Table 1Effects of ACE inhibitors and AT1R blocker administration on DENA-induced changes in rat hepatic growth factors; VEGF, TGF-b1 and FGF.

Groups VEGFpg/ml

TGF-b1pg/ml

FGFpg/ml

Control 144.43 � 3.06 132 � 26 1005þ56Captopril 142.8 � 802 87 � 13.5 865 � 81Perindopril 133.3 � 6.22 150 � 20 1024 � 41Losartan 122 � 4.4 124.4 � 17.7 1035 � 54DENA 210 � 5.08* 158.5 � 44.9 1444 � 52*DENAþ Captopril 129.2 � 5.04# 123.7 � 28.5 1149 � 57DENAþ PerindoprilDENAþLosartan

144.7 � 7.8#

128.13 � 3.9#169.8 � 18.774 � 10.4

1156 � 83#

1025 � 116#

All data represent mean values � SEM (n¼10). ACE inhibitors and AT1Rblocker were given in drinkingwater for 15 consecutive days before DENAadministration and continue during the experimental period.

* Significant difference from control group.# Significant difference from DENA group. P < 0.05.

Table 2Effects of ACE inhibitors and AT1R blocker administration on DENA-induced changes in rat hepatic MMP-2, TIMP-1 and hydroxyproline.

Groups MMP-2ng/ml

TIMP-1pg/ml

Hydroxy-prolineng/ml

Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248166

factor (FGF). Moreover a marked increase in matrix metal-loproteinase-2 and hydroxyproline content were alsoobserved. Hepatocarcinogenesis was further confirmed bya significant decrease in hepatic endostatin and metal-lothonein level. Long-term administration of the selecteddrugs for 2 weeks before and throughout the experimentalperiod produced a significant protection against hepaticcarcinogenesis. The present results claimed that differentdoses of the selected drugs succeeded in normalization ofserum tumor markers. Furthermore, the drugs reduced theelevated level in the hepatic growth factors, matrix metal-loproteinase-2 and hydroxyproline induced by the hep-atocarcinogen. Moreover, the amelioration was alsoaccompanied by augmentation of hepatic content of metal-lothionein and endostatin. Histopathological examination ofliver tissues of rats treated with DENA–CCl4 correlated withthe biochemical observations.

Conclusions: These findings suggest a similar protectiveeffect of ACE inhibitors; captopril; perindopril and AT1Rblocker, losartan against premalignant stages of livercancer in the DENA initiated and CCl4 promoted hep-atocarcinogenesis model in rats. Therefore, RAS especiallyangiotensin II (Ang II) and AT1R interaction plays a pivotalrole hepatocarcinogenesis development.

Fig. 3. Liver from rat treated with DENA and CCl4 showing necrotic hepa-tocytes with occasional dysplastic nuclei (arrow head). The adjacent portaltracts were infiltrated by numerous inflammatory cells including manyeosinophils. H&E X 400.

Fig. 2. Effects of ACE inhibitors and AT1R blocker pretreatment before carcin-ogen intoxication onhepaticmetallothionein andendostatin. Bothweremeasureby ELISA kits. The results were expressed as ng/mL. Each column represents themean of 10 rats with a vertical bar showing the SEM. *Significant difference fromcontrol group (p<0.05). #Significant difference from DENA group (p<0.05).

Control 3.47 � 0.23 945 � 22 509 � 18Captopril 3 � 0.24 716 � 79 534 � 40Perindopril 2.4 � 0.47 852 � 118 547 � 56Losartan 3.3 � 0.44 753 � 50 500 � 40DENA 14 � 0.966* 1419 � 157 893 � 73*DENAþ Captopril 4.66 � 0.57# 1021 � 55 468 � 24#

DENAþ PerindoprilDENAþLosartan

5.3 � 0.57#

5.17 � 0.94#1856 � 147*1312 � 157

497 � 24#

621 � 32#

All data represent mean values � SEM (n¼10).ACE inhibitors and AT1R blocker were given in drinking water for 15consecutive days before DENA administration and continue during theexperimental period.

* Significant difference from control group.# Significant difference from DENA group. P < 0.05.

Keywords: losartan, captopril, perindopril10.1016/j.toxicon.2012.04.140

140. Identification, Pharmacological and Structuralcharacterization and Engineering of Three-finger Toxinsinteracting with GPCRs

Guillaume Blanchet 1, Gilles Mourier 1, Elodie Marcon 1,Bernard Gilquin 2, Nicolas Gilles 1, Denis Servent 11 Service d'Ingénierie Moléculaire des Protéines, Biologie Structurale etMécanismes, Gif-sur-Yvette, France2 Service de Bioénergétique, Biologie Structurale et Mécanismes.Gif-sur-Yvette, FranceE-mail address: denis.servent@cea.fr (D. Servent).

Background: To subdue their prey or protect themagainst predators, venomous animals have selected toxinsthat primarily interact with voltage-gated and ligand-gatedion channels, targets which play crucial roles in severalbiological functions. Nevertheless, some toxins are known

Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248 167

to recognize other molecular target families, such as theG-Protein Coupled Receptors (GPCRs). The first part of thetoxins interacting with GPCRs can be considered as func-tional mimetics of natural agonists of the receptor whileother toxins display structure and pharmacological profilesunrelated to any natural endogenous ligands.

