SC IE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 4 8–1 53
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v
Hemoglobin system of Sparus aurata: Changes in fishes farmed
under extreme conditions
Salvatore Campo a , Giancarlo Nastasi a , Angela D'Ascola a , Giuseppe M. Campo a ,
Angela Avenoso a , Paola Traina a , Alberto Calatroni a , Emanuele Burrascano b ,
Alida Ferlazzo b , Giulio Lupidi c , Rosita Gabbianelli c , Giancarlo Falcioni c,⁎
a
Department of Biochemical, Physiological and Nutritional Sciences, School of Medicine, University of Messina, Policlinico Universitario,
Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy
b
Department of Morphology, Biochemistry, Physiology and Animal Production, School of Veterinary Medicine, University of Messina,
Polo Universitario Annunziata, 98168 Messina, Italy
c
Department of MCA Biology, University of Camerino, Via Gentile III da Varano, 62032 Camerino MC, Italy
AR TIC LE I N FO
ABS TR ACT
Article history:
In order to gain more knowledge on the stress responses of gilhead seabream (Sparus aurata)
Received 29 February 2008
under extreme conditions, this study investigated the functional properties of the
Received in revised form 19 May 2008
hemoglobin system and globin gene expression under hypoxia and low salinity. The
Accepted 20 May 2008
oxygen affinity for the two hemoglobin components present inside the S. aurata erythrocyte
Available online 27 June 2008
was practically identical as was the influence of protons and organic phosphates (Root
effect). The quantification of S. aurata hemoglobin fractions performed by HPLC and the data
Keywords:
on gene expression of globin chains assayed by PCR indicate that under hypoxia and low
Hemoglobins
salinity there is a change in the ratio between the two different hemoglobin components.
Root effect
The result indicating that the distinct hemoglobins present in S. aurata erythrocyte have
Globins
almost identical functional properties, does not explain the adaptive response (expression
Gene expression
change) following exposure of the animal to hypoxia or low salinity on the basis of their
Salinity
function as oxygen transporter. We hypothesize that other parallel biological functions that
Hypoxia
the hemoglobin molecule is known to display within the erythrocyte are involved in
adaptive molecular mechanisms. The autoxidation–reduction cycle of hemoglobin could be
involved in the response to particular living conditions.
© 2008 Elsevier B.V. All rights reserved.
1.
Introduction
The functional properties of hemoglobin are characterized by
the presence of both homotropic and heterotropic interaction
phenomena which have been studied over the years.
Cooperativity between subunits is achieved through the
conformational transition between the deoxy-low-affinity
state (or T state) and the oxy-high-affinity state (or R state),
which accounts for the sigmoidal shape of the oxygen binding
curve. In addition, the oxygen affinity of hemoglobin is
influenced by different effectors such as chlorides, protons
(Bohr effect), CO2 and organic phosphates. All these effectors
bind preferentially to the T state of hemoglobin lowering the
overall O2 affinity of the molecule.
Many fish species have multiple Hb components and this
Hb multiplicity is related to the greater variability in oxygen
regimes to which animals are subjected to. The presence of
different hemoglobin components correlates also with the role
of O2 release against high hydrostatic pressure inside the
swim bladder. Hemoglobins involved in this function display a
⁎ Corresponding author. Tel.: +39 0737 403211; fax: +39 0737 403290.
E-mail address: [email protected] (G. Falcioni).
0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2008.05.027
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 4 8–1 53
very marked Bohr effect (called Root effect). The oxygen
binding properties of Hbs that exhibit Root effect are
characterized by a very strong dependence of both affinity
and cooperativity on pH and organic phosphates. The
essential feature of this phenomenon appears to be a proton
induced stabilization of the low affinity state of the molecule
(Brunori, 1975). At the structural level, the Root effect has been
correlated (Perutz and Brunori, 1982) with two additional Hbonds involving the protonated form of His HC3 (146β) and a
serine in position F9 (93β) which, in Root effect hemoglobins,
substitutes the cysteinyl residue normally found in mammals.
