DOI: https://doi.org/10.52973/rcfcv-e32119
Received: 19/02/2021 Accepted: 04/04/2022 Published: 17/05/2022
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Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32119, 1 - 13
ABSTRACT
A quantitative model to expose the adrenal gland sub-cellular
alterations produced by crotamine-like (C-L) from rattlesnake venom
during 3, 6 and 24 hours (h), and also qualitative changes on mice
neuromuscular structures in vivo were observed and calculated
by transmission electron microscopy. A pure crotamine-like (C-L)
isoform was obtained using a cationic exchange chromatography
column from the rattlesnake Crotalus durissus cumanensis venom.
The C-L SDS-PAGE (15.5%) under non-reduced conditions exhibited
a molecular mass of ~3 kDa single band. The C-L in vivo qualitative
experiments induced ultrastructural changes in mouse neuromuscular
structures at 3, 6 and 24 h, such as reduction in the number of
acetylcholine vesicles, disorganisation of the secondary synaptic
clefts, enlargement of the sub-sarcolemma space and alteration
of the mitochondria morphology, number and cristae. Regarding
neurotoxic actions in vivo, the animals injected with C-L presented
spastic paralysis of the hind limbs. The quantitative alterations studied
on the capillaries, the nucleus, the mitochondria the lipid inclusions,
and the smooth endoplasmic reticulum were observed from 3 to 24
h after C-L injection. As far as it is known from the literature review,
there are no quantitative records of similar sub-cellular alterations
caused by crotamine.
Key words: Crotamine-like peptide; Crotalus durissus cumanensis;
mitochondria; motor- endplate; myotoxin, rattlesnake;
smooth endoplasmic reticulum
RESUMEN
Se observó y calculó mediante microscopía electrónica de transmisión,
un estudio cuantitativo para exponer las alteraciones subcelulares
de la glándula suprarrenal producidas por crotamina-similar (C-L) del
veneno de serpiente de cascabel, durante 3; 6 y 24 horas (h), y también
los cambios cualitativos in vivo en las estructuras neuromusculares
de ratones. Se obtuvo una isoforma pura similar a la crotamina (C-L)
usando una columna de cromatografía de intercambio catiónico del
veneno de Crotalus durissus cumanensis. La SDS-PAGE (15,5%) de
la C-L en condiciones no reducidas exhibió una masa molecular de
∼3 kDa de banda única. Los experimentos cualitativos in vivo de C-L
indujeron cambios ultraestructurales, bajo microscopía electrónica en
constituyentes neuromusculares de ratón a las 3; 6 y 24 h, tales como
reducción en el número de vesículas de acetilcolina, desorganización
de las hendiduras sinápticas secundarias, agrandamiento del espacio
sub-sarcolema y alteración de las mitocondrias. En cuanto a las
acciones neurotóxicas in vivo, los animales inyectados con C-L
presentaron parálisis espástica de las extremidades posteriores.
Las alteraciones cuantitativas estudiadas en los capilares, el núcleo,
las mitocondrias, las inclusiones lipídicas y el retículo endoplásmico
liso se observaron de 3 a 24 h después de la inyección de C-L. Hasta
donde se conoce por la revisión de la literatura, no existen registros
cuantitativos de alteraciones sub-celulares similares provocadas
por la crotamina.
Palabras clave: Péptido similar a la crotamina; Crotalus durissus
cumanensis; mitocondrias; placa motora terminal;
miotoxina, serpiente de cascabel; retículo
endoplásmico liso
A new crotamine-like from the rattlesnake (Crotalus durissus cumanensis)
venom causing damages: Qualitative and Quantitative Cytotoxic Studies on
subcellular and neuromuscular structures
Una nueva crotamina similar de la serpiente de cascabel (Crotalus durissus cumanensis) causante
de daños: estudios citotóxicos cualitativos y cuantitativos en estructuras sub-celulares y
neuromusculares
Estefanie García
1
, Héctor José Finol
1†
, Roschman González
1
and Alexis Rodríguez–Acosta
2
*
1
Universidad Central de Venezuela, Faculty of Sciences, Electron Microscopy Centre. Caracas, Bolivarian Republic of Venezuela.
2
Universidad Central de
Venezuela, Anatomical Institute, Laboratory of Immunochemistry and Ultrastructure. Caracas, Bolivarian Republic of Venezuela.
*Email: rodriguezacosta1946@yahoo.es
Crotamine-like actions on subcellular structures / Garcia et al.______________________________________________________________________
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INTRODUCTION
In many Countries that are located in the tropical and sub-tropical
areas, the ophitoxemia represents a serious Collective Health problem
[13], and they are unfortunately included in the neglected tropical
diseases. In the Neo-tropics, rattlesnakes cause about 12% of snake
envenomation [27] with signicant mortality rates. One of the most
neurotoxic and myotoxic components of these venoms is crotamine,
causing symptoms of muscle damage and spastic paralysis in patients
bitten by these species.
