Effect of tributyltin on trout blood cells: changes in mitochondrial morphology and functionality

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Biochimica et Biophysica Acta 1640 (2003) 105–112

Effect of tributyltin on trout blood cells: changes in mitochondrial

morphology and functionality

Luca Tianoa,*, Donatella Fedelia, Giorgio Santonib, Ian Daviesc, Giancarlo Falcionia

aDepartment of Biology MCA, University of Camerino, Camerino (MC), ItalybDepartment of Pharmacology and Experimental Medicine, University of Camerino, Camerino (MC), Italy

cFisheries Research Services, Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen AB11 9DB, UK

Received 16 May 2002; received in revised form 19 November 2002; accepted 14 February 2003

Abstract

The aquatic environment is the largest sink for the highly toxic organotin compounds, particularly as one of the main sources is the direct

release of organotins from marine antifouling paints. The aim of this study was to investigate the mitochondrial toxicity and proapoptotic

activity of tributyltin chloride (TBTC) in teleost leukocytes and nucleated erythrocytes, by means of electron microscopy investigation and

mitochondrial membrane potential evaluation, in order to provide an early indicator of aquatic environmental pollution. Erythrocytes and

leukocytes were obtained from an inbred strain of rainbow trout (Oncorhynchus mykiss). Transmission electronic micrographs of trout red

blood cells (RBC) incubated in the presence of TBTC at 1 and 5 AM for 60 min showed remarkable mitochondrial morphological changes.

TBTC-mediated toxicity involved alteration of the cristae ultrastructure and mitochondrial swelling, in a dose-dependent manner. Both

erythrocytes and leukocytes displayed a consistent drop in mitochondrial membrane potential following TBTC exposure at concentrations >1

AM. The proapoptotic effect of TBTC on fish blood cells, and involvement of mitochondrial pathways was also investigated by verifying the

release of cytochrome c, activation of caspase-3 and the presence of ‘‘DNA laddering’’. Although mitochondrial activity was much more

strongly affected in erythrocytes, leukocytes incubated in the presence of TBTC showed the characteristic features of apoptosis after only 1 h

of incubation. Longer exposures, up to 12 h, were required to trigger an apoptotic response in erythrocytes.

Crown Copyright D 2003 Published by Elsevier Science B.V. All rights reserved.

Keywords: Tributyltin chloride; Organotin compound; Rainbow trout

1. Introduction

Organotin compounds are pollutants of primarily anthro-

pogenic origin [1]. Their presence in the environment is due

to their use in many industrial applications [2] and as

agricultural biocides. Originally, organotin compounds (par-

ticularly butylated tin compounds) were developed as ther-

mal stabilisers in the synthesis of chlorinated polymers such

as PVC. However, their biocidal properties lead to new uses.

As a consequence of this expansion, concern increased over

their possible environmental and health effects.

The aquatic environment represents the largest sink for

accumulation of these xenobiotics, particularly from their

use in marine antifouling paints. Alkyltin compounds are a

0167-4889/03/$ - see front matter. Crown Copyright D 2003 Published by Elsev

doi:10.1016/S0167-4889(03)00025-9

* Corresponding author. Tel.: +39-737-403-208; fax: +39-737-636-

216.

E-mail address: [email protected] (L. Tiano).

significant hazard to aquatic organisms, through neurotoxic,

hepatotoxic, immunosuppressive and hormone disruptive

activities [3].

In the last few years, we have investigated the effects of

different organotin compounds on aquatic biota, using

nucleated trout erythrocytes as an experimental model [4].

As well as playing a central role in the physiology of fish

respiration, these cells represent an outstanding model to

study xenobiotic-induced damage to different cellular com-

partments. Despite their structural simplicity, the erythro-

cytes of lower vertebrates preserve the nucleus and mito-

chondria, unlike the anucleated erythrocytes of mammals.

The effects of alkyltin on trout red blood cells (RBCs) were

studied by monitoring the hemolytic process, by measuring

steady-state fluorescence anisotropy of different probes on

isolated membranes, by evaluating the stability of trout

hemoglobins and lastly by investigating their genotoxic

effects using the single-cell gel electrophoresis ‘‘Comet

Assay’’. The results obtained [5,6] indicated that incubation

ier Science B.V. All rights reserved.