Comparative studies on the structural and functional
properties of hemoglobins permit to speculate on the adaptive
molecular mechanisms developed to satisfy particular needs
of oxygen demand due to different environmental conditions
to which animals may be subjected to. Environmental factors
can include variations in temperature, pH, salinity, oxygen
tension, etc.
In intensive fish culture systems, a reduced availability of
dissolved oxygen in water is often observed. This is ascribed to
a high fish density and to the feeding practices; algal blooms
and elevated temperatures can contribute as well.
This lack of oxygen can induce responses and the typical
metabolic adjustments caused by the hypoxic stress are to
maintain oxygen supplies in the critical organs and to reduce
consumption of oxygen.
In order to cope with periods of hypoxia, many fish species
have evolved new molecular responses (Nikinmaa, 2002;
Nilsson and Renshaw, 2004; Cossins and Crawford, 2005;
Nikinmaa and Rees, 2005). Some adaptive mechanisms can
change fish gene expression with the aim of saving oxygen
(Gracey et al., 2001; Ton et al., 2003; Van der Meer et al., 2005).
The patterns of hypoxic differential gene expression are not
well known. Understanding the tissue-specific and temporal
changes in gene expression in fishes exposed to hypoxia could
reveal new mechanisms of tolerance to the lack of oxygen and
they could shed light on the evolution of this adaptive
response in vertebrates. Hemoglobin and myoglobin are
respiratory proteins that link and store oxygen. Under hypoxic
conditions gene expression of globins could be modified.
Salinity can also contribute to increase environmental
stress. Most fish live in an osmotic disequilibrium between the
environment and their blood plasma and body tissues, therefore they must be able to maintain an internal osmotic
environment across a wide range of salinity. Alterations in
the environmental amount of salt may affect electrolyte levels
(i.e. Na+ and Cl−) and, as a consequence, the acid–base balance
(Junsen et al., 1998).
There is enormous variety in the types of environmental
stressors; it is generally agreed that stress in fish evokes an
ordered sequence of well-defined physiological changes.
The Sparidae Family occupies an important position
among the Teleostei of the Mediterranean basin. In fact,
some fishes of this Family have a special role in the
gastronomic, industrial and economic fields. Amongst the
different species, Sparus aurata is the most capable of adapting
to different environmental conditions, like those of fish farms.
The aim of this study was to investigate the stress responses
of S. aurata under hypoxia and low salinity, in particular with
regards to the hemoglobin system and globin gene expression.
149
Three adult globin genes have been found in S. aurata; two
different α-globin genes and one β-globin gene have been
identified and their nucleotide and aminoacid sequences have
already been submitted to the GeneBank database (accession
numbers DQ520839; DQ520840 and DQ452379 respectively).
The oxygen binding properties of the two purified hemoglobin
components as a function of pH and organic phosphates were
also investigated.
2.
Materials and methods
2.1.
Sample collection
Six specimens of S. aurata obtained from an Ippocampus fishfarmer (Villafranca Tirrena, ME, Italy) were stabulated into a
300 L aquarium under standard conditions, at the temperature
of 20 °C, and pH 8.0. The concentration of oxygen was kept
at 6.5–7.0 mg/L (Titolation kit — Hanna, USA), by a pump
oxygenator, whereas the nitrates/nitrites were monitored and
maintained below 0.05 mg/L (Ascol test, Read Sea, and
Nitrification Active Bacteria kit — Aquaristica, Italy). After
10 days stabulation, blood was collected from the caudal vein
of each specimen, using MS-222 40 mg/L as anesthetic, for no
longer than 4 min, and 50 mM Na2EDTA as anticoagulant.
Blood samples were immediately processed.
Subsequently, the fishes were stabulated for an additional
20 days during which oxygen concentration was progressively
reduced to 2.5 mg/L towards the end. A new blood sample was
then collected under the same conditions.
Finally, the fishes were stabulated for further 20 days at
normal oxygen concentration, while progressively reducing the
salt concentration from normal 38 ppm to 28 ppm. The third
blood sample was then collected under the same conditions.