The symptoms of the ophitoxemia produced by the Crotalus snake’s
venoms are the consequence of the combination of several toxins
effects and enzymes that framework the involved species. It is
necessary to investigate their neurotoxic, myotoxic and haemostatic
effects at subcellular level. Each venom of a viper species constitutes
a unique mixture of peptides and proteins chosen by natural
selection to act on the vital systems of its prey. These qualitative and
quantitative differences between venoms exist in families, genera,
species and even intra-species [1].
The venoms of Crotalus snakes are responsible for pathological
sub-cellular lesions, which have been described by electron microscopy
techniques in several previous works [2, 8, 25, 29]. It is common that
in venoms scientic research there is a relationship between the
enzymatic and clinical pathological pictures; however, when adding
the sub-cellular level of description that offers a tool such as Electron
Microscopy (EM), the understanding and the pathological analysis is
necessarily improved. Indeed, the existence of studies in EM that
have allowed to describe the alterations caused by venoms in various
organs have been established, nevertheless, as far as it was known
from the literature review, there are no quantitative records of similar
sub-cellular alterations caused by crotamine.
Also, in the present work, a quantitative study measuring the
action of crotamine-like (C-L) from the common rattlesnake Crotalus
durissus cumanensis (Cdc), on the capillary endothelium, capillary
lumen, nucleus area, nucleus envelope, number of mitochondria,
Areal number density of mitochondria, number of mitochondrial
cristae, number of lipid inclusions, areal number density of lipids
inclusions and cisternae of the smooth endoplasmic reticulum (REL)
from adrenal gland cells have been studied. On the other hand, a
qualitative revision demonstrating a dynamic evolution of damages
on neuromuscular structures from 3, 6 to 24 hours (h) was carried out.
MATERIALS AND METHODS
Snakes
Four males and four females of common rattlesnake (Crotalus
durissus cumanensis) were captured at Santa Teresa del Tuy Town,
Miranda State, Venezuela. Snakes were placed and well-kept at the
Serpentarium of the Tropical Medicine Institute, Caracas, Venezuela.
Venom
Venom extraction was achieved by permitting the snake to chew
into a para-lm stretched over a disposable plastic cup. The venom
was pooled, centrifuged (Allegra®X-30 Centrifuges, Beckman Coulter,
USA) (500 × g for 10 minutes (min), and ltered by means of 0.45
micrometres (μm) lter, under positive pressure. The pooled venom
was lyophilised and stored (Frigidaire FGVU21F8QF Vertical Freezer,
USA) at –30°C until use.
Mice
Ultrastructural studies were carried out using 18 adult female NIH
strain mice (Mus musculus) (18-22 grams – g-) purchased from the
vivarium of the National Institute of Hygiene “Rafael Rangel”, Caracas,
Venezuela. Animals were provided with water and food ad libitum,
until used.
Crotamine-like (C-L) purication
Purication of C-L from the rattlesnake venom was carried out by
one chromatographic step [29]. Crude venom (Cdc) (30 milligrams-mg
by protein estimation) [33] was diluted to 1.0 millilitres (mL) of 50 milli
Molar (mM) Tris–HCl buffer, pH 8.2, and then applied on a Mono S HR
10/10 column (GE Healthcare Biosciences Ltd, USA) equilibrated with
an equivalent buffer. Supporting proteins were eluted with a 0–1 M
NaCl linear gradient in equivalent buffer over 60 min at a ow rate of
1.5 mL·min-1. Proteins were checked at 280 nanometres (nm). The
chromatogram showed 8 fractions, which were identied agreeing
to their elution. Well-dened spastic hind-limb paralysis action on
mice was ostensible in fraction 5 (C-L), which was dialysed against
water at 4°C, lyophilised and stored at –30°C and selected for the
experimental assays.
Polyacrylamide gel electrophoresis (SDS-PAGE)
To support the purity of the chromatographic peaks, an electrophoresis
of the Cdc crude venom (20 micrograms (µg) and the C-L (60 µg) was
performed on analytical discontinuous gels with 12.5% separation
and non-reducing conditions. The gels were stained with a Coomassie
blue staining solution. Densitometry of the gels was performed using
a GS-690 Densitometer (BIO-RAD, USA) and the analysis of the prole
of the proteins and their molecular masses were determined using the
Multi-Analyst version 1.1 program (BIO-RAD, USA).
Protein concentration
Protein concentration of C-L was spectrophotometric measured,
accepting that 1 unit of absorbance/centimeters (cm) of wavelength
at 280 nm corresponds to 1 mg protein·mL
-1
[33].
Procedure for qualitative study of specimens by electron microscopy
(TEM)
For the qualitative study, 18 adult female NIH strain mice (18-22 g)
were divided in two groups: the normal control (n=9) in which mice
were injected intravenously (i.v.) (in the central tail vein) with 0.1 mL of
saline solution and the experimental group (n=9) also injected via i.v.
with 20 μg·mL
-1
of C-L in 100 μL of phosphate buffer saline (PBS). After
3, 6 and 24 h, three mice from each group were prepared for motor
end-plate of striated muscle (muscles of the hind-limb) biopsies.