L. Tiano et al. / Biochimica et Biophy106

in the presence of triorganotin induced a plasma membrane

perturbation and accelerated the precipitation process of Hb

IV, which represent 60% of the whole pigment and is

characterised by pH-dependent oxygen affinity (Root effect)

[7]. Moreover, experimental data demonstrated the ability of

tributyltin chloride (TBTC) to readily induce DNA single-

strand breaks [8]. Although DNA damage in nucleated

erythrocytes can be related to a metHb-dependent (i.e.

product of Hb oxidation or autoxidation) oxidative stress

[9], this was not the case in TBTC-mediated DNA damage.

In fact, single-strand breaks were not prevented by stabili-

sation of Hb in the carbomonoxy derivative.

In this paper, we report an attempt to investigate in

greater detail the mechanism of toxicity of alkyltin com-

pounds, particularly the effects of these xenobiotics on

mitochondria, because of the key role of these organelles

in the mechanism of cell death [10–15]. This may be due

to mitochondria being both the source and the final target

of free radicals. Mitochondrial ultrastructure was inves-

tigated using transmission electron microscopy (TEM).

Mitochondrial functionality was also monitored, at the

single-cell level, by means of flow cytometric mitochon-

drial membrane potential (Dcm) determination using the

fluorescent probe JC-1 [16–20], a delocalized lipophilic

cation which accumulates in charged membranes. JC-1

has advantages over other potential-sensitive probes, such

as rhodamines and other carbocyanines, since it changes

colour from green to orange as the membrane potential

increases to values greater than about 80–100 mV. This

property arises from the reversible formation of JC-1

aggregates on membrane polarisation, which causes shifts

in the emitted light from 530 nm (i.e. emission of JC-1

monomeric form) to 590 nm (i.e. emission of J-aggre-

gate).

Mitochondria-related features of apoptosis, such as cyto-

chrome c release and caspase-3 activation were also eval-

uated by means of Western blot analysis. The aim of this

study was to follow the effects of organotin compounds, at

sublethal concentrations, on mitochondria of peripheral

blood cells in relation to their involvement in triggering

DNA fragmentation and apoptotic cell death. The results

provide new information on the effect of organotins in

inducing apoptosis, and could be useful for developing an

early biosensor for aquatic pollution.

2. Materials and methods

All reagents were of analytical grade. Organotin com-

pounds were obtained from Aldrich, lymphoprep for sepa-

ration of RBCs and leukocytes was obtained from (Nycomed

Pharma AS, Oslo, Norway) and JC-1 was purchased from

Molecular Probes (Eugene, OR) and stored at � 20 jC as a

1-mM stock solution in DMSO. Cell culture media and

reagents were obtained from GIBCO, and molecular biology

reagents were obtained from SIGMA.

2.1. Samples

The cells used in this study were obtained from rainbow

trout (Oncorhynchus mykiss). The fish were kept in tanks

containing water from the Scarsito River, Italy, and fed

with commercial fish food. Experiments were performed

using blood from fish of the same age (f 24 months old),

and weighing between 180 and 300 g. Blood was with-

drawn from the lateral tail vein with a syringe into an

isotonic medium (0.1 M phosphate buffer, 0.1 M NaCl,

0.2% citrate, 1 mM EDTA, pH 7.8). Blood was held at

4jC and any further treatment was conducted within 2 h.

