Invest Clin 62(4): 325 - 338, 2021 https://doi.org/10.22209/IC.v62n4a04

Corresponding author: Alexis Rodríguez-Acosta. Laboratorio de Inmunoquímica y Ultraestructura, Instituto Ana-

tómico “José Izquierdo”, Universidad Central de Venezuela, Caracas, República Bolivariana de Venezuela. Tel.+58

212 4917243. Email: rodriguezacosta1946@yahoo.es

Clinical cardiac alterations and hemostatic

toxicities caused by scorpion (Tityus

discrepans) venom and its puried fractions

on zebrash (Danio rerio) larvae.

Aurora M. Álvarez,1, Marco Álvarez2, Lourdes Perdomo2 and Alexis Rodríguez-Acosta3

1Centro de Biociencias y Medicina Molecular, Instituto de Estudios Avanzados (IDEA),

Caracas, República Bolivariana de Venezuela.

2Laboratorio de Microscopia Electrónica, Instituto Anatómico “José Izquierdo”

de la Universidad Central de Venezuela, Caracas, República Bolivariana de Venezuela.

3Laboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico “José Izquierdo”

de la Universidad Central de Venezuela, Caracas, República Bolivariana de Venezuela.

Key words: cardiotoxicity; Danio rerio; scorpions; Tityus discrepans; venom; zebrafish.

Abstract. Envenomation by the Venezuelan scorpion Tityus discrepans is

typified by local and systemic alterations. The current work investigated the

in vivo hemostatic processes, cardiac dysfunction and tissue destruction trig-

gered by Tityus discrepans purified toxins 1 (3 kDa) and 2 (5 kDa) fractions.

These fractions were obtained by C-18-HPLC chromatography. The hemostat-

ic and cardiovascular toxicities in zebrafish of both fractions was assessed by

means of specific phenotypic expressions and larvae behavior at 5, 15, 30, 40

and 60 min post-venom-treatment. The Tityus discrepans venom fractions 1

and 2 produced disseminated intravascular coagulation (presence of thrombus)

in the central vein of the larva, heart rate/rhythm alterations, and necrotic

events in more than 90% of all the larvae under their action. The outcomes have

established the potential hemostatic and cardiovascular toxicities by Tityus dis-

crepans venom, alerting on the possibility of cardiovascular injuries and throm-

boembolism in humans after scorpion stings envenomation.

326 Álvarez et al.

Investigación Clínica 62(4): 2021

Alteraciones clínicas cardiológicas y toxicidades hemostáticas

causadas por el veneno del escorpión (Tityus discrepans) y sus

fracciones puricadas en las larvas del pez cebra (Danio rerio).

Invest Clin 2021; 62 (4): 325-338

Palabras clave: cardiotoxicidad; Danio rerio; escorpión; pez cebra; Tityus discrepans;

veneno.

Resumen. El envenenamiento por el escorpión venezolano Tityus discre-

pans se caracteriza por alteraciones locales y sistémicas. El trabajo actual inves-

tigó los procesos hemostáticos in vivo, la disfunción cardíaca y la destrucción

tisular desencadenada por las fracciones de toxinas 1 (3 kDa) y 2 (5 kDa) puri-

ficadas. Estas fracciones se obtuvieron mediante cromatografía C-18-HPLC. La

toxicidad hemostática y cardiovascular en el pez cebra de ambas fracciones se

evaluó mediante expresiones fenotípicas específicas y comportamiento de las

larvas a los 5, 15, 30, 40 y 60 min post-tratamiento con veneno. Las fracciones

1 y 2 del veneno de Tityus discrepans produjeron coagulación intravascular

diseminada (presencia de trombos) en la vena central de la larva, alteraciones

de la frecuencia/ritmo cardíaco y eventos necróticos en más del 90% de todas

las larvas bajo su acción. Los resultados han establecido las posibles toxicidades

hemostáticas y cardiovasculares del veneno de Tityus discrepans, advirtiendo la

posibilidad de lesiones cardiovasculares y tromboembolismo en humanos des-

pués del envenenamiento por picaduras de escorpiones.

