Analysis of stress-induced gene expression in trout red blood cells following

tributyltinchloride exposure

Luca Tiano1,*, Ian Davies2, John Craft3 and Giancarlo Falcioni11Department of Biology MCA, University of Camerino, Via Camerini 2, I-62032, Camerino, MC, Italy;2Fisheries Research Services, Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen, AB11 9DB,UK; 3School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road,Glasgow, G4 0BA, UK; *Author for correspondence (Phone: +39-0737-403213; Fax: +39-0737-636216;E-mail: luca.tiano@unicam.it)

Accepted: 28 May 2005

Key words: hsp70, tributyltin, trout erythrocyte, xenobiotic

Abstract

Submicromolar concentrations of tributyltinchloride (TBTC), an organotin compound, is able to impairmitochondrial activity in trout erythrocytes, as detected by ultrastructural transmission electron micros-copy investigations and exhibits pro apoptotic activity in a variety of cells including nucleated red bloodcells. Here we show that TBTC is able to induce the expression of hsp70 in rainbow trout (Onchorynchusmykiss) erythrocytes in a time- and dose-dependent manner. This induction was demonstrated by quan-tification of the relative abundance of mRNA, and the synthesis of hsp70 was detected by means of animmunological test. This study could provide a practical assay of environmental stress at sublethal toxicantconcentrations.

Introduction

Organotin compounds result from the addition oforganic moieties to inorganic tin and thereforeinvolve one or more tin-carbon bonds. Suchcompounds have a wide range of industrial andagricultural applications and are ubiquitous envi-ronmental contaminants. Organotins have beenfound in water and sediments of both freshwaterand estuarine environments (Fent and Hunn 1991;Schebeck et al. 1991; Wuertz et al. 1991).

The possibility of organotin compounds beingconcentrated through the food chain as a resultof bioaccumulation is a significant concern.Bioconcentration factors may range between 100and 500,000 for organisms including microorgan-isms, macroalgae, invertebrates, fish, birds, andmammal (Laughlin 1996).

Tributyltin (TBT) compounds are used as bio-cides. The presence of TBT in the environment has

attracted the most attention, especially because ofits use in antifouling paints used to coat surfacessubmerged in water, most notably the hulls ofboats. The biocide is released from the paintcoating with time, resulting in the formation of athin layer relatively enriched in TBT immediatelyadjacent to the painted surface, which repels orkills nuisance organisms such as barnacles. How-ever, TBT can diffuse further, resulting in con-tamination of adjacent water and sediments, andexposure of non-target organisms. This hazard ledto restrictions of its use in antifouling paints.France was the first country to implement restric-tions in 1982/the UK followed in 1985 and laterthe United States introduced the ‘‘OrganotinAntifouling Paint Control Act of 1988.’’ Thisprohibited painting of vessels shorter than 25 m(aluminum hulls exempted) and required paints toleach at a rate no greater than 4 lg TBT cm)2 day(Huggett et al. 1992a, b).

Fish Physiology and Biochemistry (2004) 30: 231–240 � Springer 2005DOI 10.1007/s10695-005-8244-5

Nevertheless, organotins still occur at elevatedlevels in higher organisms, even after legislation. Infact, the persistence of organotins in sediments isof continuing importance.

In fact, while in the water columns the half lifeof TBT in sediments is in the range of days orweeks (Hoch 2001) TBT can accumulate in sedi-ments following strong association with naturalsorbents. Moreover, the anoxic environment andthe absence of light, strongly reduces breakdownof these molecules due to oxidation and photolysis(Fent and Hunn 1991). As a consequence, highlevels of TBT have been found in freshwater andcoastal sediments, with concentrations up to sev-eral mg/kg. Remarkably in Switzerland, the firstcountry to introduce a complete ban in 1988, itwas demonstrated a reduction in the TBT con-centrations in the surface water, but no significantdecrease in sediment samples or mussels tissueswas observed over 4 years following legal restric-tions (Becker and Tarradellas 1995). Finally,contamination of sediments rises great concernsince TBT sorption is reversible (Dowson et al.1993), indicating that contaminated sediments canrelease TBT to overlying waters. Mechanical dis-turbance, such as storms or anthropogenic activi-ties such as dredging may facilitate this process.

