Toxins 2014, 6, 1062-1079; doi:10.3390/ oxins6031062
oxins
ISSN 2072-6651
www.mdpi.com/jou nal/ oxins
A icle
In luence o Two Depu a ion Pe iods on he Ac i i y and
T ansc ip ion o An ioxidan Enzymes in Tilapia Exposed o
Repea ed Doses o Cylind ospe mopsin unde
Labo a o y Condi ions
Vic o ia Ríos, Remedios Guzmán-Guillén, Isabel M. Mo eno, Ana I. P ie o, Ma ía Pue o,
Angeles Jos and Ana M. Cameán *
A ea o Toxicology, Facul y o Pha macy, Uni e si y o Se illa, C/P o eso Ga cía González 2,
Se illa 41012, Spain; E-Mails: ic o ia [email protected] (V.R.); [email p o ec ed] (R.G.-G.);
[email p o ec ed] (I.M.M.); anap ie [email protected] (A.I.P.); ma iapue [email protected] (M.P.); angele[email p o ec ed] (A.J.)
* Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed];
Tel.: +34-954-556-762; Fax: +34-954-556-422.
Recei ed: 12 No embe 2013; in e ised o m: 25 Feb ua y 2014 / Accep ed: 27 Feb ua y 2014 /
Published: 13 Ma ch 2014
Abs ac : The cyanobac e ial oxin Cylind ospe mopsin (CYN), a po en p o ein syn hesis
inhibi o , is inc easingly being ound in eshwa e bodies in es ed by cyanobac e ial
blooms wo ldwide. Mo eo e , i has been epo ed o be implica ed in human in oxica ions
and animal mo ali y. Recen ly, he al e a ion o he ac i i y and gene exp ession o some
glu a hione ela ed enzymes in ilapias (O eoch omis nilo icus) exposed o a single dose o
CYN has been epo ed. Howe e , li le is known abou he e ec s induced by epea ed
doses o his oxin in ilapias exposed by imme sion and he po en ial e e sion o hese
biochemical al e a ions a e wo di e en depu a ion pe iods (3 o 7 days). In he p esen
s udy, ilapias we e exposed by imme sion o epea ed doses o a CYN-con aining cul u e
o Aphanizomenon o alispo um du ing 14 days, and hen we e subjec ed o depu a ion
pe iods (3 o 7 days) in clean wa e in o de o examine he po en ial e e sion o he
e ec s obse ed. The ac i i y and ela i e mRNA exp ession by eal- ime polyme ase
chain eac ion (PCR) o he an ioxidan enzymes glu a hione pe oxidase (GPx) and soluble
glu a hione-S- ans e ases (sGST), and also he sGST p o ein abundance by Wes e n blo
analysis we e e alua ed in li e and kidney o ish. Resul s showed signi ican al e a ions
in mos o he pa ame e s e alua ed and hei eco e y a e 3 days (GPx ac i i y, sGST
ela i e abundance) o 7 days (GPx gene exp ession, sGST ac i i y). These indings no only
OPEN ACCESS
Toxins 2014, 6 1063
con i m he oxida i e s ess e ec s p oduced in ish by cyanobac e ial cells con aining
CYN, bu also show he e ec i eness o depu a ion p ocesses in mi iga ing he
CYN-con aining cul u e oxic e ec s.
Keywo ds: cylind ospe mopsin; depu a ion; glu a hione ans e ase; glu a hione
pe oxidise; ilapia
1. In oduc ion
Cyanobac e ial oxins ha e become ecognized as a po en ial haza d in d inking wa e wo ldwide [1].
Among hem, Cylind ospe mopsin (CYN) is an eme ging oxin [2], which can be p oduced by se e al
known eshwa e cyanobac e ia species, such as Cylind ospe mopsis acibo skii,
Aphanizomenon o alispo um, Raphidiopsis cu a a, and Umezakia na ans [3–5]. This oxin is a
icyclic alkaloid comp ised o a guanidine en i y along wi h a u acil moie y po en ially esponsible o
he oxici y [6]. I is s able in a ying hea , ligh and pH condi ions and also highly wa e -soluble [7].
