oxins
A icle
S uc u e Elucida ion and Biological E alua ion o
Mai o oxin-3, a Homologue o Gambie one,
om Gambie discus belizeanus
And ea Boen e-Juncal 1, Me cedes Ál a ez 2,Ál a o An elo 2, Inés Rod íguez 2,
Ke in Calab o 3, Ca men Vale 1, Oli ie P. Thomas 3and Luis M. Bo ana 1,*
1
Depa amen o de Fa macología, Fa macia y Tecnología Fa macéu ica, Facul ad de Ve e ina ia, Uni e sidad
de San iago de Compos ela, 27002 Lugo, Spain; [email p o ec ed] (A.B.-J.);
[email p o ec ed] (C.V.)
2Labo a o io CIFGA S.A., Plaza de San o Domingo no. 20, 5a plan a, 27001 Lugo, Spain;
[email p o ec ed] (M.Á.); al a [email p o ec ed] (Á.A.); [email p o ec ed] (I.R.)
3
Ma ine Biodisco e y, School o Chemis y and Ryan Ins i u e, Na ional Uni e si y o I eland Galway (NUI
Galway), Uni e si y Road, H91 TK33 Galway, I eland; ke in.calab [email protected] (K.C.);
oli ie . [email protected] (O.P.T.)
*Co espondence: [email p o ec ed]; Tel.: +34-982-822-233
Recei ed: 30 Decembe 2018; Accep ed: 28 Janua y 2019; Published: 1 Feb ua y 2019
Abs ac :
Gambie discus species a e he p oduce s o he ma ine oxins cigua oxins and mai o oxins
which cause wo ldwide human in oxica ions ecognized as Cigua e a Fish Poisoning. A deep
chemical in es iga ion o a cul u ed s ain o G. belizeanus, collec ed in he Ca ibbean Sea, led o
he iden i ica ion o a s uc u al homologue o he ecen ly desc ibed gambie one isola ed om he
same s ain. The s uc u e was elucida ed mainly by compa ison o NMR and MS da a wi h hose o
gambie one and asce ained by 2D NMR da a analyses. G a i yingly, a close inspec ion o he MS da a
o he new 44-me hylgambie one sugges s ha his oxin would ac ually co espond o he s uc u e
o mai o oxin-3 (MTX3, m/z1039.4957 o he p o ona ed adduc ) de ec ed in 1994 in a Paci ic s ain
o Gambie discus and ecen ly shown in ou ine moni o ing p og ams. The e o e, his wo k p o ides
o he i s ime he chemical iden i ica ion o he MTX3 molecule by NMR. Fu he mo e, biological
da a con i med he simila ac i i ies o bo h gambie one and 44-me hylgambie one. Bo h gambie one
and MTX3 induced a small inc ease in he cy osolic calcium concen a ion bu only MTX3 caused cell
cy o oxici y a mic omola concen a ions. Mo eo e , ch onic exposu e o human co ical neu ons
o ei he gambie one o MTX3 al e ed he exp ession o iono opic glu ama e ecep o s, an e ec
al eady desc ibed be o e o he syn he ic cigua oxin CTX3C. Howe e , e en when gambie one and
MTX3 a ec ed glu ama e ecep o exp ession in a simila manne hei e ec on ecep o exp ession
di e ed om ha o CTX3C, since bo h oxins dec eased AMPA ecep o le els while inc easing
N-me hyl-d-aspa a e (NMDA) ecep o p o ein. Thus, u he s udies should be pu sued o cla i y
he simila i ies and di e ences in he biological ac i i y be ween he known cigua oxins and he new
iden i ied molecule as well as i s con ibu ion o he neu ological symp oms o cigua e a.
Keywo ds:
mai o oxin-3; 44-me hylgambie one; gambie one; mai o oxin; Gambie discus belizeanus;
cy osolic calcium concen a ion; glu ama e ecep o s; neu o oxici y; cigua e a
Key Con ibu ion:
The 3D s uc u e o MTX-3 ound in Gambie discus species has inally been
elucida ed, and co esponds o a homologue o he ecen ly desc ibed gambie one.
Toxins 2019,11, 79; doi:10.3390/ oxins11020079 www.mdpi.com/jou nal/ oxins
Toxins 2019,11, 79 2 o 19
1. In oduc ion
Cigua e a Fish Poisoning (CFP) is a human oodbo ne illness caused by he inges ion o ma ine
ish con amina ed wi h cigua oxins (CTX) and o he s uc u ally ela ed ladde -shaped polye he
oxins adi ionally iden i ied as mai o oxins (MTX) [
1
]. Cigua e a has ecen ly become a h ea o
ish consume s in non-endemic egions mainly due o he expanding in e na ional ade in sea ood
om opical ishe ies and o he p oli e a ion o Gambie discus as a consequence o ocean wa ming
and clima e change [
2
–
4
]. Human in oxica ions by cigua e a a ec annually 10,000 o 500,000 people
wo ldwide [
4
], al hough he p e alence o his disease could be highe [
3
,
4
]. T adi ionally, CFP was
hough o a ec mainly opical and sub- opical a eas bu p esen ly i has expanded wo ldwide [
2
].
In ac , he p esence o Gambie discus was p e iously associa ed wi h opical a eas, bu p esen ly
CFP in oxica ions ha e been iden i ied in Eu ope du ing ecen decades, especially in he Cana y
Islands [5,6] and Madei a [7,8].