Received: 23-04-2021 Accepted: 09-09-2021

INTRODUCTION

Scorpion envenomation is a Collec-

tive Health problem in numerous countries

in the tropics and subtropics geographical

regions, with important mortality in the

severe forms producing numerous organ

collapses. No other than scorpions of the

Tityus genus are of medical significance in

Venezuela, and Tityus discrepans is respon-

sible for the most severe cases of envenom-

ation and deceases.

On the other hand, during the last

years, zebrafish has become the most rel-

evant biological model among vertebrates,

after the mouse (1). It has been judged as a

vertebrate experimental model for the study

of developmental biology and genetics. The

zebrafish is a member of the Danio genus

(Cyprinidae family). Experimentation in ze-

brafish has demonstrated that most of the

important biological processes are highly

analogous between this species and mam-

mals. Amid the correspondences is already

known that the zebrafish genome is highly

similar to the human genome, with approxi-

mately 87% similarity and also the type of

proteins used to form diverse portions of the

body (2). The anatomical, physiological (in-

cluding the coagulation system) and molec-

ular physiology ranks, which explicates the

achievement of the zebrafish as a biological

model in disparity to other species (2). Tox-

in binding sites are generally well conserved

Cardiac alterations and hemostatic toxicities caused by scorpion venom 327

Vol. 62(4): 325 - 338, 2021

between zebrafish and man; the cardiovascu-

lar, nervous systems and metabolic pathways

are extremely comparable with the anatomi-

cal, physiological and molecular aspects of

mammals; their pharmacological reaction

is analogous to humans, being useful in the

identification of theoretically therapeutic

tests (3, 4). Likewise, zebrafish permits ob-

taining a high quality recognition of target

substances and a validation in vivo of these

constituents during clinical trials in humans

(5).

In the present work, we have explored

this specific vertebrate model to evaluate

the toxicity of relevant toxins of the scorpi-

on (Tityus discrepans), through the observa-

tion of the adverse effects to their exposure,

identifying the end point of toxicity, mecha-

nisms of toxicity and the determination of

the toxic-dynamics of toxins, among others

(6). Here, we have estimated the cardiovas-

cular system of the zebrafish, a significant

guide to toxicity, since the venom fractions

caused simply observable effects, such as the

alterations in the circulation of blood flow

(thrombosis), the heart rate rhythmicity,

the cardiac output, the cardiac morphology,

the pericardial morphology, among others.

These results provided rapid, reproducible

and quantifiable information (7).

MATERIALS AND METHODS

Reagents

Chemicals and reagents used in this

research were obtained from Sigma-Aldrich

Co. (St Louis, MO, USA), Thermo Fisher

Scientific (Waltham, MA, USA), and BD Bio-

sciences (San Jose, CA, USA). Low-range

“rainbow” molecular mass markers (38 to

3,5 kDa) were purchased from Amersham;

GE healthcare companies, USA.

Mice

Male mice (Mus musculus) C57/BL6

strain weighing 20 to 22 grams (g), from the

vivarium of the Instituto de Estudios Avan-

zados (IDEA), were used to determine lethal

activity (LD50). Animals were supplied with

water and food ad libitum, until use.

Scorpions Tityus discrepans and venom

collection

A pool of venoms was obtained from 42

scorpions specimens kept in captivity, hydrat-

ed with distilled water and fed with caelifer-

ous larvae (grasshoppers). These scorpions

come from the vicinity of the municipality

El Hatillo, Los Salías (Fire Department) and

Baruta (Hoyo de la Puerta), Miranda state,

Venezuela. The scorpion’s ecological area

is situated in a climatic bioregion of humid

forest, located 1200 m up of sea level, with

an average annual temperature of 21°C and

relative humidity of 85% or more. The flora

is represented for an exuberant vegetation of

humid forest formation (8).

The Tityus discrepans scorpion venoms

were collected and pooled in the Vivarium of

the Instituto de Estudios Avanzados (IDEA),

(Miranda state, Venezuela). They were man-

ually milked by electrical stimulation of the

telson. Two electrodes connected to a stim-

ulator (SD5-Grass ™) were placed; one at

the base of the aculeus and the other on a

pedipalp. The contact area of the scorpion

was humidified and got in touch with the

electrodes. Three electric pulse trains with a

frequency of 6/pps, duration of 100 ms, and

60-volt intensity were applied (SD5-Grass

™: SM6-stimulator Gras Model = GM6, sig-

nal M2010x5, Instrument CO, Quincy Miss,

USA).