Because of their lipophilicity, organotins can beregarded as membrane-active, and the cytoplasmis an obvious initial site of action. Organotins maybecome embedded at different levels in lipid bi-layers, as indicated by alterations in membranefluidity in trout erythrocytes (Ambrosini et al.1996; Santroni et al. 1999). Organotins also actintracellularly, and processes dependent on intactinternal organelles, including respiration andphotosynthesis, may be disrupted (Avery et al.1991; Mooney and Patching 1995). Consequencesat the ecosystem level normally display a longresponse time and, if effects eventually occur, it isusually too late for effective countermeasures to betaken. Before such damage becomes apparent,pollutant exposure may lead to decreased growthrates, elevated levels of infections and disease anddecreased reproduction rates. It is generallyaccepted in ecotoxicology that even these respon-ses are preceded by effects at the molecular andcellular levels in individual organisms.

Biomarkers are defined as biological measuresof sub-lethal responses to, and effects of, pollutantsin living organisms (Peakall 1994). Biomarkers can

provide powerful and cost-effective approaches toobtaining information on the state of the environ-ment and the effects of pollution on living biolog-ical resources (McCharty and Shugart 1990;Peakall 1992; Wester et al. 1994; Depledge et al.1995). The basis of biomarker responses is theutilization of known biochemical and physiologicalevents, which precede visible organismal and pop-ulation changes, as sub-lethal indicators of earlystages of toxicity and pathology. They are thereforeable to act as biological early warning systems ofadverse effects, in line with the ‘‘precautionaryprinciple’’ adopted in various international fora(Goksøyr et al. 1994). Living cells respond to stresswith specific biochemical changes. One of the mostprofound and best-studied biochemical indicatorsof stress is the rapid induction of a class of proteinsknown as heat shock proteins (HSP) (Nove 1984;Lindquist 1986; Morimoto et al. 1990). Thisresponse was first described by Ritossa (1962) inDrosophila, who used heat shock and 2,4-dinitro-phenol as inducing stressors. Subsequent studiesfrom a number of laboratories have shown that theheat shock response is ubiquitous, and that themajor HSPs are conserved through evolution atthe protein and DNA levels. Despite their biolog-ical relevance and wide interest in ecotoxicologystudies, HSPs lack specificity being induced by avariety of disturbances. Therefore, they are notreliable biomarker, nonetheless they could providea useful tool in in vitro ecotoxicity prescreening,revealing the ability of complex toxicant mixturespresent in the environment to evocate a stressresponse (Bierkens 2000).

In the last few years, our research group hasinvestigated the effect of different organotins ontrout nucleated erythrocyte (Falcioni et al. 1996;Falcioni and Zolese 2000; Tiano et al. 2001) insome detail. The effects were studied by followinghemolytic process, measuring steady-state fluores-cence anisotropy of different probes on isolatedmembranes, evaluating the stability of trouthemoglobins. Genotoxic effects were detected bymeans of the ‘‘comet assay’’. The results obtainedindicate that incubation in presence of triorganotin(tributyltin chloride and triphenyltin chloride)induced a plasma membrane perturbation, accel-erated the precipitation process in HbIV andinducedDNA single strand breaks to a great extent.

Fish red blood cells (RBCs) were chosen asthe cellular model because, unlike anucleated

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mammalian RBCs, the nucleated RBCs of lowervertebrates preserve both nucleus and mitochon-dria and can provide an attractive ‘‘strippeddown’’ model to study the effect of organic pol-lutants on cellular compartments. Furthermore,fish RBCs are capable of protein synthesis andpossess several unique characteristics making theman ideal system to study the adaptive significanceof stress protein synthesis (Currie and Tufts 1997;Lund and Tufts 2003).

(1) Unlike their anucleated counterparts, fishRBCs have high levels of aerobic metabolicactivity, which supports many cellularprocesses, including protein synthesis.

(2) Blood is easily collected and experimentallymanipulated, and many cellular functionsand experimental approaches have beendescribed in the literature.

(3) Blood sampling and analysis could serve asa valuable, non-terminal means of assessingstress in fish in natural or aquacoltureenvironments.

(4) The role of stress proteins within the RBCsof an active, highly temperature-sensitivespecies may be particularly critical to pre-serve RBC function under stressful condi-tions and to ensure adequate delivery ofoxygen to the tissues.

This study is important in elucidating new aspectsof stress protein synthesis in nucleated erythro-cytes and it could result in the development ofbiosensors for application in the aquatic environ-ment.

