del
CSIC – Tenerife
Spain
[email protected]
s
Michele
MELCHIORRE
Consiglio Nazionale delle
Ricerche – ISOF – Bologna
Italy
[email protected]
r.it
Branka
MIHALJEVIĆ
Ruder Boskovic Institute –
Radiation Chemistry and
Dosimetry Lab. – Zagreb
Croatia
[email protected]
Athanassios
NICOLAIDES
University of Cyprus –
Department of Chemistry,
Lefkosia
Cyprus
[email protected]
Cyril
OLLIVIER
Université Pierre et Marie
Curie, Univ Paris 06 – Institut France
Parisien de Chimie Moléculaire
Tomris
ÖZBEN TOMASI
Guillaume
POVIE
Akdeniz University Medical
Faculty – Department of
Biochemistry, Antalya
University of Veterinary
Medicine, Vienna – Institute of
Physiology, Pathophysiology
and Biophysics
University of Bern –
Department of Chemistry &
Biochemistry
Naila
RABBANI
University of Warwick – Clinical
U.K.
Sciences Research Institute
[email protected]
Jean-Luc
RAVANAT
CEA Grenoble –
INAC/SCIB/LAN
France
[email protected]
Michela
SALAMONE
Università di Roma "Tor
Vergata" – Dip. di Scienze e
Tecnologie Chimiche
Italy
michela.salamone@
uniroma2.it
Anna
SANSONE
Consiglio Nazionale delle
Ricerche – ISOF – Bologna
Italy
[email protected]
Shlomo
SASSON
The Hebrew University – Dept.
Israel
of Pharmacology – Jerusalem
Elena
POHL
7
[email protected]
Turkey
[email protected]
Austria
[email protected].
at
Switzerland
[email protected].
ch
[email protected]
c.il
Myriam
SEEMANN
Université de Strasbourg –
Institut de Chimie
France
[email protected]
Babes-Bolyai University –
Radu
Faculty of Chemistry and
SILAGHI-DUMITRESCU Chemical Engineering
Romania
[email protected]
Ioulia
SMONOU
University of Crete –
Department of Chemistry
Greece
[email protected]
Gábor
SPEIER
University of Pannonia –
Department of Chemistry –
Veszprem
Hungary
[email protected]
Armido
STUDER
Universität Münster –
Germany
Organisch-Chemisches Institut
[email protected]
Ivana
TARTARO BUJAK
Rudjer Boskovic Institute –
Radiation Chemistry and
Dosimetry Lab. – Zagreb
Croatia
[email protected]
Michail A.
TERZIDIS
Consiglio Nazionale delle
Ricerche – ISOF – Bologna
Italy
[email protected]
Armida
TORREGGIANI
Consiglio Nazionale delle
Ricerche – ISOF – Bologna
Italy
[email protected]
.it
Luca
VALGIMIGLI
University of Bologna –
Department of Organic
Chemistry“A. Mangini”
Italy
[email protected]
Abel
VIEIRA
New University of Lisbon –
Department of Chemistry
Portugal
[email protected]>
Neven
ZARKOVIC
Rudjer Boskovic Institute –
LabOS – Zagreb
Croatia
[email protected]
Notes
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8
Abstracts of Seminars
9
May 6th 2013 - 9:50 - WG2: Models of DNA damage
Recent Aspects of Radiation-Induced DNA Lesions
Jean-Luc RAVANAT
Laboratoire Lésions des Acides Nucléiques, INAC-SCIB UMR E-3 CEA-UJF, CEA Grenoble,
17 rue des Martyrs, 38054 Grenoble cédex 9, France
During the last three decades considerable efforts have been made to
determine the nature and quantify the amount of lesions produced in
double stranded (ds)DNA exposed to ionizing radiation. Thus an almost
complete decomposition pathway of the four DNA bases mediated by
hydroxyl radicals produced during irradiation is now available. About 70
different DNA lesions have been identified and about 15 of them have
been quantified in cells treated with ionizing radiations .
Regarding the chemical aspects of the undergoing reactions, the
mechanisms of decomposition of the DNA bases determined at the
nucleoside level are most of the time similar in dsDNA. However,
differences exist highlighting the fact that the 3D structure of DNA
somehow plays a role in the decomposition of the initially generated
radicals.
Recently we have demonstrated that in dsDNA complex DNA lesions could
be significantly generated trough a single oxidation event. These include
tandem DNA lesions that could be produced in significant levels through
peroxidation reactions and inter-strand crosslinks that are generated
through the initial formation of reactive aldehydes arising from
decomposition of the 2’-deoxyribose moiety. These two examples indicate
that the described mechanisms of decomposition of the DNA bases could
be different in dsDNA compared to free nucleosides and thus additional
work has to be done to improve our knowledge of these reactions in
dsDNA. Moreover, this also indicates that in a cellular environment,
biomolecules surrounding DNA could also play a role in the mechanisms
of decomposition DNA radicals produced by ionizing radiation. In that
respect additional work is required to study DNA-protein crosslinks.
10
May 6th 2013 - 10:10 - WG2: Models of DNA damage
Processing AP Sites as Single Lesions and within Clustered
Damaged Sites when Present in Mononucleosomes
Martine E. LOMAX
CRUK-MRC Gray Institute for Radiation Oncology and Biology, Department of Oncology,
University of Oxford, Oxford, OX3 7DQ, UK
A signature of ionising radiation exposure is the induction of DNA
clustered damaged sites, defined as two or more lesions within one to two
helical turns of DNA by passage of a single radiation track. DNA within
cells is associated with histone proteins and compacted, with
nucleosomes being the first order of higher level structure.
Mononucleosomes were reconstituted using DNA with abasic (AP) sites
present as single lesions or contained within clustered DNA damaged sites.
The AP site was either cleaved or repaired in in vitro assays using purified
APE1 or CHO-K1 nuclear extract. Cleavage of a single AP site present in a
nucleosomal template by purified APE1 is significantly retarded compared
to cleavage of an AP site in naked DNA, however this retardation is
alleviated by incubation with CHO-K1 nuclear extract. No subsequent
repair of the AP site is seen when the AP site is present in a
mononucleosome, in contrast to efficient repair of the AP site when
present in naked DNA. Efficient cleavage is observed by CHO-K1 nuclear
extract of bistranded AP sites within a clustered DNA damage site in a
nucleosomal template, resulting in the formation of double strand breaks
(DSB). In contrast, only the AP site is cleaved within a clustered damaged
site containing bistranded AP site and 8-oxoG residues but with reduced
efficiency compared to an isolated AP site, thus no DSB are formed.
These studies give insights into the processing of ionising radiation
induced clustered damaged sites.
11
May 6th 2013 - 10:30 - WG2: Models of DNA damage
The Dynamic Structure of cis-Platinum (CPT) Intra- and InterStrand Cross-Linked DNA and the Interactions with Hydrated
Electrons
Tsvetan G. GANTCHEV
Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
We are applying molecular modeling, nanosecond molecular dynamics
(MD) and supercomputing resources to investigate the dynamic structure
of intra- and inter-strand cross-linked by cis-platinum (CPT) DNA and the
interactions of solvated electrons, eaq– with DNA. This project stems from
recent findings that CPT acts as a radiosensitizer during radiation therapy
leading to multiplied DNA damages (numerous strand breaks). The
results from our ongoing studies show the presence of specific global and
local deformations in CPT-DNA, which are significantly different for intravs. inter-strand cross-linked DNA, and include type B↔A DNA transitions,
polymorphous DNA dynamic structure (conformation) at the damage site
as a result of orchestrated interactions with counter-ions, and not the last,
large DNA total bending up to 40-50o provoked by the CPT-crosslinks (3038o per helical turn with the CPT at the middle). The figure below
illustrates the large opening of the minor groove at the intra-strand G-PtG site and the DNA bend as compared to unmodified DNA. We believe our
structural dynamics findings will help in better understanding of a range
of biological effects, related to bulky DNA damages, such as DNA
interactions and damage recognition by nucleotide excision repair (NER)
proteins, and specifically in the case of CPT-DNA, its interactions with
HMGB chromatin remodeling proteins. In addition, we show explicitly that
eaq– , modeled as Kevan’s
[H2O]6 anionic water cluster has preferential
localization near the CPT-site, i.e. directly interacts with the Pt-atom and
is probably involved in a redox reaction Pt(II) → Pt (I) leading to Pt-atom
dissociation and generation of free radicals at the DNA site.
Figure legend: Superimposed averaged structures after ca. 40 ns MD of
normal DNA (green ribbon), normal DNA axis (blue beads) and CPT-DNA
with 1,2-Pt intrastrand crosslink (orange ribbon), CPT-DNA axis (redorange beads), cross-linked GG nucleotides (sticks, atom color), Pt-atom
(yellow sphere).
