Toxins 2014, 6, 96-107; doi:10.3390/ oxins6010096
oxins
ISSN 2072-6651
www.mdpi.com/jou nal/ oxins
A icle
Su ace Plasmon Resonance Biosenso Me hod o Paly oxin
De ec ion Based on Na+,K+-ATPase A ini y
Ampa o Al onso 1, Ma ía-José Pazos 2, And ea Fe nández-A aujo 1, A aceli Tobio 1,
Ca men Al onso 4, Me cedes R. Viey es 3 and Luis M. Bo ana 1,*
1 Depa men o Pha macology, Ve e ina y School, Uni e si y o San iago de Compos ela,
27002 Lugo, Spain; E-Mails: ampa [email p o ec ed] (A.A.);
[email p o ec ed].es (A.F.-A.); [email p o ec ed].es (A.T.)
2 Con ocal and Elec onic Mic oscopy Uni , RIAIDT, Uni e si y o San iago de Compos ela,
27002 Lugo, Spain; E-Mail: [email p o ec ed] (M.-J.P.)
3 Depa men o Physiology, Ve e ina y School, Uni e si y o San iago de Compos ela,
27002 Lugo, Spain; E-Mail: [email p o ec ed]
4 Ci ga Labo a o y, San o Domingo Squa e, 20, 5ª, 27001 Lugo, Spain;
E-Mail: [email p o ec ed]
* Au ho o whom co espondence should be add essed; E-Mail: lu[email p o ec ed];
Tel./Fax: +34-9828-22233.
Recei ed: 4 No embe 2013; in e ised o m: 17 Decembe 2013 / Accep ed: 18 Decembe 2013 /
Published: 27 Decembe 2013
Abs ac : Paly oxin (PLTX), p oduced by dino lagella es om he genus Os eopsis was
i s disco e ed, isola ed, and pu i ied om zoan hids belonging o he genus Paly hoa.
The de ec ion o his oxin in con amina ed shell ish is essen ial o human heal h
p ese a ion. A b oad ange o s udies indica e ha mammalian Na+,K+-ATPase is a high
a ini y cellula ecep o o PLTX. The oxin con e s he pump in o an open channel ha
s imula es sodium in lux and po assium e lux. In his wo k we de elop a de ec ion me hod
o PLTX based on i s binding o he Na+,K+-ATPase. The me hod was de eloped by using
he phenomenon o su ace plasmon esonance (SPR) o moni o biomolecula eac ions.
This echnique does no equi e any labeling o componen s. The in e ac ion o PLTX o e
immobilized Na+,K+-ATPase is quan i ied by injec ing di e en concen a ions o oxin in
he biosenso and checking he binding a e cons an (Kobs). F om he ep esen a ion o
Kobs e sus PLTX concen a ion, he kine ic equilib ium dissocia ion cons an (KD) o he
PLTX-Na+,K+-ATPase associa ion can be calcula ed. The alue o his cons an is
KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In his way he PLTX-Na+,K+-ATPase
OPEN ACCESS
Toxins 2014, 6 97
associa ion was used as a sui able me hod o de e mina ion o he oxin concen a ion in a
sample. This me hod ep esen s a new and use ul app oach o easily de ec he p esence o
PLTX-like compounds in ma ine p oduc s using he mechanism o ac ion o hese oxins
and in his way educe he use o o he mo e expensi e and animal based me hods.
Keywo ds: paly oxin; Na+,K+-ATPase; su ace plasmon esonance biosenso ;
Os eopsis siamensis
1. In oduc ion
Paly oxin (PLTX), isola ed om he ma ine so co al (genus Paly hoa), is one o he mos
poisonous non-p o ein subs ances known o da e. I is common in opical and sub opical wa e s and
may accumula e a e y high le els in ish and c abs [1]. Animals may inco po a e PLTX by il e ing
and he e o e en e ing he oxin in he ood chain [2]. PLTX is ex emely po en h ough in a enous,
in ape i oneal, and in a acheal exposu e, and less po en by di ec in agas ic exposu e [3]. Due o
co-occu ence wi h o he sea ood oxins, such as cigua oxins, saxi oxins, and e odo oxin, i has been
di icul o assess he ue isk o PLTX poisoning h ough sea ood consump ion in humans [4]. Toxin
quan i ica ion and iden i ica ion in sea ood has elied on di e en me hodologies, mainly LC-MS,
mouse bioassay, hemolysis neu aliza ion assay and ELISA assays [5–11]. The la ge spa ial expansion
o his oxin has led o in ensi ica ion o esea ch owa ds op imiza ions o me hods o de e mina ion
o PLTX p esence and oxici y. This oxin is a la ge, e y complex molecule wi h bo h lipophilic and
hyd ophilic a eas, and has he longes chain o con inuous ca bon a oms known o exi in a na u al
p oduc . Se e al molecules ela ed wi h PLTX ha e been desc ibed: paly oxin-b, homopaly oxin,
bishomopaly oxin, neopaly oxin, deoxypaly oxin, 42-hyd oxypaly oxin, os eocin-d, o a oxin-a, -b,
-c,-d, -e and - and masca eno oxin-a, -b and -c. Howe e only he chemical s uc u es o PLTX,
os eocin-d, o a oxin-a and 42-hyd oxypaly oxin ha e been cha ac e ized [12–18].