The whole blood was diluted 1:1 with the isotonic buffer

and this suspension was stratified on a solution of lym-

phoprep prior to being centrifuged for 20 min at 3000 rpm

at 4 jC. Peripheral blood lymphocytes (PBLs) were

separated from erythrocytes and both were washed with

RPMI medium. RBC and PBL suspensions were adjusted

to a density of 3.1�106 cells/ml in complete medium

supplemented with 10% foetal calf serum and incubated

for 1 h at 15 jC in the absence (control) or presence of

TBTC. A second set of experiments was conducted by

prolonging the incubation of RBCs up to 12 h at 15 jC in

the presence of TBTC at 0.1 and 1 AM. Organotin

compounds dissolved in 100% ethanol were added to the

erythrocytes (10 Al/ml of erythrocyte suspension) to final

concentrations of 0.1, 0.5, 1, 5 and 10 AM. Ten micro-

meters was used as the maximum concentration because,

in our experience, both hemolysis and met-Hb formation

are virtually absent at this concentration. Solvent control

experiments were performed by incubating the cells with

medium containing only ethanol.

An aliquot of treated erythrocytes was fixed in 2.5%

glutaraldehyde/0.1 M sodium cacodylate buffer pH 7.4 for 4

h at 4 jC, and postfixed in 1% osmium tetroxide in distilled

water for 1 h at room temperature. Cells were subsequently

dehydrated in ethanol and embedded in TAAB embedding

resin (medium grade). Ultrathin sections were cut using a

Reichert-Jung Ultracut ultramicrotome and then stained

with uranyl acetate and lead citrate. Stained sections were

observed using a Philips 301 transmission electron micro-

scope at 80 kV.

After exposure to pollutants, the erythrocyte suspension

was adjusted to a density of 1.5� 105 cells/ml and

incubated with 10 Ag/ml of JC-1 for 10 min at room

temperature in the dark for Dcm determination. A suspen-

sion of 1�106 cells/ml from each subfraction was ana-

lysed for relative fluorescence intensity using a FACScan

flow cytometer (Becton Dickinson, Mountain View, CA,

USA) equipped with a single 488 argon laser. The filter in

front of the fluorescence 1 (FL1) photomultiplier transmits

at 530 nm, and the filter used in the FL2 channel transmits

at 617 nm. The values of PMT were logarithmically set.

Red fluorescence (FL2) corresponds to the J-aggregate

form of JC-1 and is proportional to Dcm. Compensation

FL1–FL2 was 4% and compensation FL2–FL1 9.5%. A

sica Acta 1640 (2003) 105–112

L. Tiano et al. / Biochimica et Biophysica Acta 1640 (2003) 105–112 107

minimum of 13,000 cells per sample were acquired and

analysed using WINMDI 2.8 software on an IBM compat-

ible computer.

For Western blot analysis, leukocytes and erythrocytes

were treated with organotin 1 AM at 15 jC for up to 12 h.

Cells (1.5� 107) were washed once in 5 ml ice-cold phos-

phate buffer saline (PBS). For the determination of cyto-

chrome c release, cells were further centrifuged and the pellet

was resuspended in 250 Al of ice-cold homogenisation buffer

(20 mM Hepes pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM

EDTA, 1 mM EGTA, 1 mM DTT) supplemented with

protease inhibitors (0.1 mM PMSF, 5 Ag/ml pepstatine, 10

Ag/ml leupeptine, 2 Ag/ml aprotinine). After being kept on ice

for 15 min, cells were lysed by douncing 20 times in an

Eppendorf douncer. After centrifugation at 1000� g for 5

min at 4 jC, the supernatants were further ultracentrifuged at24 c (90,000� g) for 40 min at 4 jC in an airfuge ultra-

centrifuge (Beckman). For caspase-3 immunodetection, after

Fig. 1. Transmission electronic micrographs of mitochondria from trout erythrocyt

foetal calf serum at 15 jC and in the presence of 1 AM (c, d), or 5 AM (e, f) TBTC

f = 0.4 Am; d = 2.7 Am.

washing in ice-cold PBS, 1.5� 107 cells were lysed in 200 Alof RIPA buffer (0.1% nonidet-p40, 1 mM CaCl2, 1 mM

MgCl2, 0.1% sodium azide, 1 mM PMSF, 0.03 mg/ml

aprotinin, 1 mM NaVO4). Protein concentration in lysates

was measured using the Bio-Rad protein assay with albumin

as a standard. Protein extracts (20 Ag) were loaded onto 14%SDS polyacrilamide gels, electrophoresed at 200 V and than

transferred to immobilion-p membranes (Millipore, Bedford,

MA, USA) at 250 mA for 1 h. Membranes were blocked in

5% BSA and than incubated with a mouse anti-cytochrome c

monoclonal antibody (1/400) or mouse anti-caspase-3 mono-

clonal antibody (1/500) (Medical and Biological Lab, Japan)

overnight at 4 jC, followed by incubation with HRP-con-

jugated sheep antimouse antibody (1/10,000) (Amersham).

