545

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(2): 545-556, April-June 2021 (Published Apr. 19, 2021)

Action of heparin and acetylcholine modulators on the neurotoxicity

of the toad Rhinella schneideri (Anura: Bufonidae) in Brazil

Sandro Rostelato-Ferreira

1,2

; Orcid: 0000-0002-8987-434X

Orlando B. Vettorazzo

; Orcid: 0000-0003-4731-4771

Natália Tribuiani

; Orcid: 0000-0002-7661-475X

Allan P. Leal

; Orcid: 0000-0002-4689-4615

Cháriston A. Dal Belo

3,4

; Orcid: 0000-0001-7010-650

Léa Rodrigues-Simioni

; Orcid: 0000-0002-8712-6639

Rafael S. Floriano

; Orcid: 0000-0003-0759-5863

Yoko Oshima-Franco

*; Orcid: 0000-0002-4972-8444

1. Institute of Health Sciences, Paulista University (UNIP), Sorocaba, São Paulo, Brazil; sandrorostelato@yahoo.com.br

2. Graduate Program in Pharmaceutical Sciences, University of Sorocaba (UNISO), Sorocaba, São Paulo, Brazil;

orlandovettorazzo@hotmail.com, natitribuiani@hotmail.com, yofranco@terra.com.br (*Correspondence)

3. Graduate Program in Toxicological Biochemistry, Federal University of Santa Maria (UFSM), Santa Maria, Rio

Grande do Sul, Brazil; allan-leal@hotmail.com

4. Laboratory of Neurobiology and Toxinology (Lanetox), Federal University of Pampa (UNIPAMPA), São Gabriel, Rio

Grande do Sul, Brazil; charistonbelo@unipampa.edu.br

5. Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas

13083-970, São Paulo, Brazil; simioni@unicamp.br

6. Graduate Program in Health Sciences, University of Western São Paulo (UNOESTE), Presidente Prudente, São Paulo,

Brazil; rafael@unoeste.br

Received 17-XI-2020. Corrected 11-III-2021. Accepted 23-III-2021.

ABSTRACT

Introduction: Rhinella schneideri is a toad widely distributed in South America and its poison is characterized

by inducing cardiotoxicity and neurotoxicity. Objective: In this work, we investigated pharmacological strate-

gies to attenuate the peripheral neurotoxicity induced by R. schneideri poison in avian neuromuscular prepara-

tion. Methods: The experiments were carried out using isolated chick biventer cervicis preparation subjected to

field stimulation for muscle twitches recordings or exposed to acetylcholine and potassium chloride for contrac-

ture responses. Results: Poison (10 μg/ml) produced complete neuromuscular blockade in chick biventer cervi-

cis preparation within approximately 70 min incubation (times for 50 and 90 % blockade: 15 ± 3 min and 40 ±

2 min, respectively; P < 0.05, N= 5); contracture responses to exogenous acetylcholine and KCl were unaffected

by poison indicating no specificity with postsynaptic receptors or myotoxicity, respectively. Poison (10 μg/ml)-

induced neuromuscular blockade was not prevented by heparin (5 and 150 IU/ml) under pre- or post-treatment

conditions. Incubation at low temperature (23-25 °C) abolished the neuromuscular blockade; after raising the

temperature to 37 °C, the complete neuromuscular blockade was slightly slower than that seen in preparations

directly incubated at 37 °C (times for 50 and 90 % blockade: 23 ± 2 min and 60 ± 2.5 min, respectively; P <

0.05, N= 4). Neostigmine (3.3 μM) did not reverse the neuromuscular blockade in BC preparation whereas

3,4-diaminopyridine (91.6 μM) produced a partial and sustained reversal of the twitch responses (29 ± 7.8 %

of maximal reversal reached in approximately 40 min incubation; P < 0.05, N= 4). Conclusions: R. schneideri

poison induces potent peripheral neurotoxicity in vitro which can be partially reversible by 3,4-diaminopyridine.

Key words: amphibian poison; paratoid gland secretion; chick biventer cervicis preparation; neuromuscular

blockade; reversal.