Materials and methods

Animals, cell lines, erythrocyte isolation and colturemedium

Rainbow trout (Oncorhynchus mykiss) obtainedfrom a fish farm at Cruden Bay, Scotland, UKwere used as experimental organism. Fish werekept in circular tanks at 10±1 �C with constantaeration and water circulation.

Fish were sacrificed by a blow to the head andbled from the caudal sinus into heparinized vacu-tainers. Erythrocytes were isolated by centrifuga-tion of diluted blood in isotonic medium (0.1 Mphosphate buffer pH 7.8, 0.1 M NaCl, 0.2% cit-

rate, 1 mM EDTA) at 3000 rpm for 10 min. Afterremoval of plasma and buffy coat, the red bloodcells were further washed three times in the samebuffer and concentrated by centrifugation underthe same conditions.

RTG-2, a fibroblast-like line originally derivedfrom mixed gonadal tissue of male and femalerainbow trout, was used as an additional, in vitromodel. Cells were grown in flasks incubated at22 �C. The colture medium consisted of Lebovitz-15 medium supplemented with 100 U of penicillin–streptomycin per ml, and 10% fetal bovine serum(all from GIBCO Ltd.).

The hydrophobicity of TBTC presented someproblems in determining dose–response curves incell colture systems. As proposed by other authors(Zhang and Liu 1992), variance can often beattributed to the use of serum-supplemented vs.serum-free media in different experimental proto-cols. All the experiments presented in this studywere carried out in L-15 medium without addedserum, unless otherwise specified.

In vitro heat shock treatment and TBTC exposure

After washing, erythrocytes were suspended in L-15 medium and kept for 24 h at 12 �C in order tominimize the heat shock response due to handlingstress. Cells were further resuspended in L-15medium in presence of tributyltinchloride at ofconcentration of 1 nM–5lM. for 6 h at 12 �C formRNA quantification, and up to 12 h for hsp70determination. An aliquot of approximately1 · 107 cells was collected hourly, pelleted andimmediately frozen in liquid nitrogen. In addition,a temperature rise of 10 �C was applied tountreated cells and used as a positive control.Samples were collected between times 0 and 6 h.

Construction of a cDNA hsp70 probe forOnchorynchus mykiss

Tissues and cells (liver, brain, red blood cells andleukocytes) were collected from heat shockedrainbow trout. Heat shock was obtained bytransferring the animals acclimated at 10 �C to atank containing water at 19 �C. Fish were sacri-ficed after 2 h of thermal shock, cells and organswere collected and immediately frozen in liquidnitrogen. A hsp70 cDNA probe of 238 bp wasgenerated by one-step reverse transcription

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polymerase chain reaction (RT-PCR) usingReady- To-Go PCR beads from Amersham Bio-science. An hsp70 probe was designed on theoverlapping region from the rainbow trout cDNAsequences pTHS70.7 (A.N. K02549) andpTHS70.14 (A.N. K02550), as reported in the lit-erature (Kothary et al. 1984) (Figure 1). Follow-ing total RNA extraction, a one step reversetranscriptase procedure was performed using theforward primer 5¢-CTT ACC TGG GCC AGAAGG T, and reverse primer 5¢-ATG GTC AGGATG GAC ACG T. The reaction mixture wasincubated at 42 �C for 30 min and, after denatur-

ation of reverse transcriptase at 95 �C for 5 min,30 amplification cycles were executed at anannealing temperature of 52 �C.

PCR products were visualized by gel electro-phoresis, which confirmed the amplification of afragment of the predicted size. Amplified bandswere subsequently excised from the gel and DNAwas extracted using GFX PCRDNA andGel BandPurification Kit from Amersham Bioscience.Purified cDNA fragments were finally ligated intothe pGEM-T easy vector (Promega) and clonedinto competent DH 5 a E. coli. Plasmid DNA fromtransformed coltures was isolated using Quiaprep

Figure 1. Nucleotide sequences for THS10.7 and THS70.14 cDNAs. The overlap region between the two different cDNA ishighlighted.

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plasmid mini kit (Quiagen) and sequenced usingM13 primers and the ABI prism dye terminatorcycle sequencing ready reaction kit (Perkin ElmerApplied Biosystems).