Se e al s udies indica es ha PLTX binds o he Na+,K+-ATPase in he plasma memb ane o
animal cells and opens a ca ion pa hway h ough he pump [19–21]. The e o e, he Na+,K+-ATPase has
been p oposed as o he oxin ecep o . PLTX has an e ec on p ima y neu onal cul u es o ce ebella
g anule cells (CGC), leading o a la ge inc ease in he cy osolic calcium concen a ion and o a la ge
in acellula acidi ica ion o hese neu ons [22,23]. Se e al symp oms like sc a ching, jumping,
pa alysis o hind limbs, espi a o y dis ess, cyanosis also b ings nausea, i edness, dia hoea and
omi ing ollowed by dizziness, in animals ha e been desc i ed [24]. In humans PLTX poisoning is
called paly oxicosis o clupeo oxicosis [25]. This p ocess is associa ed also o in es inal symp oms,
muscle spasms, b ea hing di icul ies ollowed in some cases by dead in humans ha e been desc i ed
as consequence o con amina ed ood consop ium [24,26,27].
The use o op ical biosenso s o s udy molecula in e ac ions is a well accep ed me hod. This
echnology has been used o measu e in eal ime he binding kine ics be ween a mac omolecule in
solu ion and a ecep o immobilized. In his way, undamen al in o ma ion o e biospeci ic
in e ac ions can be ob ained. Many app oaches ha e been done o de elop ma ine oxin and ood
Toxins 2014, 6 98
con aminan s de ec ion me hods employing he biosenso s echnology by using ei he an ibodies o
oxin ecep o s wi h high success [28–33].
In his pape , we used a apid su ace plasmon esonance (SPR) biosenso assay o s udy he
in e ac ion o PLTX and ouabain, as con ol, wi h immobilized Na+,K+-ATPase om dog kidney and
o de elop a new me hod o de ec he oxin in shell ish. The echnique does no equi e any labeling o
he in e ac ing componen s and he in e ac ions a e measu ed in eal ime.
2. Resul s and Discussion
We ha e shown ha ouabain in e ac s wi h a senso su ace-a ached Na+,K+-ATPase, howe e no
in e ac ion be ween he ATPase and PLTX was obse ed [23]. Howe e , he echnology used, an Iasys
A ini y Senso , and he chemical eac ions needed o a ach he p o ein p e en ed o show any
in e ac ion. In he p esen pape , we used ano he biosenso , Biaco e X SPR, and di e en chemical
app oaches in o de o s udy and o measu e he binding be ween PLTX and he Na+,K+-ATPase. The
Na+,K+-ATPase was used as he ligand a ached o he senso su ace and PLTX in solu ion was used
as he liga e.
Ini ially he Na+,K+-ATPase was immobilized o e a CM5 senso chip p e iously ac i a ed. Amine
coupling is he mos gene ally applicable coupling chemis y because mos mac omolecules con ain
many g oups whose can pa icipa e in he amine coupling eac ion, and he e o e he immobiliza ion is
usually easy [34]. Howe e , as i was shown his kind o immobiliza ion p e en ed PLTX binding [23].
In his sense, he e a e si ua ions whe e o he coupling me hods may be p e e able because ligands
ha e ac i e si e ha include pa icula ly eac i e amino g oups ha may lose biological ac i i y on
immobiliza ion. In hese cases, a coupling by hiol-disulphide exchange, by in oducing an ac i e
disulphide on he senso chip su ace and exchanging wi h in insic hiol g oups in he ligand is used [35].