The ejected venom was collected in

non-heparinized capillary tubes and trans-

ferred to Eppendorf tubes, where it was

resuspended with 18-megaOhm water by

constant vortexing for 30 seconds (s) and

centrifuged at 20.000 g in a refrigerated

(5°C) centrifuge (Eppendorf 5417R) for

5 min. Then, the supernatant was filtered

using 0.22 µm millex-GS. It was labelled

with the date, group and number of milked

scorpions.

328 Álvarez et al.

Investigación Clínica 62(4): 2021

Protein concentration of the scorpion

Tityus discrepans venom by

bicinchoninic acid method

The Tityus discrepans venom protein

concentration was measured by the bicin-

choninic acid (BCA) method (BCA ™ Pro-

tein Assay Kit, Pierce, Illinois, USA) kit.

The procedure was carried out in 96-

well culture plates, adding 25 µL of the sam-

ples from both the standard curve and the

test sample, plus 200 µL of the BCA reagent

(prepared as indicated by the commercial

company: 50 parts of reagent A with a part of

reagent B). The plate was shaken, while in-

cubated at 37°C for 30 min. Finally, the plate

was allowed to cool for 5 min at room tem-

perature and the absorbance at 562 nm was

measured with the ELISA reader (Biotek,

Synergy ™ model, USA).

Protein determination from fractions

1 and 2 of the Tityus discrepans venom

Protein concentration of 1 and 2 frac-

tions were spectrophotometrically estimat-

ed by assuming that 1U of absorbance/cm of

wavelength at 280 nm corresponds to 1mg

protein/mL (9).

Determination of Lethal Dose Fifty (LD50)

The LD50 for Tityus discrepans crude

venom was determined (48 h) by intraperi-

toneally (i.p.) venom injection in mice (18-

22 g) and calculated according to the Spear-

man-Kärber method (10). Five mice per dose

(4.5 mg/kg to 1.25 mg/kg) were used. These

doses were selected on basis of the previous

reports of lethality of the Tityus discrepans

crude venom in our laboratory (11). The

LD50 tests from fractions 1 and 2 were not

carried out, since the test required signifi-

cant quantities of fractions and we have not

the necessary amount of these fractions.

Ethical statement

Expert personnel set all the experimen-

tal techniques concerning the use of live an-

imals, including zebrafish embryos, larvae,

juveniles, adults and scorpions. Venezuelan

pertinent regulations as well as institutional

guidelines, according to protocols ratified by

the Institute of Anatomy Ethical Committee

of the Universidad Central de Venezuela in

accordance with the ethical principles in an-

imal research adopted by the World Health

Organization (12).

Fractionation of the scorpion

Tityus discrepans venom by HPLC

The Tityus discrepans venom (2 mg)

was fractionated using the high-resolution

chromatography method (HPLC). A Zorbox

3000SB-C18 chromatographic column was

used as the separating matrix. The proteins

were eluted off the column at room tempera-

ture with an acetonitrile linear gradient of

0%–80% (v/v) in 0.12% TFA for 60 min. A

Waters 1525 binary HPLC pumps with a Wa-

ters 2487 dual k absorbance detector (280

nm) were employed. Samples for biological

assays were lyophilized twice to remove po-

tential trace amounts of solvent. Ten differ-

ent HPLC runs were performed keeping the

same conditions. The fractions were collect-

ed at 1.5 mL/tube. All fractions were tested

for any activity on the larvae (type of move-

ment, presence or not of paralysis, blood

flow characteristics and/or death).

In the well-defined fractions could be

observed the fractions 14 and 55, with reten-

tion times of 11.87 min and 25.58 min, re-

spectively, which showed the highest activity

and purity. They were dialyzed against water

at 4°C and lyophilized to perform the subse-

quent assays in the zebrafish model.

Gel electrophoresis

Polyacrylamide gel (15%) electrophore-

sis following the (13) method was used to

determine the Tityus discrepans venom be-

sides 1 and 2 fractions molecular masses.