Mai o oxins a e among he la ges na u al non-polyme ic compounds and he mos oxic ma ine
compounds iden i ied o da e [
9
]. These oxins a e p oduced by dino lagella es o he gene a
Gambie discus and Fukuyoa [
10
,
11
]. Up o now, ou oxin analogs designa ed as mai o oxin-1 (MTX1),
mai o oxin-2 (MTX2), mai o oxin-3 (MTX3) and mai o oxin-4 (MTX4) isola ed om di e en s ains
o hese dino lagella es ha e been iden i ied in his oxin g oup [
1
,
10
,
12
,
13
]. So a , mai o oxin
(MTX1) is he la ges non-polyme ic ca bon chain molecule in na u e [
14
,
15
]. Addi ionally, species
o Gambie discus and Fukuyoa ha e been shown o p oduce o he non-s uc u ally ela ed polye he
analogs such as gambie ic acids [
16
], gambie oxide [
17
], gambie ol [
18
], and he ecen ly iden i ied
gambie one [
19
]. The di e si y in he chemical s uc u es and biological ac i i ies o he molecules
in ol ed in CFP could e lec hei di e en mechanisms o ac ion. Thus, while MTX-like ac i i y is
associa ed wi h a massi e calcium in lux and apid cell dea h [
20
–
23
], cigua oxins and gambie one
cause ol age-ga ed sodium channel ac i a ion a nega i e memb ane po en ials leading o cell
depola iza ion a es and o he dis up ion o pe iphe al and cen al ne e ansmission [
3
,
19
]. Finally,
gambie ol ac s mainly by blocking ol age dependen po assium channels [24,25].
Among he oxins in ol ed in CFP in Aus alia, a pu a i e mai o oxin-3 (p-MTX3) was equen ly
encoun e ed in mos species o Gambie discus [
26
]. Fo many yea s, p-MTX3 has been de ec ed in
Gambie discus species ha showed low oxici ies in unc ional bioassays [
27
,
28
], bu he chemical
s uc u e o his molecule emained o be elucida ed due o he low amoun o biomass a ailable.
Mass spec ome y analyses o s ains o Gambie discus e ealed he p esence o p-MTX3 wi h a
majo MS peak a m/z 1039.5 [
29
]. P esen ly, ou ine moni o ing o oxins (pa icula ly cigua oxins)
by mass spec ome y e ealed he almos ubiqui ous p esence o his mass in posi i e ioniza ion
mode [
27
,
28
,
30
]. MTX3 was ini ially isola ed by Holmes e al. [
10
] om cul u es o he Gambie discus
s ain WC1/1. The molecule was depic ed as di-sul a ed wi h a MW = 1060.5 Da ( o di-sodium sal )
and m/z 1039.5 as he mos in ense peak obse ed in IonSp ay Mass Spec ome y (ISMS). MTX3 was
ound o be oxic o mice [
10
] and despi e hei conside able di e ences in molecula size (mai o oxin-3
is abou one hi d he MW o mai o oxin-1), bo h molecules showed simila
in i o
ac i i y in mice
o hei dea h- ime s. in ape i oneal dose [
10
]. Recen ly, he s uc u e o a new mai o oxin analog,
named MTX4, was iden i ied om a s ain o Gambie discus excen icus. MTX4 was epo ed o exhibi
a oxic e ec simila o he one o MTX1 in neu oblas oma cells [
13
]. The p esence o he p-MTX3 wi h
he same MS da a was also desc ibed in his s ain bu again no chemical s uc u e could be assigned.
Ini ial s udies indica ed ha MTX3 p oduced simila symp oms and biological e ec s o hose elici ed
by MTX1 and MTX2 al hough wi h lowe po ency han MTX1 [
10
]. The educ ion in po ency a e
sol olysis o hese mai o oxins led o sugges s ha sul a e moie ies we e impo an o he biological
ac i i y o each mai o oxin analog [10,29].
So a , i e compounds p oduced by Gambie discus we e ound o con ain a leas one sul a e
g oup: MTX1, MTX2, MTX3, gambie one and gambie oxide. The p esence o a sul a e g oup in he
molecula skele on o mai o oxin is no synonymous o a ypical mai o oxin-like biological ac i i y.
In ac , ecen analysis o he biological ac i i y o gambie one e ealed ha his compound did no
Toxins 2019,11, 79 3 o 19
exhibi simila cellula e ec s o hose o mai o oxin. Ra he , he biological ac i i y o gambie one
esembled he e ec o he ol age-ga ed sodium channel ac i a o CTX3C al hough he biological
po ency o gambie one was lowe han ha o he syn he ic cigua oxin [
19
]. The biological ac i i y
o gambie oxide, he o he sul a e-con aining polye he compound ela ed o mai o oxin, has no ye
been assessed.
Du ing ou e o s o isola e cigua oxin om G. belizeanus we ound a la ge chemical di e si y
and we decided o ocus on wo majo me aboli es wi h wo p ominen ions a m/z 1025.6 and
1039.5 in elec osp ay ioniza ion (ESI)(+)-MS. The me aboli e wi h he i s mass has ecen ly been
iden i ied as a no el ladde -shaped polye he compound named gambie one [
19
]. The o he ion was
elu ed in he mai o oxin egion in a ypical e e se phase ch oma og aphy. We epo he ein he
s uc u e o he me aboli e associa ed wi h he mass a m/z 1039.5. All NMR da a a e consis en wi h
44-me hylgambie one (1) (Figu e 1). This s uc u e he e o e ep esen s he candida e s uc u e o
MTX3. We also epo he compa a i e biological ac i i ies o his new me aboli e and gambie one
bo h isola ed om G. belizeanus.
Toxins 2019, 11, x FOR PEER REVIEW 3 o 20
gambie oxide, he o he sul a e-con aining polye he compound ela ed o mai o oxin, has no ye
been assessed.
Du ing ou e o s o isola e cigua oxin om G. belizeanus we ound a la ge chemical di e si y
and we decided o ocus on wo majo me aboli es wi h wo p ominen ions a m/z 1025.6 and 1039.5
in elec osp ay ioniza ion (ESI)(+)-MS. The me aboli e wi h he i s mass has ecen ly been iden i ied
as a no el ladde -shaped polye he compound named gambie one [19]. The o he ion was elu ed in
he mai o oxin egion in a ypical e e se phase ch oma og aphy. We epo he ein he s uc u e o
he me aboli e associa ed wi h he mass a m/z 1039.5. All NMR da a a e consis en wi h 44-
me hylgambie one (1) (Figu e 1). This s uc u e he e o e ep esen s he candida e s uc u e o
MTX3. We also epo he compa a i e biological ac i i ies o his new me aboli e and gambie one
bo h isola ed om G. belizeanus.