The e iciency o hiol coupling is e y high, and he condi ions o immobiliza ion a e o en less
c i ical han wi h amine coupling. This was he s a egy ollowed in he p esen pape . As Figu e 1
shows, 100 µg mL−1 o Na+,K+-ATPase om dog kidney dissol ed in sodium ace a e was added,
410 s, o he senso chip pe o med by in oducing an ac i e disulphide on he senso chip su ace.
In hese condi ions, a ypical co alen binding cu e was ob ained. Then, he su ace was washed wi h
HBS bu e low and no all in he signal was obse ed, indica ing ha Na+,K+-ATPase was s ongly
immobilized on o he su ace o he senso chip. Finally cys eine/NaCl was injec ed (600 s o 740 s) o
deac i a e eac i e disulphides excess and emo e non-co alen ly bound p o ein.
Nex , o check he ac i i y o immobilized Na+,K+-ATPase, di e en concen a ions o ouabain
we e added a 25 °C, by using HBS-EP as unning bu e a low a e o 10 µL min−1. In his case,
ypical associa ion cu es we e obse ed. As Figu e 2 shows, he esponses a e 120 s ouabain
addi ion ollow a ypical associa ion cu e p o ile. In he p esence o 2 mM ouabain he signal is
87.04 RU, while in he p esence o 12 mM ouabain he esponse eaches 178.99 RU. The indi idual
binding cu es om Figu e 2 we e analyzed o de e mine he kine ic cons an s o
ouabain-Na+,K+-ATPase binding, namely, he obse ed a e cons an (Kobs), he associa ion a e
cons an (Kass), he dissocia ion a e cons an (Kdiss), and he kine ic equilib ium dissocia ion cons an
(KD). A equilib ium, by de ini ion, Kdiss/Kass = KD. The pseudo- i s -o de associa ion a e cons an
Kobs (s−1) was de e mined o each ouabain concen a ion by using he 1:1 Langmui associa ion model
o he BiaE alua ion so wa e (BiaCo e, Uppsala, Sweden). Figu e 3 shows a ep esen a ion o each
Toxins 2014, 6 99
Kobs agains he co esponding concen a ion o ouabain ( ep esen a i e o one expe imen ). This plo
ollows a linea co ela ion coe icien , = 0.99. F om he equa ion o his ep esen a ion, Kass, M−1 s−1,
g adien o he plo , and Kdiss, s−1, in e cep o he plo we e ob ained. Wi hin hese wo alues, he
kine ic equilib ium dissocia ion cons an KD (Y-in e cep /slope) o he ouabain-Na+,K+-ATPase
binding was ob ained. The alue o his cons an was 4.2 × 10−3 ± 8.4 × 10−4 M ouabain (a e age o a
leas h ee expe imen s). This alue is simila o he alue ob ained in ou labo a o y o he same
expe imen in di ec binding assays wi h a esonan mi o biosenso [23].
Figu e 1. Senso su ace ac i a ion and Na+,K+-ATPase immobiliza ion. Ac i a ion: i s
a ow shows he addi ion o EDC/NHS o ac i a e he CM5 dex an. De i a isa ion: he second
a ow shows he addi ion o 80 mM PDEA o he su ace o de i a iza e. Immobiliza ion:
he hi d a ow indica es he addi ion o 100 µg mL−1 Na+,K+-ATPase om dog kidney.
Blocking: he emaining ac i a ed si es we e blocked wi h cys eine/NaCl, ou h a ow.
Figu e 2. Ouabain-Na+,K+-ATPase associa ion. Associa ion cu es a e addi ion o
di e en amoun s o ouabain o immobilized Na+,K+-ATPase. Di e en Ouabain
concen a ions we e injec ed using HBS-EP as unning bu e and a low a e o
10 µg mL−1. The associa ion cu es we e ob ained a e sub ac ion o hei espec i e
sol en con ol. Rep esen a i e o 4 expe imen s.
0
5000
10000
15000
20000
25000
30000
35000
0 200 400 600 800 100
0
R
esponse un
i
(
R
U
)
Time (sec)
NHS/EDC
PDEA
Na,K-ATPasa
L-Cys eine-NaCl
0
50
100
150
200
0 20 40 60 80 100 120
Response uni
(
RU
)
Time
(
sec
)
12 mM
6 mM
4 mM
2 mM
Ouabain
Toxins 2014, 6 100
Figu e 3. Analysis o ligand binding. Kine ic plo o appa en associa ion a e cons an
Kobs (s−1) ob ained om plo in Figu e 2 (calcula ed by he BiaE alua ion so wa e) e sus
ouabain concen a ion. Rep esen a i e o 4 expe imen s.