12
May 6th 2013 - 10:50 - WG2: Models of DNA damage
Computational Modeling of ROS/Radical-induced DNA Damage
Processes
Leif A. ERIKSSON
Department of Chemistry and Molecular Biology, University of Gothenburg, 412 96
Göteborg, Sweden
We have for more than 15 years studied the effects of high energy
radiation and reactive oxygen species on nucleobases and small model
systems, based on density functional theory (DFT) methodology.
An overview of our studies is presented that includes both computations
of EPR parameters for intermediate radicals formed, as well as reaction
mechanisms leading to different lesions such as strand breaks and base
release processes in DNA and RNA model systems.
We will also present work relating to molecular dynamics simulations of
larger systems, such as intercalation of photosensitizers in DNA, and
interactions between DNA and hydroxyl radical generating anticancer
drugs bleomycin and anthramycins.
13
May 6th 2013 - 11:40 - WG1: Radical Enzymes
How Do Microbes Degrade Hydrocarbons in the Oxygen-Free
Biosphere?
Bernard T. GOLDING
School of Chemistry, Newcastle University, Bedson Building, Newcastle upon Tyne, NE1
7RU, UK
In collaboration with German colleagues, we are studying the mechanism
of dioxygen-free microbial attack on alkanes initially using hexane as a
representative saturated hydrocarbon. The pathway discovered may
represent an evolutionary ancient mode of microbial hydrocarbon
utilisation, which was originally widespread, but upon the rise of
atmospheric dioxygen was confined to subsurface habitats including
petroleum reservoirs and the deep sea. Saturated hydrocarbons (alkanes)
are widespread natural organic compounds that are the main constituents
of fossil organic matter and play a fundamental role in the global carbon
cycle. They are generated by thermal reactions deep within sedimentary
basins and, over geologic timescales, may accumulate in petroleum
reservoirs. Hydrocarbons represent a major substrate pool for
microorganisms (anaerobes) living in the subterranean oxygen-free
biosphere. The degradation of hydrocarbons by these anaerobes requires
attack on a strong carbon-hydrogen bond, which is performed by a
'radical enzyme' using fumaric acid in place of dioxygen. The destruction
of oil spills also depends on the activity of such anaerobes. Whereas
biodegradation of hydrocarbons with dioxygen is long known and
understood in much detail, the dioxygen-independent process was poorly
understood until the present study.
Using (R,R)- (S,S)- and meso-2,5-dideuterohexanes, we have found that
an enzyme’s cysteinyl radical stereospecifically abstracts the pro-S
hydrogen atom from C-2 of hexane in a possible concerted reaction in
which the hex-2-yl moiety adds to fumarate (Figure 1). The ensuing
sequence leads to (R)-4-methyloctanoate (coenzyme A ester), which is
further degraded (Figure 2). Elucidation of this pathway was aided by the
design of novel synthetic routes to reference compounds. Current work is
focussed on mechanistic studies with decane and toluene, as well
attempts to devise functional model systems for the remarkable
biochemistry described. Understanding how radical enzymes functionalise
hydrocarbons could lead to new industrial processes utilising hydrocarbon
feedstocks.
Reference: Stereochemical investigations reveal the mechanism of the
bacterial activation of n-alkanes without oxygen, Angew Chem Int Ed,
2012, 51, 1334-1338.
14
Figure 1: Concerted mechanism for
combination of hexane with fumarate.
Figure 2: Complete pathway for hexane
degradation.
15
May 6th 2013 – 12:00 - WG1: Radical Enzymes
Inhibitory Effect of Flavonoids on Catalase. Structure – Activity
Relationship
Justyna Krych, Jerzy L. GEBICKI and Lidia Gebicka
Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of
Technology (TUL), Lodz, Poland
Catalase, a four subunits ferriheme enzyme belongs to the antioxidant
defence system of the cell. It catalyzes decomposition of H2O2 into water
and molecular oxygen. The reaction proceeds in two steps. First, H2O2
oxidizes the heme iron of the resting enzyme to oxoferryl derivative with
a π-cationic porphyrin radical, called compound I. This step is followed by
the oxidation of a second molecule of H2O2 by compound I. With
appropriate substrates, however, compound I can undergo a single
electron reduction to form compound II in which porphyrin radical is
reduced, but the oxoferryl metal center is retained. This compound is
unreactive to H2O2. We have found that some natural polyphenols,
flavonoids, inhibit catalase activity via one-electron reduction of catalase
compound I. Structure-activity relationship for this process has been
observed. Among investigated flavonoids myricetin appears to be the
most potent inhibitor. The biological consequences of flavonoid-catalase
interactions will be discussed.
16
May 6th 2013 - 12:20 - WG1: Radical Enzymes
Impacts of Manganese (II) Pentaazomacrocyclic Superoxide
Dismutase Mimic in Diabetes. Adipose Tissue as a Target
Aleksandra JANKOVIC
University of Belgrade, Department of Physiology, Institute for Biological Research
“Sinisa Stankovic”, Bulevar despota Stefana 142, 11060 Belgrade, Serbia
Lipids overflow from adipocytes and altered synthesis of adipokines such
as adiponectin and resistin compromise overall lipid and glucose
homeostasis, i.e. normal insulin sensitivity. We examined whether
changes in the adipose tissue function of diabetic rats are associated with
tissue redox changes, and whether these could be modulated by synthetic
superoxide dismutase (SOD) mimic-Mn(II)(pyane)X2. For this purpose,
rats were divided into nondiabetic and alloxan-induced diabetic groups,
both additionally subdivided into nontreated and the group treated for 7
days with Mn(II)(pyane)X2. There were no significant changes in the
expression levels of SODs and catalase. However, compared to untreated
diabetic group, Mn(II)(pyane)X2 normalizes increased levels of
glutathione-peroxidase (GSH-Px) and endothelial nitric oxide synthase
(eNOS) protein expression observed in diabetic untreated rats. Besides,
extensive increase of resistin protein level in adipose tissue of diabetic
rats was restituted to control values after 7-day Mn(II)(pyane)X2
treatment. Such results lead to the hypothesis that Mn(II)(pyane)X2 may
ameliorate insulin sensitivity and metabolic homeostasis in diabetes,
through normalization of resistin synthesis, and that this effect of SOD
mimic may involve nitric oxide.
17
May 6th 2013 - 12:40 - WG1: Radical Enzymes
Reduction of Aromatic Rings by Dearomatizing Arylcarbonyl-CoA
Reductases
Matthias BOLL
Institute of Biology II, University of Freiburg, Germany
In anaerobic bacteria the over most part of aromatic growth substrates
are degraded via the key intermediate benzoyl-CoA that serves as
substrate for dearomatizing reductases. These enzymes catalyze a
“Biological Birch Reduction” by transferring two electrons to the aromatic
ring yielding a conjugated cyclic 1,5-dienoyl-CoA. As no natural reductant
exists to serve as donor (E°’= -622 mV), benzoyl-CoA reduction has to be
coupled to an exergonic process. Class I benzoyl-CoA reductases contain
three [4Fe-4S] clusters and couple electron transfer to a stoichiometric
ATP hydrolysis. Class II benzoyl-CoA reductases contain an active site
tungsten-cofactor. Here electron transfer is most possibly driven by an
electron bifurcation process involving eight subunits with >20 FeS cluster,
3 FAD and selenocystein as cofactors. With 2-naphthoyl-CoA reductase,
the prototype of the class III arylcarbonyl-CoA reductases was recently
identified. This enzyme belongs to the old yellow enzyme family of flavin
containing oxidoreductases. It catalyzes the four electron reduction of 2naphthoyl-CoA to 5,6,7,8-tetrahydronaphthoyl-CoA without a coupling to
an exergonic process.
Reference: Fuchs G, Boll M, Heider J (2011) Nat Microbiol Rev 9, 803-813.
18
May 6th 2013 – 13:00 - WG1: Radical Enzymes
GcpE and LytB, two Fe/S Enzymes Involved in the Biosynthesis of
Isoprenoids
Myriam SEEMANN
Université de Strasbourg, Institut de Chimie, UMR CNRS UDS 7177, 4 rue Blaise Pascal,
67070 Strasbourg, France
In many bacteria, including Mycobacterium tuberculosis responsible for
tuberculosis, in the plant chloroplasts and in the malaria parasite
Plasmodium falciparum, the biosynthesis of isoprenoids occurs according
to the methylerythritol phosphate (MEP) pathway, an alternative to the
well-known mevalonate pathway. The MEP pathway (Scheme 1) does not
exist in humans and is therefore a valuable target for the development of
new specific antibacterial and antiparasitic drugs.
The first steps leading from pyruvate and D-glyceraldehyde 3-phosphate
(GAP) to methylerythritol 2,4-cyclodiphosphate (MEcPP) are rather well
documented. The last steps converting MEcPP into IPP and DMAPP are
respectively catalyzed by GcpE and LytB, two oxygen sensitive iron-sulfur
enzymes. The mechanisms of these enzymes are still under investigation
and may involve radical intermediates.