Figu e 1. S uc u e o he me aboli es isola ed om Gambie discus belizeanus.
2. Resul s
Gambie discus belizeanus was g own in 20 L bags and scaled up o 2000 L. The cells we e ha es ed
by il a ion and ex ac ed wi h MeOH unde ul asonica ion. The me hanolic ex ac was hen
subjec ed o successi e sepa a ion s eps and ul ima ely pu i ied by HPLC using a Re e se Phase (RP)
phenylhexyl column yielding pu e compound 1 (6 mg) and gambie one (2, 15 mg).
2.1. S uc u e Elucida ion o Compound 1
Compound 1 was isola ed as a whi e solid wi h he molecula o mula C52H78O19S as deduced
om High Resolu ion Mass Spec a (HRMS) da a wi h a majo ion peak a m/z 1039.4957 [M+H]+. A
i s inspec ion o he 1H NMR spec um o 1 in CD3OD con i med i s polyke ide na u e wi h he
p esence o oxyme hine and me hyl signals (Figu e S1). The 13C and HSQC NMR spec a o 1
con i med 52 ca bon signals in he o m o i e me hyls ( ou single s and one double ), 17 me hylenes
(including 14 non-oxygena ed, one oxygena ed, and wo unsa u a ed), 23 me hines (one non-
oxygena ed, 18 oxygena ed and ou unsa u a ed), one ke al a δC 100.7 (C-4), one ke one a δC 211.6
(C-40), h ee non-p o ona ed and oxygena ed ca bons and one unsa u a ed qua e na y ca bon a δC
136.0 (C-44) (Table 1, Figu es S2,S3 and S6). The p esence o a sul a e was demons a ed by he
p esence o an in ense agmen a m/z 941.5299 co esponding o [M+H-H2SO4]+ The molecula
o mula p oposed o 1 co esponded o a s uc u al homologue o gambie one (2) and indeed he
agmen a ion pa e n was e y simila be ween bo h compounds sugges ing he p esence o an
addi ional me hylene in he s uc u e o 2 compa ed wi h he one o 1. While he signals o he p o ons
o he ladde -shaped wes e n pa o 2 we e clea ly main ained in 1, some di e ences we e obse ed
in he eas e n pa o he molecule. Indeed, he lack o one ole inic p o on and he p esence o a new
me hyl a δH 1.74 (s, 3H, H-52) and he unsa u a ed qua e na y ca bon we e shown in he HSQC NMR
spec um (Figu e S6). The posi ion o he new me hyl on he ole inic ca bon C-44 was es ablished
h ough he key H-52/C-42, C-43 and C-44 HMBC co ela ions (Figu e S7). The ela i e con igu a ion
o he C-43/C-44 double bond was deduced as E om a key H2-42/H3-52 Oe.
Figu e 1. S uc u e o he me aboli es isola ed om Gambie discus belizeanus.
2. Resul s
Gambie discus belizeanus was g own in 20 L bags and scaled up o 2000 L. The cells we e ha es ed
by il a ion and ex ac ed wi h MeOH unde ul asonica ion. The me hanolic ex ac was hen
subjec ed o successi e sepa a ion s eps and ul ima ely pu i ied by HPLC using a Re e se Phase (RP)
phenylhexyl column yielding pu e compound 1(6 mg) and gambie one (2, 15 mg).
2.1. S uc u e Elucida ion o Compound 1
Compound
1
was isola ed as a whi e solid wi h he molecula o mula C
52
H
78
O
19
S as deduced
om High Resolu ion Mass Spec a (HRMS) da a wi h a majo ion peak a m/z1039.4957 [M+H]
+
.
A i s inspec ion o he
1
H NMR spec um o
1
in CD
3
OD con i med i s polyke ide na u e wi h he
p esence o oxyme hine and me hyl signals (Figu e S1). The
13
C and HSQC NMR spec a o
1
con i med
52 ca bon signals in he o m o i e me hyls ( ou single s and one double ), 17 me hylenes (including
14 non-oxygena ed, one oxygena ed, and wo unsa u a ed), 23 me hines (one non-oxygena ed,
18 oxygena ed and ou unsa u a ed), one ke al a
δC
100.7 (C-4), one ke one a
δC
211.6 (C-40),
h ee non-p o ona ed and oxygena ed ca bons and one unsa u a ed qua e na y ca bon a
δC
136.0
(C-44) (Table 1, Figu es S2, S3 and S6). The p esence o a sul a e was demons a ed by he p esence o an
in ense agmen a m/z941.5299 co esponding o [M+H-H
2
SO
4
]
+
The molecula o mula p oposed
o
1
co esponded o a s uc u al homologue o gambie one (
2
) and indeed he agmen a ion pa e n
was e y simila be ween bo h compounds sugges ing he p esence o an addi ional me hylene in
he s uc u e o
2
compa ed wi h he one o
1
. While he signals o he p o ons o he ladde -shaped
wes e n pa o
2
we e clea ly main ained in
1
, some di e ences we e obse ed in he eas e n pa o
he molecule. Indeed, he lack o one ole inic p o on and he p esence o a new me hyl a
δH
1.74 (s, 3H,
H-52) and he unsa u a ed qua e na y ca bon we e shown in he HSQC NMR spec um (Figu e S6).
The posi ion o he new me hyl on he ole inic ca bon C-44 was es ablished h ough he key H-52/C-42,
Toxins 2019,11, 79 4 o 19
C-43 and C-44 HMBC co ela ions (Figu e S7). The ela i e con igu a ion o he C-43/C-44 double
bond was deduced as E om a key H2-42/H3-52 Oe.