A e se ing up hese condi ions we de eloped a me hod o s udy he binding PLTX-Na+,K+-ATPase.
In his case, di e en concen a ions o PLTX we e dissol ed in me hanol-wa e (1:1) and added on o
he immobilized Na+,K+-ATPase. As Figu e 4 shows, ypical associa ion cu es we e ob ained when
PLTX was added. A e 100 s, esponses om 30 o 170 RU, depending on he PLTX concen a ion,
we e ob ained. The PLTX-Na+,K+-ATPase in e ac ion ollows a pseudo- i s -o de kine ics whe e Kobs
can be calcula ed. The alue o his cons an is inc easing wi h PLTX concen a ion. When Kobs was
ep esen ed agains he co esponding concen a ion o PLTX a linea eg ession wi h a co ela ion
coe icien o = 0.9986 was ob ained, Figu e 5. F om his ep esen a ion he kine ic equilib ium
cons an o he PLTX-Na+,K+-ATPase binding was ob ained KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX.
This alue is in he ange o KD o unions be ween ac i e biological species (10−11 and 10−4 M) [36]. A e
hese esul s, he me hod was used o de ec PLTX in con amina ed samples. The amoun o oxin in
wo Os eopsis siamesis ex ac s and one P o ocen um e icula um (P. e icula um) cul u e, as
nega i e con ol, was quan i ied and he esul s compa ed wi h he amoun ob ained by luo esce
pola iza ion (FP) o he PLTX de ec ion assay ecen ly desc ibed [5]. Os eopsis spp. ha e been
desc ibed as PLTX-like compounds p oduce s [37]. As Figu es 6 and 7 show, he ypical associa ion
binding cu e we e ob ained om Na+,K+-ATPase-PLTX union when Os eopsis spp. cul u es we e
added. Howe e , as Figu e 7 shows P. e icula um cul u e did no induce any signal inc ease. The Kobs
alues calcula ed om hese cu es can be ans o med in PLTX concen a ion using he equa ion
ob ained om he calib a ion cu e (Figu e 5). In his case, he PLTX concen a ions ob ained wi h he
biosenso we e 56.2.8 ± 8.83 µM and 670 ± 31.6 µM and wi h he FP assay 68.8 ± 9.8 µM and
761.1 ± 4.5 µM (Table 1). The e o e his me hod is eliable, he esul s ob ained a e compa able wi h
FP me hod, and has a high deg ee o epea abili y. The biosenso me hod a oids he e hical p oblems
o he o icial mousse bioassay and he alse-posi i e and alse-nega i e esul s. In addi ion, he
biosenso me hod he e p oposed is a as and accu a e me hod whe e only PLTX o like-PLTXs
compounds a e de ec ed [7,24,38]. The limi o de ec ion (LOD) o SPR me hod is 3.73 pg PLTX and
he limi o quan i ica ion (LOQ) is 11.2 pg PLTX. These limi s a e compa able wi h o he biological
PLTX de ec ion me hods as he luo escen pola iza ion assay [5,7], he cy oly ic assay [8] o he

Toxins 2014, 6 101
sandwich ELISA assay [11], bu maybe no as good as he hemolysis o he EILISA assays [9,10]. In
addi ion, a oxic dose in humans would be be ween 2.3 and 31.5 µg [39], which is highe han he
lowe limi o de ec ion o his assay.
Figu e 4. PLTX-Na+,K+-ATPase associa ion. Associa ion cu es a e addi ion o di e en
amoun s o PLTX o immobilized Na+,K+-ATPase. Di e en PLTX concen a ions we e
injec ed o e he CM5 chip using HBS-EP as unning bu e and a low a e o 10 µL min−1.
The associa ion cu es we e ob ained a e sub ac ion o hei espec i e sol en con ol.
Rep esen a i e o 4 expe imen s.
Figu e 5. Analysis o ligand binding. Kine ic plo o appa en associa ion a e cons an
Kobs (s−1) ob ained om plo in Figu e 4 (calcula ed by he BiaE alua ion so wa e) e sus
ouabain concen a ion. Rep esen a i e o 4 expe imen s.
Table 1. Concen a ion o PLTX in Os eopsis siamensis and P. e icula um ex ac s.
