25th Congress of the newEuropean Society ofComparative Biochemistry and Physiology

Ravenna (Italy) - Sept 7-11, 2008

New challenges in integrative physiology and biochemistry:From molecular mechanisms to environmental adaption

Cellular homeostasis and the environment: Towards a better understanding of interactionsof multiple stressors

SESSION OPENING LECTURE

1.The modulating effect of individual differences on the impact ofmultiple stressors on homeostasis

S.E.Wendelaar Bonga (RadboudUniversity, Nijmegen, The Netherlands)

The stress response is an integrated response to external threats(stressors) originating from physical, chemical or biological factors.Stressors induce typical patterns of activation in the brain and theendocrine system, leading to increased cardiac and respiratoryactivity, followed by stimulated energy mobilization. When chronic,stress is associated with decreased growth, reproduction and diseaseresistance. Since the stress response is essentially a uniform response,irrespective of the type of stressor, exposure to more than one stressorcauses effects that (within certain limits) are cumulative. Undernatural conditions, and more in particular in farming or aquaculture,several factors may have mild stress effects that may be subliminal foran observer. However, under such conditions an additional stressor ormanagement activity, that has normally no serious effects on theanimals, may then easily lead to unexpected losses of appetite andweight or even serious disease outbreaks andmortality. Moreover, theimpact of stressors is modulated by individual differences betweenanimals. Life stage (larval, juvenile or adult), reproductive phase aswell as, in social animals, hierarchical position are important factorsthat can modulate considerably the impact of stressors on anindividual or a group of animals. As a result, the effects of e.g.experimental exposure of animals to stressors may show largefluctuations between experiments, even under experimental condi-tions considered as standardized. This complicates the determination

of for instance optimal farming conditions or the determination of noeffect levels of exposure to toxic chemicals. Such problems will beillustrated by differences observed between tilapia of high and lowhierarchical position in the impact of chemical and other stressors.Such individual differences, which in mammals have been linked togenetically determined bimodality in (proactive and reactive) copingstyles, are also present in birds, reptiles and fish, and are associatedwith low and high cortisol responses to stressors.

doi:10.1016/j.cbpa.2008.05.074

ORAL PRESENTATIONS

2.Use of the land snail Helix aspersa as sentinel organism formonitoring ecotoxicological effects of atmospheric pollution inurban areas

S.Gorbi, A.Notti,D. Fattorini, R. Bocchetti,M.Benedetti, F. Regoli (IstitutodiBiologia e Genetica, Università Politecnica delle Marche, Ancona, Italy)

The increase of vehicular traffic and emissions are majorcontributors to air pollution and a matter of growing importance inurban areas. Despite normative limits indicate themaximum levels fora number of individual parameters (i.e. PM10, benzene, carbonmonoxide, sulphur oxides), the toxicological risks of atmosphericpollutants remain relatively unexplored in field conditions.

In this work the impact of urban emission was analyzed with anintegrated ecotoxicological approach using the terrestrial gastropodsHelix aspersa as bioindicator species. Organisms were caged in various

S19Comparative Biochemistry and Physiology, Part A 151 (2008) S19–S30

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A

j ourna l homepage: www.e lsev ie r.com/ locate /cbpa

sites of Ancona town, and a significant accumulation of trace metalsand polycyclic aromatic hydrocarbons was observed after 4 weeks,depending on the different levels of traffic intensity. Chemical datawere integrated with the analyses of several molecular and biochem-ical biomarkers, including the induction of detoxification pathwaysand the onset of cellular damages which confirmed the biologicalreactivity of accumulated contaminants. Such biological responsesrevealed a significant induction of metallothioneins and peroxisomalproliferation, while a limited activity of biotransformation enzymes.Efficiency of antioxidant defenses, total oxyradical scavenging capacity(TOSC), lysosomal membrane stability and DNA integrity were alteredin organisms caged in sites with more elevated traffic intensity.

The overall results of this study confirmed the utility of land snailsas a model sentinel species, suggesting that toxicological andbiochemical studies, integrated with data from air quality monitoringstations, could provide important information about the impact ofatmospheric pollutants on human health and ecosystems.

doi:10.1016/j.cbpa.2008.05.075

3.Perfect adaptation to cope with metal stress and trace elementhomeostasis: Metal specificity is a fundamental structural featureof snail metallothioneins

S. Atrian, A. Pagani, (Universitat de Barcelona, Spain); M. Capdevila,O. Palacios, (Universitat Autonoma de Barcelona, Spain); M. Egg,M. Höckner, R. Dallinger (University of Innsbruck, Austria)

The metallothionein (MT) isoforms from the terrestrial snail Helixpomatia prove high metal specificity and thus cell- and organ-specificfunctions which have so far not been described in any other animalspecies. The Cd-binding MT isoform accounts for detoxification, whilethe Cu-binding isoform plays a role in Cu homeostasis. In our previousexperiments, nearly pure metal-specific isoform variants were purifiedfrom snail tissues in vivo by High Performance Liquid Chromatography(HPLC),with characteristic features uponUVandCircularDichroism (CD)spectroscopy, and Electrospray ionization mass spectrometry (ESI–MS).