Thus, he concen a ion o CYN dissol ed in wa e can cons i u e as much as 90% o o al CYN
a ailable [8,9]. The en i onmen al le els o CYN de ec ed wo ldwide a e dependen on he season and
he a ea o de ec ion and he oxin p oducing species. Thus, in Aus alia, he le els o CYN p oduced
by na u al blooms o C. acibo skii and A. o alispo um oscilla ed be ween 0.3 µg/L o 92 µg/L and
4–120 µg/L, espec i ely. Highe le els we e measu ed in aquacul u e ponds and a m dams p oduced
by A. o alispo um (589–800 µg/L). In Eu ope, he CYN le els de ec ed in na u al wa e s we e lowe
han in Aus alia, hus, in Ge many CYN was ound in maximum concen a ions o 1.80 µg/L and
11.75 µg/L p oduced by C. acibo skii and A. g acile in di e en lakes. In F ance, CYN was de ec ed
in ele en wa e bodies wi h concen a ions up o 1.95 µg/L. In I aly, he p esence o CYN was
obse ed in le els in a ange om 0.41 o 42.3 µg/L and in Spain CYN was measu ed in a le el up o
9.4 µg/L. In Flo ida (USA), CYN was posi i ely quan i ied in a ange om 8.07 o 97.12 µg/L [10].
Di e en s udies ha e e ealed he en i onmen al oxici y o CYN owa ds di e en g oups o
o ganisms, including amphibians [11], gas opods [12], ish [13,14], plan s [15], and aqua ic
mac ophy es [16], as well as an ibac e ial ac i i y [17]. CYN exposu e has hepa o oxic, neph o oxic
and gene al cy o oxic e ec s and can also lead o e al oxici y, umo ini ia ion, mic onucleus
induc ion and ch omosome loss [4,5]. The oxici y o CYN is due o he inhibi ion o glu a hione and
p o ein syn hesis, he inhibi ion o cy och ome P450, and di ec in e ac ion wi h DNA [18,3].
A deple ion o hepa ic glu a hione has been p o ed in ish in i o [19] and in i o [20]. Mo e ecen ly,
some s udies ha e shown ha pu e CYN is able o induce oxida i e s ess in ilapia ish
(O eoch omis nilo icus) when hey a e exposed o a single dose o CYN by o al ou e [13,19] and
in ape i oneal (i.p.) injec ion [14]. Al e a ions in he ac i i y and gene exp essions o
glu a hione-S- ans e ases (GST) and glu a hione pe oxidase (GPx) in esponse o a single dose o
CYN, has been p e iously in es iga ed in ou labo a o y in ilapia ish [13,21]. Resul s showed ha
ilapia exposed by ga age o 200 and 400 µg/kg bw o pu e CYN and sac i iced a e 24 h had
al e a ions in di e en bioma ke s including ac i i y and gene exp ession o he enzymes GPx and
GST [13]. Gu ié ez-P aena e al. [21] p o ed ha he ype o exposu e and he ime o sac i ice played
Toxins 2014, 6 1064
a ole in he ac i i y and ela i e mRNA exp ession by eal ime PCR o GPx and GST and he sGST
p o ein abundance.
Unde na u al condi ions, ish a e exposed o cyanobac e ial blooms o sub-ch onic pe iods, bu
CYN oxicological s udies o long exposu e pe iods a e e y sca ce a he ime [22], especially in he
case o ish. In his sense, Guzmán-Guillén e al. [23] showed an in ol emen o oxida i e s ess as a
mechanism o oxic ac ion o CYN in ilapia a e sub-ch onic exposu e o cyanobac e ial cells
con aining CYN (10 and 100 μg CYN/L), o 7 and 14 day by imme sion ou e, mimicking na u al
exposu e). I was demons a ed ha subch onic exposu e o low concen a ions o cyanobac e ial cells
con aining CYN exceeding 10 μg/L should be conside ed o pa icula ly high isk o ish, because
e iden his opa hological changes we e ound om his concen a ion [24].
Se e al au ho s ha e s udied he e ec o di e en depu a ion pe iods on s ess bioma ke le els in
ish and clams exposed o con aminan s [25–29], e ealing ha depu a ion educes he oxida i e s ess.
Howe e in he case o cyanobac e ial oxins, depu a ion s udies in aqua ic o ganism a e ela i ely
sca ce. Ozawa e al. [30] s udied he accumula ion and depu a ion o mic ocys ins (MCs) in eshwa e
snails, and Vasconcelos e al. [31] and T ica ico e al. [32] in he c ay ish P ocamba us cla kii.