Rostelato-Ferreira, S., Vettorazzo, O.B., Tribuiani, N., Leal, A.P.,

Dal Belo, C.A., Rodrigues-Simioni, L., Floriano, R.S., Oshima-

Franco, Y. (2021). Action of heparin and acetylcholine modulators

on the neurotoxicity of the toad Rhinella schneideri (Anura:

Bufonidae) in Brazil. Revista de Biología Tropical, 69(2), 545-

556. DOI 10.15517/rbt.v69i2.44539

DOI 10.15517/rbt.v69i2.44539

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Rhinella genus (Bufonidae) comprises 94

species distributed in Central (Panama) and

South America (Paraguay, Bolivia, Argenti-

na, Uruguay and Brazil), with approximately

30 of them found in Brazil (Pereyra et al.,

2016; Wake, 2020). The secretion produced

by amphibian paratoid glands is composed by

numerous active components such as biogenic

amines, peptides, steroids (bufadienolides and

bufotoxins), alkaloids (batrachotoxin ‘tetrodo-

toxin’), and proteins (Gregerman, 1952; Zel-

nik, 1965; Mahan & Biggers, 1977; O’Rourke,

Chen, Hirst, Rao, & Shaw, 2004), compris-

ing the main protection mechanism against

potential predators (Clarke, 1997; Kowalski,

Marciniak, Rosiński, & Rychlik, 2018). These

components, especially those bufadienolides

and bufotoxins, have been considered potential

therapeutic tools for exhibiting cancer inhibi-

tory activity (Meng et al., 2009), apoptosis

suppressive action (Qi et al., 2010) and anti-

microbial activity (Tempone et al., 2008). The

paratoid gland secretion and their components,

e.g., non-enzymatic presynaptic active toxin

(730.6 Da) isolated from R. schneideri poison

(Rostelato-Ferreira et al., 2018), can induce

neurotoxicity characterized by suppression of

the motor acetylcholine release (Rostelato-

Ferreira, Dal Belo, Cruz-Höfling, Hyslop, &

Rodrigues-Simioni, 2011; Rostelato-Ferreira,

Dal Belo, Leite, Hyslop, & Rodrigues-Simioni,

2014; Rostelato-Ferreira et al., 2018), includ-

ing cardiotoxicity consisted in arrhythmia and

ventricular failure mostly by antagonizing Na

ATPase of cardiomyocytes (Toledo & Jared,

1995; Sakate & Oliveira, 2000; Gadelha, Lima,

Batista, Melo, & Soto-Blanco, 2014; Leal

et al., 2020). R. marinus and R. vulgaris are

responsible for causing the most cases of

envenomation in domestic animals (Sakate &

Oliveira, 2000; Sakate & Oliveira, 2001).

In recent years, several biological proper-

ties have been characterized from Rhinella

schneideri ‘Schneider’s toad’ poison (Rostela-

to-Ferreira et al., 2011; Rostelato-Ferreira et al.,

2014; Abdelfatah, Lu, Schmeda-Hirschmann,

& Efferth, 2019; Leal et al., 2020), a species

widely distributed in Brazil (Wake, 2020),

including the chemical identification and struc-

tural characterization of bufadienolides, e.g.,

marinobufagenin, bufalin, telocinobufagin, hel-

lebrigenin, 20S21R-epoxymarinobufagin and

β-sitosterol, which exhibit cytotoxic activity,

modulatory action on the complement cascade,

anti-inflammatory activity, inhibitory action

of serine protease, presynaptic neuromuscular

blocking activity and anticonvulsant action

(Cunha-Filho et al., 2010; Anjolette et al.,

2015; Sousa-Filho et al., 2016; Shibao, Anjo-

lette, Lopes, & Arantes, 2015; Freitas et al.,

2017; Rostelato-Ferreira et al., 2018; Baldo et

al., 2019; Zheng et al., 2020).