Scheme 1. MEP pathway
19
May 6th 2013 - 14:30 - WG3: Membrane Stress, Signaling and Defences
The Impact of Nutritional Overload-Induced Phospholipid
Remodeling on Pancreatic β-cell Function and Dysfunction
Shlomo SASSON
Institute for Drug Research, Dept. of Pharmacology, Faculty of Medicine, The Hebrew
University, 91120 Jerusalem, Israel
Hyperglycemia and high concentrations of fatty acids (i.e., palmitic acid,
16:0) are detrimental to β-cells. The combination of both deteriorates βcell function in a phenomenon termed glucolipotoxicity. A recent analysis
we performed with Dr. Carla Ferreri’s group (Bologna, Italy) of membrane
phospholipids in INS-1E cells (a rat pancreatic-derived β-cell line)
revealed significant high glucose-induced remodeling of saturated (SFA)-,
mono-unsaturated (MUFA)- and poly-unsaturated fatty acids (PUFA). Of a
particular interest is the release of arachidonic- (20:4, AA) and linoleic
acids- (18:2, LA) to the cell interior. Both molecules undergo free radicalpropagated peroxidation and generate 4-hydroxynonenal (4-HNE), which
at high levels is cytotoxic. Furthermore, we have found that exposure of
INS-1E cells to palmitic acid increased the content of this fatty acid in
phospholipids in a dose-dependent manner. This was also accompanied
with its enhanced desaturation to palmitoleic acid (16:1), which was also
directed to phospholipids. Recent studies from our laboratory indicate
that palmitoleic acid may act as pro-proliferative mediator in β-cells. Of
importance is also the finding that palmitic acid significantly and dosedependently reduced AA and LA content in phospholipids: maximal effects
(~40% reduction) were observed already in cells exposed for 2 h to 50125 µM palmitic acid in the presence of 5, 11 or 25 mM glucose. Cell
viability was not affected under these conditions, while glucosestimulated insulin secretion was 30-40% higher than in the control cells.
Cytotoxic effects of the combination of glucose and palmitic acid were
apparent only in cells maintained at 11 or 25 mM glucose in the presence
of 250-500 µM of palmitic acid: cell number and total cell insulin content
were reduced by 30-40% after 12-16 h exposure to this combination.
There was a strong correlation between these effects and an increased
generation of 4-HNE. Interestingly, these detrimental effects of palmitic
acid were not observed in cells maintained 5 mM glucose, in which 4-HNE
level remained below the cytotoxic threshold. Finally, careful
examinations of the lipidomic data showed additive glucose- and palmiticinduced changes in the content of various ω-3-PUFA, MUFA and SFA.
These results highlight the central role of phospholipid remodeling in
mediating regulatory and cytotoxic interactions induced by glucose and
20
free fatty acids in β-cells. Specifically, our findings on the liberation of
fatty acids from phospholipids to the cell interior call for thorough
investigations of this process and the subsequent generation of myriad
lipid mediators that affect β-cells function and dysfunction.
21
May 6th 2013 - 14:50 - WG3: Membrane Stress, Signaling and Defences
Protein Activation by Reactive Aldehydes
Elena E. POHL
Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary
Medicine, Vienna
Oxidative stress and lipid peroxidation of cell membrane phospholipids
have been implicated in the aetiology of many pathological states such as
metabolic, cardiovascular and inflammatory failures. Uncoupling proteins
(UCPs) is a subfamily in a family of mitochondrial membrane anion
carriers. One of the most controversially discussed UCP function is their
ability to regulate mitochondrial ROS production and/or to be regulated
by ROS or ROS derivatives. Our current studies pursue the goal to reveal
(i) whether the aldehydic product of lipid peroxidation 4-hydroxy-2nonenal (HNE) but also other reactive aldehydes (RA, malondialdehyde,
4-oxo-2-nonenal, 4-hydroxy-2-hexenal and 4-oxo-2-hexenal) activate
UCP1 and UCP2, and (2) to evaluate the involved mechanism of HNE
action. In course of our investigations we use two complementary
experimental systems, bilayer lipid membranes reconstituted with
recombinant uncoupling proteins1,2,3 and primary neuronal cell cultures.
Based on the results we discuss two main hypotheses concerning the
mechanism of the proton transport mediated by RA: (i) RA influence lipid
membrane parameters, leading to the facilitation of fatty acid transport
(e.g. membrane fluidity, dipole or surface membrane potential) or/and
(ii) RA binding to the protein leads to transient proton channel formation
due to a conformational change of the protein. Understanding ROS action
mechanisms is crucial for the development of potent therapeutic
strategies in the treatment of the diseases mentioned above.
(1)
(2)
(3)
Beck, V. et al. (2006) Biochim Biophys Acta 1757(5-6): 474-9.
Beck, V. et al. (2007) FASEB J. 21(4): 1137-44).
Rupprecht, A. et al. (2010) Biophysical Journal, 98(4):1-9.
22
May 6th 2013 - 15:10 - WG3: Membrane Stress, Signaling and Defences
Novel Biomarkers for Radical Stress Attack in Mitochondria and
Plasma: mtDNA Lesions, trans-PUFA in Membrane Phospholipids
and Isoprostanes
Manar AOUN1,2, G Fouret2, C Lauret, F Michel1, C Coudray2, MA
Carbonneau1, C Feillet-Coudray2, JP Cristol1
1- Institut Universitaire de Recherche Clinique, Université de Montpellier 1, 34000
Montpellier, France. 2- INRA UMR 866, Dynamique Musculaire et Métabolisme, INRA,
34060 Montpellier, France
Metabolic syndrome (MS) is a collection of risk factors that includes
obesity, insulin resistance (IR) and dyslipidemia. The incidence of MS has
reached epidemic proportion worldwide due primarily to a modern
lifestyle that includes overeating and underactivity. Consequently, MS has
emerged as a major cause of type 2 diabetes, cardiovascular (CVD), and
non-alcoholic fatty liver (NAFLD) diseases in the industrialized societies.
MS is often characterized by oxidative stress and the relationship between
chronic oxidative stress and human diseases has moved from association
to essentially cause and effect. Biomarkers of oxidative stress have the
potential to help establish pathogenic stages and the risk for MS
associated diseases. To be truly useful, the biomarker must have some
degree of predictive validity, but nowadays full substantiation of this
relation and biomarker’s specificity are still lacking. On one hand,
mitochondria are both a major site for fat metabolism and the main
source of reactive oxygen species in tissues and are postulated to play a
central role in the pathogenesis of nutritional associated diseases such as
NAFLD and IR. On the other hand, the MS is also associated with a high
cardiovascular risk. The hypothesis that oxidized LDL (ox-LDL), a marker
of lipoprotein-associated oxidative stress, in the vessel wall intima is a
key event in atherogenesis and CVD has been widely accepted. Thus,
determination of lipid damages in mitochondria and LDL particles may be
an interesting tool to evaluate early radical stress. In earlier studies, we
demonstrated that dietary fatty acids influenced significantly fatty acids
composition of membrane phospholipids from rat liver mitochondria with
NAFLD, and thus altered mitochondria functions, lipid metabolism and
redox status. Moreover, we detected mono-trans polyunsaturated fatty
acids (PUFA) that could be the result of endogenous isomerization process
linked to a radical stress directly occurring in the biological environment,
a pathway consistently different from the dietary contribution. Since
radical stress can cause damages to lipids, proteins and DNA, and
mitochondrial DNA (mtDNA) damage is more extensive and persists
longer than nuclear DNA damage in human cells following free radical
attack, our future study aims to detect, in addition to lipid damages,
oxidative mtDNA lesions for better understanding the implications of
mitochondrial dysfunctions in oxidative stress and MS. Secondly, our
study aims to study the relation between ox-LDL and the MS. In a current
23
study, we performed in vitro oxidation of isolated LDL from human
plasma in the presence of copper (Cu2+), 2-2’ azobis(2-amidinopropane)
hydrochloride (AAPH) or 3-morpholinosydnonimine (SIN-1). LDL
oxidizability will be assessed by measuring the conjugated dienes
absorbance as well as antioxidant activities. Moreover, we are interested
in mono-trans fatty acids and isoprostanes determination in ox-LDL which
might be more specific and suitable than malondialdehyde (MDA) that are
also correlated with the severity of disease. Thus, the identification and
the quantification of trans-PUFA from LDL phospholipids and of transcholesteryl esters isomers connected to radical stress, proposed here for
the first time in LDL, in addition to isoprostanes determination, can
suggest novel biomarkers in health applications.
24
May 6th 2013 - 15:30 - WG3: Membrane Stress, Signaling and Defences
Radical Scavenging Activity of trans-Resveratrol: Photophysical
and Photochemical Properties of Resveratrol
Branka MIHALJEVIC, Iva Džeba
Ruđer Bošković Institute, Radiation Chemistry and Dosimetry Laboratory,
Bijenička 54, 10000 Zagreb, Croatia
Resveratrol (ArOH), a natural compound has attracted considerable
interest because of its biological activities against a collection of diseases.