2.2.
Separation of homogeneous hemoglobin components
by preparative column chromatography
To obtain purified Hb components, a total of about 4 mL of
blood was collected from different animals. Blood from sea
bream S. aurata was withdrawn from the caudal vein into an
isotonic medium constituted by 0.1 M phosphate buffer, 0.1 M
NaCl, 0.2% citrate, 1 mM EDTA, pH 7.8. The red blood cells were
separated from the plasma by centrifugation for 10 min at
1000 g and were washed four times with the same isotonic
buffer. Hemolysis was accomplished by adding 3 volumes of
distilled water to the red cells; after 30 min the solution was
centrifuged for 20 min at 10,000 g and the supernatant was
dialysed against 0.1 M Tris–HCl buffer at pH 9.1 containing
EDTA 5 × 10− 4 M. The solution of hemoglobin previously
dialysed was applied to a DEAE (diethylaminoethylcellulose)
Sephadex A-50 column (dimensions 40 × 2 cm) equilibrated
with the same buffer. A pH gradient elution was carried out
using two containers, one with 250 mL of 0.1 M Tris–HCl
buffer + EDTA 5 × 10− 4 M at pH 9.1 under stirring and the other
with 1 L of 0.1 M KH2PO4, the former being connected directly
to the column. The flow rate was maintained at about 30–
40 mL/h; 3–4 mL fractions were collected. Representative
fractions were examined by cellulose acetate electrophoresis
and stained with a solution of red poinceau (Fig. 1).
150
SC IE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 4 8–1 53
2.6.
S. aurata globin gene expression by PCR real time
Oxygen equilibrium curves were determined spectrophotometrically by the method of Rossi Fanelli and Antonini (1958).
The fractional saturation with oxygen of hemoglobin components in air (pO2 = 155 mm Hg) as a function of pH was followed
with a Cary 219 spectrophotometer in the visible region.
10 ng of S. aurata total RNA was retrotranscribed by using
hexameric random primers and the High Capacity cDNA
Archive kit (Applied Biosystems, USA), according to the manufacturer's instruction, to estimate the variation of the globin
mRNAs under normal and extreme condition fish-farming.
Primers and probes for PCR real time were designed and
supplied by Proligo (France). All probes, containing LNA bases
(Linking Nucleic Acid), were labeled with 5′-FAM reporter dye and
3′ Black Hole Quencher 1 (BHQ1) (Table 2). For each globin and for
β-actin (as endogenous control) the reactions were carried out in
triplicate and in monoplex on the mod. 7500 RT-PCR real time
System (Applied Biosystems, USA) by using the TaqMan Universal
PCR Mastermix kit (Applied Biosystems, USA) as suggested by the
manufacturer, and a standard curve prepared side by side with
scalar dilutions of the previously obtained clone. After normalization, the results were expressed as relative amounts vs. normal
controls (normal stabulated fish samples).
2.4.
Identification and quantification of S. aurata
hemoglobin fractions
3.
Fig. 1 – Cellulose acetate electrophoresis of Sparus aurata
hemoglobin components isolated by column chromatography;
a) hemoglobin preparation applied to the column, b) component I, c) component II.
2.3.
Spectrophotometric measurements
An aliquot of each collected blood sample was diluted in an
appropriate volume of distilled water (DDW) and cells lysed by
freezing–thawing. The solution was then centrifuged (3000 g, 4 °C)
to remove the membrane debris and diluted to a final hemoglobin
concentration of 1 g/dL, spectrophotometrically (Biomate 3 —
Thermo Electron Corporation, USA) evaluated at 412 nm.
The hemoglobin fractions were separated by HPLC with a
gradient of increasing ionic strength, from 10 mM Bis–Tris/HCl,
pH 5.9 to 270 mM Bis–Tris/HCl, pH 5.6 in 6 min. The detection was
performed spectrophotometrically at 412 nm and acquired on PC
by a dedicated software (mod. Binary Pump 1525, Waters, USA),
by ToSo cation exchange resin (5 μm particle diameter, 10 μm).