Immunoreactivity was detected with the enhanced chemilu-

minescence system (Amersham) using the CL detector

Chemidoc (Bio-Rad) and analysis software Quantity One

(Bio-Rad). As a reference for the amount of proteins, blots

es. Control (a, b), erythrocytes suspended in RPMI supplemented with 10%

, for 1 h. N, nucleus; m, mitochondria; pm, plasma membrane. Bar a, b, c, e,

Fig. 2. Flow cytometric analysis of JC-1 red fluorescence proportional to

Dcm in trout (A) erythrocytes and (B) leukocytes suspended in RPMI

supplemented with 10% foetal calf serum at 15 jC for 1 h in the presence or

absence of TBTC. Abscissa indicates JC-1 red fluorescence intensity in

arbitrary units proportional to mitochondrial membrane potential. Ordinate

the relative cell number.

L. Tiano et al. / Biochimica et Biophysica Acta 1640 (2003) 105–112108

were probed with a mouse anti-a tubuline monoclonal anti-

body.

Apoptotic DNA laddering was investigated by DNA

extraction and standard agarose gel electrophoresis. The

procedure was optimised to separate apoptotic DNA from

high molecular weight, intact genomic DNA. Briefly, follow-

ing rapid lysis of cell samples and inactivation of nucleases,

small DNA samples were recovered by centrifugation. DNA

was incubated with 100 Ag RNase A for 1 h at 37 jC and

subsequently with 150 Ag Proteinase K at 50 jC overnight.

This was followed by precipitation in isopropanol, separation

by standard 1.7% agarose gel electrophoresis and staining

with ethidium bromide.

Fig. 3. Flow cytometric analysis of JC-1 red fluorescence proportional to

Dcm in (a) trout erythrocytes and (b) trout leukocytes suspended in RPMI

supplemented with 10% foetal calf serum at 15 jC and in the presence or

absence of 10 AM TBTC. (A) Control experiments in absence of TBTC; (B)

after 30 min of exposure; (C) after 1 h of exposure. Abscissa indicates JC-1

red fluorescence intensity in arbitrary units proportional to mitochondrial

membrane potential. Ordinate the relative cell number.

3. Results

The analysis of tributyltin-mediated damage to isolated

cells showed that both erythrocytes and PBLs were affected

by xenobiotic exposure in a dose-dependent manner at

concentrations >1 AM. After 1 h of incubation at 15 jC in

complete medium in the presence of TBTC, erythrocyte

mitochondrial morphology, and mitochondrial functionality

in both cell types, were significantly compromised.

Transmission electronic micrographs showed TBTC con-

centration-dependent alterations to the mitochondrial mor-

phology of trout RBCs. Untreated erythrocytes displayed

small, rod-shaped mitochondria, localized in the proximity

of the nucleus, with normal cristae ultrastructure (Fig. 1a,b).

After 1 h of incubation in the presence of 1 AM TBTC,

mitochondria appeared swollen, although mitochondrial in-

tegrity was preserved (Fig. 1c,d). The effects were more

severe following incubation with 5 AM TBTC. This causes a

perturbation of the inner mitochondrial membrane and a

consequent loss of cristae organization (Fig. 1e,f).

Similarly, mitochondrial membrane potential, quantified

by the measurement of JC-1 red fluorescence, indicated

mitochondrial depolarisation in cells treated with 5 and 10

AM TBTC after 1 h of incubation at 15 jC in complete

medium (Fig. 2). The drop in Dcm was much more

pronounced in trout erythrocytes (Fig. 2a) than in leuko-

cytes, which showed a more gradual decrease in potential

(Fig. 2b).