Results: Here we first presented our last results relatedto the identification and pharmacological characterizationof new three-finger fold toxins interacting with alpha-adrenergic receptors. Two toxins: r-Da1a and r-Da1b, wereisolated from the Dendroaspis angusticeps snake venom andcharacterized for their high affinity and specific interactionwith a1a- and a2-adrenoceptors, respectively. The particularpharmacological profile of these toxins allows envisioningtheir exploitation as imaging or therapeutical agents, as forexample for r-Da1a in the treatment of benign prostatichyperplasia. In addition, structure-function studies ofvarious toxin-receptor complexes were performed inorder to identify at the molecular level the origin of thespecificity of these interactions. Our results reveal howMT7toxin recognizes the muscarinic M1 receptor and proposesa structural model of this complex in which the MT7interacts with a dimeric form of the receptor. This modelstructurally supports the high affinity and selectivity of theMT7-hM1 interaction, shows the atypical mode of interac-tion of this allosteric ligand with the GPCR receptor and canbe used as a starting point to design new ligands withpredetermine pharmacological property. Thus, MT7 engi-neering using block permutations lead to the generation oftoxins with new muscarinic/adrenergic functional profiles.

Conclusion:Our results identifymolecular determinantsinvolved in the affinity, selectivity and functional property ofaminergic toxins and highlight the ability of the three-fingertemplate to support various GPCRs interacting profiles.

References

C. Marquer et al. Structural model of ligand-G protein-coupled receptor (GPCR) complex based on experimentaldouble mutant cycle data: MT7 snake toxin bound todimeric hM1 muscarinic receptor, J Biol Chem 286 (2011)31661-31675.C. Rouget, et al. Identification of a novel snake peptide toxindisplaying high affinity and antagonist behaviour for thealpha2-adrenoceptors, Br J Pharmacol 161 (2010) 1361-1374.L. Quinton, et al. Isolation and pharmacological character-ization of AdTx1, a natural peptide displaying specificinsurmountable antagonism of the alpha1A-adrenoceptor,Br J Pharmacol 159 (2010) 316-325.

Keywords: three-finger toxins, GPCRs, bio-engineering10.1016/j.toxicon.2012.04.141

141. Lipid Bilayer Condition Abnormalities FollowingMacrovipera lebetina obtusa and Montivipera raddeiSnake Envenomation

Naira M. Ayvazian, Narine A. Ghazaryan, Lusine GhulikyanLaboratory of Toxicology, Institute of Physiology of NAS RA, Yerevan, ArmeniaE-mail address: taipan@ysu.am (N.M. Ayvazian).

Background: Viper bites are an endemic public healthproblem in Armenia, even in the cities. Venoms producedby snakes of the family Viperidae contain proteins thatinterfere with the coagulation cascade, the normal hae-mostatic system and tissue repair, and human envenoma-tions are often characterized by clotting disorders,hypofibrinogenemia and local tissue necrosis.

Methods: Studies on the interaction of snake venomand organized lipid interfaces have been conducted usinga variety of systems, including BLMs, SUVs and LUVs. Giantunilamellar vesicles (GUVs) with a mean diameter of 30 mmhave a minimum curvature and mimic cell membranes inthis respect. GUVs were formed from the total lipid fractionfrom bovine brain by the electroformation method (Ange-lova and Dimitrov, 1987). Macrovipera lebetina obtusa andMontivipera raddei venom was added to the samplechamber before the vesicles were formed. The membranefluorescence probes, ANS and pyrene, were used to assessthe state of the membrane and specifically mark thephospholipid domains. Fluorescent spectra were acquiredon a Varian fluoremeter instrument.

Results: The membrane fluorescence probes, ANS andpyrene, were used to assess the state of membrane andspecifically mark the phospholipid domains. Independent oftheir lipid composition, all GUVs modified by Macroviperalebetina obtusa venom were enlarged in size as venom-dependent lipid hydrolysis proceeded. In contrast, liposomesmodified with Montivipera raddei venom demonstrate a socalled “oval deformations” and venom-dependent shrinkingresponse. In addition to the visible morphological changes,ANS and pyrene also allows us to quantify the fluiditychanges in the membrane by measuring of the fluorescenceintensity. Thepresenceof vipervenom inGUVsmedia revealsa noticeable decreasing of membrane fluidity compare thecontrol, while the binding of fluorophores with GUVsmodified by venom lead to appearance of channel activity.

Conclusions: These studies emphasize the importanceof a membrane surface curvature for its interaction withenzymatic components of venom.

Keywords: BLM, GUV, electroporation, snake venomics, artificialmembranes10.1016/j.toxicon.2012.04.142

142. From alpha-Conotoxins and alpha-Neurotoxins toEndogenous “Prototoxins" and Binding Sites inNicotinic Acetylcholine Receptors

Victor I. Tsetlin, Yuri N. Utkin, Igor E. Kasheverov,Ekaterina N. LyukmanovaShemyakin-Ovchinnikov Institute of Bio organic Chemistry, Russian Academyof Sciences, Moscow, RussiaE-mail address: vits@mx.ibch.ru (V.I. Tsetlin).

Background: Snake venom alpha-neurotoxins helpedto isolate a muscle-type nicotinic acetylcholine receptor(nAChR) and more recently the acetylcholine-bindingprotein (AChBP), an excellent model for the ligand-bindingdomains of all nAChR subtypes. Alpha-conotoxins fromConus snails are more selective in distinguishing neuronal