The cancer chemoprevention effects of ArOH has been related to its
antioxidant activity, because free radical mediated peroxidation of
membrane lipids and ensuing oxidative damage are assumed to play a
causative role in cancer. However, it is somewhat surprising that spectral
and kinetic data of the transient and mechanism of ArOH reaction with
most relevant radical species are scarce, particularly with radicals which
are involved as mediators in lipid peroxidation process.
trans-3,5,4'-trihydroxystilbene,ArOH
ArO●
The antioxidant activity of ArOH is referred to the ease of the donation of
the phenolic H-atom to the attacking free radical, but it is not established
whether radical scavenging occurs by hydrogen atom transfer, electron
transfer or radical adduct formation leading to the formation of phenoxyl
radical (ArO•). The kinetics and mechanism of ArOH reaction with reactive
oxygen species are still controversial and systematic investigations are
lacking. We have accordingly investigated antioxidative properties of
ArOH obtained in a model system of mixed micelles of linoleic acid. Some
photophysical and photochemical properties of ArOH in acetonitrile at
room temperature using laser flash photolysis will be presented.
25
May 6th 2013 - 15:50 - WG3: Membrane Stress, Signaling and Defences
Free Radical Stress and Fatty Acid-Based Lipidomics
C. Ferreri, M. MELCHIORRE, A. Sansone, A. Torreggiani, C. Chatgilialoglu
ISOF, Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy
Free radical stress targets lipids, the main chemical transformations being
lipid peroxidation and isomerization which involve the unsaturated fatty
acid moieties of natural lipids. In the isomerization process the natural cis
geometry is transformed in the corresponding trans configuration,
causing an impairment of structural and functional properties, in
particular regarding the role of cell membrane lipids. Mono-trans isomers
of the natural fatty acids have been developed as biomarker of free
radical stress in cell, animal and human models in connection with the
formation of sulfur-centered radical species during stress. Lipid
transformations are relevant for molecular medicine, since the change in
the quality and quantity of fatty acids can be crucial for the cell fate,
going from biophysical consequences to signaling and metabolic effects.
Moreover, the relationship with metabolism and nutritional contributions
makes lipids a very interesting research subject in the context of health
care and prevention.
Our group provides a chemical biology approach for the synthesis of
mono-trans molecular libraries with full characterization by GC, NMR, IR
and Raman analyses, and develops them as biomarkers of free radical
stress in biological samples. In this approach fatty acid-based lipidomics
is also implemented in order to investigate several aspects of the lipid
turnover and reactivity under different physiological and pathological
conditions. On this basis the term of nutrilipidomics has been coined, as
an interdisciplinary topic which links cell membrane status with a
personalized nutritional and nutraceutical strategy with health protective
effects.
26
May 6th 2013 - 16:40 - WG4: Bio-Inspired Synthetic Strategies
Oxidative Biomimetic Carbene Catalysis
Armido STUDER
Westfälische Wilhelms-University, Corrensstrasse 40, 48149 Münster, Germany
In the presentation applications of TEMPO[1] and diquinones as
environmentally benign oxidants in combination with NHC catalysis will be
discussed. We will first focus on the use of these organic SET reagents as
mild oxidants in biomimetic aldehyde oxidations.[2] Oxidative
esterifications and amidations will be discussed.[3] Reactions occur via
acylazolium ions which show unusual chemoselectivities in the reaction
with amines and alcohols.[4] α,β-Unsaturated acylazolium ions can also be
used as formal Michael acceptors.[5]
[1] Tebben, L.; Studer, A. Angew. Chem. Int. Ed. 2011, 50, 5034.
[2] Guin, J.; De Sarkar, S.; Grimme, S.; Studer, A. Angew. Chem. Int. Ed.
2008, 47, 8727.
[3] De Sarkar, S.; Studer, A. Org. Lett. 2010, 12, 1992.
[4] De Sarkar, S.; Grimme, S.; Studer, A. J. Am. Chem. Soc. 2010, 132,
1190.
[5] De Sarkar, S.; Studer, A. Angew. Chem. Int. Ed. 2010, 49, 9266.
Biswas, A.; De Sarkar, S.; Fröhlich, R.; Studer, A. Org. Lett. 2011, 13,
4966. Biswas, A.; De Sarkar, S.; Tebben, L.; Studer, A. Chem. Commun.
2012, 48, 5190. Samantha, R. C.; Maji, B.; De Sarkar, S.; Bergander,
K.; Fröhlich, R.; Mück-Lichtenfeld, C.; Mayr, H.; Studer, A. Angew. Chem.
Int. Ed. 2012, 51, 5234.
27
May 6th 2013 - 17:00 - WG4: Bio-Inspired Synthetic Strategies
Catalytic Antioxidants
Lars ENGMAN
Department of Chemistry - BMC, Box 576, SE-751 23 Uppsala, Sweden
All organic materials exposed to air undergo oxidative degradation.
Reducing the rate of such processes by utilizing low concentrations of
“antioxidants” is of paramount importance for aerobic organisms as well
as for producers of most kinds of commercial products. In medicine the
term ”oxidative stress” has been introduced to describe a situation
characterized by an elevation in the cellular steady-state concentration of
reactive oxygen-derived species. ”Antioxidant pharmacotherapy” has
emerged as a possible remedy for pathological conditions caused by
excessive radical production. Vitamin E is the most important lipophilic,
chain-breaking antioxidant in vivo. Thus, it serves to protect important
biomolecules from becoming oxidatively damaged in radical processes. αTocopherol (α-TOH), the most reactive component of Vitamin E, is known
to trap two peroxyl radicals before it is converted into non-radical
products. It would therefore seem wise for Nature to provide a system for
its regeneration and catalytic mode of action. It is generally accepted that
ascorbate could serve this function and donate a hydrogen atom to the αtocopheroxyl radical, α-TO., which results from the interaction of a-TOH
with peroxyl radicals (Scheme 1).
Scheme 1. Catalytic chain-breaking activity of α-tocopherol
We are interested in the antioxidative properties of organochalcogen
compounds. In addition to their capacity to catalyze hydroperoxide
decomposition, they may also, just like α-tocopherol, serve as chainbreaking antioxidants. Interestingly, when assayed in a two-phase model
for lipid peroxidation, some of these antioxidants perform in a catalytic
fashion in the presence of stoichiometric amounts of a thiol reducing
agent. The presentation will describe our efforts to prepare and
understand the catalytic multifunctional (chain-breaking and peroxide
decomposing) antioxidant activity of these materials.
28
May 6th 2013 - 17:20 - WG4: Bio-Inspired Synthetic Strategies
Catechol Derivatives in Radical Chain Reactions
Guillaume POVIE, Philippe Renaud
Universität Bern, Departement für Chemie und Biochemie, Freiestrasse 3, CH-3012 Bern,
Switzerland
As dietary polyphenols[1] or polymerization inhibitors,[2] phenol
derivatives are widespread chain breaking antioxidants. This behavior
relies on their good hydrogen atom donor ability combined with the low
reactivity of the resulting phenoxyl radicals, which generally disrupt chain
processes through recombination/disproportionation reactions.[3] In
contrast, we recently proposed that o-semiquinone radicals may react
with B-alkylcatecholboranes by substitution of an alkyl residue at the
boron atom.[4] This behavior appeared to be general for most of
organoboranes reactive in homolytic substitutions, which led to the
development of a method for the formal protonolysis of B–C bonds.[5]
Applying this concept to the reduction of alkyl iodides, the hydrogen atom
transfer step was found to be sensitive to the polarity of the attacking
radical. While alkyl substituted radicals are efficiently reduced, estersubstituted radicals slowly react with catechol, giving them sufficiently
long lifetime to undergo cyclisation/addition reactions. The Arrhenius
parameters for the hydrogen atom transfer from 4-tert-butylcatechol to
different alkyl radicals could be determined using substituted 5-hexenyl
radical clocks. The variations of the activation energy in function of the
radical’s polarity demonstrate a particularly favored reaction with
electronrich alkyl radicals.
References:
[1] E. T. Denisov, T. G. Denisova, Russ. Chem. Rev. 2009, 78, 1047–
1073. S. Quideau, D. Deffieux, C. Douat-Casassus, L. Pouységu, Angew.
Chem. Int. Ed. 2011, 50, 586–621.
[2] E. V. Kolyakina, D. F. Grishin, Russ. Chem. Rev. 2011, 80, 683–704.
[3] G. Litwinienko, K. U. Ingold, Acc. Chem. Res. 2007, 40, 222–230. M.
Lucarini, G. F. Pedulli, Chem. Soc. Rev. 2010, 39, 2106–2119.
[4] G. Povie, G. Villa, L. Ford, D. Pozzi, C. H. Schiesser, P. Renaud, Chem.
Commun. 2010, 803–805.
[5] G. Villa, G. Povie, P. Renaud, J. Am. Chem. Soc. 2011, 133, 5913–
5920.
29
May 6th 2013 - 17:40 - WG4: Bio-Inspired Synthetic Strategies
New Synthetic Strategies Based on C–, O– and N–centered
Radicals.