The lyophilized venom was reconstitut-

ed in denaturing buffer (10mM Tris-HCl, 2%

SDS, 0.1M DTT, 0.01% blue bromophenol,

and 1mM EDTA, pH 8.0). Low-range “rain-

bow” molecular mass markers (38.0 to 3.5

kDa) were used as reference for SDS elec-

Cardiac alterations and hemostatic toxicities caused by scorpion venom 329

Vol. 62(4): 325 - 338, 2021

trophoresis. Proteins were stained with Coo-

massie blue stain.

Breeding of zebrafish (Danio rerio) larvae

Zebrafish were bred and keep up in the

Electronic Microscopy Section, Anatomical

Institute ‘‘José Izquierdo,’’ Universidad Cen-

tral de Venezuela (Caracas, Venezuela), by an

adjusted method (14). The fishes were pre-

served in 20-L tanks, previously filled with

newly prepared filtered tap water (FTW), at

28°C, pH 6.6 - 7.0, with 12-h light/12-h dark

cycle. For reproduction reasons, six to eight

pairs of adult zebrafish were set up in 10-L

tanks with a mesh and allowed to breed with-

out restrictions. During the reproduction

period, 100 and 150 eggs were collected per

pair. They were gathered, washed and trans-

ferred to Petri dishes. Eggs containing dead

or decreased quality larvae were discarded.

The larvae were cleaned and well-arranged

by stage of development 5 d after fertiliza-

tion (dpf) to perform toxicity assays. The

fish larvae were located in FTW into microti-

ter plates, and then the fractions achieved

by the C-18 HPLC chromatography were ten

larvae per well tested, with fraction concen-

trations of 0.76 µg/100 µL/FTW. Negative

controls with FTW were utilized.

Larvae observation

Fully alive larvae pictures were taken-

with an Olympus IX-71 Series (Olympus, Ja-

pan) inverted microscope. Specific software

to capture images (MetaMorph-Microscopy

Automation & Image Analysis Software) and

a Sony SRS-PC71 device camera with micro-

color filter, adapted to 200 squares/s with 40

X magnification that was fix to quantify the

length of the images structures were used.

Zebrafish larvae under toxicity analysis

Inspection of the larvae under Tityus

discrepans venom fractions toxicity was usu-

ally carried out in 24-well plates. Larvae at

the 5 dpf stage (weight of 5 dpf larvae was

∼ 0.01 g) were hoarded and transferred to

assay wells (normally 10 larvae/well), by ten-

der pipetting to reduce injury to the larvae.

Then, its development was monitored hour-

ly post-fertilization and visually inspected

at room temperature, under a microscope

equipped with a digital video camera. All

abnormal morphological and performing

changes, including lethality, were recorded

and kept as exhaustive pictures and videos.

Ten wells have been examined for each frac-

tion, and the authors performed technical

and biological replicates.

Morphological modifications and larvae

behavior

Body larvae irregularities and motion

comportments were visualized during early-

and late-stage of development. The observa-

tion was reiterated, in order to validate any

detected harm, to confirm that the damages

and conducts were reliable and reproduc-

ible. The injuries and the mobility behaviors

were explicitly noted as ‘‘positive’’, when

remarked in >80% (i.e., 8 of 10) of tested

larvae.

Hemorrhages and thrombosis

Looking for thrombus and/or hemor-

rhages presence, the larvae zebrafish ven-

tricle and cardinal vein (cardiovascular sys-

tem) was considered. It is accepted that

hemorrhages and/or thrombosis originate

from fissure of blood circulatory vessels in

any organ.

Cardiovascular evaluation

Cardiac frequency (heart rate) (pulsa-

tion per minute: ppm) from ten 5 dpf zebraf-

ish larvae were scattered into a microtitre

plate, in which Tityus discrepans 1 and 2

venom fractions were dispensed and tested

for 15, 30, 40, and 60 min at room tempera-

ture and examined under a Olympus IX-71

Series microscope. After incubation, ven-

tricular and atrial rates (ppm) were counted

during 30 s with a stopwatch and a counter

(7). The number of contractions was multi-

plied by two to calculate heart rate in pulsa-

tions per minute (7). Ten tests were taken,

330 Álvarez et al.