Sequencing of the vector showed the presenceof the required 238 bp region, identical to theclone THS70.7 conserved cDNA sequence repor-ted in literature. All the sequences amplified fromdifferent tissues were identical.

mRNA quantification, northern and slot blot

Total cellular RNA was prepared from treatedrainbow trout erythrocytes and the RTG-2 cell lineusing RNAzolB (AMS Biotechnology), a guani-dine isothiocyanate based extraction medium,isopropanol precipitated and resuspended indiethylpyrocarbonate (DEPC)-treated water.Purified RNA was treated with 10 U DNase I(Ambion) per ml RNA sample at 37 �C for30 min. Subsequently, RNA was quantified byspectrofluorometry using Ribogreen RNA quan-tification kit from Molecular Probes. For North-ern blots, aliquots of 20 lg were electrophoresedunder denaturing conditions on a 1% agarose/formaldehyde gel. After electrophoresis, the RNAwas transferred to a positively charged nylonmembrane (Roche) using a passive, slightly alka-line, downward elution. This procedure is muchfaster than upward transfer, and therefore resultsin tighter bands and stronger signal. Slot blotswere performed using a microsample filtrationmanifold.

The transferred RNA was UV crosslinked andhybridized with a rainbow trout hsp70-PCRderived cDNA probe. Probes were labeled withdigoxigenin (DIG) using a PCR probe synthesiskit from Roche. Hybridizations were performedusing UltraHyb ultrasensitive hybridization bufferfrom Ambion at 42 �C for 18 h. Finally, two lowstringency washes (2 · SSC, 0.1% SDS) for10 min at room temperature, and two high strin-gency washes (0.1 · SSC, 0.1% SDS) for 15 minat 42 �C, were applied prior to chemiluminescentdetection using anti-digoxigenin, alkaline phos-phatase conjugates and the chemiluminescentsubstrate CSPD (Roche). All glassware and solu-tions were treated with 0.1% DEPC and baked orautoclaved, respectively, before use. Semiquanti-tative analysis was performed by quantification of

density of autoradiographies using the analysissoftware Quantity One (Biorad).

Hsp70 detection

Hsp70 in cell lysate was quantified after xenobioticexposure for up to 12 h using a commercial ELISAhsp70 detection kit from Stressgen, Victoria,Canada. After incubation at 12 �C in presence ofTBTC, 1 · 107 cells from each sample were lysed.Cell lysates were normalized according to proteincontent to 0.3 mg/ml and assayed. The assayconsisted of a quantitative sandwich immunoas-say. A mouse monoclonal antibody, specific forinducible hsp70 was precoated onto the wells of amicrotitre plate. Inducible hsp70 was captured bythe immobilized antibody and detected with ahsp70 specific, biotinylated rabbit polyclonalantibody. The biotinylated detector antibody issubsequently bound by an avidin horseradishperoxidase conjugate. The assay is developed withtetramethylbenzidine substrate and a blue colordevelops in proportion to the amount of capturedhsp70. The color development is stopped byaddition of an acid solution, which converts theendpoint color to yellow. The intensity of the coloris measured in a microplate reader at 450 nm.Hsp70 concentrations in samples are quantified byinterpolating absorbance readings using a stan-dard curve generated using the hsp70 proteinstandard provided with the kit.

Statistical analyses

Significant differences (P<0.05) within treatmentswere detected using one-way ANOVAs and iden-tified using the Student–Newman–Keuls tests.Significant differences (P<0.05) between treate-ments were detected using paired Student t-tests.

Results

Quantification of relative abundance of mRNAfor hsp70 and hsp70 immunological detectionTo study the hsp response at the level of geneexpression, we measured the amount of hsp70mRNA in heat shocked trout erythrocytes and incells exposed to TBTC. Both stimuli resulted in anincrease in the expression of hsp70. After incuba-tion of the cells at 22 �C, the amount of hsp70

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mRNA increased significantly compared to basalvalues after 1 h of incubation (P<0.05) and reacheda maximum after 2 h. mRNA levels returned tovalues comparable with the control after 4 h (Fig-ure 2). Therefore, there was a marked increase inhsp70 gene expression in response to elevated tem-perature. TBT-mediated hsp70 gene expressionpresented a complex time and dose dependentresponse, as shown in the slot blot in Figure 3. After1 h of incubation in presence of TBTC, mRNAexpression profiles presented a clear dose dependentresponse in the range 1 nM/5 lM. However, thekinetics of expression were different at differentconcentrations of TBTC.

The level of heat shock mRNA induction seemsto reflect the intensity of the inducing stimulus.The levels of hsp70 mRNA varied with the con-centration of TBTC used; hsp70 levels increasedwith exposure concentration up to approximately100 nM TBTC.