Heparin has been successfully used to

neutralize the neurotoxic and myotoxic activi-

ties of Bothrops (Viperidae: Crotalinae) ‘pit-

viper’ snake venoms (Melo & Suarez-Kurtz,

1988a; Melo & Suarez-Kurtz, 1988b; Melo &

Ownby, 1999; Calil-Elias, Martinez, & Melo,

2002; Rostelato-Ferreira et al., 2010) and their

major phospholipase A

(PLA

) (Lomonte,

Moreno, Tarkowski, Hanson, & Maccarana,

1994a; Lomonte, Tarkowski, Bagge, & Han-

son, 1994b; Rodrigues et al., 2004a; Perchuc et

al., 2010; Rostelato-Ferreira et al., 2010). The

action of heparin against the effects produced

by amphibian poisons on the motor neurotrans-

mitter release has not been previously investi-

gated, however, the acid-basic characteristics

of steroid derivatives and biogenic amines

found in toad venoms may suggest an even-

tual interaction with heparin (Zelnik, 1965). In

addition, acetylcholine (ACh) release modulat-

ing drugs, e.g., neostigmine, an acetylcholin-

esterase inhibitor, and 3.4-diaminopyridine, a

voltage-gated potassium channel blocker, show

to be effective in counteract the neuromuscular

paralysis caused by animal venoms and iso-

lated toxins by promoting the increase of ace-

tylcholine release in motor nerve terminal (Ng

et al., 2017; Floriano et al., 2019; Neely, Sabir,

& Kohli, 2020). In this work, we have tested

different pharmacological strategies in order to

counteract the neuromuscular action induced

by R. schneideri poison in avian isolated prepa-

ration by applying heparin and acetylcholine

(ACh) release modulating drugs.

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MATERIALS AND METHODS

Reagents and poison: Acetylcholine chlo-

ride, neostigmine methylsulfate, 3.4-diamino-

pyridine and heparin sodium salt were obtained

from Sigma-Aldrich Chemical Co. (St. Louis,

MO, USA). All salts used to prepare the physi-

ological solutions were of analytical grade. The

methanolic extract of R. schneideri poison was

provided by Universidade Federal do Pampa

(UNIPAMPA, São Gabriel, RS, Brazil) through

Dr. Cháriston A. Dal Belo. R. schneideri poi-

son was collected from a single specimen by

manual compression from parotid glands and

an amount of 2 mg was then included in 50 ml

of methanol for three days at room temperature

(Gao et al., 2010). The methanolic extract was

then lyophilized and maintained under refrig-

eration until used. Aliquots of the lyophilized

extract were dissolved in Krebs solution prior

to testing in the neuromuscular preparations.

Animals: This study was approved by an

institutional Committee for Ethics in Animal

Use (CEUA/UNISO, Protocol no. 096/2016)

and the experiments were carried out accord-

ing to the general ethical guidelines for animal

use established by the Brazilian Society of

Laboratory Animal Science (SBCAL). Male

chicks (4-8 days old, HY- line) were provided

by Aviculture Santa Barbara Ltda. (Sorocaba,

SP, Brazil) and housed in metal cages with a

sawdust substrate at 25 °C on a 12 h light/dark

cycle with lights turned on at 6 a.m. The ani-

mals had free access to food and water.

Chick biventer cervicis (BC) prepara-

tion: Chicks were previously euthanized with

isoflurane and the biventer cervicis muscles

were removed and mounted under a resting

tension of 1 g in a 5 ml organ bath (Panlab, Bar-

celona, Spain) containing aerated (95 % O

and

5 % CO

) Krebs solution (composition, in mM:

NaCl 118.7, KCl 4.7, CaCl

1.88, KH

1.17,

MgSO

1.17, NaHCO

25.0 and glucose 11.65,

pH 7.5) at 37 °C, as described elsewhere (Gins-

borg & Warriner, 1960). The preparations were

exposed to a field stimulation (0.1 Hz, 0.2 ms,

4-6 V) with stimuli being delivered from a

LE 12406 TC stimulator (Panlab, Barcelona,

Spain) and the muscle twitches recorded using

an MLT0201 force-displacement transducer

coupled to a Quad Bridge Amp and PowerLab

4/35 data acquisition system connected to a

LabChart Pro v. 6.0 (ADInstruments Pty Ltd.,

2020) software (all from ADInstruments Pty

Ltd., Sydney, Australia). Muscle contractures

to exogenous acetylcholine (ACh, 110 μM) and

potassium chloride (KCl, 20 mM) were evoked

in the absence of electric stimulation, before

and after treatments with poison (10 mg/ml)

and heparin (1-30 ml/ml), to assess the function

of postsynaptic nicotinic receptors and muscle

membrane integrity, respectively (Harvey et

al., 1994); the final muscle contractures to ACh

and KCl were expressed as a percentage of the

resting state, considered as 100 % of response.