In the present work, the two isoforms were recombinantly expressedas fusion proteins (GST/MT) after metal supplementationwith Zn2+, Cd2+

or Cu2+ of the E .coli culture media. The pure metal–MT complexes wereanalysed by ICP-AES, UV and CD spectroscopy and ESI–MS, confirmingtheir metal specificity. The metal-specific physiological performance ofthe two isoformswas demonstrated byexpression inDTY4p424 yeastMTknockout cells, where the snail Cd-MT (but not Cu-MT) conferred to thecells an elevated Cd tolerance, whilst the snail Cu-MT (but not Cd-MT)was able to protect the cells from Cu toxicity. We conclude that metalspecificity is a fundamental structural feature of the two MT isoformsfrom Helix pomatia, suggesting that the two peptides may have beenshaped during evolution in a unique manner to perform metal-specificphysiological tasks.

doi:10.1016/j.cbpa.2008.05.076

4.Biochemical characterization of metallothionein isoforms interrestrial snails: Relationship between amino acid sequence andmetal binding specificity

D. Schuler, R. Dallinger (University of Innsbruck, Austria); F. Hispard,A. De Vaufleury (University of Besancon, France)

Terrestrial helicid snails (Helicidae) accumulate and store hugeamounts of metallic trace elements (Cu, Cd) directly from theirenvironment by binding the metals to Metallothioneins (MTs).Previous studies with different helicid species (Helix pomatia, Ar-ianta arbustorum) have revealed that these animals possess twostrictly metal-specific MT isoforms. While one of them is directlyinvolved in the maintenance of intracellular Cu+ homeostasis, thesecond isoform is strictly associated with Cd2+ and plays an importantrole in detoxification of this harmful metal.

The purpose of the present studywas to identify and characterizeMTisoforms from another helicid species,Cornu aspersum. Three full-lengthMTcDNAswere characterized from this species. Twoof themexhibited ahigh degree of identity with the formerly known Cu+- and Cd2+-specificMT isoforms from Helix pomatia. The third isoform isolated from C.aspersum seems to be associated with bothmetals (Cd and Cu). Analysisof the translated aminoacid sequence of the intermediate Cd/Cu isoformexhibited major differences in the primary sequence compared to theother two isoforms. The three aminoacidsArg, Lys andAsn (prevailing inthe other two isoforms) were replaced by the neutral amino acid Ser.

We suggest therefore, that the molar content and position of theamino acids mentioned within the sequence of the three isoformsmay contribute significantly to metal specificity. Computationalanalysis of several biochemical parameters indicates, moreover, thatthe novel Cd/Cu isoform may represent an intermediate evolutionarystage between the two other, metal-specific isoforms.

doi:10.1016/j.cbpa.2008.05.077

5.Understanding the functioning of a Gene: Ability of theCd-Metallothionein of Roman snail (Helix pomatia L.) to cope withMultiple Environmental Stress

R. Dallinger, M. Egg, M. Höckner, D. Schuler (University of Innsbruck,Austria)

Metallothioneins (MTs) are low-molecular mass, metal-chelatingproteins. They are induced in most animal species upon environmentalexposure to certain heavymetals (Cd2+, Cu2+), protecting their hosts fromharmful effects due to interactions of metal ions with cellular structures.Terrestrial helicid gastropods, for example, possess Cd-detoxifying MTisoforms which confer to these animals the ability to survive at highlyelevated Cd concentrations in the soil. Our recent experiments haveshown that apart fromCdexposure, these isoforms are also induceduponexposure to non-metallic stressors playing an important role in the snail'sadaptation to rapidly fluctuating environmental conditions (desiccation,starvation). Obviously, the induction pattern of these isoforms ismeticulously regulated at the transcriptional level.

We can show that the Cd-MT gene of Helix pomatia exhibits severalremarkable features not common to most other MT genes. Apart from apromoter region with typical arrangements of Metal-Responsive Ele-ments (MREs), the gene contains several intronic cassette-like structuresconsisting of repeats of putative Transcription Factor Binding Sites (TFBs)for stress-involved transcription factors, and for transcriptional enhancersand activators. A bioinformatic analysis demonstrates that among all MTgenes known so far, the Cd-MT gene of H. pomatia exhibits the highestrelative number of stress factor binding sites per nucleotide sequence.

We suggest that owing to these particular features, the Cd-MTgeneof H. pomatia may have adapted specifically to coping with multi-farious environmental stress in the particular chemical environmentin which terrestrial snails thrive.

doi:10.1016/j.cbpa.2008.05.078

S20 Abstracts / Comparative Biochemistry and Physiology, Part A 151 (2008) S19–S30