Figure 3 illustrates the time course of effect ofTBTC. At lower concentrations (1–100 nM), agradual and significant increase in expressionoccurred up to 6 h of exposure, compared to basalvalues (P<0.05). On the contrary, expressionpeaked already after 1 h of exposure at higherconcentrations (1–5 lM), and did not show anysignificant variations over time.

Overall, significant differences between treat-ment concentrations were detectable up to 100 nMconcentration and for exposure times below240 min (P<0.05; Figure 3c) indicating thatexperimental conditions outside these ranges leadto a saturation of the signal.

Northern blot analysis provided a better visu-alization of the induction of the specific gene, asshown in Figure 4. By repeating the experimentwith another in vitro model, RTG-2 cells, incu-bated in presence of TBTC at the same range ofconcentrations, no significant increase in the levelof hsp70 mRNA was detected in relation to dif-ferent concentrations of TBT or different periodsof exposure (Figure 5). Immunological detectionof hsp70 in trout erythrocytes seems to vary sim-ilarly (Figure 6). Hsp70 levels in erythrocytesincubated in presence of 100 nM TBTC increasedafter 4 h and reached a maximum after 8 h.However, at 1 lM, hsp levels peaked immediatelyafter exposure to TBTC and remained elevated incomparison to controls. Trout hsp70 was detect-able at low levels in uninduced cells by this tech-nique, possibly due to the presence of hsp70-likegenes that are expressed at a low level. In fact,since the mouse monoclonal antibody is specificfor the inducible hsp70 in the rodent, it might alsorecognize constitutive Hscs in rainbow trout.

Discussion

TBT has long been known to be extremely toxic toa wide variety of terrestrial and aquatic organisms(Fent 1996). The aim of this study was to assess thevalidity of hsp70 levels in nucleated trouterythrocytes as a biomarker of TBT-mediatedstress. Stress proteins play a primary role in pro-tecting cells from injuries caused by a variety ofpollutants, and stress protein levels have potentialuse in environmental monitoring (Sanders 1990;Huggett et al. 1992a, b; Sanders 1993). There havebeen numerous studies of stress proteins as bio-markers, Stress protein induction has been shownto be caused by many pollutants at environmen-tally realistic levels (Sanders et al. 1991; Bradley1993; Dyer et al. 1993). Moreover, given thediverse functions of hsps, the induction of hsps byTBTC in nucleated trout erythrocytes may providea basis for improved understanding of the effectsof TBTC at molecular and cellular levels.

Figure 2. Induction of hsp70 mRNA by heat shock. Slot blot:Erythrocytes were treated with a 22 �C heat shock for varioustime periods. No recovery time was allowed. After total RNAextraction, slot blot was performed as described above. mRNAlevels for hsp70 are significantly increased compared to basalvalues between 60 and 120 min (*P<0.05; n=3).

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Figure 3. Induction of hsp70 mRNA with TBTC. Slot blot (a): Erythrocytes were exposed to TBTC at concentrations of 1 nM–5 lMfor various times. No recovery was allowed. After total RNA extraction slot blot was performed as described above. (b) Significantdifferences were detectable up to 100 nM TBTC and for exposure times below 240 min, indicating that conditions outside these rangeslead to a saturation of the signal (c) c Control C; 100 nM TBTC n 5 lM TBTC. Signficancy refers to 100 nM TBTC concentration(*significant increase compared to basal values; **Significant differences between treatment concentrations; P<0.05; n=3).

Figure 4. Induction of hsp70 mRNA with TBTC. Northern blots: Erythrocytes were exposed to TBTC at concentrations of 1 nM–1 lM for 60 min. (a) or to a single concentration of TBTC 0.1 lM for various times (b). No recovery was allowed. After total RNAextraction Northern blots were performed as described above, Quantitative and qualitative control was performed by evaluatingrRNA 18S.

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We have examined the kinetics of hsp70 syn-thesis in trout red blood cells induced by elevatedtemperature and by sub-micromolar concentra-

tions of TBTC. Both stimuli result in rapid syn-thesis of trout hsp70 mRNA. Prolonged exposure(up to 6 h) to TBTC, however, causes continuedincreases in hsp70 mRNA levels, whereas contin-ued heat shock beyond 2 h results in a decrease inhsp70 mRNA levels. This could be due to a dif-ference in either the regulation of transcription bythese two inducers or in their inductionmechanisms.