The fragments were obtained from control and experimental mice
immediately after to be sacriced by cervical dislocation. Samples
were straightaway in situ xed with 3% glutaraldehyde and 1% OsO
4
(both xatives diluted in 320 mM phosphate buffer saline, pH 7.4),
dehydrated in ethanol and embedded in LX-112 resin (Ladd Research
Inc.). Ultrathin sections were stained with uranyl acetate and lead
citrate and observed with a FEI, TecnaiSpirit 12G2 model, (University
of Minnesota, College of Engineering, USA) transmission electron
microscope with an accelerating voltage of 100 kilovolts (kV).
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Procedure for quantitative study specimens by principal
component analysis (PCA)
Principal component analysis (PCA) consisted of expressing a
collection of variables in a set of linear combinations of factors not
correlated with each other [12, 38]. This method allowed the original
data (individuals and variables) to be represented in a space with a
dimension less than the original space [12, 38].
Digitisation of the image
In order to carry out a descriptive quantitative study of the
biological sample (The capillaries, the nucleus, the mitochondria
the lipid inclusions, and the smooth endoplasmic reticulum) from
a micrograph, the magnication handled for their registration was
pondered, as well as the magnication factor, with which the revealed
physical image was acquired. By means of the “mouse” (Mouse laser
Bluetooth ThinkPad, China) it was delineated points, the length, the
angle, the area, among others, in the image acquired directly from
the TEM, so that the object in the image could be determined in real
time. Thus the images pick up directly from the electron microscope
were used to the quantitative study. The structures appraised with
the TEM were processed with the computer to analyse and classify
the data using statistical programs (see below). The structures were
included and preserved sizes according to the original magnication,
with which it was reached. The images obtained by the FEI Quanta 250
FEG electron microscopy (USA) were gathered digitally in a computer
for further study.
Considered usage of the results
Image J
From the micrographs procured in the thin and thick sections,
morphometric magnitudes were done through the Image J software
(National Institutes of Health, Bethesda, MD, USA).
The images obtained from the FEI Quanta 250 FEG equipment
were analysed by using the IMAGE J program, which from the micro-
mark supplied in each image allows carried out morphometric
measurements. From each sampling point, 30 digital images that
generated 40 measurements were obtained at each time of each
treatment (3, 6 and 24 h).
Statistical analysis
The statistics were carried out by ANOVA statistical technique,
which is used to associate a probability, with the notion, that the
mean of a group of scores is different, from the mean of another
group of scores. Through the comparison, it was tested a hypothesis
of difference between more than two groups [6]. To apply the ANOVA
it was used a priori Levene’s tests (Levene’s Test for Homogeneity
of Variances). To corroborate the homogeneity of variance and for
normality, the Kolmogorov-Smirnov test with Lilliefors correction
was used and the histograms were also veried.
To consider the dependent morphometric and other variables
analysed: Capillary endothelium, Capillary lumen, Nucleus area, Nucleus
envelope, Number of mitochondria·µm
-2
, Areal number density of
mitochondria, Mitochondria cristae, Number of lipid inclusions·µm
-2
,
Areal number density of lipids inclusions, Diameter smooth endoplasmic
reticulum (SER), two-way analysis of variance (2-way ANOVA) was used
by Sokal and Rohlf [18, 31]. Two independent variables (2 factors) were
taken in each analysis: the rst “the group” (Control and C-L and the
second “the time” (6,12 and 24 h). For the application of each ANOVA,
the prior assumptions or tests of normality and homogeneity were
corroborated, using a condence of 95% (signicance 0.05). Obtaining
a total of 6 treatments (2 groups with 3 time each). In the cases in which
the ANOVA indicated statistically signicant differences, the posterior
test of Minimal signicant differences (MSD) was used. The results of
these tests indicated in tables 1 to 10 show letters that specify the 6
treatment groups that have statistically signicant differences. These
tables also show the descriptive statistics for each variable (arithmetic
mean and standard error) (STATISTICA V.8.0 Program).
Principal component analysis (PCA)
The PCA was dened by the expressing of a set of variables, in
an arrangement of linear combinations of factors, which were not
correlated among them. This process allowed to symbolise the original
data (individuals and variables), in a space of fewer dimensions than
the original space. The variables, for the quantitative description of
the thickening of the capillary endothelium, area of capillary lumen,
swelling of the perinuclear space, nucleus envelope, number of
mitochondria, Areal number density of mitochondria, number of
mitochondrial cristae, number of lipid inclusions, areal number density
of lipids inclusions and cisternae diameter of the smooth endoplasmic
reticulum (REL) were considered (MVSP V3.0 Program).
RESULTS AND DISCUSSION
Knowing that the effects of snake venom appear in a short time
in the patients and that a high rate of damage is exhibited [27], in
the current work, it was planned to show the damages produced
by the C-L, not only in a specific time, but then by including a
chronological variable, allowing us forming a kinetics to establish
an approximate time, when the cellular alterations obtained by ME
occur, and comparing the variability of the alterations among the
treatments with the toxin, in relation to the time. To achieve the
aforementioned, three intervals were taken into account, at 3, 6 and
24 h post C-L injection. In this way, ultrastructural alterations were
observed throughout the experiment, being represented in different
types of sub-cellular damage. These changes were necessarily due
to the action of C-L during the kinetic study, and were related to
the type of ultrastructure observed and certainly associated to the
symptoms and the pathology generated by the toxin [1].
