SN 1071-5762 Volume 41, Supplement 1 (October 200~

Abstracts of the SFRR Europe 2007 Meeting Vilamoura, Algarve, Portugal

1 0-13 October 2007

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antioxidant activity (fAA) was measured by the ABTS - decolouriza­tion method. Serum a - and )• -tocopherol and malondialdehyde (M DA) were d~termincd by HPLC. COJ\'IT Val l 081158M et polymorphism was detected by tetraprimer ARMS-PCR. Specifically the COJY!T h igh activity allele (Val) was "'sociated with a d~cr~ascd scrum antioxidant s tatus at E2max respecting E2min, expre"'ed by: (a) increased scrum oxida tion rates in the lag- and propagation phase>, and a decreased lag time; (b) decreased TAA; (c) reduced ·x - and )' -tocopherol content; and (d) increased MDA. We conclude that the COMT low activity genotype (Nlctl!Vle t) coniers antioxidant protection respecting the high activity ailele dur ing the ovarian stimulation achieved in IVF.

Supported by 1v!SC (FIS!FEDER PI02/0233), Gobierno Vasco (SAIO­T EK), M EC (grant to IA) and U PV (gram to AR) .

P -010 Protection of an thocyanins a gains t human LDL oxida tion and th dr s tructure -activity r ela tionship : A l<ey c omponent in t h e F rench paradox Crisrina Azevcdo, Leonor Almeida, Joao Laran jinha, & Teres. Dinis Laboratol)' of Biochmustl~\', Faculry of Plrarma'y a11d Ccuw· for Ncwm­ciCI!c,;s, Univ.:rsit;)• of Coimbra, 3000-295 Cvimbra, Pcmugai

An increased interest in anthocyan ins and the ir biological effect> hns emerged in the iast year». They arc a <ob-group of fhvonoids responsible fo r the coloU1' and most of the benefits of moderate consumption of red wine. The present study was designed to evah:arc and compare the antioxidarivc properne' of fo ur s tructurally related amhocyanins - pdargonidin, cyanidin, malvidin and malvidin-3-glucoside against human LDL oxidation promoted either by AAPH-gcnerated peroxyl radicals, or two physiologically relevant oxidants, ferrylmyoglobin and pcroxynin·ite. T heir ability to recycle u -cocopherol (ct -TOH), the most abundant LDL- lipoph ilic antioxi­d ant, was also s tudied. When LDL ox.idanon was initiated e ither by AAPH or ferrylmyoglobin, as determined by the fluorc>cence decay of incorporated cis-parinaric acid and con jugated dicncs fo rmation, those amhocyanins strongly in hibit LDL oxidativc damage, cyanid in and m ah•idin being far more efficient as compared with pd argonidin. Also, malvidin-3-glucoside exhibited a stronger antioxidant activity than malv1din, the non-gly~o<i lated der ivative. Peroxynitrite-promotcd LDL apoprotcin modificatic>m, as evaluated by apoB net s ur face charge alt eration>, were efficiently mhibited by cyanidin, malvidin or malvidin-3-glucoside, while almost no df~cts were obs~rved with pdargonidin. M oreover, a ll the anthocyan in> significantly d~creascd peroxynitrit~­mediatcd carbonyl groups formation ia L D L. cl'R measurements of ~ -LOcop heroxyl rad ical showed that the anthocyanins strongly reduce the signal intensity of that radica l pointing to the1r highest abilities 10

recycle 1-TOH, a lthough ma!vidin-3-glucoside was far less effective. Our results corroborate the relevance of patterns of hydroxyl or methoxyl sub<tirution and glycosylarion to the modulation of antiox­idant .1ctivities of amhocyanins . Also, th ey suggest that the consump­tion of amhocyanins through the intake of red wine may greatly contribute to protect LDL from oxidativc d amage and, therefore, may be a key component in the French paradox.

Supported by FCT (POCI/AGR/599 19/2004) .