Angeles MARTIN, Antonio J. Herrera
Instituto de Productos Naturales y Agrobiología del CSIC
Avda. Astrofísico Francisco Sánchez 3, 38206 La Laguna – Tenerife, Spain.
Our last research programs have been based on the development of new
selective and environmentally friendly synthetic methodologies for the
synthesis of chiral synthons and novel bioactive compounds. We have
introduced the use of new oxidizing systems based on hypervalent iodine
reagents for the generation of O- and N-centered radicals and the use of
visible light or sunlight to generate C-radical by photoexcitation of 1,2diketones. These protocols provide challenging ways to perform chemical
modifications on carbohydrates and amino acids, affording a wide variety
of substructures present in natural products and their analogues, as well
as enable to study the behavior of transient radicals postulated in
biological processes such as DNA damage.
In this talk, we will show an overview of our main research lines, focused
on the application of different kind of reactions and reactive species,
including Hydrogen Atom Transfer (HAT) reactions promoted by O- and
N-centered radicals, β-fragmentation reactions of O-centered radicals,
and HAT reactions of photoexcited 1,2-diketones.
30
May 7th 2013 - 9:30 - WG2: Models of DNA damage
Purine 5’,8-Cyclo-2’-Deoxynucleoside Lesions
Chryssostomos Chatgilialoglu, Annalisa Masi, Michael A. TERZIDIS
ISOF, Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy
The majority of HO• attacks toward the sugar unit of DNA occurs at H5'
with the formation of C5' radical, that gives an intramolecular attack to
the C8 position of the purine base, generating a unique cyclic base-sugar
adduct (purine 5',8-cyclo-2'-deoxynucleosides). These tandem-type
lesions are observed among the DNA modifications and have also been
identified in mammalian cellular DNA in vivo. Inspired by the mode of
formation of these lesions, the synthetic approach to the full library of
purine 5',8-cyclo-2'-deoxynucleosides has been thoroughly investigated
by radical cascade protocols. In particular, the diastereomeric forms of
5',8-cdAdo (1 and 2) and 5',8-cdGuo (3 and 4) have been synthesized
and fully characterized, as well as their corresponding phosphoramidates.
This facilitates the quantitative determination of these lesions in biological
samples as biomarker of free radical damage and the preparation of
modified oligonucleotides for biochemical/biophysical studies related to
specific diseases and impairment of enzymatic repair.
31
May 7th 2013 - 9:50 - WG2: Models of DNA damage
Pulse Radiolysis: a Tool for Investigating Radical Processes in
Biological Molecules
Krzysztof BOBROWSKI
Centre of Radiation Research and Technology, Institute of Nuclear Chemistry and
Technology, 03-195 Warszawa, Poland
Pulse radiolysis (first introduced in the 1960) has found a broad range of
important applications in chemistry and biochemistry. The method is
based on the excitation and ionization of solvent molecules by intensive
pulses of high energy electrons where sufficiently high concentration of
radicals is generated in a short time in order to follow their reaction
directly. The potential role of pulse radiolysis for studying radical
processes, particularly in biological macromolecules, has been recognized
rather early. Important applications of pulse radiolysis are connected with
understanding of radical processes that are of particular interest in
biology and medicine [1]. Relevant examples include radical processes
connected with a damage of biological material, oxidative stress,
repairing and protective mechanisms, aging, inflammation processes, and
various diseases including cancer, and neurodegenerative diseases [2]. In
most cases, pulse radiolysis method has been valuable in identification of
radicals, establishing their structures and exploring their reactivities [3].
For radicals in solution the most commonly used technique is timeresolved UV/vis spectroscopy. The others, although less widely employed,
include time-resolved conductivity, electron paramagneic resonance,
vibrational resonance Raman spectroscopy, and microwave absorption
spectroscopies, circular dichroism, and very recently infrared
spectroscopy. They can provide kinetic and spectral information that are
not acccessible via optical measurements. The nanosecond pulse
radiolysis facility based on the electron linear accelerator installed at the
INCT, Warszawa constructed in 1999 will be presented. The LAE 10 is
solely dedicated to pulse radiolysis experiments with the following
nominal parameters: pulse duration (4−10 ns, 100 ns) and electron
energy (10 MeV) [4].
For those who are not familiar with radiation chemistry, in order to show
briefly the potential use of water radiolysis, formation of •OH, O2•-, RO2•,
RO•, NO•, NO2•, CO3•- has been summarized. The scope of this lecture is
limited to the selected contributions of pulse radiolysis to better
understanding the role of free radical processes in peptides and
nucleobases. Stabilization of the monomeric sulfur radical cations
(MetS+•) derived from methionine (Met) through three-electron bond
formation with either the oxygen and/or the nitrogen atoms of the
adjacent peptide bonds in linear model peptides containing Met residues
and the reaction paths of •OH radicals with 2’-deoxyguanosine will be
presented [5,6].
32
References:
[1] R. V. Bensasson, E. J. Land, T. G. Truscott, Excited States and Free
Radicals in Biology and Medicine, Oxford Science Publications, 1993
[2]
C.
Houée-Levin,
K.
Bobrowski,
J.
Prot.
2013,
http://dx.doi.org/10.1016/j.jprot.2013.02.014
[3] K. Bobrowski, Radiation induced radical reactions. In: Encyclopedia of
radicals in chemistry, biology, and materials (C. Chatgilialoglu, A. Studer,
eds.) John Wiley&Sons Ltd, 2012
[4] K. Bobrowski, Nukleonika 2005, 50, S67-76
[5] K. Bobrowski, C. Houée-Levin, B. Marciniak, Chimia 2008, 62, 728-34
[6] C. Chatgilialoglu, M. D’Angelantonio, G. Kciuk, K. Bobrowski, Chem.
Res. Toxicol. 2011, 24, 2200-06
33
May 7th 2013 - 10:10 - WG2: Models of DNA damage
Laser Flash Photolysis as a Tool for the Study of Hydrogen Atom
Transfer and Electron Transfer Reactions of Oxygen Centered
Radicals
Massimo BIETTI, Michela Salamone
Dipartimento di Scienze e Tecnologie Chimiche, Università "Tor Vergata", Via della
Ricerca Scientifica, 1 I-00133 Rome, Italy.
Oxygen-centered radicals are known to react with biomolecules mainly
through hydrogen atom transfer, electron transfer and addition reactions.
These include, among other species, the highly reactive hydroxyl (HO•)
and alkoxyl (RO•) radicals.
Laser flash photolysis (LFP) represents one of the most powerful and
effective methods for the kinetic study of the reactions of these radicals in
chemical and biological systems. In a typical LFP experiment, a laser
source provides a single wavelength excitation with nanosecond
resolution that is used to initiate a chemical reaction. The reactive
intermediates thus generated can be directly monitored spectroscopically,
providing quantitative information on the reactivity of the system under
investigation. HO• is however undetectable with conventional timeresolved techniques and kinetic information can be obtained indirectly by
the use of a suitable probe that allows the determination of the rate
constants by means of competitive kinetics.
Alkoxyl radicals have often served as general models for the study of the
reactivity of oxygen centered radicals in chemical and biological systems,
and offer a number of advantages over HO•. They are less reactive and
more selective, can be easily generated by photolysis of parent peroxides,
are characterized (when bearing aromatic groups bound to the α-carbon)
by absorption bands in the visible region of the spectrum, and can
tolerate a wide range of experimental conditions. Taken together, these
features make alkoxyl radicals particularly convenient for in situ
generation and tracking, allowing for the direct kinetic study of their
reactions with biomolecules, thus providing quantitative kinetic
information on the susceptibility of biomaterials to radical damage and
the role of medium effects on these processes.
An overview of our recent time-resolved kinetic studies on hydrogen atom
transfer and electron transfer reactions from a variety of substrates to
alkoxyl radicals will be presented, highlighting the important role of
structural and medium effects on these processes.
34
May 7th 2013 - 10:30 - WG2: Models of DNA damage
Nucleosome Remodeling and Base Excision Repair
Dimitar ANGELOV
Laboratoire de Biologie Moléculaire de la Cellule, LBMC UMR CNRS/ENS 5239, Ecole
Normale Supérieure de Lyon, 46, allée d’Italie, 69007 Lyon
Eukaryotic cells are constantly subjected to oxidative stress leading to a
tremendous number of insults, which have to be efficiently repaired. In
the DNA, 8-oxo-7,8-dihydroguanine (8-oxoG) is the major lesions induced
upon oxidative stress, which is repaired by the Base Excision Repair
(BER) pathway. BER uses a limited number of enzymes and how BER
functions on naked DNA template is well characterized. 8-oxoG is
recognized and removed by the 8-oxoguanine DNA glycosylase (OGG1),
which exhibits both a glycosylase and an apurinic/apyrimidinic (AP) lyase
activity. How BER operates on chromatinized templates is far from being
clear. In general, the presence of nucleosomes interferes with BER but
the DNA interacting proteins involved appeared to be differently affected
by the nucleosome structure.