Investigación Clínica 62(4): 2021

and their median was statistically consid-

ered. In the tests, the ppm for 1 and 2 Tityus

discrepans venom fractions were measured

up to equivalent normal control larvae under

identical conditions.

Statistics

Statistical analyses were carried out

employing an unpaired Student’s t-test (*p

< 0.05; ***p < 0.001).

RESULTS

Protein concentration of the scorpion

Tityus discrepans venom by

bicinchoninic acid

The Tityus discrepans venom protein

concentration by BCA was 7.5 mg/mL.

Meanwhile, the fractions 1 and 2 tested

were 0.25 and 0.13 mg/mL, respectively, as-

suming that 1U of absorbance/cm of wave-

length at 280 nm corresponds to 1mg pro-

tein/mL (9).

Fractionation of the scorpion Tityus

discrepans venom by HPLC

The Tityus discrepans venom was frac-

tionated using the high-resolution chroma-

tography method. In Fig. 1, well-defined

fractions can be observed. Fractions 14 (now

fraction 1) and 55 (now fraction 2), with re-

tention times of 11.87 min and 25.58 min,

respectively, were chosen to perform in the

zebrafish larvae the subsequent tests, such

as it was stablished above.

Electrophoretic profile of venom by 15%

SDS-PAGE

Fig. 2 shows the Tityus discrepans

venom electrophoretic profiles. The distri-

bution of protein bands occurred within gel

regions corresponding to narrow range mo-

lecular masses. In Tityus discrepans crude

venom ~11 protein bands were evident. The

high intensity bands accorded with the ∼

42, 35, 25, 8 and 3 kDa molecular masses.

Other lower intensity bands corresponded to

Fig. 1. Tityus discrepans venom separation by HPLC. Y axis indicates the absorbance at 280 nm, the X axis the

elusion time of the fractions. Fractions 14 (now fraction 1) and 55 (now fraction 2), with retention

times of 11.87 min and 25.58 min, respectively.

Cardiac alterations and hemostatic toxicities caused by scorpion venom 331

Vol. 62(4): 325 - 338, 2021

proteins up to ∼ 42 kDa. Fractions 1 and 2

showed a single band of ∼3 and ∼ 5 kDa, re-

spectively.

Lethality: determination of the LD50

of the Tityus discrepans crude venom

The LD50 of the Tityus discrepans crude

venom tested in mice was 3.5 mg/kg.

Larvae observation

From the relevant fractions (HPLC), the

doses were prepared to be confronted by the

larvae, and observed with an Olympus mi-

croscope, which allowed the analysis of their

physical, organic and behavioral changes. A

tool (Table I) was created to keep records of

the analyses.

Zebrafish larvae under toxicity analysis

The purest fractions that had the high-

est amount of proteins were tested. Frac-

tions 1 and 2 were chosen for study in the

zebrafish model showing several and differ-

ent effects (Table II and Figs. 3 and 4).

Cardiac and circulatory thrombosis

For untreated larvae, mortality and

spontaneous changing dysfunction naturally

happened at comparatively low frequency

(approximately < 3% of larvae).

Larvae developing in vivo toxicity by

Tityus discrepans fraction 1 showed after

15 min, visible internal organ damage, in-

cluding thrombus formation in the ventricle

and the cardinal veins (Fig. 5). This cardiac

Fig. 2. SDS-PAGE (15%) profile from Tityus discre-

pans crude venom stained with Coomassie

blue. (1) “Low-range rainbow” molecular

mass markers (38 to 3 kDa). (2) Tityus dis-

crepans crude venom (5µg). (3) Fraction 1

(5 µg). (4) Fraction 2 (5 µg).

TABLE I

DATA RECORDING FOR ASSAY ANALYSIS.

Fractions Tested doses Observations

(1h)

Fraction 1 0.76µg Types of motion

Paralysis presence

(0 or +)

Characteristic

of blood flow

Death (0 or +)

Other observations

Fraction 2 0.76µg

TABLE II

RESULTS FROM MORPHOLOGICAL

MODIFICATIONS AND BEHAVIOUR, OBTAINED

FROM THE ACTION OF Tityus discrepans

FRACTIONS (1 AND 2) ON 10 ZEBRAFISH

LARVAE, VIA OLYMPUS MICROSCOPE WITH

PHASE CONTRAST VISION.