P-Oll Antioxjda n t st a tus of h uma n follieula r fluid: Implications in female infertility F adil Bakk.1li1

, David Roura1, Sandra Gonzalcz2, Jose Luis De Pablo~,

Zaloa Larreategui2, M. L uisa Hermindez 1, M. Bcgofia Ruiz-Larrea 1, & Jose Ignacio Ruiz-Sanz 1 1 Dcparlllto!lll of Physiolof<y, JV! cdicir1.: al'ld Dcmi.m~v S chool, University of the Basq11<! Cmmtrv, 2 IVI Bzlbao, Spain

The aim of this work was to determine the antioxidant s tat u> in foUiculnr fl uid and assess its involvement in woman in fertility. ORAC (oxygen radical absorbance capacity) and TAC (total antioxidant capacity) were measured in .fo llicular fluid aspira ted from follicles during oocyte pickup from women enrolled in IVF the rapy (11 - 30) unci were compared with the activities in follicular fluid aspirated from healthy control donor> (11 = 30). O R..'\ C was measured by a-scssing the area under the fluorescence decay curve (AUC) of fluorescein wiLI-t .'\AP!-1 as free radical initiator in the p resence of the sample as

compared to that in the blank in which no amioxid am is present. T he ORAC value was also determined in the soluble fraction after acetone deprotcinizarion. TAC was measured by the ABTS + radica l cation decolourization method. The fo llicular flu id of subfcnile women exhibited a significant lower OR AC value compared with control donors {5783 ± 1237 vs 6492 ± 1066 pM T rolox, p = 0.021 ). N o differences in either the ORAC value in dcproteinizcd samples or TAC were found between b oth groups. In conclusion, the reduced antioxidant activity in the follicular fluid suggests a role for free radica ls in women infe rti lity, probably contributing to impairment of reproduc­t ion in these P<Hicnts .

Supported by Gobierno Vasco (S:\lOT EK S-PE06UN03 and grant to

F B) .md !l·almet/Errasmik (grant to DR).

P-012 Fr ee t•mlieal scavengin g a c t ivity of d ifferent almond (P••u11us dulcis) val"ic ties joiio C. M . Barreira 1'1, Isabel C. F. R. Fcrrcira 1, Bcatriz O!iveira2

, & Jose Alberta Pereira 1

1C JMO-ESrlR, Imtitwo Polir~wico & Bragam;a, Campus d.: Sta. .-lpolimia, .rlpartado 11 72, 5301-855 B ragallfa, Porrugal, 2 REQUIMTEI Servivo de Brommologia, Fawldadc de Famuicia da Univ<rsidad~ do Porto, Rua llllibnl Cw1fw, 164, 4099-030 Porro, Porwgal

Reactive oxygen species a rc known to be implicated in many cell d isorders and in the development of many diseases including cardio­,·ascular disea,es, a therosclcrosb, cataracts, chronic inflammation or neurodegencrative diseases such "" Alzhc im~r'• or Pnrkinson '< disease. Thus, s)'nthetic antioxidants arc widely u;,cd in the food industry, but, because of their toxic and carcinogenic effects, their U>C i> being restricted. T he pursuit for novel natural sources of bioactivc com­pounds, namely those who present antioxidant activity, hm. been acquiring higher significance, since t hese compounds may contribute to the prevention of d i,eases in which free radicals are implicated. In this study, the antioxidant properties of different almond varieties (Cu.w1w1.w , Dnro ltaliallo, Ferradud , Fen·an/:J:,, f ',:,rrcd STar, Gtwra , kfolar, Ordha de klula and P~gari11hus ) were evaluated through several biochemical assays: D PPH (2,2-diphenyl- J-picrylhydrazyl) radical scavenging activity, reducing power, inhibition of p -carotene bleaching, inhibition of oxid ati\·c haemolysis in erythrocytes, induced by 2,2'­azobis(2-nmidinoprop;;ne)dihydrochloridc (AAPH) and inhibition of lipid pcroxidation in pig brain tissue through formation of thiobarbi­turic acid reactiw subotunces (TBARS). Fur all the methods, EC 50