After the initial assessments with ACh and

KCl, the preparations were extensively washed

and electrically stimulated for at least 20 min to

allow them a stabilization period before treat-

ments. Muscle twitches were recorded for up

to 120 min or until complete blockade; some

preparations were maintained in Krebs solution

alone for 120 min to obtain muscle response

control. Other preparations were incubated at

22-24 °C to assess the influence of temperature

on poison-induced (10 mg/ml) neuromuscular

blockade. Some pharmacological approaches

using 3.4-diaminopyridine (91.6 mM) and neo-

stigmine (3.3 mM) were applied to understand

the effects of methanolic extract of R. schnei-

deri poison on the avian peripheral neurotrans-

mission. The heparin neutralizing activity on

the R. schneideri poison-induced neuromus-

cular blockade was assessed under two exper-

imental conditions: 1) pre-treatment-poison

(10 mg/ml) was previously pre-incubated with

heparin (5-150 IU/ml) for 30 min at 37 °C and

then tested in the BC preparations; 2) post-

treatment-heparin (1 and 30 ml/ml) was added

into the recording bath after 10 min exposure to

poison (10 mg/ml). The experiments were car-

ried out using four isolated preparations (N= 4)

for every protocol described above.

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Statistical analysis: The results (myo-

graphic recordings) were expressed as the

mean ± SEM of four isolated preparations from

different animals for each experimental proto-

col. Changes in the twitch-tension responses

of BC preparations were considered as a per-

centage relative to baseline (time zero) values.

Statistical comparisons were performed using

Student’s t-test or ANOVA followed by the

Tukey-Kramer test, with P < 0.05 indicating

significance. All data analyses were done using

Origin 8 SR4 v.8.0951 (Microcal Software Inc.,

2020) or Prism 5 (GraphPad Software Inc.,

2020) software.

RESULTS

Effect of heparin on the R. schneideri

poison-induced neuromuscular blockade in

chick BC preparation: R. schneideri poison

(10 μg/ml) caused complete neuromuscular

blockade in indirectly stimulated preparations

at 37 °C approximately 70 min after incubation

(times for 50 and 90 % blockade: 15 ± 3 min

and 4 ± 2 min; P < 0.05 compared to control

preparations, N= 5); poison (10 μg/ml)-induced

neuromuscular blockade was not prevented by

heparin (5 and 150 IU/ml) under pre-treatment

experimental conditions (times for 50 and 90

% blockade: 13 ± 2.5 min and 57 ± 3 min

Fig. 1. Neuromuscular blockade induced by R. schneideri (10 µg/ml) poison alone and pre-treated with heparin (5 and 150

IU/ml) in BC preparations. A. Heparin (5 and 150 IU/ml) failure to prevent the poison (10 µg/ml)-induced blockade. B.

Unaffected muscle contractures to exogenous ACh and KCl after complete neuromuscular blockade induced by poison alone

and pre-treated with heparin. Symbols in section A. and columns in section B. are the mean ± SEM (N = 4). In A. *P < 0.05

compared to control preparations; the superposed (*) represent each point within that time interval. In B. the values were

expressed as a percentage of the responses observed in basal contractures recorded before the treatments.

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(5 IU/ml); 9 ± 1.3 min and 29 ± 2 min (150 IU/

ml), respectively; P < 0.05 compared to con-

trol preparations, N= 4) (Fig. 1A). There were

no important changes in the contractures to

exogenous ACh and KCl after incubation with

poison (10 μg/ml) alone (106 ± 26 % (ACh)

and 120 ± 36 % (KCl) compared to basal; N=

4) or pre-treated with both heparin concentra-

tions (5 and 150 IU/ml) (150 ± 1 (ACh) and

80 ± 1 (KCl) for 5 IU/ml; 81.6 ± 1.7 (ACh)

and 68.6 ± 11.4 (KCl) for 150 IU/ml, N= 4);

these values were expressed as a percentage of

the basal contractures, considered as 100 % of

response (Fig. 1B).