Mitochondrial functionality of both RBCs and PBLs was

compromised very rapidly after incubation with tributyltin.

Fig. 4. Increased release of cytochrome c to the cytosol in trout erythrocytes and leukocytes upon apoptotic stimulation with TBTC 1 AM in RPMI medium at

15 jC up to 6 and 12 h for leukocytes and erythrocytes, respectively. After ultracentrifugation normalized samples were subjected to 14% SDS-PAGE and

transferred to nitrocellulose filter. The filter was probed with a monoclonal anti-cytochrome c antibody. As a reference for the amount of proteins, blots were

probed with a mouse anti-a tubuline monoclonal antibody as described in Materials and methods.

L. Tiano et al. / Biochimica et Biophysica Acta 1640 (2003) 105–112 109

There were no significant changes in Dcm between 30 and

60 min incubation with 10 AM TBTC (Fig. 3).

Mitochondrial impairment and mitochondrial membrane

depolarisation, clearly evident in trout erythrocytes in our

experimental conditions, represent early events in apopto-

sis. It has been shown in isolated mitochondria [21] and

Jurkat cells [22], that TBTC induced a rapid loss of Dcm,

which is associated with the release of cytochrome c,

important for subsequent apoptosis. Here, we tested

whether TBTC induced the release of cytochrome c from

mitochondrial membrane into the cytosol of erythrocytes

and leukocytes. As shown by Western blot analysis of

cytosolic extracts, 1 AM TBTC induced cytochrome c

release in both cell types (Fig. 4). Interestingly, the release

of cytochrome c exhibited different kinetics in the two

cell types: in leukocytes cytosolic cytochrome c content

increased significantly after 2 h of incubation and peaked

after 6 h of exposure, whereas in erythrocytes a gradual

increase was detected up to 12 h of exposure. In accord-

ance with cytochrome c levels, caspase-3 activation was

sustained only 12 h after exposure of trout erythrocytes

(Fig. 5). In mammalian cells, caspase-3 is synthesised as a

32 kDa zymogen that is processed to mature 20/17 kDa

and 12 kDa subunits by cleavage at Asp9, Asp28 and

Asp175 [23]. In trout erythrocytes we found that the size

Fig. 5. Caspase-3 activation in trout erythrocytes by TBTC 1 AM. Cells

were incubated in complete medium at 15 jC in the presence of TBTC,

harvested at different time periods and lysed. Normalized samples were

subjected to 14% SDS-PAGE and transferred to nitrocellulose filter. The

filter was probed with a monoclonal anti-caspase-3 antibody, as described

in Materials and methods. Caspase-3 is synthesised as a 27 kDa zymogen

that is processed to mature 16.8 kDa subunit.

of zymogen and activated caspase were slightly different

from those in mammals, 27 and 16.8 kDa, respectively.

This difference is not surprising taking into account that

sequences of caspase-3 reported for other teleost (Acces-

sion No. BAC00949.1, BAC00948.1) are significantly

smaller than for mouse caspase-3. Nevertheless, the spe-

cificity of antibody reaction was tested on rat thymocyte

submitted to apoptotic stimuli (data not shown). Finally,

we isolated the low molecular weight DNA fraction and

used this to verify the presence of internucleosomal cleav-

age, a characteristic late feature of apoptosis. After 1 h of

incubation in the presence of TBTC, PBLs displayed DNA

laddering at xenobiotic concentrations of 1 AM or greater

(Fig. 6a). In contrast, a fragmented, low molecular weight

DNA fraction was absent from RBCs exposed in the same

experimental conditions (Fig. 6b). Nevertheless, on a lon-

ger time scale, TBTC was able to trigger apoptotic cell

death in erythrocytes in accordance with cytochrome c, and

caspase-3 results. DNA laddering was evident after 8–12 h

Fig. 6. Agarose gel electrophoresis of low molecular weight DNA isolated

from (a) trout leukocytes and (b) erythrocytes treated for 1 h with TBTC at

0.1 AM (lane 1/1V), 0.5 AM (lane 2/2V), 1 AM (lane 3/3V), 5 AM (lane 4/4V), or10 AM (lane 5/5V). Exposures were conducted at 15 jC in RPMI

supplemented with 10% foetal calf serum complete medium. DNA markers

(M/MV) were electrophoresed as a base pair reference.