Having the opportunity and possibility of describing an innovative
study about the ultrastructural alterations that C-L generates on
sub-cellular structures and being able to quantitatively measure them,
the opportunity of observing qualitative collateral studies, related to
the action of C-L on the motor endplate could not be missed.
In recent years, several neuro and myotoxins have been isolated
from snake venoms, and the mechanism of action of their components
has been investigated [2, 3, 8, 29], since crotamine is one of the
important components of Crotalidae venom that combines cytotoxic
and neurotoxic properties
[5], it was carried out this study of the
alterations in the neuro-motor plates produced by the action of
puried C-L as described in results sections.
First of all, it was started with the C-L purication and isolation
from Cdc venom, which was separated by Mono S HR 10/10 cationic
exchange column, which produced 8 well-dened fractions (FIG.1).
The spastic inferior limbs paralysis action (conventional sign of
Crotamine-like actions on subcellular structures / Garcia et al.______________________________________________________________________
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crotamine presence) was noticed in fraction 5, named crotamine-
like (C-L). SDS-polyacrylamide gel electrophoresis (SDS-PAGE)[5] of
C-L fraction (10–20%) under non-reduced conditions showed a single
band of ~3 kilodaltons (kDa) molecular mass (FIG. 2).
The action of crotamine had been studied in skeletal muscle from
a pathological point of view, showing that the toxin, once inoculated
intramuscularly, induced muscle contraction both in vivo (spastic
inferior limbs paralysis) and in vitro [5] experiments. Crotamine,
isolated from Crotalus snake venom act on skeletal muscle cells,
combining cytotoxic and neurotoxic properties
[10, 20].
The ability of crotamine to penetrate the cell membrane varies
among the different isoforms that have been described
[14, 26,
35]. In these works, it was shown that the toxinological action of
crotamine was carried out through nerve tissue voltage gated sodium
and potassium channels [23, 24], in addition to the activation of
sodium influx at muscle cells [4]
; its action on skeletal muscle
has represented the rst snake venom peptide classied as a cell
penetrating peptide and antimicrobial peptide (CPP and AMP), also
demonstrating that crotamine induces skeletal muscle contraction
both in vitro/in vivo, and damages in the motor endplate [24].
In the normal image of the muscle motor endplate were observed
the presence of acetylcholine vesicles in an axonal terminal,
mitochondria with normal ultrastructure and a secondary synaptic
cleft as corresponding to a normal motor endplate structure (FIG. 3.1).
After 3 h post-C-L injection, the micrograph showed a terminal
axon with few acetylcholine vesicles, secondary synaptic clefts.
Mitochondria without cristae were also observed (FIG. 3.2).
After 6 h post-C-L injection, a terminal axon with scarce acetylcholine
vesicles, disordered secondary synaptic cleft, free polysomes and an
enlarged sub-sarcolemma region were noticed (FIG. 3.3).
After 24 h post-CL injection, the micrograph showed: (FIG. 3-4a) oblique
cuts images of muscle bres, with an enlarged sub – sarcolemma space
and a terminal axon with absence of acetylcholine vesicles; secondary
synaptic clefts and morphological altered mitochondria. (FIG. 3-4b) A
terminal axon with cellular debris and absence of synaptic vesicles
were seen. Electrondense mitochondria were also observed (FIGS.
3-4A and 4B). In the FIG. 3, it has also been included an endplate
image scheme. The sequential alterations description produced by
C-L at the level of the motor endplate was possible to appreciate the
signicant reduction in the number of acetylcholine vesicles, which
expresses the severe damage to cellular function. The decrease in
these acetylcholine vesicles, being involved in the contraction and
relaxation processes of the muscle, could be a possible explanation
for the muscular paralysis that the mouse presents a few min after
inoculation with C-L. This neurotoxic action showed that the injected
animals were sensitive to C-L, since they presented spastic paralysis
of the hind limbs, five min after intravenous injection [22]. The
mice also exhibited muscle spasms, likely caused by considerable
neurotransmitter release, changes in ion channels, and inhibition of
vesicular recycling [23]. These authors also proposed that crotamine
acts primarily on mammalian skeletal muscle and secondarily on other
excitable organs, nerve and heart, which are regularly unaffected. This
paralysis presented in the mice was not permanent and disappears
within a few min later. Rodent studies have shown that the motor
nerve terminals [7] and muscles are particularly susceptible to
activities caused by myotoxins
[21].
Others subcellular components detected in the current work, such
as the endoplasmic reticulum (sarcoplasmic reticulum in muscle)
were severely damaged. This organelle represents the largest
intracellular accumulation of calcium in skeletal muscle, and plays
a fundamental role in the regulation of the phenomenon of contraction
and excitation, inducing intracellular calcium concentrations during
muscle contraction and relaxation [9].