values were calcula ted in order to evaluate the antioxidant efficiency of each variety. T he total phenols and fl avonoid contents were also ob tained and correlated wid1 antioxidant activity. Fcn·cd S rar and Dum lcalrano revealed better antioxidant properties, prescnring lower ECso valuco, particularly for lipid peroxidarion inhibition in TBARS assJy. The highest antioxidant comenrs (phcnols and fl avonoid>) were also found for these varieties.

F oundation for Science and Tcchnol<lg)' (Portugal) gave financial suppor t to J,C.M. Uarreirn (SFIUI/BD/29060/2006), and Program INTERREG IliA, Project P IREFI.

P-0 13 F r ee t•adical scaven ging ac tivity an d bioactive c om pounds of five A gm ·icu s s p. edible mushroom s Lillian Bnrros, Paula B nptista, D aniela M. CotTeia, & lsabel C. F. R. F erreira C IMO-J:.'SAB, In.stiwro Polit<wico de Bragau~a, Campus de S tu. rlpolonia, Apartado 1172, 530!-855 Brngallfa, Portugal

Reactive oxygen and free radicals play nn important role in cellular in jury and the ageing proc~~~ and a lso are considered to induce the lipid pcroxidation that causes the deterioration of food>. Although organisms have endogenous antioxidant defences produced during normal cell aerobic resp1ra tion against d1e reactive oxygen r.pecics, other antiox­idants a rc taken from rh~ diet, both from natural or syn d tc tic origin. Thus, synd1ctic antioxidanrs are widely used in food industry, but because of their toxic and carcinogenic effects, their u se is being restricted. Individual tocopherol profile of five AgariCIIs mushroom species, widely consumed in Portugal, was obtained by high perfor­mance liquid chromatography coupled to a fluorescence d etector

(HPLC/tluorcsccncc). 13ioactive compounds such as phcnols, !lavo­noids, ascorbic acid, fJ -carotene and lycopcnc were also determined. The liptd peroxidation inhibition capaciry of the edible mushrooms was acccsscd by biochemical ns~ays used as models for the lipid peroxida­tion damage in biomcmbrancs, namclly inhibition of (!-carotene bleaching in the presence of linoleic acid radicals, inhibition of erythrocyte~ haemolysis mediated by peroxyl radicals and inhibition of thiobnrbituric acid reactive substances (TBARS) formation in brain cells. T heir antioxidant properties were also evaluated through the reducing power determination and radical scavenging activity of 2,2-diphenyl- 1-picrylhydrazyl (DPPH) radicals. A . silvaricw revealed bcuer antioxidant properties (lower EC 50 values) than the o ther ,1garicw specie:. (d. arvmsis , A. bisporw, ,1 . rollwLrytesii , A. silvicola ) which is in agreement with the higher content of bioactive compounds found in the first ~pecie . :<-tocopherol and P -tocophcrol were found in the samples, while ;• - and ,; -tocopherols were not detected.

Rcscar<:h project POCI/AGR/56661 ,2004 (FCT- Portugal).

P-01-1 Effect~ of two n ew di(hetero)arylan~incs on p revention of ox.id.uive stress induced in two different biological models D. Pinto Basto 1

,]. P. Silva 1, M. ]. Queirozz, :\. ]. Moreno' & 0 . P.