As a model system we have used precisely positioned dinucleosomes
assembled with linker histone H1. A single 8-oxoG was inserted either in
the linker or the core particle DNA within the dinucleosomal template. We
found that in absence of H1 the removal of 8-oxoG from the linker DNA
by OGG1 proceeds as efficiently as in the naked DNA. In contrast, the
presence of histone H1 resulted in close to ten-fold decrease in the OGG1
efficiency in linker DNA independently of linker DNA length. At the same
time, the very strongly inhibited removal of 8-oxoG within the core
particle DNA remains independent on H1. Chaperone-induced uptake of
H1 restored the efficiency of the glycosylase from linker DNA, but not
from the octamer wrapped DNA. Finally, we show that chaperone-assisted
removal of histone H1 and ATP-dependent nucleosome remodeling are
both necessary and sufficient for an efficient initiation of 8-oxoG repair
within nucleosomal DNA. A model for a quasi-stochastic mechanism of
BER within chromatin will be discussed.
35
May 7th 2013 - 10:50 - WG2: Models of DNA damage
Radiation-Induced Clustered DNA Lesions: Induction and Repair
Alexandros G. GEORGAKILAS
Department of Physics, School of Applied Sciences, National Technical University of
Athens, Zografou Campus, GR 15773 Athens, Greece
In cells and tissues there is a constant radiolytic attack that has
exogenous or endogenous (intracellular) oxidative origin. During these
attacks, although not in all cases, the primary damage is being induced
by reactive oxygen species (ROS) and reactive nitrogen species (RNS).
The oxidatively-induced DNA damage consists of a variety of lesions of
small to high importance and dangerous for the cell i.e., isolated base
lesions or single strand breaks (SSBs) to complex lesions like double
strand breaks (DSBs) and other non-DSB oxidatively-induced clustered
DNA lesions (OCDLs). Ionizing radiation, even at low doses (1 Gy) can
induce a significant amount of clustered DNA lesions which can be very
difficult to process and repair efficiently. This has various effects and
implications also in radiation therapy and in the study of the so called
bystander or non-targeted effects. In this presentation, I will discuss the
current status of knowledge and evidence on the induction mechanisms
and repair mechanisms in human cells and biological importance.
36
May 7th 2013 - 11:40 - WG1: Radical Enzymes
Novel Inhibitors and Activators of Lipoxygenases and Their Role
in NF-κB Signaling
Frank DEKKER
Department of Pharmaceutical Gene Modulation, Groningen Research
Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV
Groningen, The Netherlands.
Lipoxygenases are non-heam iron containing enzymes that oxidize
unsaturated fatty acids by a one-electron oxidation to form radical
intermediates. These radical intermediates are converted into
leukotrienes, which are signaling molecules that play a key role in
inflammation. Recently, we identified that para-alkoxybenzyl substituted
6-hydroxysalicylates are a new class or modulators of human 5lipoxygenase. Interestingly, this compound class either inhibits or
activates the 5-lipoxygenase activity depending on the substitution
pattern. In addition, enzyme kinetics demonstrate mixed-type inhibition
or activation in which the inhibitor or activator can bind to the free
enzyme and to the substrate bound enzyme. This behavior indicates the
presence of an allosteric binding site that regulates the enzyme activity.
Preliminary experiments demonstrate that these compounds also activate
or inhibit signaling via the NF-κB pathway, which plays an important role
in inflammation. Future studies will be directed at development of probes
for activity-based profiling of lipoxygenases and cycloxoygenases under
inflammatory conditions.
37
May 7th 2013 - 12:00 - WG1: Radical Enzymes
High- and Room-Temperature Isomerizations of Dimers of Highly
Pyradalized Alkenes
Athanassios NICOLAIDES
Department of Chemistry, University of Cyprus, Nicosia, 1678, CYPRUS
The first three members (n=0,1,2) of the homologous series of
pyramidalized alkenes 1, are so reactive that even at low temperature
dimerize to give polycyclic cylobutane derivatives (2).1,2 Two of these
dimers (2, n=0,1) are known to ring-open thermally to the corresponding
dienes 3, n=0,1, in a formally forbidden reaction, and presumably via
diradical 4, n=0,1. The thermolysis of 2, n=1 gave in addition diene 5
and, [2]diadamantane (6), the latter possibly via diradical 7.
Computational Chemistry methods have been applied to understand the
thermochemistry of these compounds and the possible mechanisms of
their isomerizations.
38
May 7th 2013 - 12:20 - WG1: Radical Enzymes
Peptide Dendrimers with Bipyridine at the Core as Models of
Metaloenzymes: pH-Tuned Metal Coordination and Peroxidase
Activity
Tamis DARBRE
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012,
Bern, Switzerland.
Peptide dendrimers with a bipyridine at the core were obtained by double
thioether bond formation between 5,5’-bis(bromomethyl)-2,2’-bipyridine
and two equivalents of third generation peptide dendrimers such as N1
(Ac-Glu-Ser)8(Dap-Glu-Ala)4(Dap-Amb-Tyr)2Dap-Cys-Asp-NH2 (Dap =
branching 2,3-diaminopropanoic acid, Amb = 4-aminomethyl-benzoic
acid) to give BP1. At pH 4.0 BP1 bound Fe(II) to form the expected triscoodinated complex [FeII(BP1)3] (Kf = 2.1 × 1015 M-3). At pH 6.5 a monocoordinated complex [FeII(BP1)] was formed instead (Kf = 2.1 × 105 M-1)
due to electrostatic repulsion between the polyanionic dendrimer
branches, as confirmed by the behavior of three analogues where
glutamates were partially or completely replaced by neutral glutamines or
positive
lysines.
[FeII(BP1)]
catalyzed
the
oxidation
of
o-1
phenylenediamine with H2O2 with enzyme-like kinetics (kcat = 1.0 min ,
KM = 1.5 mM, kcat/kuncat = 90,000) and multiple turnover, while Fe2+ or
[Fe(bipy)3]2+ were inactive. The labile coordination positions allowing
coordination to H2O2 and to the substrate are likely responsible for the
enhanced peroxidase activity of the metallo-peptide dendrimer.
BP1
39
May 7th 2013 - 12:40 - WG1: Radical Enzymes
Computational Approaches on Two Free Radical Generating
Systems: the Peroxide Chemistry Specific to Bleomycin and P450,
and the Cobalt Corrins
Radu SILAGHI-DUMITRESCU
Babes-Bolyai University, Cluj-Napoca 400028, Romania
A key intermediate in bleomycin’s anti-DNA action is a species known as
activated bleomycin, ABLM, which, on the basis of spectroscopic and
theoretical studies, appears well described as a bleomycin-ferrichydroperoxo adduct (Figure below). ABLM’s instability has to some extent
precluded detailed structural characterization, but its formation is
generally accepted to entail reaction of ferrous bleomycin with molecular
oxygen, followed by a one-electron reduction to yield a formally ferroussuperoxo adduct, whose protonation would lead to ABLM. A somewhat
more stable cognate, the Co(III)-hydroperoxo bleomycin adduct, has
been characterized spectroscopically, and its inferred structural features
appear very similar to those of ABLM. Here, the structures and reactivities
of ABLM and of its cobalt analogue are discussed based on density
functional theory (DFT) data, revealing surprising similarities in the
context where the cobalt version is known to be much more stable than
the iron one. The mechanism whereby the peroxo bond is activated by
bleomycins must, most likely, be reformulated. Analogies with enzymatic
systems are drawn. Also discussed will be electronic structure elements in
cobalamin derivatives responsible for free-radical-type reactivity,
including super-reduced species.
40
May 7th 2013 - 13:00 - WG1: Radical Enzymes
Biocatalysed Reactions and One-pot Synthesis of Pharmaceutical
Precursors
Ioulia SMONOU
Department of Chemistry, University of Crete, Heraklion-Voutes 71003, Crete, Greece
The role of Biocatalysis in the development of simple and straightforward
methodology for the stereoselective synthesis of pharmaceutically
interesting
compounds
will
be
presented.
More
specifically,
stereoselective ketoreductase-catalyzed reductions of various carbonyl
compounds such as diketones, keto esters, diketo esters have resulted in
the synthesis of almost all possible stereoisomers of the corresponding βhydroxy ketones, 1,3-diols, β-hydroxy esters or dihydroxy esters in high
optical purities (>99% de, >99% ee) and chemical yields. These
compounds are key intermediates in asymmetric organic synthesis. They
represent important class of chiral synthons as they have been used for
the synthesis of many natural products,[1] pharmaceuticals [2] and other
valuable compounds.[3]
Ketoreductases are chemo-, stereo- and regioselective catalysts that
often reduce a wide range of ketones and have been proved very efficient
catalysts for the preparation of optically active keto alcohols, diols and
hydroxy esters.[4]
More importantly, biocatalytic cascade processes consisting of two
consecutive steps have been designed for the synthesis of chiral diols and
β,δ-dihydroxy esters.[5] These chiral building blocks were formed by the
addition of enzymes in the same vessel, in high stereoselectivity and
chemical yield, without the isolation of any intermediates.