2.5.
Preparation of S. aurata globin cDNA clones for the
PCR real time standard curves
For each S. aurata globin gene that was identified and for the βactin (GenBank accession number X89920), a couple of primers
were designed on the 5′ and 3′ mRNAs respectively (Table 1),
with the aim to obtain specific clones for PCR real time
standard curve preparation. RT-PCRs were carried out using
100 ng of previously extracted total RNA and Super Scrypt OneStep RT-PCR Platinum Taq HiFi (Invitrogen, USA), according to
the manufacturer's instruction. Each RT-PCR product was
cloned by using the TOPO TA cloning kit (Invitrogen, USA) as
suggested by the manufacturer; the clones were analyzed by
nucleotide sequencing (v.1.1 BigDye terminators ready to use
mix and the mod. 310 Genetic Analyzer — Applied Biosystems,
USA) and spectrophotometrically quantitatively evaluated.
Results
Fig. 2 shows the separation of the components of S. aurata
hemoglobin as obtained by pH gradient elution from DEAE
Sephadex A-50 column. Homogeneous components tested by
cellulose acetate electrophoresis of S. aurata hemoglobin were
used for studies on their functional properties.
3.1.
Oxygen binding properties
The oxygen equilibrium curve for the two hemoglobin
components at pH 7.0 was practically identical (Fig. 3); it is
cooperative (the Hill coefficient n is close to 1.75), implying
positive interactions between the oxygen binding sites.
Fig. 4 shows the effect of pH on the fractional saturation
of S. aurata Hbs in air, i.e., at constant O2 activity. This representation of the effect of pH is particularly useful to
illustrate the influence of solvent composition on the Root
effect. When the experiments were performed in the absence
of organic phosphate, the midpoint of the transition for both
proteins was pH ~5.0 (at pO2 ~155 mmHg and 25 °C). The
addition of saturating amounts of ATP (1 mM) shifted the
region where the Root effect was present towards higher pH
values: the midpoint was at pH ~ 6.2.
3.2.
Quantification of S. aurata hemoglobin fractions by HPLC
High performance liquid chromatography analysis of hemolyzate from S. aurata shows two distinct hemoglobin components (called on the basis of their relative retention times, HbI
and HbII). HbII, under standard fish-farming conditions, is the
Table 1 – Primers used to obtain Sparus aurata globins and β-actin cDNA clones for the PCR real time standard curves
α1 globin
α2 globin
β globin
β actin
F(5) 5′-gcagcatcttcttgatccattttc-3′
F(36) 5′-gaagaaaagggcagtcatgag-3′
F(2) 5′-agaagggttgcgatcaacgt-3′
F(234) 5′-ctgggatgacatggagaaga-3′
R(878)
R(829)
R(770)
R(900)
5′-aagggtttggtgacgttgct-3′
5′-ttatggttggcgacgtcatg-3′
5′-cacttgtagagctgcttcac-3′
5′-agacagcacagtgttggcat-3′
(DQ520839)
(DQ520840)
(DQ452379)
(X89920)
151
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 4 8–1 53
Table 2 – Primers and probes for PCR real time of each globin and β actin mRNAs of Sparus aurata
Forward
α1 globin
α2 globin
β globin
β actin
(693)
(667)
(115)
(778)
5′-caacctgatggtagttgatcg-3′
5′-gtcattgccatgtactacc-3′
5′-agatcgatgtgggtgaaatc-3′
5′-gccctcttccagccatcc-3′
Reverse
(770)
(787)
(311)
(899)
Probe
5′-aggaacttgtcgaatgagac-3′
5′-tttggatcagtggagtttagc-3′
5′-tgagaagtgtctttgagtcc-3′
5′-gacagcacagtgttggcatac-3′
(720)
(692)
(141)
(800)
5′-6FAM-cttCccCgcCgaCttc-3′BHQ1
5′-6FAM-actTcactGcGgaGgtc-3′BHQ1
5′-6FAM-cagGctTtgTccAggc-3′BHQ1
5′-6FAM-tcgGtatGgagTcCtgc-3′BHQ1
The oligos were designed on the basis of the GenBank database sequences (accession numbers: DQ520839 for the α1 globin, DQ520840 for the α2
globin, DQ452379 for the β globin and X89920 for the β actin). Capital letters in the probe sequences are LNA bases.
main fraction with a mean value of 65%; while the mean value
for HbI under the same conditions was 35% (Fig. 5A).