Galan i e al. [33] in es iga ed he de oxi ica ion dynamic o MC-LR in he sh imp
Palaemone es a gen inus. Mo eo e , Kankaanpää e al. [34] s udied he depu a ion mechanism o
nodula in in mussel (My ilus edulis). As a as we know, in he case o CYN, only Sake e al. [35]
in es iga ed he accumula ion and depu a ion o CYN in he eshwa e mussel Anodon a cygnea.
Taking all hese da a in o accoun , he aim o his s udy was o in es iga e he ansc ip ional and
ca aly ic esponse o he glu a hione (GSH) ela ed enzymes GPx and sGST, in ilapia ish (li e and
kidney) a e a dose epea ed exposu e (a en i onmen ally ele an concen a ion) o a
CYN-con aining cul u e o Aphanizomenon o alispo um du ing 14 days, and he in luence o wo
di e en depu a ion pe iods (3 o 7 days) in hese pa ame e s. A e CYN-exposu e and bo h depu a ion
pe iods, mRNA le els and enzyma ic ac i i ies we e measu ed o GPx and sGST, including p o ein
abundance o sGST.
2. Resul s and Discussion
2.1. Resul s
No ish died and hey exhibi ed no ob ious signs o s ess du ing he acclima ion pe iod, he
exposu e o A. o alispo um cul u e o du ing he depu a ion pe iods.
2.1.1. Glu ha ione Pe oxidase and Glu ha ione-S-T ans e ase Ac i i ies
In gene al, no changes in GPx ac i i y we e obse ed in he li e o ish exposed o
CYN-con aining cul u e (equi alen o 10 μg CYN/L) in any o he es g oups in compa ison wi h
hei con ols (Figu e 1a). In he kidneys, a signi ican inc ease in GPx ac i i y was obse ed in ish
ea ed wi h CYN and no depu a ed, when compa ing wi h he con ol g oup (Figu e 1b).
Toxins 2014, 6 1065
Figu e 1. Glu a hione pe oxidase (GPx) ac i i y (nka als/mg p o ein) in li e (a) and kidney (b)
o ish exposed by imme sion o epea ed doses o CYN-con aining cul u e (equi alen o
10 µg CYN/L, added in o aqua ia e e y wo days) o 14 days and la e sac i iced (0 day) o
depu a ed (3 o 7 days). The alues a e exp essed as mean ± SE (n = 8). The signi icance
le els obse ed a e ** p < 0.01 in compa ison wi h hei espec i e con ol g oup,
&& p < 0.01 when CYN exposed and no depu a ed ish (0 day g oup) and ish depu a ed a
di e en imes (3 o 7 days g oup) a e compa ed, and ### p < 0.001 when compa ing
CYN-depu a ed ish a 3 o 7 days o depu a ion.
Figu e 2. Glu a hione-S- ans e ase (GST) ac i i y (nka als/mg p o ein) in li e (a) and
kidney (b) o ish exposed by imme sion o epea ed doses o CYN-con aining cul u e
(equi alen o 10 µg CYN/L, added in o aqua ia e e y wo days) o 14 days and la e
sac i iced (0 day) o depu a ed (3 o 7 days). The alues a e exp essed as mean ± SE (n = 8).
The signi icance le els obse ed a e *** p < 0.001, * p < 0.05 in compa ison wi h hei
espec i e con ol g oup, &&& p < 0.001 when CYN exposed and no depu a ed ish
(0 day) and ish depu a ed a di e en imes (3 o 7 days) a e compa ed, and ### p < 0.001,
# p < 0.05 when compa ing CYN-depu a ed ish a 3 o 7 days o depu a ion.
sGST ac i i ies we e dec eased in bo h o gans du ing he exposu e ime (0 day g oup,
li e : 3.8- old and 1.4- old in kidney) and a e he ea ly days o depu a ion (3 days g oup, 1.8- old and
1.3- old in li e and kidney, espec i ely). A e 7 days o depu a ion, he sGST le els in li e and
kidney we e es o ed o con ol alues, and signi ican di e ences we e de ec ed in compa ison o ish
Toxins 2014, 6 1066
exposed o cyanobac e ial cells con aining CYN wi hou depu a ion. Mo eo e , signi ican highe alues
we e also obse ed a e 7 days o depu a ion in compa ison o 3 days o depu a ion (Figu e 2a,b).