Table 1. 1H (750 MHz) and 13C (125 MHz) NMR Da a o 1in CD3OD.
A om Numbe δH, Mul . (Jin Hz) δCn◦δH, Mul . (Jin Hz) δC
13.47, dd (11.0, 4.5)
3.42, m 67.8 27 3.51, m 77.7
2 4.10, m 69.8 28 2.19, m
1.33, m 39.8
32.00, m
1.71, dd (14.5, 10.0) 39.7 29 3.12, m 70.1
4 - 100.7 30 2.94, m 78.1
5 4.21, d (3.0) 73.0 31 1.89, m
1.54, (11.5) 34.9
6 4.70, dd (10.0, 3.0) 77.8 32 3.76, dd (8.5, 3.5) 72.5
7 3.37, m 77.3 33 - 77.3
8 3.77, m 67.9 34 2.34, d (12.5)
2.15, d (12.5) 54.1
92.17, m
1.58, (11.5) 37.9 35 - 143.5
10 3.35, m 79.6 36 2.54, d (14.5)
2.21, d (14.5) 43.3
11 3.79, m 82.4 37 - 79.9
12 5.64, d (12.5, 2.0) 132.8 38 4.06, d (10.5, 3.0) 73.1
13 5.75, d (12.5, 2.0) 133.0 39 2.61, m
2.59, m 45.8
14 3.81, m 83.0 40 - 211.6
15 3.44, dd (5.0, 2.0) 79.9 41 2.59, m 43.9
16 1.99, m
1.49, (12.0) 47.4 42 2.39, q (7.0) 23.3
17 - 76.6 43 5.45, (7.5) 132.3
18 3.01, d (11.0, 2.5) 86.8 44 - 136.0
19 1.79, m
1.62, m 25.4 45 6.33, d (17.5, 10.5) 142.6
20 1.95, m
1.80, m 34.2 46 5.09, d (17.5)
4.90, d (11.0) 111.2
21 3.53, m 87.4 47 1.20, s 16.4
22 3.54, m 75.7 48 1.00, d (7.5) 13.5
23 1.82, m
1.64, m 32.8 49 1.19, s 16.9
24 1.92, m
1.78, m 29.5 50 4.98, b s
4.85 a118.6
25 2.19, m 35.6 51 1.13, s 20.7
26 3.11, m 85.9 52 1.74, s 11.7
aO e lapped wi h HOD. d: double , m: mul iple , : iple , s: single , b : b oad.
Toxins 2019,11, 79 5 o 19
In e es ingly, compound
1
sha ed he same majo ion peak a 1039.5 in ESI(+)-LRMS wi h he one
cha ac e izing he pu a i e MTX3, since 1994 and ecen ly ound in o he s ains o Gambie discus [
31
].
The e o e, we wonde ed whe he 44-me hylgambie one (
1
) could be he ac ual s uc u e o he
long-sough p-MTX3. Fi s , MTX3 was always assumed o be disul a ed om ESI(+)-LRMS da a
when
1
has been p o en unambiguously o con ain only one sul a e g oup. The molecula o mula was
ne e ob ained o MTX3 as only low esolu ion mass spec a we e gi en wi h associa ed agmen s
and he e o e he p esence o wo sul a es was only a ague assump ion [
29
]. The agmen a ion
pa e n ob ained in ou ESI(+)-LRMS analysis o
1
is in pe ec ag eemen wi h he one ob ained a
120V o MTX3 in he i s epo o his molecule and we could ac ually co ec he p oposed ions o
MTX3 (Figu e 2, Table 2) [
29
]. We a e he e o e e y con iden o p opose he s uc u e o p-MTX3 as
(43E)-44-me hylgambie one (
1
). The absolu e con igu a ion o
1
is supposed o be he same as he one
desc ibed o gambie one (2) based on ECD calcula ions due o s uc u e simila i y [19].
Toxins 2019, 11, x FOR PEER REVIEW 5 o 20
120V o MTX3 in he i s epo o his molecule and we could ac ually co ec he p oposed ions
o MTX3 (Figu e 2, Table 2) [29]. We a e he e o e e y con iden o p opose he s uc u e o p-MTX3
as (43E)-44-me hylgambie one (1). The absolu e con igu a ion o 1 is supposed o be he same as he
one desc ibed o gambie one (2) based on ECD calcula ions due o s uc u e simila i y [19].
Figu e 2. (A). ESI(+)-LRMS and (B). ESI(+)-HRMS spec a o compound 1.
Table 2. Compa ison be ween ions and agmen s in posi i e mode o 1 and MTX3 in he li e a u e.
nd: no de ec ed.
Peaks Ion Species and F agmen s Repo ed in ESI(+)-LRMS o :
Compound 1 MTX3 by Lewis e al. [29]
1099.5 nd [M-H+2Na+K]
+
1083.5 [M-H+2Na]
+
[M-2H+3Na]
+
1077.5 [M+K]
+
[M-H+Na+K]
+
1061.5 [M+Na]
+
[M-H+2Na]
+
1056.5 [M+NH
4
]
+
nd
1055.5 nd [M+K]
+
1039.5 [M+H]
+
[M+Na]
+
1021.5 [M+H-H
2
O]
+
[M+Na-H
2
O]
+
1003.5 [M+H-2H
2
O]
+
[M+Na-2H
2
O]
+
996.5 nd [M-H+Na+K-SO
3
]
+
Figu e 2. (A) ESI(+)-LRMS and (B) ESI(+)-HRMS spec a o compound 1.
Toxins 2019,11, 79 6 o 19
Table 2.
Compa ison be ween ions and agmen s in posi i e mode o
1
and MTX3 in he li e a u e.
nd: no de ec ed.