On the other hand, it is possible that the depletion or decrease
of the synaptic vesicles from the nervous terminal is not merely a
reection of the improved exocytosis; the increased uidity of the
nerve end membrane aided by crotamine will also inhibit or retard
Crotalus durissus cumanensis
CdcCrotalus
durissus cumanensis
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the recycling of synaptic vesicles. Synaptic transmission begins
when an action potential or an electrochemical impulse reaches
the synaptic button of the presynaptic neuron and depolarises it.
In response, potential-dependent calcium channels open, allowing
extracellular Ca
2++
ions to enter and diffuse into the synaptic button.
The inuence of Ca
2++
causes that many of the presynaptic vesicles,
which store acetylcholine, fuse with the presynaptic membrane
and thus hundreds of molecules of this substance are released by
exocytosis in the cleft or synaptic space [11]. This process stops
quickly because calcium ions are rapidly removed from the cytoplasm
of the synaptic cleft by active transport mechanisms, and are
transferred to the mitochondria, vesicles, or endoplasmic reticulum
[28]; taken together, these results broaden the understanding of the
procedure of action of C-L in tissues; in addition, they release new
points of view for their biomedical application. Toxins that induce
lysosomal cell death, a self-regulating caspase pathway, could be
mostly effective in the treatment of tumour diseases.
Regarding the results of the quantitative study to calculate the C-L
effect on different subcellular structures, the C-L effect on the 10
variables were described: Areal number density of mitochondria;
Number of the cristae; Number of the mitochondria; Area of the
nucleus; Number of Lipid Inclusions; Areal number density of lipids
inclusions; Diameter of cisternae of the smooth endoplasmic reticulum;
Thickening of the capillary endothelium; Capillary lumen and Nuclear
envelope, which with an analysis of variances (ANOVA) of two factors
(95% condence) were investigated. It was found that there were
signicant differences for changes in treatments (factor 1) P=0.00
and signicant difference for the times (factor 2) P=0.00, with an
interaction of P=0.00. The graph shows the tendencies of the means
and standard deviations of the treatments over time. The results in
the tables are shown with the means, standard deviations of each
treatment, obtained in the study of the same variables, and letters
that indicate the statistically signicant differences derived from a
posteriori test of minimum signicant difference (MSD) (FIG. 4 to 14
and TABLES 1 to 10).
This analysis permitted, to make clear the sub-cellular alterations
developed during a follow out of C-L toxic action from 3 to 24 h. The
alterations were quantied by a morphometric study documenting
that the C-L action, at three different intervals had statistical meaning.
Modications of the endothelium showed an increase at 6 and 24
h compared with the normal control. Alterations of the endothelium
are constantly complicated with the occurrence of haemorrhage after
snake venom accidents [27]. In a different variable, the capillary lumen
at six h after C-L injection showed a trivial increase, which was not
statistically signicant matched with the control. Although at 24 h,
a kinetic of temporary increase related with a possible dilatation of
FIGURE 3. (1) Motor Endplate Normal Control and Endplate Scheme. The presence of acetylcholine vesicles can be observed (circle), it is a normal ultrastructure
of axonal terminal. Mitochondria with normal cristae was seen (star); secondary synaptic cleft (arrowhead) accordingly to a motor endplate. Micromark = 1
μm. (2) Motor Endplate after 3 hours post C-L injection. The micrograph shows a terminal axon with few acetylcholine vesicles (arrow), secondary synaptic
clefts (oval) and mitochondria without cristae (star) were observed Micromark = 1 μm. (3) Motor Endplate after 6 h post-C-L Injection. A terminal axon with
scarce acetylcholine vesicles (arrow), disordered secondary synaptic cleft (oval); free polysomes (Po) and an enlarged sub-sarcolemma region (circle)
were noticed. Micromark = 1 μm. (4) Motor Endplate after 24 h post-CL injection. The micrograph shows: (4a) oblique cuts images of muscle bres, with
an enlarged sub – sarcolemma space (circle) and a terminal axon with absence of acetylcholine vesicles (triangle); secondary synaptic clefts (oval) and
altered mitochondria (star) were visualised. (4b) A terminal axon with cellular debris (triangle) and absence of synaptic vesicles was seen. Electrondense
mitochondria (star) were observed Micromark = 1 μm
Crotamine-like actions on subcellular structures / Garcia et al.______________________________________________________________________
6 of 13
the capillary lumen respect to the control was detected. The changes
of the endothelium could explain the augment of the capillary lumen
at 24 h after C-L injection.
On the subject of nucleus area, at 3 and 6 h after C-L injection
was not remarkably affected since a not statistically signicant
decrease was seen, but at 24 h the nucleus area decreased nearly
50%. In the nucleus are the chromosomes and the genetic material.
Therefore, the cell regulation of the production of enzymatic proteins
using messenger RNA (mRNA), which transport the ribosomal RNA
information to the cytoplasm. Crotamine-like may induce nuclear
apoptosis over several ways, for instance collaboration with lysosomes
to activate intracellular cell Ca
2+
for only a short time; variation of
mitochondrial membrane potential and activation of enzymes,
specically caspases, which modify the typical perform of sub-cellular
assemblies and directing to cell death [16, 30]. In the observation
of the number of mitochondria, a slight variation, which was not
statistically signicant was detected at 3 and 6 h; but, at 24 h, a
strong decrease was seen. The mitochondria foremost function is
to deliver the required energy to accomplish cellular respiration [37].