2.1.2. Glu a hione Pe oxidase and Glu a hione-S-T ans e ase Gene Exp ession
Signi ican al e a ions we e obse ed in he ela i e gene exp ession o he enzyme GPx in he li e
o ish exposed o cyanobac e ial cells con aining CYN non depu a ed and depu a ed o 3 days, in
compa ison o hei espec i e con ol g oups (4- old and 3.5- old up- egula ion, espec i ely). No
s a is ical di e ences we e de ec ed in ish o he longes depu a ion pe iod (7 days) in compa ison wi h
hei con ol g oup, and signi ican di e ences we e e iden be ween his g oup (7 days) and non-depu a ed
ish and hose subjec ed o 3 days o depu a ion (Figu e 3a). In he kidney, all g oups o ish showed
signi ican inc eases in compa ison o hei espec i e con ol g oups (4.5, 1.7 and 1.5- old up- egula ion,
espec i ely) (Figu e 3b).
Figu e 3. Rela i e gene exp ession o glu a hione pe oxidase (GPx) (×1000) in li e (a)
and kidney (b) o ish exposed by imme sion o epea ed doses o CYN-con aining cul u e
(equi alen o 10 µg CYN/L, added in o aqua ia e e y wo days) o 14 days and la e
sac i iced (0 day) o depu a ed (3 o 7 days). The alues a e exp essed as mean ± SE
(n = 8). The signi icance le els obse ed a e *** p < 0.001 in compa ison wi h hei
espec i e con ol g oup, &&& p < 0.001, & p < 0.05 when CYN exposed ish and hen
sac i iced and ish depu a ed a di e en imes (3 o 7 days) a e compa ed, and ### p < 0.001
when compa ing CYN-depu a ed ish a 3 o 7days-depu a ed ish.
Rega ding o sGST, bo h depu a ion p ocesses assayed induced signi ican inc eases in he li e s o
ish in compa ison o hei espec i e con ol g oups (1.7- old and 2.8- old o 3 o 7 days,
espec i ely). Mo eo e , s a is ical di e ences we e obse ed be ween ish exposed o CYN-con aining
cul u e and no depu a ed and hose subjec ed o bo h depu a ion pe iods (1.8 and 3.5- old o 3 o
7 days, espec i ely), which we e mo e e iden as ime o depu a ion inc eased (7 days s. 3 days)
(Figu e 4a). In he kidney, a signi ican enhancemen was obse ed in all ea ed ish in compa ison
wi h hei con ol g oups (2.5- old, 1.7- old and 3- old, espec i ely). Simila o he li e , signi ican
di e ences we e obse ed be ween bo h g oups o CYN-exposed ish and hose subjec ed o di e en
depu a ion pe iods (7 days s. 3 days) (1.7- old) (Figu e 4b).

Toxins 2014, 6 1067
Figu e 4. Rela i e gene exp ession o Glu a hione-S- ans e ase (GST) (×1000) in li e (a)
and kidney (b) o ish exposed by imme sion o epea ed doses o CYN-con aining cul u e
(equi alen o 10 µg CYN/L, added in o aqua ia e e y wo days) o 14 d and la e
sac i iced (0 day) o depu a ed (3 o 7 days). The alues a e exp essed as mean ± SE (n = 8).
The signi icance le els obse ed a e *** p < 0.001 in compa ison wi h hei espec i e
con ol g oup, &&& p < 0.001 when CYN exposed ish and hen sac i iced and ish
depu a ed a di e en imes (3 o 7 days) a e compa ed, and ### p < 0.001 when
compa ing CYN-depu a ed ish a 3 o 7 days-depu a ed ish.
2.1.3. Glu a hione-S-T ans e ase P o ein Exp ession (Wes e n Blo ing)
The li e expe ienced a signi ican dec ease in he p o ein exp ession o sGST in ish exposed o
CYN and no depu a ed in compa ison o hei con ol g oup (0 day g oup). A e depu a ion in clean
wa e (3 o 7 days), no al e a ions in sGST exp ession we e obse ed (Figu e 5a). In he kidney, no
signi ican changes we e obse ed in any g oup o ish (Figu e 5b).