Peaks Ion Species and F agmen s Repo ed in ESI(+)-LRMS o :
Compound 1 MTX3 by Lewis e al. [29]
1099.5 nd [M-H+2Na+K]+
1083.5 [M-H+2Na]+[M-2H+3Na]+
1077.5 [M+K]+[M-H+Na+K]+
1061.5 [M+Na]+[M-H+2Na]+
1056.5 [M+NH4]+nd
1055.5 nd [M+K]+
1039.5 [M+H]+[M+Na]+
1021.5 [M+H-H2O]+[M+Na-H2O]+
1003.5 [M+H-2H2O]+[M+Na-2H2O]+
996.5 nd [M-H+Na+K-SO3]+
981.5 [M+Na-SO3]+[M-H+2Na-SO3]+
963.5 nd [M-H+2Na-H2O-SO3]+
959.5 [M+H-SO3]+[M+Na-SO3]+
941.5 [M+H-H2SO4]+[M+Na-H2O-SO3]+
923.5 [M+H-H2O-H2SO4]+[M+Na-2H2O-SO3]+
905.5 [M+H-3H2O-SO3]+[M+Na-3H2O-SO3]+
887.5 [M+H-4H2O-SO3]+[M+Na-4H2O-SO3]+
nd: no de e mined.
Based on MS da a and hei agmen a ion pa e ns, we also p opose a possible moni o ing
analysis o bo h he p esence o gambie one (
2
) and MTX3 (
1
) using he ollowing MRM
ansi ions (Table 3).
Table 3. Op imized MS/MS spec ome ic pa ame e s o he MRM ansi ions.
Compound Mai o oxin-3 Gambie one
ESI
Mode
Pa en
Ion Cone/V F agmen
Ion Coll/e Pa en
Ion Cone/V F agmen
Ion Coll/e
+1039.5 30
233.1 30
1025.5 30
219.1 30
277.1 60 277.1 60
803.4 30 803.4 30
941.5 30 927.5 30
1021.5 4 1007.5 4
−
899.6 70 96.8 60 899.6 70 96.8 60
1037.5 70 96.8 60 1023.5 70 96.8 60
899.6 60 899.6 60
2.2. Biological Ac i i y o MTX3 and Gambie one
The biological ac i i y o gambie one (
2
) was p e iously epo ed in undi e en ia ed human
neu oblas oma cells and in cell lines exp essing ol age dependen sodium channels iso o ms [
19
].
In hese biological sys ems
2
exhibi ed a mode a e ac i i y simila o he one obse ed o he syn he ic
cigua oxin CTX3C al hough wi h lowe po ency. In his wo k, he biological ac i i ies o syn he ic
cigua oxin CTX3C, MTX1, gambie one (
2
) and MTX3 (
1
) we e compa ed in human neu onal co ical
neu ons p e iously used o e alua e he e ec s o o he ma ine oxins in neu onal unc ion [
32
,
33
]
and in he human neu oblas oma cells used ea lie [
19
]. As indica ed in Figu e 3,
in i o
exposu e
o human co ical neu ons o 5 days o
1
,
2
o CTX3C a concen a ions anging om 0.01 nM o
20 nM did no a ec cellula iabili y in cul u es o human co ical neu ons. This esul ag ees wi h
p e ious obse a ions in p ima y cul u es o mice co ical neu ons showing no cy o oxic e ec o
Toxins 2019,11, 79 7 o 19
he syn he ic cigua oxin CTX3C [
22
]. Fu he mo e, i suppo s he p e iously epo ed simila i ies
be ween he biological ac i i ies o CTX3C and
2 [19,22]
, and addi ionally i e eals o he i s ime a
simila ac i i y o MTX3 and gambie one. I is wo h highligh ing he lack o cy o oxici y o hese
h ee compounds in human co ical neu ons while MTX1 induced comple e cell dea h a e 2 h, 24 h o
5 days ea men wi h IC50 alues below 1 nM [32].
Toxins 2019, 11, x FOR PEER REVIEW 7 o 20
Figu e 3. Compa ison o he e ec s o CTX3C, gambie one and MTX3 on cell iabili y in human
co ical neu ons. None o he oxins a ec ed CTX0E16 cell iabili y a low concen a ions. In his case,
he maximum oxin concen a ion e alua ed was 20 nM, and oxici y was e alua ed a e exposu e o
he cells o he di e en compound concen a ions o 5 days in i o. Cell iabili y was e alua ed by
he MTT assay. Resul s a e exp essed as mean ± sem o 4 independen expe imen s, each pe o med
in iplica e.
To comple e he compa ison o he biological ac i i y o gambie one and MTX3 in he same
cellula sys em, he e ec o hese compounds on he cy osolic calcium concen a ion ([Ca2+]c) was
e alua ed. Calcium homeos asis plays a main pa hophysiological ole in synap ic signaling and
neu odegene a ion [34] and a known e ec o MTX1 in mice and human co ical neu ons is o induce
a massi e calcium in lux ha leads o apid neu onal dea h [22,33]. The e o e, he e ec o
gambie one and MTX3 on [Ca2+]c was e alua ed. As indica ed in Figu e 4, exposu e o di e en ia ed
human co ical neu ons o ei he gambie one o MTX3 a 20 nM caused a small ise in he cy osolic
calcium concen a ion. In con as wi h he as and sus ained [Ca2+]c ise elici ed by 5 nM MTX1
(Figu e 4A), he e ec o ei he gambie one (Figu e 4B) and MTX3 (Figu e 4C) was conside ably
sho e and smalle , hus con i ming ha he biological ac i i y o MTX3 was o lowe po ency han
ha o MTX1 as p e iously demons a ed in i o [10] al hough u he in es iga ions should be
pu sued o iden i y he ole o his molecule on he neu ological symp oms o human CFP [10].
Figu e 3.
Compa ison o he e ec s o CTX3C, gambie one and MTX3 on cell iabili y in human co ical
neu ons. None o he oxins a ec ed CTX0E16 cell iabili y a low concen a ions. In his case, he
maximum oxin concen a ion e alua ed was 20 nM, and oxici y was e alua ed a e exposu e o
he cells o he di e en compound concen a ions o 5 days
in i o
. Cell iabili y was e alua ed by
he MTT assay. Resul s a e exp essed as mean
±
sem o 4 independen expe imen s, each pe o med
in iplica e.