Concerning the areal number density of mitochondria alterations
at 6 and 24 h after C-L injection an intense increase in the area was
noticed, which was statistically signicant compared with the control.
Increases in metabolic activity during the rst post injection should
be accompanied by increases in the number of mitochondria, since
mitochondria play an important role in the synthesis of cortical
steroids, including cortisol, a hormone that increases in stress. It is
considered the mitochondria as a valuable organelle that has severe
ultrastructural changes that appears after the C-L injection.
Crotamine-like (C-L)
3 40 0.327 ± 0.012
c
6 40 0.653 ± 0.020
b
24 40 0.733 ± 0.019
a
Sub-total (C-L)
120 0.571 ± 0.207
Normal control (N-C)
3 40 0.305 ± 0.013
c
6 40 0.308 ± 0.014
c
24 40 0.288 ± 0.013
c
Sub-total (N-C)
120 0.300 ± 0.008
Total measurements 240
h: hours; n: number; SE: Standard error;
abc
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
TABLE I
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TABLE II
TABLE III
Crotamine-like (C-L)
3 40 8.600 ± 0.167
c
6 40 9.125 ± 0.193
b
24 40 10.550 ± 0.179
a
Sub-total (C-L)
120 9.425 ± 1.400
Normal control (N-C)
3 40 8.575 ± 0.079
c
6 40 8.475 ± 0.080
c
24 40 8.775 ± 0.131
c
Sub-total (N-C)
120 8.608 ± 0.058
Total measurements 240
h: hours; n: number; SE: Standard error;
abc
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
Crotamine-like (C-L)
3 40 15.725 ± 0.268
b
6 40 13.775 ± 0.698
c
24 40 7.100 ± 0.479
a
Sub-total (C-L)
120 12.200 ± 4.909
Normal control (N-C)
3 40 17.275 ± 0.397
a
6 40 16.925 ± 0.361
a
24 40 17.275 ± 0.424
a
Sub-total (N-C)
120 17.158 ± 0.226
Total measurements 240
h: hours; n: number; SE: Standard error;
abc
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
Crotamine-like actions on subcellular structures / Garcia et al.______________________________________________________________________
8 of 13
The number of mitochondrial cristae (FIG.10) under C-L effects
showed a decrease at 6 and 24 h, which was statistically signicant
compared with the control. At 24 h an intense decrease in their number
was noticed. This organelle is in charge of numerous chemical cellular
reactions, such as cellular respiration, protein and electron transport
and oxidative phosphorylation. Its cristae outline a membranous
system that links with the internal mitochondria membrane in diverse
segments facilitating the transport of metabolites and organic
compounds to various parts of the organelle [28].
Crotamine-like (C-L)
3 40 13.975 ± 0.288
c
6 40 15.500 ± 0.139
b
24 40 18.800 ± 0.224
a
Sub-total (C-L)
120 16.092 ± 2.467
Normal control (N-C)
3 40 14.100 ± 0.128
c
6 40 14.000 ± 0.139
c
24 40 14.100 ± 0.138
c
Sub-total (N-C)
120 14.067 ± 0.077
Total measurements 240
h: hours; n: number; SE: Standard error;
abc
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
Crotamine-like (C-L)
3 40 1.525 ± 0.080
ab
6 40 1.275 ± 0.101
b
24 40 0.325 ± 0.075
c
Sub-total (C-L)
120 1.042 ± 0.749
Normal control (N-C)
3 40 1.750 ± 0.117
a
6 40 1.775 ± 0.121
a
24 40 1.775 ± 0.127
a
Sub-total (N-C)
120 1.767 ± 0.070
Total measurements 240
h: hours; n: number; SE: Standard error;
abc
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
Crotamine-like (C-L)
3 40 0.151 ± 0.010
c
6 40 0.346 ± 0.018
b
24 40 0.439 ± 0.031
a
Sub-total (C-L)
120 0.312 ± 0.180
Normal control (N-C)
3 40 0.142 ± 0.009
c
6 40 0.127 ± 0.008
c
24 40 0.141 ± 0.009
c
Sub-total (N-C)
120 0.136 ± 0.005
Total measurements 240
h: hours; n: number; SE: Standard error;
abc
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
TABLE IV
TABLE V
TABLE VI
_________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32119, 1 - 15
9 of 13
The number of lipid inclusions under C-L effects exhibited a
decrease at 6 and 24 h (FIG. 11), which was statistically signicant
compared with the control. At 24 h an intense decrease in their
number was observed. When observing the sequential changes on
lipids, this decrease in the number of lipid inclusions of the studied
cells could be explained by their utilisation, reected by the increase
in cortical activity under stress, which must begin during the rst h
post-injection of C-L.