Figu e 5. Rela i e abundance o Glu a hione-S- ans e ase (GST) p o ein in li e (a) and
kidney (b) o ish exposed by imme sion o epea ed doses o CYN-con aining cul u e
(equi alen o 10 µg CYN/L, added in o aqua ia e e y wo days) o 14 days and la e
sac i iced (0 day) o depu a ed (3 o 7 days). The alues a e exp essed as mean ± SE (n = 8).
Resul s a e exp essed as ela i e abundance %. The signi icance le els obse ed a e * p < 0.05
in compa ison wi h hei espec i e con ol g oup, & p < 0.05 when CYN exposed ish non
depu a ed and ish depu a ed a di e en imes (3 o 7 days) a e compa ed.
Toxins 2014, 6 1068
2.1.4. De e mina ion o CYN in Wa e Samples om Aqua ia
The ini ial concen a ion o CYN (a e adding he i s dose o CYN-con aining cul u e in o he aqua ia)
was 11.2 ± 0.5 µg/L and inc eased up o 42.4 ± 0.2 µg/L a e 14 days o exposu e. Once ish we e
emo ed om he aqua ia, no aces o CYN we e de ec ed in he clean wa e du ing he
depu a ion pe iods.
2.2. Discussion
P e ious s udies ha e epo ed ha oxida i e s ess migh play a ole in he pa hogenici y o CYN
on ilapia exposed o a single dose [13,21] o epea ed doses [23] o his molecule. Howe e , he
in luence o di e en depu a ion pe iods on he changes in oxida i e s ess bioma ke s induced by CYN
has no been analyzed up o da e.
The biochemical unc ion o GPx is o educe lipid hyd ope oxides o hei co esponding alcohols
and o educe ee hyd ogen pe oxide o wa e . Fou een days o exposu e o CYN-con aining cul u e
o A. o alispo um by imme sion ha e esul ed in changes o GPx ac i i y only in he kidney. An
inc eased suscep ibili y o his pa ame e o bo h pu e CYN and CYN-con aining cyanobac e ial cells
in he kidney o ish in compa ison o he li e has also been ound by o he au ho s [13,23]. This
could be ela ed o he hyd ophilic p ope ies o CYN. Humpage and Falcone [36] conside ed ha he
kidney appea ed o be he mos sensi i e o gan in mice as well. Mo eo e , s udies o he body
dis ibu ion o 14C-labeled CYN in mice ha e shown ha he main exc e o y ou e is h ough he
kidneys, wi h nea ly 50% o an in ape i oneally (i.p.) adminis e ed dose appea ing in he u ine wi hin
6 h, and 20% o he dose p esen in he li e [37].
The inc ease obse ed bo h in he GPx ac i i y and gene ic exp ession is in acco dance wi h he
highe le els o lipid pe oxida ion p oduc s induced in hese ish [38] and may indica e a de ensi e
esponse o he ish o he oxic insul . Mo eo e , he inc ease obse ed in GPx ac i i y is also ela ed
o he educed alues o GSH/GSSG obse ed in his o gan [38] as GSH is being used in he ca aly ic
ac i i y o his enzyme o sca enge eac i e oxygen species. GPx gene exp ession was signi ican ly
al e ed in bo h o gans a e 14 days o exposu e o CYN-con aining cul u e. This can be in e p e ed as
a highe sensi i i y o molecula bioma ke s. O he au ho s ha e also ound an absence o changes on
GPx ac i i y in he li e bu on he con a y signi ican changes on GPx gene exp ession, al hough
CYN concen a ions and imes o exposu e we e di e en [13,21].
Rega ding o he e ec s o he depu a ion pe iods conside ed, no in luence was ound o GPx
ac i i y in he li e as CYN-con aining cul u e did no cause any al e a ion in his o gan a e he
exposu e pe iod. Simila ly, Galan i e al. [33] did no obse ed changes in GPx ac i i y nei he in he
accumula ion pe iod (3 days) no in he de oxi ica ion pe iod (6 days) in he sh imp Palaemone es
a gen inus exposed o MC-LR unde labo a o y condi ions. Fe ei a e al. [26] ound a signi ican
dec ease o GPx ac i i y in he li e o mulle s a e one mon h o depu a ion om Dou o es ua y
con aminan s. In e es ingly, a e 4 mon hs he ac i i y inc eased and he au ho s a ibu ed his inding
o he highe empe a u es o his pe iod. In he kidney, he oxic esponse obse ed in ish exposed o
cyanobac e ial cells con aining-CYN was eco e ed al eady a e 3 days o depu a ion. This is in
Toxins 2014, 6 1069
ag eemen wi h he educ ion o LPO induc ion in hese ish and he ea ly eco e y (only 3 days) o he
GSH/oxidized glu a hione (GSSG) le els [38] du ing he depu a ion p ocess.