To comple e he compa ison o he biological ac i i y o gambie one and MTX3 in he same
cellula sys em, he e ec o hese compounds on he cy osolic calcium concen a ion ([Ca
2+
]
c
) was
e alua ed. Calcium homeos asis plays a main pa hophysiological ole in synap ic signaling and
neu odegene a ion [
34
] and a known e ec o MTX1 in mice and human co ical neu ons is o induce a
massi e calcium in lux ha leads o apid neu onal dea h [
22
,
33
]. The e o e, he e ec o gambie one
and MTX3 on [Ca
2+
]
c
was e alua ed. As indica ed in Figu e 4, exposu e o di e en ia ed human
co ical neu ons o ei he gambie one o MTX3 a 20 nM caused a small ise in he cy osolic calcium
concen a ion. In con as wi h he as and sus ained [Ca
2+
]
c
ise elici ed by 5 nM MTX1 (Figu e 4A),
he e ec o ei he gambie one (Figu e 4B) and MTX3 (Figu e 4C) was conside ably sho e and smalle
, hus con i ming ha he biological ac i i y o MTX3 was o lowe po ency han ha o MTX1 as
p e iously demons a ed
in i o
[
10
] al hough u he in es iga ions should be pu sued o iden i y
he ole o his molecule on he neu ological symp oms o human CFP [10].
The mos abundan exci a o y neu o ansmi e ecep o s in he b ain belong o he iono opic
class o glu ama e ecep o s, which media e as synap ic ansmission and eadily adap o coun e ac
changes in neu onal ac i i y [
35
,
36
]. Since ac i a ion o ol age-ga ed sodium channels by CTX3C
and he consequen change in neu onal ac i i y has been shown o egula e inhibi ion and exci a ion
in opposi e di ec ions in mice co ical neu ons [
25
], nex , we e alua ed he e ec o gambie one and
MTX3 on iono opic glu ama e ecep o s exp ession. In his sense, we ha e p e iously epo ed ha
ch onic exposu e o p ima y co ical neu ons o CTX3C elici ed long e m al e a ions in synap ic
neu o ansmission leading o he down- egula ion in he p o ein le el o iono opic glu ama e
ecep o s [
37
]. The e o e, in o de o compa e he long e m e ec s o gambie one and MTX3 o hose
o CTX3C, di e en ia ed CTX0E16 neu ons we e main ained in he p esence o 20 nM gambie one o
20 nM MTX3 o 15 days in cul u e wi h ull oxin enewal each wo days, and he exp ession o bo h
α
-amino-3-hyd oxy-5-me hyl-4-isoxazolep opionic acid (AMPA) and N-me hyl-D-aspa a e (NMDA)
Toxins 2019,11, 79 8 o 19
ecep o subuni s was e alua ed. As shown in Figu e 5A, exposu e o human neu ons o gambie one
o MTX3 dec eased he le el o he AMPA ecep o subuni s by 21.31
±
7.47% (n= 12, p= 0.0093) and
23.06
±
8.17% (n= 12, p= 0.009), espec i ely. Howe e , bo h compounds inc eased he exp ession o
he NMDA ecep o s subuni s by 83.45
±
24.51% (n= 9; p= 0.0036) and 87.11
±
26.26 (n= 7; p= 0.0051)
espec i ely (Figu e 5B). Thus, e en when he ac i i y o gambie one and MTX3 seems simila o he
biological ac i i y o CTX3C in p ima y mice co ical neu ons [
37
] bo h compounds inc eased he
p o ein exp ession o NMDA ecep o s while ch onic CTX3C dec eased he exp ession o bo h NMDA
and AMPA ecep o subuni s [37].
Toxins 2019, 11, x FOR PEER REVIEW 8 o 20
Figu e 4. E ec o MTX1 (A), gambie one (B) and MTX3 (C) on he cy osolic calcium concen a ion in
human di e en ia ed co ical neu ons. A concen a ion o 5 nM MTX1 caused a apid and sus ained
inc ease in [Ca2+]c while bo h gambie one and MTX3, a 20 nM, elici ed a sho e and smalle e ec on
[Ca2+]c. Da a ep esen means ± sem o 4 independen expe imen s in he case o gambie one and 3
independen expe imen s o MTX1 and MTX3. Ba h applica ion o he oxins is indica ed by he
a ow.
The mos abundan exci a o y neu o ansmi e ecep o s in he b ain belong o he iono opic
class o glu ama e ecep o s, which media e as synap ic ansmission and eadily adap o
coun e ac changes in neu onal ac i i y [35,36]. Since ac i a ion o ol age-ga ed sodium channels by
CTX3C and he consequen change in neu onal ac i i y has been shown o egula e inhibi ion and
exci a ion in opposi e di ec ions in mice co ical neu ons [25], nex , we e alua ed he e ec o
gambie one and MTX3 on iono opic glu ama e ecep o s exp ession. In his sense, we ha e
p e iously epo ed ha ch onic exposu e o p ima y co ical neu ons o CTX3C elici ed long e m
al e a ions in synap ic neu o ansmission leading o he down- egula ion in he p o ein le el o
iono opic glu ama e ecep o s [37]. The e o e, in o de o compa e he long e m e ec s o
gambie one and MTX3 o hose o CTX3C, di e en ia ed CTX0E16 neu ons we e main ained in he
Figu e 4.
E ec o MTX1 (
A
), gambie one (
B
) and MTX3 (
C
) on he cy osolic calcium concen a ion in
human di e en ia ed co ical neu ons. A concen a ion o 5 nM MTX1 caused a apid and sus ained
inc ease in [Ca
2+
]
c
while bo h gambie one and MTX3, a 20 nM, elici ed a sho e and smalle e ec on
[Ca
2+
]
c
. Da a ep esen means
±
sem o 4 independen expe imen s in he case o gambie one and 3
independen expe imen s o MTX1 and MTX3. Ba h applica ion o he oxins is indica ed by he a ow.