On the other hand, there was an increase in its areal number density of
lipids inclusions after 6 h post C-L injection (FIG. 12), with high increase
at 24 h. At rst glance, the results obtained in both experiments could
be contrasting, however, the decrease in the number of lipid inclusions
and the increase in their areal number density are not necessarily
correlative data [19]; these authors investigating Cushing syndrome,
which is a hormonal disorder caused by prolonged exposure to an
excess of cortisol (hormone produced by the adrenal glands)[15] explain
that the increase in the area of lipid inclusions should be due to the
increase in the activity of related enzymes in the uptake of low-density
lipoprotein (LDL)-cholesterol, which could explain the data obtained
regarding the alterations produced in lipid inclusions [34].
Crotamine-like (C-L)
3 40 28.375 ± 0.751
a
6 40 14.850 ± 0.944
b
24 40 3.950 ± 0.443
c
Sub-total (C-L)
120 15.725 ± 11.059
Normal control (N-C)
3 40 27.459 ± 0.736
a
6 40 26.211 ± 1.129
a
24 40 26.617 ± 0.912
a
Sub-total (N-C)
120 26.762 ± 0.540
Total measurements 240
h: hours; n: number; SE: Standard error;
abc
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
Crotamine-like (C-L)
3 40 0.428 ± 0.011
a
6 40 0.320 ± 0.012
b
24 40 0.167 ± 0.014
c
Sub-total (C-L)
120 0.305 ± 0.132
Normal control (N-C)
3 40 0.458 ± 0.008
a
6 40 0.445 ± 0.008
a
24 40 0.533 ± 0.089
a
Sub-total (N-C)
120 0.478 ± 0.030
Total measurements 240
h: hours; n: number; SE: Standard error;
abc
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
TABLE VII
TABLE VIII
Crotamine-like actions on subcellular structures / Garcia et al.______________________________________________________________________
10 of 13
Given the large presence of electrondense lipids in the results
observed, it was possible to assume that there was a disorganisation
in the activity of the smooth endoplasmic reticulum (SER), and based
on this assumption it was focused on a more detailed study of SER.
SER covers approximately 80% of some glandular cells, another
reason for the importance of its observation during C-L action [36].
The diameter of the SER cisternae was dramatically increased from
the early 3 h, reaching its maximum at 24 h (FIG. 13). This subcellular
structure is responsible for the synthesis of molecules and the
transport of substances. One of its functions is to deliver the proteins
synthesised to the Golgi apparatus, which will transform and send
them to the rest of the organisms. The C-L produced a strong reduction
of the SER (data not shown), but the diameter of the cisternae was
intensely increased at 6 and 24 h after C-L injection. Soulsby’s work [32]
indicated that the swelling of the SER cisternae was not necessarily
related to increased activity. It should be remembered that the C-L
could involve a nal mitochondrial degeneration process, as seen
previously in the mitochondrial alterations, this would compromise
the production of energy (ATP), modifying the active transport of ions
of the cell membrane and the sodium-potassium pump [17].
TABLE IX
TABLE X
Crotamine-like (C-L)
3 40 0.460 ± 0.006
d
6 40 0.647 ± 0.010
b
24 40 0.800 ± 0.009
a
Sub-total (C-L)
120 0.636 ± 0.149
Normal control (N-C)
3 40 0.489 ± 0.001
c
6 40 0.489 ± 0.001
c
24 40 0.490 ± 0.001
c
Sub-total (N-C)
120 0.489 ± 0.001
Total measurements 240
h: hours; n: number; SE: Standard error;
abcd
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
Crotamine-like (C-L)
3 40 0.460 ± 0.006
d
6 40 0.647 ± 0.010
b
24 40 0.800 ± 0.009
a
Sub-total (C-L)
120 0.636 ± 0.149
Normal control (N-C)
3 40 0.489 ± 0.001
c
6 40 0.489 ± 0.001
c
24 40 0.490 ± 0.001
c
Sub-total (N-C)
120 0.489 ± 0.001
Total measurements 240
h: hours; n: number; SE: Standard error;
abcd
Means with different
superscripts dier according to the test of minimal signicant dierence
(MSD) (P<0.05)
The release of Ca
2++
and the increase in its intra-mitochondrial
concentration, further inhibit oxidative phosphorylation, consequently
increasing anaerobic glycolysis and the accumulation of lactic acid
in the cytoplasm, whereby the pH falls, thus multiplying the damage
in the membranes and increased permeability.
PCA consisted of expressing a collection of variables in a set of
linear combinations of factors not correlated with each other. This
method allowed the original data (individuals and variables) to be
represented in a space with a dimension less than the original space.
The analysis has great value for studies involving different variables
[12] because it manages to create a multidimensional space where
in this case it combines the 10 morphometric variables studied.
This representation of 10 variables simultaneously analysed can
be synthesised in a two-dimensional representation (FIG. 14) that
comes from a true multidimensional graph; in the present case, the
two-dimensional representation has significance because it has
95.85% of the total variation of the multinational system (TABLE XI).
In it, the differences of C-L to control were clearly observed, but unlike
the other analyses that lead us to the same conclusion variable by
variable, in this PCA analysis it is possible to observe the reality of
95.85% of the phenomenon with the 10 variables studied and the
effect of C-L in only 1 graph. The graphical representation of the PCA
results and representation of different variables were shown in FIG. 14.