Natural products,
pharmaceuticals
References:
[1] (a) K. Tatsuta, Hosokawa, Seijiro. Chem. Rev. 2005, 105, 4707 (b) R.
N. Patel, Adv. Synth. Catal. 2001, 343, 527
[2] R. N. Patel, ACS Catalysis 2011, 1 (9), 1056-1074
41
[3] M. M. A. Mondol, J. H. Kim, M. Lee, F. T. Shahidullah, H-S. Lee, Y-J.
Lee, H. J. Shin, J. Nat. Prod. 2011, 74, 1606
[4] (a) D. Kalaitzakis, J. D. Rozzell, S. Kambourakis, I. Smonou, Org. Lett
2005, 7, 4799 (b) D. Kalaitzakis, J. D. Rozzell, I. Smonou, S.
Kambourakis, Adv. Synth. Catal. 2006, 348, 1958 (c) D. Kalaitzakis, I.
Smonou, Tetrahedron 2010, 66, 9431-9439
[5] (a) D. Kalaitzakis and I. Smonou, J. Org. Chem., 2010, 75 (24),
8658–8661 (b) A. Bariotaki, D. Kalaitzakis, I. Smonou, Org. Lett. 2012,
14(7), 1792–1795.
42
May 7th 2013 - 14:30 - WG4: Bio-Inspired Synthetic Strategies
New Catalytic Electron Transfer- Induced and Thermal Radical
Cyclizations
Ullrich JAHN
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech
Republic, Flemingovo namesti 2, 16610 Prague, Czech Republic
Radical reactions in tandem with those of other intermediates types are
an attractive strategy to generate molecular complexity. The power of
redox and intermediate diversity is demonstrated at selected examples.
The transformation of such processes into traceless catalytic processes is
shown.
Where electron-transfer induced radical chemistry meets limitations,
thermal radical cyclizations using the same precursors and the persistent
radical effect1 come to the rescue. Applications of such processes toward
the total synthesis of selected alkaloid classes will be reported
1.
A. Studer, Chem. Soc. Rev. 2004, 33, 267-273.
43
May 7th 2013 - 14:50 - WG4: Bio-Inspired Synthetic Strategies
Radical Chemistry of a Flavin Coenzyme Mimic
István Bors, Nárcisz Bagi, Gábor SPEIER, József Kaizer
Department of Chemistry, University of Pannonia, 8200 Veszprém, Hungary
Oxazaphosphazoles are formed in a [4+1] electrocyclic addition of
quinone monoimines a triphenylphosphine. The products show
tautomerism (A and B) and are unique in their formation since they
exhibit radical intermediates.
They react also with triplet molecular oxygen at ambient conditions in a
stoichiometry of 1:1. They are good catalyts for the oxygenation of
triphenylphosphine to the oxide and oxidation of thiols to disulfides (1
and 2). In these reactions they behave as flavin mimics due to their facile
activation of 3O2.
Mechanistic aspects of these flavin mimics for the reactions 1 and 2 will
be discussed.
44
May 7th 2013 - 15:10 - WG4: Bio-Inspired Synthetic Strategies
Green Photocatalytic Organic Transformations Using MesoporousMetal-Oxide-Supported Gold Nanoparticles and Polyoxometalates
Ioannis N. LYKAKIS
Department of Chemistry, Aristotle University of Thessaloniki, University Campus 54124,
Thessaloniki, Greece
For catalytic processes, an attractive approach is the use of a solid,
recyclable catalyst and environmentally friendly reagents, such as O2. The
use of supported catalysts on a well ordered metal oxide surface for
heterogeneous catalysis offers several advantages on the catalyst
reusability and the regio- and chemo-selectivity of the reaction process.
Although, AuNPs or polyoxometalates (POM) anions supported on metal
oxide materials have been used in several organic transformations,
combined AuNPs-POM-mesoporous metal oxides (MMO) materials are
scarce. Such materials are expected to exhibit superior photo- as well as
thermal-catalytic activities compare to the commercial ones. Thus, this
proposal outlines a research program aimed to gain insight into the
catalytic applications of new AuNPs-POM heterogeneous systems, in order
to afford new chemical transformations, based on the combination of
AuNPs and POM photo-irradiated properties. For this reason catalytic
reduction of nitro-aromatic compounds in absence of H2 gas, using only
mild reducing agents such as hydrazine and 1,1,3,3-tetramethyldisiloxane
(TMDS) as hydrogen donor molecules, as well as CO2 photo-chemical
reduction to methanol or formic acid, using the mesoporous AuNPs-POMhydrazine and AuNPs-POM-TMDS catalytic systems (green CO2 reduction),
will be studied. A detailed description of our results concerning the above
reaction will be presented.
45
May 7th 2013 - 15:30 - WG4: Bio-Inspired Synthetic Strategies
“Bio-Inspired” Electron Transfer Processes for Fine Chemical
Synthesis
Cyril OLLIVIER
Institut Parisien de Chimie Moléculaire (UMR CNRS 7201)
UPMC Univ-Paris 06, Sorbonne Universités, 4 Place Jussieu, C. 229, 75005 Paris, France
Compounds responsible for electron-transfer reactions have been
increasingly seen as alternative radical mediators to toxic tin hydrides,
thus contributing de facto to the development of an eco-friendly radical
synthetic chemistry.
Inspired by natural photosynthesis, we have recently started a new
program directed towards the use of solar radiation to carry out chemical
transformations involving redox processes. The use of visible light and a
photoredox catalyst has lately appeared to offer very promising
possibilities for fine chemical synthesis. Recently, we expanded the scope
of photoreduction catalysis to the generation of radicals from
ketoepoxides, ketoaziridines and also onium salts. We took advantage of
the reactivity of the photogenerated radicals to create new carbon-carbon
bonds (Eq. 1).[1]
In parallel, we were interested in exploring the reactivity of iron hydrides
as new catalytic redox mediators since Iron is abundant and participates
in numerous biological processes. We showed that iron(II) complexes
associated with sodium borohydride are efficient precatalysts for radical
cyclization of unsaturated iodide and bromide derivatives. As evidenced
by cyclic voltammetry studies, a radical mechanism is at work which
involves an anionic hydrido-iron(I) species as the key intermediate for the
activation of the substrates by electron transfer (Eq. 2).[2]
[1] M.-H. Larraufie, R. Pellet, L. Fensterbank, J.-P. Goddard, E. Lacôte, M.
Malacria, C. Ollivier Angew. Chem. Int. Ed. 2011, 50, 4463. [2] A.
Ekomié, G. Lefèvre, L. Fensterbank, E. Lacôte, M. Malacria, C. Ollivier, A.
Jutand Angew. Chem. Int. Ed. 2012, 51, 6942.
46
May 7th 2013 - 15:50 - WG4: Bio-Inspired Synthetic Strategies
Iron Complexes as Functional Models for the Soluble Methane
Monooxygenase Enzyme
Jozsef KAIZER, J. S. Pap, G. Speier
Department of Chemistry, University of Pannonia, 8200 Veszprém, Watha V. u. 1.,
Hungary
Oxygen activation by nonheme diiron enzymes occurs in numerous
metabolically important transformations, including those by soluble
methane monooxygenase (sMMO), ribonucleotide reductase (RNR), fatty
acid desaturases, human deoxyhypusine hydroxylase, etc. During the
activation of O2 activation the diiron(II) center is transformed to a
peroxodiiron(III) intermediate that in turn converts to the oxidizing
species. This intermediate typically exhibit visible features between 600750 nm and resonance Raman (rRaman) features associated with a (m1,2-peroxo)diiron(III) unit. Studies of synthetic complexes that mimic O2
activation can complement and augment the information available from
enzyme studies. Particularly useful are complexes for which intermediates
along the O2-activation pathway can be traced. We report here the
spectroscopic characterization of the peroxo-intermediates derived from
the reaction of H2O2 with Fe(II) complexes of rigid N-donor ligands.
Investigations into their catalytic reactivity with H2O2 were performed on
the oxidation of sulfides, alcohols and C-H compounds.
47
May 7th 2013 - 16:40 - WG3: Membrane Stress, Signaling and Defences
Bleomycin Increases trans Lipid Isomers in Human Testicular
Cancer Cell Membrane
A. Cort1, Tomris OZBEN1, M. Melchiorre2, C. Chatgilialoglu2, C. Ferreri2
1- Akdeniz University Medical Faculty, Department of Biochemistry, Antalya, Turkey
2- ISOF, Consiglio Nazionale delle Ricerche, Via P. Gobetti 101, 40129 Bologna, Italy
Polyunsaturated fatty acids are the major components of phospholipids,
the principal structural unit of biological membranes. Unsaturated fatty
acids have one or more double-bonds in a cis or trans configuration.