PLTX concen a ions we e ob ained by using he biosenso me hod and he luo esce
pola iza ion me hod [5].
PLTX (µM)
Biosenso assay
PLTX (µM)
FP assay
Ex ac 1 56.2.8 ± 8.83 68.8 ± 9.8
Ex ac 2 670 ± 31.6 761.1 ± 4.5
P. e icula um - -
0
50
100
150
200
0 20406080100
Response uni (RU)
Time (sec)
80 nM
250 nM
500 nM
1000 nM
PLTX
Toxins 2014, 6 102
Figu e 6. De e mina ion o PLTX concen a ion in Os eopsis siamensis ex ac 1.
Associa ion cu e a e addi ion o ex ac 1 dissol ed in me hanol-wa e o immobilized
Na+,K+-ATPase. The Kobs (s−1) alue was calcula ed om his cu e and ans o med in
oxin concen a ion using he equa ion ob ained om plo in Figu e 5.
Figu e 7. De e mina ion o PLTX concen a ion in Os eopsis siamensis ex ac 2 and
P o ocen um e icula um. Associa ion cu es a e addi ion o ex ac s dissol ed in
me hanol-wa e o immobilized Na+,K+-ATPase. F om ex ac 2 cu e he Kobs (s−1) alue
was calcula ed and ans o med in oxin concen a ion using he equa ion ob ained om
plo in Figu e 5.
3. Ma e ials and Me hods
3.1. Ins umen a ion
A Biaco e X SPR biosenso wi h Con ol So wa e and BIAe alua ion so wa e e sion 3.0 we e
pu chased om Biaco e (GE Heal heca e, Uppsala, Sweden).
3.2. Chemicals
PLTX was ob ained om Wako Pu e Chemical Indus ies (Neuss, Ge many). CM5 senso chips,
HBS-EP bu e (pH 7.4, 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% polyso ba e),
0
20
40
60
80
100
0 20406080100
Ex ac 1
Response uni (RU)
Time (sec)
Ex ac
0
20
40
60
80
100
120
0 50 100 150
Response uni (RU)
Time (sec)
Ex ac
Ex ac 2
Ex ac
P. e icula um
Toxins 2014, 6 103
amine coupling ki (1-e hyl-3-(3-dime ylaminop opyl) ca bodiimide hid ochlo ide (EDC) and
N-hyd oxysuccinimide (NHS) and 2,2-py idinyldi hio-e haneamine (PDEA) we e supplied by Biaco e
AB (Uppsala, Sweden).
L-Cys eine, Bo ic Acid, Sodium Fo ma e, Sodium Hyd oxide we e pu chased om Sigma
Chemical Co. (Mad id, Spain). All bu e ed solu ions we e degassed and il e ed h ough a Millex® GP
0.22 µm po e size il e om Millipo e Co po a ion (Bed o d, MA, USA).
Ouabain and Na+,K+-ATPase isola ed and pu i ied om dog kidney we e pu chased om
Sigma (S Louis, MO, USA).
3.3. Me hods
3.3.1. Su ace Ac i a ion and Ligand Inmobiliza ion
Senso su ace ac i a ion and ligand immobiliza ion we e pe o med using HBS-EP as unning
bu e a a low a e o 5 µL min−1 and 25 °C. The CM5 dex an ma ix chip was ac i a ed
using an amine coupling ki . Following manu ac u e ins uc ions, a mix u e (1:1, / ) o EDC and
NHS was applied o 2 min o e he senso chip. A e emo al excess solu ion, 80 mM PDEA
dissol ed in 0.1 M bo a e bu e a pH 8.5 was injec ed o 4 min o de i a ize he senso chip. Nex
he ligand, 100 µg/mL o Na+,K+-ATPase om dog kidney dissol ed in sodium ace a e 10 mM a pH 4.5,
was added. Finally cys eine/NaCl was injec ed o deac i a e excess eac i e disulphides, o emo e
non-co alen ly bound p o ein and he e o e o block emaining ac i a ed si es.
3.3.2. Binding o PLTX o Ouabain o Immobilized Na+,K+-ATPase
Se e al Ouabain concen a ions dilu ed in wa e we e added o e senso chip o bind wi h he
immobilized Na+,K+-ATPase. The du a ion o he sample injec ion was 2 min a 10 µL min−1 low
a e. Nex , dissocia ion o bounded ouabain in HBS bu e low was s udied. The bounded alkaloid o
he chip su ace was emo ed be o e he nex injec ion by adding 1 M glycine-HCl a pH 2.5 o 1 min.