Fig. 7. Agarose gel electrophoresis of low molecular weight DNA isolated

from (a) trout erythrocytes exposed to 1 AM TBTC in complete medium at

15 jC for up to 12 h; lane 1 = 1 h, lane 2 = 4 h, lane 3 = 8 h, lane 4 = 12 h.

(b) DNA isolated from trout erythrocytes incubated in complete medium at

15 jC for 12 h in the presence or absence of TBTC; lane 1V= control, lane2V= 0.1 AM TBTC, lane 3V= 1 AM TBTC.

L. Tiano et al. / Biochimica et Biophysica Acta 1640 (2003) 105–112110

of incubation in the presence of 0.1 and 1 AM TBTC

(Fig. 7).

4. Discussion

In the present study, we evaluated the impairment of

mitochondrial functionality and its relationship to apop-

totic DNA fragmentation in fish blood cells following

exposure to TBTC, a highly toxic organotin compound.

Our data show that exposure to TBTC at sublethal

concentrations, in the range 5–10 AM, can induce rapid

and clear mitochondrial impairment in both erythrocytes

and leukocytes. This is in agreement with other reports

that indicate that trialkyltin compounds can inhibit oxida-

tive phosphorylation. This inhibition is provoked by high-

affinity binding of alkyltins to sites on the mitochondrial

membrane [24–26]. It is coupled with the inhibition of a

step in the energy transfer process [26], leading to

incomplete oxygen reduction and subsequent formation

of reactive oxygen species (ROS). Therefore, an increase

in the cellular level of oxygen free radicals following

TBTC-mitochondria interactions, as documented in liter-

ature in other cellular models [27], may be the cause of

the TBT-induced DNA damage previously reported by us

in fish erythrocytes [8].

The formation of ROS, and the impairment of mito-

chondria, are known to be involved in apoptotic cell death

[28]. Apoptotic effects of organotins are well-documented

for several cellular models [29–33], even though the

molecular mechanisms involved are still unclear. Several

mechanisms have been proposed and mainly concern

disturbance of Ca2 + homeostasis [34,35]. This is a basic

mode of action of alkyltins, whereby an increase in

cytosolic calcium can activate a number of intracellular

reactions including release of neurotransmitters, protein

phosphorylation, protease activity, and, eventually, cell

death. ROS have also been proposed as possible causes

of these effects [27,35,36].

TBTC (5–10 AM) was able to destabilise mitochondrial

functionality in both cell types. However, after 1 h of

exposure, TBTC induced apoptotic DNA laddering only

in leukocytes, whereas longer exposure times (8–12 h) to

TBTC < 1 AM were required to trigger apoptosis in eryth-

rocytes.

Mitochondria may represent the initial target of orga-

notins, but they seem to be involved differently in the

death response of different cell types. In PBLs, mitochon-

drial depolarisation was significant, but gradual, in the

population. Subsequent production of ROS, induction of

mitochondrial permeability transition and cytochrome c

release in the cytosol would be responsible for the

induction of apoptosis, as indicated by DNA laddering.

By contrast, TBTC-mediated mitochondrial toxicity in

RBCs was more pronounced, but the apoptotic cascade

was activated only at low concentrations of TBTC (V1 AM)

after several hours of incubation. This may suggest that, in

heavily contaminated environments, mitochondrial impair-

ment and DNA damage are not the principal effects of

organotins on RBCs. TBTC toxicity to RBCs is manifested

earlier by membrane perturbation leading to hemolysis. On

the contrary, lower concentrations of TBTC enable RBCs

to react and carry out the programmed cell death pro-

cess.