Trans isomers are able to perturb both cell membrane arrangement and
lipid enzymatic cascades. Endogenous trans fatty acid isomers are formed
by the isomerization of fatty acids in cell membranes due to an
endogenous free radical process. Reactive oxygen species (ROS) lead to
the oxidative degradation of lipids in cell membranes, resulting in cell
damage. Cell membranes, which are structurally made up of large
amounts of PUFA, are highly susceptible to oxidative attack and,
consequent changes result in altered membrane fluidity, permeability,
and cellular metabolic dysfunction. Some chemotherapeutic agents and
all radiation therapy induce oxidative stress by generation of ROS which
might be an alternative mechanism for their cytotoxic effect via inducing
cell death. Bleomycin is used commonly in the treatment of testicular
cancer. Bleomycin is an antibiotic which affects nucleic acids and
generates high level of ROS. In this study, we aimed to compare the fatty
acid profile of human testicular cancer cell membranes during normal
culturing and bleomycin treatment. The membrane fatty acid profile
analysis in control and bleomycin treated samples was performed using
gas chromatographical (GC) analysis with external reference standards.
Lipid extraction and transesterification were performed to obtain the fatty
acid residues as the corresponding methyl esters. Incubation of the cells
with 100 µg/ml bleomycin for 24 hour, caused an increase in trans lipid
concentration. Our results indicated that bleomycin induced ROS
generation results in trans lipid isomerization in testicular cancer cell
membrane.
48
May 7th 2013 - 17:00 - WG3: Membrane Stress, Signaling and Defences
The Influence of Natural Occuring Antioxidants on Lipid
Peroxidation and Isomerization Processes in Model Micellar
System
Ivana TARTARO BUJAK, Branka Mihaljević
Ruđer Bošković Institute, Radiation Chemistry and Dosimetry Laboratory
Zagreb, Croatia
Biomimetic models provide basic molecular mechanisms studies that are
closer to the biological environment such as living organism. There are
two main classes of reactions that are known in the context of reactivity
of polyunsaturated fatty acids toward free radicals. One of the well known
process is lipid peroxidation and the second one is geometrical
isomerization of unsaturated lipids which can be catalysed by thiyl
radicals (RS•). Our previous study of these processes has been extended
in the presence of several natural occuring antioxidants which have
shown their protective properties against lipid peroxidation and trans
isomerization.
Ascorbic acid
α-tocopherol
Resveratrol
All results obtained indicate that trans isomerization depends on the
localization of the particular antioxidant in model system. In order to
protect lipid structures from the thiyl radical damage a new role of the
essential antioxidants can be assessed in the observed processes.
49
May 7th 2013 - 17:20 - WG3: Membrane Stress, Signaling and Defences
Glycation of HDL and LDL by Methylglyoxal Causes Structural
Remodelling Linked to Increased Atherogenicity
Naila RABBANI, PJ Thornalley
Warwick Medical School, Clinical Sciences Research Institute, University of Warwick,
University Hospital, Coventry CV2 2DX, U.K.
Cardiovascular disease (CVD) is a major cause of mortality worldwide.
Increased risk of CVD and atherosclerosis is associated with high levels of
LDL and low level of HDL. The processes associated with transformation
of LDL to atherogenic forms and decline in quality of HDL are not fully
understood. Glycation of plasma proteins by methylglyoxal (MG) and the
risk of CVD increase 2 – 3 fold in patients with diabetes. In recent studies
we have investigated effect of glycation of LDL and HDL by MG to
physiological, minimal extent on particle size, aortal trapping, plasma
clearance and catabolism (1).
LDL minimally modified by MG (MGmin-LDL) in vitro had decreased particle
size – small and dense. It had increased binding to proteoglycans and
increased aggregation in vitro. Cell culture studies showed that MGmin-LDL
was bound by the LDL receptor but not by the scavenger receptor, and
had increased binding affinity for cell surface heparin sulfate-containing
proteoglycan. Radiotracer studies in rats showed that MGmin-LDL had
similar fractional clearance rate in plasma to unmodified LDL but
increased partitioning onto the aortal wall. Mass spectrometry peptide
mapping identified arginine-18 as the hotspot site of apolipoprotein B100
modification in MGmin-LDL. A computed structural model predicted that
methylglyoxal modification of apolipoprotein B100 induces distortion,
increasing exposure of the N-terminal proteoglycan binding domain on
the surface of LDL. This likely mediates particle re-modelling and
increased proteoglycan binding. MG modification of LDL in vivo was ca.
5%, increasing to 16% in patients with type 2 diabetes (2).
HDL subtypes HDL2 and HDL3 modified minimally by MG had decreased
particle size and stability. Radiotracer studies in rats showed that human
MGmin-HDL2 and MGmin-HDL3 had increased plasma clearance with
increased partitioning in the kidneys and liver of rats. Mass spectrometry
peptide mapping identified critical arginine residues that were hotspot
sites of apolipoprotein A-1 modification in MGmin-ApoA-1, MGmin-HDL2 and
MGmin-HDL3.
Glycation of LDL and HDL by MG represent new non-oxidative (3) routes
to dysfunctional lipoprotein that renders LDL more atherogenic and
impairs the quality of HDL. Interventions to counter increased MG
concentration in diabetes, renal failure and ageing – dicarbonyl stress may provide a novel strategy to suppress risk of CVD.
1. Rabbani, N., Godfrey, L., Xue, M., Shaheen, F., Geoffrion, M., Milne,
R. and Thornalley, P.J. (2011) Glycation of LDL by Methylglyoxal
50
Increases Arterial Atherogenicity. A Possible Contributor to
Increased Risk of Cardiovascular Disease in Diabetes. Diabetes 60,
1973-1980.
2. Naila Rabbani, Madhu Varma Chittari, Charles W. Bodmer, Daniel
Zehnder, Antonio Ceriello and Paul J. Thornalley (2010) Increased
glycation and oxidation damage to apolipoprotein B100 of LDL in
patients with type 2 diabetes and effect of High dose metformin
therapy. Diabetes 59, 1038 – 1045
3. Rabbani, N. and Thornalley, P.J. (2011) Methylglyoxal modification
of LDL – pro-atherogenicity without oxidation opens new paths to
prevention of cardiovascular disease. Clinical Lipidology 6, 631-634.
51
May 7th 2013 - 17:40 - WG3: Membrane Stress, Signaling and Defences
Novel Causative Relationship Between Low HDL and Diet-Induced
Non-Alcoholic Fatty Liver Disease
Eleni A. Karavia1, Dionysios J. Papachristou2, and Kyriakos E. KYPREOS1
1- Pharmacology laboratory; Department of Medicine, University of Patras Medical
School, Greece
2- Anatomy, Histology and Embryology laboratory, Department of Medicine, University
of Patras Medical School, Greece
Background and Aims: During the biogenesis of HDL, lipid free or
minimally lipidated apoA-I interacts functionally with the lipid transporter
ABCA1 to form immature discoidal HDL which are then converted into
mature spherical particles by the action of lecithin:cholesterol acyl
transferase (LCAT). Here we investigated the mechanistic relationship
between low and dysfunctional HDL and diet-induced NAFLD development
using mouse models.
Methods: We employed male apoA-I-deficient (apoA-I-/-) mice that lack
classical apoA-I containing HDL and male deficient (LCAT-/-) mice that
have immature discoidal HDL.
Results: Mice were fed the standard western-type diet for 24 weeks and
then histological and biochemical analyses were performed. ApoA-I-/mice showed increased diet-induced hepatic triglyceride deposition and
disturbed hepatic histology while they exhibited reduced glucose
tolerance and insulin sensitivity. Quantification of FASN-1, DGAT-1, and
PPARγ mRNA expression suggested that the increased hepatic triglyceride
content of the apoA-I-/- mice was not due to de novo synthesis of
triglycerides. Similarly, metabolic profiling did not reveal differences in
the energy expenditure between the two mouse groups. However, apoAI-/- mice exhibited enhanced intestinal absorption of dietary triglycerides,
accelerated clearance of postprandial triglycerides, and a reduced rate of
hepatic very low density lipoprotein triglyceride secretion. In agreement
with these findings, adenovirus-mediated gene transfer of apoA-IMilano in
apoA-I-/- mice fed western-type diet for 12 weeks resulted in a significant
reduction in hepatic triglyceride content and an improvement of hepatic
histology and architecture. Similar to apoA-I-/- mice, LCAT-/- mice were
characterized by increased diet-induced hepatic triglyceride deposition
and impaired hepatic histology and architecture. Adenovirus-mediated
gene transfer of LCAT in LCAT-/- mice that were fed western-type diet for
12 weeks resulted in a significant reduction in hepatic triglyceride content
and a great improvement of hepatic histology and architecture.
Conclusions: Taken together, our data establish that the HDL metabolic
pathway is a central contributor to the deposition of dietary triglycerides
to the liver and the development of NAFLD. Our data further support that
the coexistence of reduced HDL levels and NAFLD in an individual with
metabolic syndrome may not be a mere coincidence, rather it underlays a
strong causative relationship between these two conditions.
52
53
54