Fractions

(0.76 µg/

larva)

Observations

(1h)

Fraction 1 Circular movements

Circulatory paralysis

Decreased blood flow

Disseminated intravascular

coagulation

Tremor

Death at 40 min

Fraction 2 Alteration of circulation (late)

Little cardiac alteration

Detachment of the epithelium

Tremor

Death at 60 min

332 Álvarez et al.

Investigación Clínica 62(4): 2021

thrombosis was evaluated using larvae in the

presence of 0.76µg of Tityus discrepans frac-

tion 1, in comparison with negative control

(FTW-treated) samples (Fig. 5).

Cardio-circulatory appraisal

The larvae confronted with fraction 1, af-

ter 5 min presented a reduction in irrigation

blood and intravascular coagulation (Fig. 3).

The larvae died past 40 min. On the other

hand, with respect to the fraction 2, the ef-

fect on blood circulation was late, when com-

pared with fraction 1. However, after 30 min

the blood circulation began to decrease, and

at 60 min, there was a necrotic detachment

of the epithelium (Fig. 3B), and death of the

Fig. 3. Microscopic observation of zebrafish larvae under Tityus discrepans fractions action (0.76µg). (A)

Normal control. (B) Fraction 1 showed an intravascular coagulation (arrows). (C) Fraction 2, an

epithelial necrosis was palpable (arrows).

Fig. 4. Comparison of cardiac frequency of zebrafish larvae among Tityus discrepans fractions 1 (rhombus),

fraction 2 (square) and normal control (star). The X axis represent time and the Y axis pulsations per

minute.

Cardiac alterations and hemostatic toxicities caused by scorpion venom 333

Vol. 62(4): 325 - 338, 2021

larvae. The larvae confronted with fraction 1,

after 5 min presented an intense decrease in

heart rate. Fraction 2 caused a more moder-

ated decrease in heart rate (Fig. 4).

DISCUSSION

Utilization of the zebrafish model in

toxicology research has increased in the lat-

est years, converting it in one of the best

advantageous and convenient systems for

understanding parameters of toxins on the

hemostatic and cardiovascular systems (14).

Even though there is considerable evolu-

tionary separation between zebrafish and

humans, significant genetic and phenotypic

preservation in both systems have permitted

substantial progresses in our understanding

of how natural toxins act on the different

organs and tissues of the human body. The

potential of zebrafish, in the observation of

toxins activities on the hemostasis and car-

diovascular structures, allows for the possi-

bility to carry out at in vivo real-time obser-

vations of these systems, and their altered

functions, along with the simplicity. Simi-

larly, the different toxins modifying specific

hemostatic processes or cell alterations can

be recognized and typified.

A scorpion uses its venom to paralyze

and kill its prey (usually insects) that it is go-

ing to eat, and its gland takes approximately

three weeks to replenish its venom. The ven-

om is biologically composed of toxins with

diverse actions, for instance: cardiotoxins,

nephrotoxins, hemolytic toxins, phosphodi-

esterases, phospholipases, hyaluronidases,

glycosaminoglycans, histamine, serotonin,

tryptophan, bradykinin-enhancing and cyto-

kine-releasing peptides (15).

In the current work the Tityus discre-

pans venom was obtained from 42 scorpi-

ons, whose venom dry weight yield was 51.7

mg, which represents 1.22 mg of venom per

milked scorpion. Similar results were re-

corded (16), when milking, under the same

conditions, 92 T. caripiensis scorpions, ob-

taining a dry weight of venom of 96.6 mg

that represented 1.05 mg of venom per

milked scorpion. Likewise, these data corre-

lated with those described for the scorpion

T. pachyurus from Colombia, which gave a

yield between 0.3 and 1.0 mg of venom per

specimen (17). On the other hand, it has

been described that the color of venom ob-

tained by electrical stimulation is white,

such as it was our venom and did not turn

blue after milking. Contrastingly, the venom

Fig. 5. Thrombolytic activity from Tityus discrepans fraction 1. Images of the treatment of 5 dpf larvae with

Td fraction 1 (0.76µg), from 0 to 40 min, displayed ‘‘early-stage’’ (15 min) modifications, with evi-

dent thrombosis in the ventricle. (A) Low magnification of thrombus in the cardiac area (arrow); (B)

Normal control; (C) Cardiac thrombus (arrow) Tityus discrepans fraction 1; (D) Magnified thrombus

image (arrow) at 100 x.