After fish-farming at low-oxygen concentration, the levels
of HbII decreased to the mean value of 49%, whereas the mean
level of HbI increased to 51% (Fig. 5B). In a similar way, low salt
concentration fish-farming induced a reduction in the HbII
fraction to 52% and an increase in the HbI mean amount (48%)
(Fig. 5C). A small amount of ferric hemoglobin which slightly
increased when the animals were submitted to extreme
conditions (hypoxia) was also present (data not shown).
3.3.
Under low-oxygen concentration, the levels of α2 globin
mRNA decreased (0.35 fold) while those of α1 globin increased
(2.4 fold) with respect to normal conditions. Under low salt
concentration, the values of α2 and α1 globin mRNAs still
decreased (0.7 fold) and increased (1.7 fold) respectively.
Instead, the values of the beta globin mRNA remained
unchanged.
The result in the variation of the S. aurata globin gene
expression, under the described fishI fraction is probably
composed of the tetramerous α12–β2, while the slowest HbII
fraction is a α22–β2 molecule.
Globin gene expression
The gene expression of globin chains assayed by PCR real time
was in agreement with the data obtained by HPLC analysis
here reported on hemoglobin fractions (Fig. 6).
4.
Discussion
Our results indicate that the two major components of
S. aurata hemoglobin are, at least under our experimental
conditions, functionally very similar and both display Root
effect (a similar behaviour has been reported for carp, where
there are three structurally distinct hemoglobins whose
functional properties are almost identical to one another)
(Gillen and Riggs, 1972; Tan et al., 1973). In other teleost fish
(salmon, eel, loach and trout) the oxygen equilibrium of at
least one hemoglobin present inside the erythrocyte is
characterized by homotropic interactions and by the complete
insensitivity to protons and organic phosphates and they have
different oxygen affinity (Weber and Jensen, 1988). In fish
inhabiting low-oxygen water areas, the hemoglobin–oxygen
Fig. 2 – pH gradient elution from DEAE Sephadex A-50
column. Linear gradient: Tris/HCl 50 mM pH 9.1+EDTA
0.5 mM and KH2PO4 50 mM.
Fig. 3 – Hill plot of the oxygen binding curves of HbI (○) and HbII
(□). Conditions: potassium phosphate buffer 0.1 M pH 7.0;
T = 20 °C.
Fig. 4 – Oxygen fractional saturation (Y) in air (pO2 = 155 mmHg)
of HbI and HbII as a function of pH in the presence and absence
of 1 mM ATP. ■ HbI in Bis–Tris or MES buffer 0.05 M, ▲HbII in
Bis–Tris or MES, X HbI+ATP, ○ HbII+ATP. Other conditions: Bis–
Tris or MES 0.05 M; T = 20 °C.
152
SC IE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 4 8–1 53
Fig. 5 – Cation exchange HPLC fractionation of Sparus aurata hemoglobins under: A) normal conditions (HbI 35% HbII 65%).
B) low-oxygen concentration conditions (HbI 51% HbII 49%). C) low salt concentration conditions (HbI 48% HbII 52%).
affinity is higher than that of fish in other regions (Nikinmaa,
2001; Powers et al., 1979). Changes in hemoglobin patterns
have been reported due to hypoxia conditions (Marinsky et al.,
1990). The result indicating that the distinct hemoglobins
present in the S. aurata erythrocyte have functional properties
almost identical to one another, does not explain the adaptive
response (expression change) following exposure of the
animal to hypoxia or low salinity on the basis of their function
of oxygen transport. The physiological significance of
this change is unclear. The expression change of the two
hemoglobin components does not alter the function of oxygen
transport even in the presence of a modified acid–base
balance (eventually due to changes in external salinity) in
plasma. However, responses for fish living in adverse osmotic
conditions have to take into account demand required to
balance the inevitable loss or gain of water by osmosis (Kidder
et al., 2006).