Toxins 2019,11, 79 9 o 19
Toxins 2019, 11, x FOR PEER REVIEW 9 o 20
p esence o 20 nM gambie one o 20 nM MTX3 o 15 days in cul u e wi h ull oxin enewal each
wo days, and he exp ession o bo h α-amino-3-hyd oxy-5-me hyl-4-isoxazolep opionic acid
(AMPA) and N-me hyl-D-aspa a e (NMDA) ecep o subuni s was e alua ed. As shown in Figu e
5A, exposu e o human neu ons o gambie one o MTX3 dec eased he le el o he AMPA ecep o
subuni s by 21.31 ± 7.47% (n = 12, p = 0.0093) and 23.06 ± 8.17% (n = 12, p = 0.009), espec i ely.
Howe e , bo h compounds inc eased he exp ession o he NMDA ecep o s subuni s by 83.45 ±
24.51% (n = 9; p = 0.0036) and 87.11 ± 26.26 (n = 7; p = 0.0051) espec i ely (Figu e 5B). Thus, e en when
he ac i i y o gambie one and MTX3 seems simila o he biological ac i i y o CTX3C in p ima y
mice co ical neu ons [37] bo h compounds inc eased he p o ein exp ession o NMDA ecep o s
while ch onic CTX3C dec eased he exp ession o bo h NMDA and AMPA ecep o subuni s [37].
Figu e 5. Long e m exposu e (15 days in cul u e) o human di e en ia ed co ical neu ons o ei he
gambie one o MTX3, a 20 nM, a ec ed he p o ein le el o glu ama e ecep o subuni s. (A) Bo h
compounds dec eased he p o ein le el o he AMPA ecep o subuni s. Wes e n blo bands showing
he exp ession o GluR2,3,4 subuni p o eins a e shown on he le panel and he co esponding
quan i ica ions o he bands a e shown on he igh . (B) The same ea men s we e pe o med o
e alua e he e ec o he compounds on he exp ession o NMDA ecep o subuni s NR2A/B.
Rep esen a i e wes e n blo bands showing NR2A/B le els in di e en ia ed CTX0E16 neu ons a e
shown on he le panel and band in ensi ies quan i ica ions a e shown on he igh . Fi een days
exposu e o human co ical neu ons o 20 nM gambie one o 20 nM MTX3 inc eased NR2A/B le els,
and e ec opposi e o ha p e iously desc ibed o CTX3C in mice co ical neu ons. The numbe o
independen de e mina ions is shown in pa en hesis. ** p < 0.01 e sus con ol.
In iew o he sub le di e ences be ween he ch onic neu onal al e a ions p oduced by CTX3C
in mice co ical neu ons [37] and hose p oduced by gambie one and MTX3 epo ed he e he e ec s
o bo h gambie one and MTX3 on cell iabili y we e addi ionally e alua ed a highe concen a ions
in human neu oblas oma cells since his cellula model was ini ially employed o assed he biological
e ec s o gambie one [19]. Cell iabili y was e alua ed using he MTT assay which e alua es he
abili y o he nico inamide-adenine-dinucleo ide (NAD(P)H) coenzyme and dehyd ogenases om
me abolically ac i e cells o educe e azolium sal s and o m s ongly colo ed and lipophilic
o mazan p oduc s which a e hen quan i ied by abso bance o luo escence [38]. The amoun o
colo ed o mazan p oduc is in linea co ela ion wi h a heal hy mi ochond ial unc ion and wi h he
Figu e 5.
Long e m exposu e (15 days in cul u e) o human di e en ia ed co ical neu ons o ei he
gambie one o MTX3, a 20 nM, a ec ed he p o ein le el o glu ama e ecep o subuni s. (
A
) Bo h
compounds dec eased he p o ein le el o he AMPA ecep o subuni s. Wes e n blo bands showing
he exp ession o GluR2,3,4 subuni p o eins a e shown on he le panel and he co esponding
quan i ica ions o he bands a e shown on he igh . (
B
) The same ea men s we e pe o med o e alua e
he e ec o he compounds on he exp ession o NMDA ecep o subuni s NR2A/B. Rep esen a i e
wes e n blo bands showing NR2A/B le els in di e en ia ed CTX0E16 neu ons a e shown on he
le panel and band in ensi ies quan i ica ions a e shown on he igh . Fi een days exposu e o
human co ical neu ons o 20 nM gambie one o 20 nM MTX3 inc eased NR2A/B le els, and e ec
opposi e o ha p e iously desc ibed o CTX3C in mice co ical neu ons. The numbe o independen
de e mina ions is shown in pa en hesis. ** p< 0.01 e sus con ol.
In iew o he sub le di e ences be ween he ch onic neu onal al e a ions p oduced by CTX3C in
mice co ical neu ons [
37
] and hose p oduced by gambie one and MTX3 epo ed he e he e ec s o
bo h gambie one and MTX3 on cell iabili y we e addi ionally e alua ed a highe concen a ions in
human neu oblas oma cells since his cellula model was ini ially employed o assed he biological
e ec s o gambie one [
19
]. Cell iabili y was e alua ed using he MTT assay which e alua es he
abili y o he nico inamide-adenine-dinucleo ide (NAD(P)H) coenzyme and dehyd ogenases om
me abolically ac i e cells o educe e azolium sal s and o m s ongly colo ed and lipophilic o mazan
p oduc s which a e hen quan i ied by abso bance o luo escence [
38
]. The amoun o colo ed o mazan
p oduc is in linea co ela ion wi h a heal hy mi ochond ial unc ion and wi h he cell iabili y [
39
].