334 Álvarez et al.

Investigación Clínica 62(4): 2021

collected manually rapidly becomes blue af-

ter milking (18).

In the Tityus discrepans venom HPLC

fractionation, around 60 well-defined frac-

tions were observed; those with the high-

est concentration were those belonging to

retention times in the range of 20 to 25

min. Previous studies (19) of Tityus discre-

pans venom fractionated with HPLC (∼ 65

fractions) had shown that the peaks elute

at similar retention times, where there were

groups of compounds with similar size and

activity.

Fractions that eluted during the first 10

min had molecular masses between 20 and

200 kDa; among these are toxins that have

Xa inhibitory factor, amidolytic and plas-

min inhibitory activity (20). The toxins that

elute between 10 and 30 min had masses be-

tween 3 and 5 kDa; In this range, there are

toxins with activity on potassium channels

(21, 22). The fractions that eluted between

30 and 40 min had masses ranging from 5

to 8 kDa; this range includes toxins capable

of modulating sodium channels in the mem-

brane (23). Less hydrophilic proteins with

high molecular weights elute sometimes >

40 min; the latter contains a curarizing pep-

tide TdFI33 and enzymes such as serine and

metalloproteases (20, 21).

This venom is a mixture ∼ 80 differ-

ent toxins, mostly of low molecular masses,

isolated and recognized by chromatography

and electrophoresis. Opposition assays have

proven their actions on the voltage-depen-

dent ion channels (mainly Na +, Ca ++,

K + and Cl-), and on excitable membranes

(glandular, nervous and muscular tissue),

transforming their ionic permeability, depo-

larizing them and yielding neurotransmitter

discharges in the post-ganglionic endings, of

the sympathetic and parasympathetic ner-

vous system. However, non-all of these frac-

tions are venomous to humans (24). Only

about 10 act on mammals and 3 of these

subtypes are toxic (containing between 61

and 62 amino acids). The venom is speedily

absorbed, agreeing to the findings of several

studies, since from the first 5 to 15 min obvi-

ous clinical signs of the action of the venom

were observed (21).

In our study, when facing the selected

Tityus discrepans venom fractions, numer-

ous changes were observed in the organic

structure and behavior of the specimens,

such as circular movements, tremor, de-

tachment of the epithelium, disseminated

intravascular coagulation, decrease in heart

rate and death. This gives us an indication

of the effect of these fractions, especially in

the circulatory system. The toxic effects of

scorpion envenomation are probably due to

a considerable discharge of sympathetic and

parasympathetic neurotransmitters; the se-

riousness is associated to cardiac and hemo-

dynamic alterations, with cardiogenic shock

and pulmonary edema causative of the cru-

cial causes of death (25).

It is also important to note that to date;

no other studies are known to use the zebraf-

ish model to assess the toxicity and lethal-

ity of the Tityus discrepans scorpion venom.

However, we had assayed this methodology,

characterizing a cardiotoxic effect in zebraf-

ish larvae, produced by a toxin (Mutacytin-1)

present in the venom of the Lachesis muta

muta snake (14) and Bothrops venezuelen-

sis venom. This toxin has coagulant activ-

ity (verified with the formation of a fibrin

meshwork in platelet-poor human plasma

and direct observation of clot formation in

the heart of the animal model) and induces

compromise of the cardiac system (decrease

in cardiac frequency and output) (44).

Electrophoretic analysis of Td venom

by 15% SDS-PAGE indicated that the Tityus

discrepans crude venom sample showed one

major band of protein with molecular mass-

es averaging around 8 to 10 kDa. D’Suze et

al. (22) had described proteins in the Tityus

discrepans venom of 3 and 5 kDa. According

to Diaz et al. (23), they found proteins from

5 to 8 kDa in the scorpion venom.

In the present work, the LD50 determi-

nation for Td crude venom was of 3.5 mg/kg,

demonstrating high lethality by the intraper-

Cardiac alterations and hemostatic toxicities caused by scorpion venom 335

Vol. 62(4): 325 - 338, 2021

itoneal route in C57/BL6 mice. When com-

paring the LD50 obtained in this work, with

those detected (2.5 mg/ kg) with the same

species, by other authors (11, 26), differ-

ences in the levels of lethality were observed.