Heparin (5 and 150 IU/ml) has also failed

to prevent the R. schneideri poison (10 μg/ml)-

induced neuromuscular blockade under post-

treatment experimental condition with either

the lowest or highest concentrations (times

for 50 and 90 % blockade: 10 ± 1.5 min and

46 ± 2.5 min (5 IU/ml); 18 ± 2 min and 50 ±

3 min (150 IU/ml), respectively; P < 0.05, N=

4) (Fig. 2A). The contractures to ACh and KCl

were not altered either in those preparations

incubated with poison and subjected to post-

treatment with 5 and 150 IU of heparin/ml (65

± 1 (ACh) and 73 ± 1 (KCL) for 5 IU/ml; 112.8

± 27 (ACh) and 130 ± 30 (KCl) for 150 IU/ml,

Fig. 2. Neuromuscular blockade induced by R. schneideri (10 µg/ml) poison alone and post-treated with heparin (5 and

150 IU/ml) in BC preparations. A. Heparin (5 and 150 IU/ml) failed to prevent the poison (10 µg/ml)-induced blockade. B.

Unaffected muscle contractures to exogenous ACh and KCl after complete neuromuscular blockade induced by poison alone

and treated with heparin. The symbols in section A. and columns in section B. are the mean ± SEM (N= 4). In A. *P < 0.05

compared to control preparations; the superposed (*) represent each point within that time interval. In B. the values were

expressed as a percentage of the responses observed in basal contractures recorded before the treatments.

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N = 4); these values were expressed as a per-

centage of the basal contractures, considered as

100 % of response (Fig. 2B).

Effect of temperature on the R. schnei-

deri poison-induced neuromuscular block-

ade in chick BC preparation: The poison (10

μg/ml)-induced neuromuscular blockade was

abolished in BC preparations maintained at a

low-temperature conditions (23-25 °C) for 60

min incubation; however, raising rapidly the

temperature to 37 °C after 60 min incubation

(without exchanging the physiological media),

the poison produced complete neuromuscular

blockade which was slightly slower than that

seen with BC preparations directly incubated

at 37 °C (times for 50 and 90 % blockade: 15 ±

1.4 vs. 23 ± 2

min and 40 ± 3.2 vs. 60 ± 2.5

min for preparations directly incubated at 37

°C and those incubated initially at 23-25 °C

and then 37 °C, respectively; P < 0.05 com-

pared to times for 50 and 90 % blockade from

those preparations directly incubated at 37 °C,

N= 4) (Fig. 3).

Effect of neostigmine (NEO) and

3.4-diaminopyridine (3.4-DAP) on the R.

schneideri poison-induced neuromuscular

blockade in chick BC preparation: In some

BC preparations, NEO and 3.4-DAP were test-

ed after complete poison (10 μg/ml)-induced

neuromuscular blockade to assess whether they

could contribute to reverse the neuromuscular

blockade. NEO (3.3 μM) and 3.4-DAP (9.2

μM) did not cause a reversal of the com-

plete poison (10 μg/ml)-induced neuromuscu-

lar blockade; however, increasing tenfold the

concentration of 3.4-DAP (91.6 μM) there was

a partial and sustained reversal of the twitch

responses from 10 min incubation (29 ± 7.8 %

of maximal reversal reached in approximately

40 min incubation; P < 0.05, N= 4) (Fig. 4).

DISCUSSION

R. schneideri poison is already known to

induce potent blockade of the motor neuro-

transmission in avian neuromuscular prepara-

tions in vitro. The negative modulatory activity

induced by R. schneideri poison is preceded by

facilitation of the twitch responses, seen with

lower concentrations (3 and 10 μg/ml), whilst a

highest concentration (30 μg/ml) induces only

rapid complete blockade of neuromuscular

twitches approximately 20 min, with no evi-

dence of facilitation (Rostelato-Ferreira et al.,

2011). Here, we have observed that the metha-

nolic extract of R. schneideri poison (10 μg/ml)

Fig. 3. Influence of temperature on the neuromuscular blockade induced by R. schneideri (10 µg/ml) poison in BC

preparations. Incubation at 22-24 °C prevented the poison (10 µg/ml)-induced blockade in indirectly stimulated preparations.

The symbols are the mean ± SEM (N= 4). *P < 0.05 compared to the latest point recorded at 22-24 °C; P < 0.05 compared

to the time for 50 % blockade from preparations incubated directly at 37 °C.