As indica ed in Figu e 6, exposu e o undi e en ia ed human neu oblas oma cells o 4 days o ei he
gambie one o MTX3 a concen a ions anging om 10 o 1000 nM led o di e en e ec s on cell
iabili y (Figu e 6A). As expec ed om ou p e ious wo k [
19
] gambie one did no cause cell dea h
e en a concen a ions o 1
µ
M while MTX3 a he highes concen a ion e alua ed dec eased cell
iabili y by mo e han 70% wi h an IC
50
o 7.02
×
10
−7
M (95% con idence in e als om 6.09
×
10
−7
o
8.1
×
10
−7
M). In con as , 24 hou s exposu e o human neu oblas oma cells o MTX1 caused comple e
cell dea h a concen a ions as low as 0.01 nM (Figu e 6B) while nei he CTX3C, no gambie one o
MTX3 a ec ed cell iabili y a e 24 hou s (Figu e 6C). Thus, hese esul s p o ide addi ional da a
indica ing ha MTX3 exhibi s cigua oxin-like ac i i y a he han a mai o oxin-like oxici y.
Toxins 2019,11, 79 16 o 19
4.6.6. Wes e n Blo
The p o ein le el o glu ama e ecep o s was e alua ed in cul u es o ma u e human co ical
neu ons main ained o 15 days in he p esence o gambie one o MTX3, bo h a 20 nM, wi h hal
medium changes and oxin eplacemen each wo days. A e ea men he cells we e washed h ee
imes wi h cold PBS and cell lysa es we e ob ained using RIPA lysis bu e (The mo ishe ), con aining
25 mM T is-HCl (pH 7.6), 150 mM NaCl, 1% NP-40 (nonyl phenoxypolie hoxyle hanol), 1% sodium
deoxychola e and 0.1% SDS supplemen ed wi h comme cial phospha ase and p o ease inhibi o s
cock ails (The mo ishe ), and s o ed a
−
20
◦
C when needed. To al p o ein concen a ion in cell lysa es
was measu ed in iplica e by he B ad o d assay, using bo ine se um albumin (BSA) as s anda d.
Fo SDS-PAGE elec opho esis cell lysa es samples con aining 30
µ
g o o al p o ein we e dena u alized
in comme cial 4
×
Laemmli sample bu e (Bio-Rad, con aining 277.8 mM T is-HCl, pH 6.8, 4.4% SDS,
44.4% glyce ol, and 0.02% b omophenol blue, supplemen ed wi h 2.5% 2-me cap oe hanol (Bio-Rad
Labo a o ies S.A., Ba celona, Spain)) and esol ed in 10% polyac ylamide gels a a cons an ol age o
200 V o 38 minu es. A e wa ds, p o eins we e ans e ed o PVDF memb anes (10 V o 30 min)
using a semi-d y ans e cell (Bio-Rad). The memb anes we e blocked o 1 hou wi h 5% BSA and
incuba ed o e nigh a 4
◦
C wi h p ima y polyclonal an ibodies agains AMPA ecep o subuni s
(GluR2,3,4 a a dilu ion o 1:500,) o NMDA ecep o subuni s 2A/B (1:500) bo h om The mo ishe .
P o ein bands we e de ec ed using he Supe signal Wes Pico chemiluminescen subs a e (Pie ce,
The mo Fishe Scien i ic, Mad id, Spain) and he Di e si y 4 gel documen a ion and analysis sys em
(Syngene, Camb idge, UK). Chemiluminescence was quan i ied wi h he Di e si y GeneSnap so wa e
(Syngene).
β
-ac in (Millipo e, Me ck Chemicals & Li e Science S.A., Mad id, Spain) was used as
con ol o lane loading and o no malize chemiluminescence alues. Each condi ion was analyzed in
duplica e wells o each ea men .
4.6.7. S a is ical Analysis
All da a a e exp essed as means
±
SEM o n de e mina ions. S a is ical compa ison was by
S uden ’s es . p alues < 0.05 we e conside ed s a is ically signi ican .
Supplemen a y Ma e ials:
The ollowing da a a e a ailable online a h p://www.mdpi.com/2072-6651/11/2/
79/s1, Figu e S1:
1
H NMR spec um o
1
a 750 MHz in CD
3
OD. Figu e S2:
13
C NMR spec um o 1 a 125 MHz in
CD
3
OD. Figu e S3: DEPT135 NMR spec um o 1 a 125 MHz in CD
3
OD. Figu e S4: COSY NMR spec um o 1 a
750 MHz in CD
3
OD. Figu e S5: zTOCSY NMR spec um o
1
a 750 MHz in CD
3
OD. Figu e S6: CRISIS-HSQC
NMR spec um o
1
a 750 MHz in CD
3
OD. Figu e S7: HMBC spec um o 1 a 750 MHz in CD
3
OD. Figu e S8:
ROESY NMR spec um o 1 a 750 MHz in CD3OD.
Au ho Con ibu ions:
Chemical analyses: M.Á., Á.A., K.C., I.R., O.P.T. Biological e alua ion: A.B.-J., C.V., L.M.B.
All he au ho s pa icipa ed in he design o expe imen s and he w i ing and p oo eading o he manusc ip .
Funding:
And ea Boen e-Juncal is ecipien o a ellowship om Minis e io de Educación, Cul u a y Depo e,
Spain. The esea ch leading o hese esul s has ecei ed unding om he ollowing FEDER co unded-g an s.
F om Conselle ia de Cul u a, Educacion e O denación Uni e si a ia, Xun a de Galicia, 2017 GRC GI-1682 (ED431C
2017/01). F om CDTI and Technological Funds, suppo ed by Minis e io de Economía, Indus ia y Compe i i idad,
AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2, ITC-20161072.
F om Eu opean Union POCTEP 0161-Nanoea e s -1-E-1, In e eg Ale oxNe EAPA-317-2016, In e eg Ag i ox
EAPA-998-2018, and H2020 778069-EMERTOX.
Con lic s o In e es :
The au ho s decla e no con lic o in e es . The unde s had no ole in he design o he
s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip , o in he decision o
publish he esul s.
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