Exposure to TBTC did not increase the levelsof hsp70 mRNA in rainbow trout RTG-2 cell line.This data is not surprising if it is noted that bothtissue and species-specificity of hsp70 synthesis inbrain, gill and liver has been reported in four dif-ferent fish species (Dietz and Somero 1992).Moreover, the protein expression pattern of cellstaken from fish exposed to a stressor may differfrom the response of stressed primary cell colturesor immortalized cell lines (Kultz 1996). These dataemphasize the suitability of trout erythrocytes forthis assay.

The heat shock response has been extensivelystudied, yet the molecular signals that inducesynthesis of hsp remain unclear. Several hypothe-ses can be postulated to describe the molecularmechanisms that link TBTC stress and increased

Figure 5. Induction of hsp70 mRNA with TBTC. Northernblots: RTG-2 cells were exposed to TBTC at concentrations of1 nM–1 lM for 60 min. (a) or to a single concentration ofTBTC 0.1 lM for various times (b). No recovery was allowed.After total RNA extraction Northern blots were performed asdescribed above.

Figure 6. Induction of hsp70 in trout erythrocytes following incubation for up to 12 h in presence of TBTC 0.1 lM, 1 lM and inuntreated control samples.

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hsp70 levels. Transcription of hsp genes appears tobe influenced by the presence of denatured pro-teins, but since hsps have diverse functionsinvolving stress, pathophysiological states andnon-stressful conditions, it is very likely that thereare multiple pathways involved in this complexsystem.

Because of their lipophilicity, organotins can beregarded as membrane active, and the cytoplas-mic membrane is clearly a possible initial site ofaction. In fact, data in the literature indicate thatTBT is able to alter the structure and functionof lipid bilayers both at plasma membrane andsubcellular level. Remarkably, it has been shownthat organotin compounds may affect Na+/H+

exchangers activity in erythrocyte membrane(Tufts and Boutilier 1990; Virkki and Nikinmaa1993).

Disruption of membrane integrity may occurbecause of organotin binding, or as a consequenceof insertion of the compound into the membrane.As we have previously reported, organotins maybecome embedded at different levels in lipid bi-layers of trout erythrocytes, as indicated by alter-ation in membrane fluidity (Santroni et al. 1999;Falcioni et al. 1996). In this context, it has beenproposed that heat shock response may be trig-gered by changes in the physical state of cellularphospholipid membranes. Physical strains inmembrane microenvironments may make certainphospholipids available to phospholipases associ-ated with the membrane, causing their activity toincrease, resulting in release of lipid mediators.These in turn could interact with known domainsof HSF, driving their activation, DNA bindingand subsequent hsp synthesis (Samples et al.1999).

Organotins also act intracellularly, and may dis-rupt processes dependent on intact organelles,including respiration and photosynthesis (Averyet al. 1991; Mooney and Patching 1995). We repor-ted that TBTC impairs the activity of mitochondriaand leads to gross swelling of mitochondrial mem-branes (Tiano et al. 2003). This presumably leads tointerference with oxidative phosphorilation andconsequent reduction in ATP synthesis andincreased formation of reactive oxygen species(ROS). If the TBTC insult is prolonged it is also ableto trigger apoptotic cell death. Impairment of mito-chondrial activity by TBT could be another bio-chemical signal leading to hsp activation. ROS may

induce HSP as indicator of tissue damage; in factoxidative damage to protein and consequent alter-ation in conformation is one of the final commonstressors leading to heat shock factor (HSF) activa-tion. Moreover, ATP depletion is thought to causeaccumulation of denatured proteins (Nguyen andBensaude 1994) and aggregation of constitutivehsp70 that ultimately triggers the heat shockresponse at non-heat-shock temperatures (Ananthanet al. 1986; Baler et al. 1992; McDuffee et al. 1997).

In conclusion, our data show that TBTCexposure increases the level of hsp70 mRNA innucleated trout erythrocytes. Trout erythrocytesare more sensitive than some other coltured celllines to TBT as inducer of hsp. Trout erythrocytesoffer several practical advantages (non-lethal mi-crosampling, almost direct exposure to the exter-nal environment), such that erythrocyte hsp70mRNA quantification is a potential practical assayof environmental stress at sublethal toxicant con-centrations.

Acknowledgement

This work was supported by a CNR grant to G.F.

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