Differences in the kinetics of cell death are related to

early events in the apoptotic cascade, as indicated by the

differences in mitochondrial depolarisation. Taking into

account membrane-directed toxicity of TBTC and the differ-

ent physicochemical properties of membranes of the differ-

ent cell type studied, it is likely that the different behaviour

observed in erythrocytes and leukocytes could be due to

differences in the uptake of the triorganotin derivative by the

cellular membrane.

Organotin derivatives are assumed to be taken up across

the plasma membrane via passive diffusion processes by

partitioning into hydrophobic biological membranes. Phys-

icochemical properties of membranes play an important role

as indicated by differential accumulation in different cell

types [37] and in cells of different ages [38]. Consequently,

different amounts of alkyltin available in the cytosol may

affect mitochondria in different ways. This is in agreement

with recent observations [21] which indicate that TBTC

may effect mitochondria by two discrete mechanisms

depending on its concentration. At low concentrations,

TBT induces gradual loss of mitochondrial membrane

potential and mitochondrial swelling, whereas at higher

concentration a rapid induction of MPT was observed.

Moreover, at high concentrations of TBT, cells are forced

into a necrotic death as TBT inhibits the catalytic activity of

caspase-3 [22].

L. Tiano et al. / Biochimica et Biophysica Acta 1640 (2003) 105–112 111

In addition to the toxicological relevance of our obser-

vations, a particular consideration can be made of the

modalities of cell death in nucleated erythrocytes, in com-

parison to mammalian erythrocytes. In fact, as mature

erythrocytes lack cellular organelles and because they can

survive conditions that induce programmed cell death,

RBCs have been considered to be the only mammalian cells

that lack programmed cell death processes. However, the

senescence involved in erythrocyte death and removal is

characterised by distinct morphological changes very sim-

ilar to the morphological characteristics of programmed cell

death, including cell shrinkage, plasma membrane micro-

vesiculation and phosphatidilserine externalisation. Follow-

ing the recent work of Bratosin et al. [39] and Berg et al.

[40], aspects of the programmed death of mammalian

erythrocytes has been reinvestigated in light of the discov-

ery of components of the cell death machinery in mature

RBCs. Mammalian erythrocytes contain procaspase-3 and

procaspase-8 at levels comparable with those found in

nucleated cells. However, their role is ambiguous, since

erythrocytes lack other elements of the apoptotic machinery,

such as APAF 1 cytochrome c and caspase-2, -6, -7 and -9.

An alternative role has been proposed for caspase-3 in

mammalian RBCs as inhibitor of flippase activity: it has

been shown that under oxidative stress, procaspase-3 is

activated leading to impairment of aminophospholipid trans-

locase, phosphatidilserine externalisation and increased

erythrophagocytosis [41].

On the contrary, nucleated erythrocytes of lower verte-

brates are less specialised than their non-nucleated counter-

parts, by retaining nucleus and organelles after the

reticulocyte stage. These organelles are not cellular relicts,

they are active and can influence the physiology of the cell.

Little is known about programmed cell death of nucleated

erythrocytes. Chicken erythrocyte are reported to be able to

react to proapoptotic stimuli such as serum deprivation,

staurosporine and cycloheximide by showing characteristics

of apoptosis [42], but caspase activation in this cell type is

uncertain and apparently decreases with cell age. In trout

erythrocytes Moyes et al. [43] recently reported that pro-

oxidants and mitochondrial inhibitors failed to induce pro-

grammed cell death, suggesting that mitochondrial pathways

are not able to trigger apoptosis in this cell type.

Our study demonstrates, for the first time, that nucleated

trout erythrocyte are able to commit apoptotic cell death via

a mitochondria-mediated route. Tributyltinchloride is

known to be mitochondria-active and to be able to induce

cytochrome c release in isolated mitochondria. TBTC

expresses toxicity not only through the induction of formal

membrane permeability transition, but also in a relatively

non-specific manner that involves mild loss of Dcm and

induction of swelling [21]. Our data indicate that the low

mitochondrial content of erythrocytes may release sufficient

amounts of proapoptotic proteins, and consequently that

mitochondrial pathways are able to trigger apoptosis in this

cell type.

Acknowledgements

This work was supported by CNR (National Research

Council) fund to G.F.

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