Coutinho' 1 Oeparmrmr of BivloJ:.V, -D.:parrmmr of Chemisrry , Urrit·.:rsiry rJj J\lz11hv, Br..z .; •. z, Pr>rw~al, 1 Dcpamnmt of Zoology, Univasity of Coimbra, C:oimbra, Porwgal

We invc~tigatcd the antioxidnnt properties of two new dinrylamines from orgamc >ymhe; is (,\1JQ I and !v1JQ2) , whose basic srru~tur~ is similar to oth~ro, with reported antioxidant capacity, assesst:d by ch~mical test~, Jnd biological activity against microorganism5. In thi;, study we induc~d lipid peroxidation, in isolated rat liver mitochondria with ADP/Fe·' and the dbrylamine effects were examined by O~'Ygen con>umpllon Jnd by TBARS method. The 3nti-pcroxidati\ e effec t was maximal for r\11JQ J at 50 n.\ 1 (higher than the one reported for trolox) and for MJQ2 :n bC )iM. At these same concentrations none or them depressed the transmembrane pmemial 0 'I') developed by mitochon­dria, ne ither the RCR nor the ADP/0 ratio values For 2-fold con~enrracion~ both diarylamines were effective in the prevention of mitochondrial 6'1' collapse observed on respiring mitochondria, with the TPP - dectrodt!, which means a stabilizauon action on mitochon­drial mnt!r membrane. T he resulrs ob!aincd were confirmed tn who le cells. The compounds did nor show toxi~ iry to the L929 cell line, evaluated by the MTT reducing test and clc.trly protected from liptd pcroxidation, induced by the oxidant pair ascorbatc/Fe: ', to the PC 12 cell model, at the concentrations where maximal antioxidant effect was observed tn mitochondria. 1l1e new diarylamine~ were revealed as very good antioxidants at \'cry low concentrations, both in mitochondria and in whole cells. The results suggest a specific action site, for MJQ2 , at mitochondrial complex Jlevel. \\'le arc funher exploring other intracel­lular targets for these new compounds that seem very promising :1gainst pathologies where oxid:uive stress is involved.

Supported by FCT gram SFRH/BD/ 17 17-t/2004.

P-015 Nitric oxid e reg ulates Foxo3a activity to modulate mitochondr ial ROS produ ction Sara Borniquel 1

, Inmaculada Valle2, Yolanda Olmos 1

, Esrrd13 Soria3,

Santiago Lnmas 1, & !'vl aria .'vlonsalve 1

1Cmrro Nacional de brwsn'~:acioues Cardiovas.:ulares (CNIC), 1Hadrid, Spa m, : Oiaber~s Cemer, San Frmzcisco, CA, USA, 1Ccmm de lnvcsril(a­.:zollt'S Biologicas (CIB-CSIC) , lvfadnd, Spain

lt ha:. long been established rhat, depending on tl1c biological context, nitric oxide (NO) can play either" pro-oxidant or :m anti-oxidant role; however, the underlying molecular mechanism< have only recen tly begun eo be elucidated. Our studies hHvc ;;hown that NO can both positively and neg~tivc ly regulate the expression of the main proteins involved in mitochondrial ROS detoxification. Thi~ regulation involv~s

NO-mediated modulation of the mRNA levels of the transcriptional couctivator J>GC-1 2 via the activation of the: sGC/PKG pathway f I ). PGC- 1 x is tl1e master transcriptional regulator of mitochondrial