GPx gene exp ession in he li e equi ed 7 days o depu a ion o dec ease he enhancemen
induced by CYN-con aining cul u e and o es o e he basal alues. Howe e , in he kidney, 7 days
we e no enough, sugges ing again a highe suscep ibili y o his o gan in compa ison o he li e . No
o he epo s ha e been ound in he scien i ic li e a u e dealing wi h he in luence o depu a ion
pe iods on he gene ic exp ession o oxida i e s ess bioma ke s o his oxin.
GPx ac i i y and gene exp ession showed he same pa e n in he li e o in oxica ed ish, wi h an
inc ease om day 0 o day 3 and a dec ease om day 3 o 7. Howe e , he e was no coincidence in o he
cases (exposu e and depu a ion). This could sugges a pos - ansc ip ional egula ion o he enzyme o
he combined e ec o di e en GPx genes, as a ious o ms o GPx a e ound in e eb a es [39,40].
GST is a well-known enzyme o Phase II o he me abolism o de oxica ion which conjuga es
glu a hione o ce ain xenobio ics compounds o o hei me aboli es [41]. Ne e heless i has been
demons a ed ha GST shows pe oxidase ac i i ies and may ac in he an ioxida i e de ence [42]. In
his case, GST ac i i y showed a signi ican dec ease bo h in he li e and kidney. This inding is in
ag eemen wi h he educ ion o GSH/GSSG obse ed in hese ish [38]. I is no known i GST is
in ol ed in CYN me abolism. Ac ually, he epo ed CYN me abolic ou e is a hepa ic mixed unc ion
oxida ion (CYP450) esul ing in s ill unknown me aboli es wi h a p ominen ole in CYN oxici y [43].
Conside ing his, he changes obse ed in GST ac i i y could be a ibu ed o i s an ioxida i e ole, as
CYN-con aining cul u es ha e been demons a ed o inc ease he oxida ion o lipids, DNA and
p o eins [38]. P e iously, we had ound a di e en esponse, namely an inc ease o he GST ac i i y,
bu ish we e exposed o pu e CYN by acu e ga age and in ape i oneal injec ion [13,21]. Changes o
he esponse o his pa ame e can be obse ed e en in he same expe imen . Thus, Galan i e al. [33]
epo ed an inc ease in he ac i i y o he memb ane bound GST exposed o MC-LR, whe eas no
change was obse ed in he soluble GST. Mo eo e , addi ional s udies epo ed di e en sGST and
mGST esponses a e MC-LR exposu e in o he o ganisms [44,45].
sGST gene exp ession showed a di e en pa e n in compa ison o he ac i i y, wi h no changes in
he li e and an inc ease in he kidney o ish exposed o CYN-con aining cells o 14 days.
Pue o e al. [13] also ound he same esponse in he kidney bu no in he li e . Again, di e en CYN
concen a ions and exposu e ime lead o di e en esul s. Mo eo e , esponses o li e and kidney can
be di e en , because he ansc ip ion o GST iso o ms a ies in di e en ways wi hin an o gan and
among o gans, as Li e al. [46] deduced om a s udy pe o med on gold ish exposed o MC-LR. The e
a e di e en iso o ms o GSTs, as hey a e a mul iple gene amily o dime ic enzymes, which can
explain he di e ences obse ed be ween he enzyma ic ac i i y and he gene exp ession o e en he
di e en ial esponse o he o gans. Also, he absence o changes in GST exp ession a e he
in oxica ion pe iod does no imply he same esponse a e 14 days. He e al. [47] obse ed an inc ease
o GST exp ession in ilapia exposed o MC-LR a e 8 h o in oxica ion and, unexpec edly, a dec ease
a e 24 h.