_________________________________________________________________________Revista Cientica, FCV-LUZ / Vol. XXXII, rcfcv-e32119, 1 - 15
11 of 13
TABLE XII
TABLE XI
Principal Component Analysis (variables correlations)
Auto-values 9.089 0.496
Percentage 90.892 4.962
Accumulated percentage 90.892 95.854
The principal component 1 (PC1); X axis of FIG. 14 describes 90.89%
of the information (variable system) and the principal component 2
(PC2), Y axis; 4.9% of this information (TABLE XI). The graph express
95.85% of the variables, which is considered satisfactory.
The correlated variables are shown (TABLE XII). Thus. it is observed
that there is a high positive correlation between the areal number
density of mitochondria, the areal number density of lipids inclusions,
the diameter of the cisternae of the smooth endoplasmic reticulum, the
thickening of the capillary endothelium, the lumen of the capillary and
the nuclear envelope on Axis 1; and positive correlation between the
number of mitochondria, number of lipid inclusions, thickening of the
capillary endothelium, capillary lumen and nuclear envelope on Axis 2
Areal number density of mitochondria (Mit-A) 0.325 -0.106
Cristae number (Mit-NC) -0.328 -0.032
Mitochondria number (Mit) -0.319 0.175
Nucleus Area (N-A) -0.322 -0.208
Lipid Inclusions Number (Lip-NI) -0.287 0.658
Areal number density of lipids inclusions (Lip-A) 0.322 -0.257
Diameter of the Smooth Endoplasmic
Reticulum Cisternae (SERcd)
0.325 -0.075
Capillary Endothelium Thickening (Ce) 0.315 0.12
Capillary lumen (CL) 0.301 0.494
Nuclear Envelope (Ne) 0.316 0.388
CONCLUSIONS
In summary, this work point toward describing the quantitative
and qualitative expressions of the general ultrastructural variations
happening in the subcellular components of cells of envenomed mice,
highlighting the crotamine-like action on the above mentioned cellular
vital structures related to the cellular function, which play an essential
protagonist role in the homeostasis and physiological regulation of the
human body. In closing stages, even though muscle necrosis and/or
apoptosis is the mainly substantial pathological problem associated
with small myotoxins, such as crotamine, the search put forward that
the medical impact of this molecule is related to their ability to cause
intense damages at subcellular level, subject to their pharmacokinetic
properties. In view of the above, it was concluded that the injection
of C-L in mice induced acute inammatory damages.
This toxin exhibited different sub-cellular targets, already talk over in
the discussion as well as a number of activities, including neurotoxicity
and myotoxicity. Its myotoxic capability feasibly is mainly related to
the electrophysiological changes in sodium and potassium channels,
changes in mitochondrial calcium homeostasis, alteration in the
SER and degeneration of myobrils, with resulting structural injury
to muscle cells. Furthermore, investigations have revealed that the
process of action of crotamine is not limited to the muscle tissue, if not,
as it has seen in this work it involves other tissues, mainly adrenal gland,
liver and kidneys. The cytotoxic effects of C-L have been demonstrated
in vivo and in vitro employing adrenal gland cells and muscular motor
endplate permitting the study of the mechanisms by which the C-L can
modify cellular homeostasis by making injury to sub-cellular organelles
such as mitochondria, SER, nucleus, and so on.
AUTHOR CONTRIBUTIONS
All experiments were performed at the Centro de Microscopia
Electrónica, Facultad de Ciencias de la Universidad Central de
Venezuela and the Laboratorio de Inmunoquímica y Ultraestructura,
Instituto Anatómico de la Universidad Central de Venezuela, Caracas,
Crotamine-like actions on subcellular structures / Garcia et al.______________________________________________________________________
12 of 13
Venezuela. HJF, RG and ARA designed the study. EGL, RG and ARA
performed the experiments. HJF, RG, EGL and ARA analysed and
interpreted the data. EGL and ARA wrote the draft of the manuscript.
All authors reviewed the manuscript and approved the nal version
for publication.
†During the evaluation process of this paper, Prof. Héctor. J. Finol
passed away. Prof. Finol taught us his philosophical aspect of how to
approach the study of cellular ultrastructure, in addition the devotion
to his disciples and his general righteousness.
FINANCIAL SUPPORT (FUNDING)
The research was partially funding by grant from the Consejo de
Desarrollo Cientíco y Humanístico de la Universidad Central de
Venezuela (# PG: 09-8760-2013). Caracas. Venezuela.
ETHICAL STATEMENT
The project was accepted by the Institute of Anatomy Ethical
Committee of the Universidad Central de Venezuela (April 26, 2019),
under certication number: # 260420, carried out by the norms
from the ARRIVE (EU Directive 2010/63/EU) for animal experiments
guidelines, in agreement with the U.K. Animals (Scientic Procedures)
Act, 1986 and associated guidelines.
DECLARATION OF COMPETING INTEREST
The people responsible for the completion of this paper declare
that they have no competing nancial interests to inuence the work
reported here. The funders had no responsibility in the plan of the
study; in the assembly, scrutinises, or analysis of data; in the writing
of the document, or in the resolution to publish the results
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