The level of nucleotide triphosphates in fish red cells plays
a vital role in adaptation to hypoxia. A variety of fish acquire
an increase in red cell oxygen affinity when placed in hypoxic
water (Weber et al., 1975; Weber and Lykkeboe, 1978). The level
of red blood cell nucleotide triphosphates falls within a few
days after fish are exposed to hypoxic water. Conversely,
when fish are displaced from their hypoxic deep-water habitat
to normoxic water, the level of organic phosphate in their red
cells rises (Wood et al., 1975). Erythrocytes of teleost fish use
adenosine triphosphate (ATP), guanosine triphosphate (GTP)
or inositol pentaphosphate and probably also lactate as
allosteric effectors (Gillen and Riggs, 1977; Isaacks et al.,
1977). GTP has been found to lower oxygen affinity of carp
hemoglobins twice as much as ATP (Weber and Lykkeboe,
1978); in trout IV hemoglobin the influence of the allosteric
effectors on oxygen affinity resulted the same (Gronenborn
et al., 1984), even though the only difference in the lining of the
allosteric effector sites lies in the replacement of glu NA2β in
carp by Asp in trout IV hemoglobin. Probably, other parallel
biological functions that the hemoglobin molecule is known to
display within the erythrocyte (Giardina et al., 1995) are
involved in the adaptive molecular mechanisms. The pre-
sence of different hemoglobin components in the same
erythrocyte may be of importance for the stability, and thus
the life-span, of the cell itself and may help to prevent
precipitation or crystallization inside the red cell.
For trout hemoglobins, we previously reported (Fedeli et al.,
2001) a peroxidase activity which is greater for HbIV
(the hemoglobin component with Root effect) than for trout
HbI (it binds oxygen cooperatively but is insensitive to pH and
other allosteric effectors). It is also necessary to point out
that hemoglobins with Root effect are often only partially
saturated with oxygen and thus more easily oxidizable with
respect to the fully oxygenated derivative.
Comparative evaluation has shown that respiratory pigments in fish are less resistant to oxidation than in higher
vertebrates, specifically in mammals (Jensen, 2001). However,
in most fish species, the met-Hb concentration in blood is in
the range of 3–13% and regular changes in met-Hb concentration in fish blood are present during the annual cycle (Soldatov
and Maslova, 1989). During the autoxidation of human
hemoglobin, the alpha chains are preferentially oxidized
Fig. 6 – Sparus aurata alpha and beta globin gene expression
variations under extreme farming conditions vs. normal
farming conditions; (□) low-oxygen concentration, (■) low salt
concentration.
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 4 8–1 53
(Mansouri and Winterhalter, 1973; Tomoda et al., 1981). A
typical feature of fish hemoglobins is represented by their
stability as tetramers and therefore, all the possible oxidation
intermediates (Colosimo et al., 1982), i.e., tetramers containing
ferrous and ferric hemes, could originate from the autoxidation of the hemes in the circulating blood. These oxidized
intermediates have lower cooperativity and higher affinity,
associated with a reduction in the total oxygen binding
capacity per mass of protein. The autoxidation–reduction
cycle of hemoglobin could be involved in the response to
particular living conditions (i.e. low-oxygen availability) even
if the response to abnormal conditions has been attributed to
modifications in the intracellular concentration of modulator
compounds like protons and ATP.
Certainly, information on the heterotropic effect of the
different organic phosphates versus the hemoglobin components, on their intracellular levels and on the fraction of metHb in the circulating blood could contribute to better understand the molecular mechanisms involved in adaptive
processes.
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