In he case o sGST ac i i y, he depu a ion p ocess inc eased he alues and allowed he es o a ion
o he basal le els bo h in he li e and kidney in a ime-dependen manne . This inding could be
linked o he es o a ion o GSH/GSSG le els du ing he depu a ion p ocess epo ed by
Guzmán-Guillén e al. [38], as he ac i i y o his enzyme depends on a s eady supply o GSH.
Toxins 2014, 6 1070
A simila esponse was obse ed by Özcan O uç [27] in ilapia, in which case an inc ease o he GST
ac i i y was obse ed a e 15 days o depu a ion ollowing chlo py i os exposu e. In P. a gen inus
Galan i e al. [33] also obse ed a signi ican inc ease o sGST and mGST du ing he de oxi ica ion
pe iod. Kankaanpää e al. [34] e alua ed he in luence o a 144 h depu a ion pe iod in My ilus edulis
exposed o 24 h o nodula in, bu hey did no obse e any change in he exposu e pe iod o in he
depu a ion ime. sGST gene exp ession o ish on he con a y, was no in luenced by he depu a ion
p ocess in he kidney and esul ed in highe alues in he li e . This could be explained because
be ween he gene exp ession o a pa icula enzyme and i s inal ac i i y he e a e se e al molecula
p ocesses ha can con ibu e o he di e ences obse ed.
sGST p o ein abundance dec eased in he li e and did no show changes in he kidney. The
educ ion obse ed in he li e is in acco dance wi h a well-known oxic mechanism o CYN, he
inhibi ion o p o ein syn hesis [48,49]. Thus, Humpage e al. [50] epo ed ha he CYN pa en
compound was esponsible o he inhibi ion o p o ein syn hesis. This esul is no in ag eemen wi h
p e ious s udies pe o med by Pue o e al. [13] and Gu ié ez-P aena e al. [21]. Howe e , in hose
s udies only a single dose o pu e CYN was used and he obse a ion pe iod was sho e . The au ho s
sugges ed ha he dose employed was no enough o ha he e ec migh need mo e ime o be
obse ed in i o. In his ega d, Kinnea [7] epo ed ha CYN had a delayed oxici y in ol ing
mul iple o gan sys ems, p incipally he li e and kidneys. In his case, he con inued exposu e o
CYN-con aining cul u es (equi alen o 10 µg CYN/L) o 14 days was enough o induce his oxic e ec .
The ela i e abundance o sGST p o eins did no co ela e in all cases wi h he ac i i y and he gene
exp ession o he enzyme, sugges ing ha he e is a egula ion a he ansc ip ional and ansla ional
le el o pos ansla ional modi ica ions.
The depu a ion pe iod o his pa ame e showed a posi i e in luence on he li e , as alues we e
es o ed o he basal le els al eady a e 3 days. The e ec on he kidneys could no be obse ed due o
he ac ha cyanobac e ial cells ha had been exposed o CYN did no a ec his bioma ke .
The o e all posi i e e ec s o he depu a ion pe iods on he oxici y induced by CYN on oxida i e
s ess bioma ke s ha e been epo ed p e iously o o he con aminan s [28,33,34,51]. These epo s
sugges ha he ime necessa y o de ec he in luence o he depu a ion p ocess is di e en o e e y
con aminan , and i depends among o he ac o s on he in ensi y o he oxic exposu e, he de oxi ying
mechanisms a ailable, he elimina ion kine ic o he oxic subs ance and he bioma ke in es iga ed.
3. Expe imen al Sec ion
3.1. Chemicals and Reagen s
CYN s anda d (pu i y > 95%) was supplied by Alexis Co po a ion (Lausen, Swi ze land). S anda d
solu ions o CYN we e p epa ed in wa e milli Q (100 μg/mL, Millipo e, Bed o d, MA, USA) and
dilu ed as equi ed o hei use as wo king solu ions (0.08–5.0 μg/mL). All chemicals and eagen s
used o he di e en assays and analysis we e pu chased om Sigma-Ald ich (Mad id, Spain) and
VWR In e na ional Eu olab S.L. (Se ille, Spain). Deionized wa e (>18 MΩ cm−1 esis i i y) was
ob ained om a Milli-Q wa e pu i ica ion sys em (Millipo e, Bed o d, MA, USA). BOND ELUT®
Toxins 2014, 6 1077
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