S19

function and ROS protection systems (2]. Our currcm work focuses on understanding the mechanism that mediates NO-induced down­regulation of PGC- I :t expresston in the vascul:lr endothelium. Since some NO effects are known to be mediated via activation of the Pl3K/ AKT pathway, we have examined tl1c effect of inhibiting PI3K. Pre­treatment of primary endmhelial cells with PIJK inhibitors prevented down-regulation of PG C - l a expression in response to the N O donor DET A-NO or the PKG activator 8-Br-G/VlPc, indicati ng that PBK acts downstream of PKG. Similarly, pre-treatment with the AKT inhibitor AKT IV blocked N O-induced PGC-la down-regulation, whereas DETA-NO increased the phosphorylation (activation) of AKT, supporting a role for the PI3K/AKT pathway in NO regulation of PGC-1 x expression. Point mutation in a functional IRS box of the mouse PG C-l ·" promoter cancelled NO activity, sugges ting the involvement of a FOXO transcription facror as a mediator of NO action. 1mportamly, FOXO factors arc phosphorylated and inactivated by AKT. lu further experiments we h:n·e shown that trcaunem wirh DET:\-NO lead' to phosphorylauun .tnd inacrivauon of rhe FOXO factor Foxo3a. To determine wheth~r l"oxo3a is in fact a transcriptional regulator of PGC-la we have u~ed a series of experimental approachc• . Over-cxprc.sion of a constitutive form or Foxo3a dramatically uprcgu­lated PGC-I:t levels, whereas a ~iRNA directed againt.t Ft1xo3a had the opposite effecr. ChiP analy>is showed that Foxo3a d irect:ly associates with the PGC-1 "' promoter and transient tr:msfcction U%ays indicated that Foxo 1a activates the PG C-l :.t promoter through the 'am.: binding sire thar is required fo r NO activirv. We therefore conclude that NO down-regulates PGC-l:.c expression through t:l1c Pl3KJAKT.Foxo3a pathway.

References

f I] Borniquel e t al. FASEB J 2006; 20: 1889 91. [2] Valle et al. C ardiovasc Res 2005;66:562 7'3.

P-016 Bilirubin induces oxidativc str ess in neurons, which is pt·cvcntcd by n NOS inhibition Maria A. Brito, :\na R. Vaz, Silvia Lima, Adelaide Fcmandcs, Ana S. Falcfio, Sandra L. Silva, Rui 1-. M . Silv:t, & Dora 13ritcs Cemro de Patogimcs~ Moi.:ctzlar- i.iH~d. UL, Facu/dade de Famzacza, Unh•.,·siry of Lisbon, Porwgal

High levels of unconjugaced bilirubin (UCB) can be toxic to the central nervous system and oxidative stre~~ is emerging as a rclt:vant event in the mechanisms of UCB encephalopathy. We itWC>tigatcd if exposure of rat neurons to UCB leads eo disruption of the redox status by a mechanism involving nNOS activation. Rat cortical neuronal cultures nt !l-days in vir.m were exposed to 50 or 100 ~LM UC13, for 4 h, at 37"C . To assess the role of NO, neurons were eo-incubated with I 00 pM UCB and 100 pM L-NAME, an inhibitor nf nNOS. All the experiments were performed in the presence of I 00 11•\1 human serum albumin, used to

solubilize UCB. Formation of prQ[ein carbonyls was assessed by slo t blot analysis, reduced glutathione (GSH) by an cnzymatic recycling assay and cell death was dete rmined by quantification of LDH release using a commercial kit. NO production was evaluated by quantification of nitrite levels using Gricss reagent, while the activity of nNOS was estimated by Western blot. Keuronal exposure to 50 or 100 11•"1 UCB led to protein oxidation (p < 0.05) and decrease in GSH content (p < 0.05), resulting in enhanced cell death (p < 0.01 ). Oxidative disruption was accompanied by nNOS activation (p < 0.05), with consequent increase in :--.:0 production (p < 0 .0 1) . All UCB-induced effects were significantly counteracted by co-im:ubution with L-NAJ\IlE. This ~tudy reinforces the involvement of oxidativc s tress in neuronal damage induced by UCB and demonstrates thnr cell lesion is mediated by nl'-:OS acrivation, therefore, pointing to NO <t> a key dcmem.

Funded bv 1-C'H'OCVSAU -MM0/55955/200'1.

P-017 Resverat t·ol prevents pcro:..'Ynitritc·induccd endothelial cells apoptosis by dis t·upting the rnitochotH:ll·ial pmbway Paula Brito, Nuria Sim<'ks, Leonor A!mcidu, & Tercsa Dini~ LabvratVno de BioquimicaJ Fw:uldadt! de! Fa.nuacia and Cenrro de Ncurocihtczas < Riologia Ce/u/ar d< Comzbra, 3000 Co1111bra, Porwgal