A ange o PLTX concen a ions we e dilu ed in me hanol-wa e and added o e senso chip o
s udy he binding be ween PLTX and Na+,K+-ATPase immobilized. The dissocia ion was done as be o e.
Con ol solu ions (wi h me hanol/wa e ) we e injec ed in he second low cell in he same way and
subsequen ly sub ac ed om liga e esponse signal.
Since oxin dissocia es almos comple ely, we used he associa ion phase o quan i y he
oxin-Na+,K+-ATPase in e ac ion.
3.4. Dino lagella e Ex ac s
Cul u es o Os eopsis siamensis, we e g own (2 L glass conical lask) in seawa e en iched wi h L1
medium and GSe medium ( o P o ocen um e icula um cul u e) wi hou silica es. The salini y, 33‰,
was adjus ed a hese p opo ions by he addi ion o eshwa e o seawa e and emo ing chlo ine by
ae a ion. The expe imen s we e ca ied ou a 18–19 °C and he cul u es we e subjec ed o pho ope iod
wi h day-ligh lamps on a 16:8 h ligh -da k pho o-cycle. Cells we e shaken manually wice a day. The
s ain g ow h was di ided in s eps by p og essi ely inc easing he cul u e olume. The dino lagella es
we e kep unde hese condi ions un il he beginning o he s a iona y phase which was eached 30 days
Toxins 2014, 6 104
a e inocula ion. The cells we e coun ed using an U e möhl came a. The whole cul u e was il e ed,
he cells we e esuspended in me hanol/wa e (1:1), sonica ed o hei comple ely homogeniza ion and
s o ed ozen un il use.
3.5 S a is ical Analysis
All expe imen s we e ca ied ou a leas h ee imes, each by duplica e and da a we e no malized.
Resul s we e analyzed using he S uden ʼs - es o unpai ed da a. A p obabili y le el o 0.05 o
smalle was used o s a is ical signi icance. Resul s we e exp essed as he means ± s anda d e o o
he mean (SEM).
4. Conclusion
The use o SPR chip su aces is a new, easy and use ul ool o de ec ion o se e al ma ine oxins.
By using a SPR chip and an amine coupling no binding be ween PLTX and Na+,K+-ATPase was
p e iously obse ed sugges ing ha addi ional equi emen s could be necessa y o s udy he in e ac ion
be ween bo h molecules. The lack o binding was p obably due o some s e ic impedimen o he la ge
molecule, PLTX MW 2680, o o he blockade o some liga e g oup in o he su ace chip essen ial o
he binding [23]. In he p esen pape , by using a di e en coupling, hiol-disulphide exchange, he
in e ac ion be ween PLTX and he Na+,K+-ATPase can be demons a ed. This in e ac ion has alue o
KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX, indica ing a high a ini y be ween hese wo molecules. Wi h
his design a de ec ion me hod o he oxin is de eloped. The me hod epo ed suppo s he alue o
biosenso assays as sc eening me hods ha would help o educe he use o o he mo e expensi e and
e hically ques ionable me hods wi hou pu ing a isk human heal h p o ec ion.
Acknowledgmen s
The esea ch leading o hese esul s has ecei ed unding om he ollowing FEDER
co unded-g an s: F om Minis e io de Ciencia y Tecnología, Spain: AGL2009-13581-CO2-01,
AGL2012-40485-CO2-01. F om Xun a de Galicia, Spain: 10PXIB261254 PR.
F om he Eu opean Union’s Se en h F amewo k P og amme managed by REA—Resea ch
Execu i e Agency h p://ec.eu opa.eu/ esea ch/ ea (FP7/2007–2013) unde g an ag eemen
Nos. 211326-CP (CON IDENCE), 265896 BAMMBO, 265409 µAQUA, and 262649 BEADS,
315285 Cigua ools and 312184 Pha maSea. F om he A lan ic A ea P og amme (In e eg IVB
T ans-na ional): 2008-1/003 (A lan ox) and 2009-1/117 (Pha ma lan ic).
Re e ences
1. I o, E.; Yasumo o, T. Toxicological s udies on paly oxin and os eocin-D adminis e ed o mice by
h ee di e en ou es. Toxicon 2009, 54, 244–251.
2. Gleibs, S.; Mebs, D. Dis ibu ion and seques a ion o paly oxin in co al ee animals. Toxicon
1999, 37, 1521–1527.