Concerning hemostatic and cardio-

circulatory considerations, previous in vi-

tro trials carried out in human plasmas,

demonstrated that in the venom of Tityus

discrepans an anti-procoagulant fractions

exist (27, 28). This venom also contains

compounds capable of degrading fibrino-

gen (28). Furthermore, this venom pro-

duces an activity similar to the Xa factor

(procoagulant activity) (29) that was found

in fraction 1; this finding is consistent with

the synergy in amidolytic activity, described

by the commercial Xa factor. Probably, the

components with the activity similar to Xa

factor present in the venom and fraction 1,

induced the shortening of the coagulation

times. It is recognized that the α2β1 inte-

grin situated on the exterior of platelets

membranes are essential for thrombus for-

mation on exposed collagen, at locations of

vascular endothelium injury (30).

Toxicological responses of the heart to

Tityus discrepans toxins were analyzed and

interpreted, to establish the central axis of

this research in the field of cardiotoxicity.

The autonomous innervation of the heart

regulates the contraction and the force of

cardiac muscle. Catecholamines, released

by sympathetic stimulation produce the car-

diac chronotropic action (accelerating ac-

tion), but the inotropic action is the force of

cardiac contraction, which is controlled by

the ß-adrenergic receptors that are essential

for sustaining the rate and strength of con-

traction of the heart muscle (31). While the

adrenergic stimulus in supporting heart rate

has been described in adult zebrafish, the

precise responsibility of adrenergic regula-

tion in zebrafish larvae is still poorly known.

Several authors (32, 33) proposed that ze-

brafish larvae start to display a chronotropic

response to adrenergic agonists at 4 or 6-day

post-fertilization, since at that time zebra-

fish larvae have ß1 adrenergic and ß2 adren-

ergic receptors, both associated to the in-

crease in chronotropic and inotropic actions

of the cardiac muscle.

The Tityus discrepans fraction 1 in-

duced a severe decrease in the cardiac fre-

quency (negative chronotropic action)

leading the larvae to death. Fraction 2 also

presented a negative chronotropic effect,

but less intense than fraction 1. We cannot

determine whether the activity of both tox-

ins have a direct effect on the ß1 adrenergic

and ß2 adrenergic receptors, both linked to

increased chronotropism and inotropism of

the cardiac muscle. These actions are due to

the stimulating action on Protein G, which

increases adenyl cyclase activity, causing

high levels of cyclic AMP (34-37). Gibbins

(38) has suggested that the principal factor

to take in consideration about the cardiotox-

ic effect has been the modulation of heart

frequency that can be regularly measured by

a direct optical examination, by video-edge

detection systems.

It has been reported that the early

clinical signs are principally occasioned by

venom-induced adrenergic and cholinergic

effects (30, 39). Adrenergic expressions

are secondary to catecholamine discharge,

which include cardiac failure and arrhyth-

mias, tachycardia, arterial hypertension and

shock. Symptomatology usually starts with

further transient parasympathetic stimula-

tion. However, severity, on the other hand,

is largely established by the continuing im-

pacts of high catecholamine concentrations

in the cardiovascular system (40-42).

Finally, we have evidenced the useful-

ness of this kind of in vivo assay estimat-

ing histologically and functionally zebrafish

hearts in real-time, which allowed the evalu-

ation of responses to toxins in the short and

medium term. We want to propose the use

of the zebrafish model, to evaluate the scor-

pion toxins on the cardiac system, trying to

extrapolate the observed damages to that

occurring in humans, all through many epi-

demiological studies (43).

336 Álvarez et al.

Investigación Clínica 62(4): 2021

ACKNOWLEDGMENTS

We acknowledge the use of the Zebraf-

ish Core Facility of the Laboratorio de Mi-

croscopía Electronica, Instituto Anatómico

de la Universidad Central de Venezuela.

This study was supported by the Funding

grant from the Science and Technology Fund

(FONACIT) programs (PEI 201400352)

(Universidad Central de Venezuela, Dr. A.

Rodríguez-Acosta).

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