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The mutagenic effects of ivermectin in germinal cells and serum protein of the mouse

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Ivermectin (IVM) is a broad-spectrum anti-parasite agent. It is extremely toxic to fish and aquatic life. Some animals showed reduction in the fertility, the number of variable fetuses and sperm count following treatment with (IVM). Therefore, the objective of the current work was to investigate the mutagenicity of IVM on meiotic chromosomes of mice. The variations in protein fractions of blood serum were also studied using sodium Dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).Animals received single injections only of 200ug/kg b.wt. for meiotic chromosome study. Whereas single and double treatment for serum protein examinations. Analysis of the treated samples revealed significant increase in meiotic aberrations, 33.83% vs 5.8% for the control (P < 0.001). Single injection induced much variation in the percentage area of the separated protein than that produced by double treatment. These findings supports the mutagenicity of IVM, accordingly cautious use of IVM is advisable. © 2019 Pakistan Journal of Pharmaceutical Sciences. All Rights Reserved.
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Pak. J. Pharm. Sci., Vol.32, No.2, March 2019, pp.637-646
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The mutagenic effects of ivermectin in germinal cells and serum
protein of the mouse
Karima Mohammad Sweify, Iman Abd El Moneim Darwish*
and Dalia Demerdash Abd El Monem Hafez
Women′s College for Arts, Science and Education, Ain Shams University, Cairo, Egypt
Abstract: Ivermectin (IVM) is a broad-spectrum anti-parasite agent. It is extremely toxic to fish and aquatic life. Some
animals showed reduction in the fertility, the number of variable fetuses and sperm count following treatment with
(IVM). Therefore, the objective of the current work was to investigate the mutagenicity of IVM on meiotic chromosomes
of mice. The variations in protein fractions of blood serum were also studied using sodium Dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE).Animals received single injections only of 200ug/kg b.wt. for meiotic
chromosome study. Whereas single and double treatment for serum protein examinations. Analysis of the treated samples
revealed significant increase in meiotic aberrations, 33.83% vs 5.8% for the control (P < 0.001). Single injection induced
much variation in the percentage area of the separated protein than that produced by double treatment. These findings
supports the mutagenicity of IVM, accordingly cautious use of IVM is advisable.
Keywords: Ivermectin (IVM), meiotic preparations; quadrivalent chain (CIV), quadrivalent ring (RIV), Autosome
univalent (A-U), hexavalent chain (CVI), sex univalent (X-Y U), Sodium Dodecyl Sulphate Polyacrylamide Gel
Electrophoresis (SDS-PAGE), percentage area (% area).
INTRODUCTION
Ivermectin is an antiparasitic drug, a derivate of
avermectins, and a product of fermentation of an
actinomycete, Streptomyces avermitilis. Ivermectin is
presently used in mass treatment of onchocerciasis, other
filariasis, some intestinal nematode infections, but also in
scabies, and more rarely in resistant head lice (Chosidow
and Gendrel, 2016). It is used extensively globally for
treatment of helminthic and ectoparasitic infections in
animals and humans (Derua et al., 2016).
Single treatment, whether topical or oral, is associated
with high cure rate in a week post IVM treatment.
However, repeating treatment after one week may be
required to achieve 100% cure (Ahmad et al., 2016).
Ivermectin was effective, safe and well tolerated (Ali et
al., 2015). New uses for IVM are identified regularly,
including possible antibacterial, antiviral, and anticancer
potential (Omura and Crump, 2014).
Ivermectin is found effective when administered through
oral and subcutaneous routes (Panigrahi et al., 2016). The
synergistic effect of combinations of antibiotics and
ivermectin could be used to achieve complete eradication
of lice (Sangaré et al., 2016). Two applications of topical
ivermectin provided a cure rate of 63.1% at the 2-week
follow-up, which increased to 84.2% at the 4-week
follow-up after repeating the treatment (Goldust et al.,
2013). A single application of ivermectin was more
effective than vehicle control in eliminating head-louse
infestations at 1, 7, and 14 days after treatment (Pariser et
al., 2012). A 200 microg/kg/d ivermectin dose was an
adequate therapeutic regimen in the treatment of
uncomplicated strongyloidiasis in children (Ordónez and
Angulo, 2004).
IVM induced higher mortality, reduced fecundity, feeding
difficulty, and incomplete ecdysis (Sheele and Ridge,
2016) and caused population decline and biodiversity loss
(Verdú et al., 2015). Dominant clinical symptoms of
adverse effects and toxicity of ivermectin in animals are
tremor, ataxia, central nervous system (CNS) depression
and coma which often results in mortality (Trailovic et al.,
2011) and caused renal tubular necrosis (DeMarco et al.,
2002). In addition, reports on adverse events are rare
although this drug can cause cardiac dysfunction and liver
disease (Sparsa et al., 2006), dizziness (Nontasut et al.,
2005) and encephalopathy in individuals heavily infected
with Loa loa microfilariae who were treated with
ivermectin against onchocerciasis (Esum et al., 2001).
On the other hand, Dou et al. (2016) indicated that
ivermectin is a potential option of the treatment of breast
cancer. Mangia et al. (2016) reported transporter proteins
(P-glycoproteins, PgP) expression of an Ixodes ricinus-
derived tick cell line following treatment of ivermectin.
Raza et al. (2016) noticed significant increase in
transcription of some ATP binding cassette (ABC)
transporter genes following 3 h exposure to both
ivermectin (IVM) and levamisole (LEV) in the resistant
isolate only. Molinari et al. (2013) observed a time-
dependent increase in ivermectin (IVM) and IVM-
*Corresponding author: e-mail: imandarwish73@yahoo.com
The mutagenic effects of ivermectin in germinal cells and serum protein of the mouse
Pak. J. Pharm. Sci., Vol.32, No.2, March 2019, pp.637-646
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containing technical formulation Ivomec® (IVO; 1%
IVM)-induced DNA damage.
Furthermore, co-administration of both ivermectin and
verapamil induced genotoxicity in both dam and
embryonic cells indicated by reduced mitotic index,
increased number of micro nucleated erythrocytes in both,
and increased different types of chromosomal aberrations
in dam cells, while ivermectin alone showed some
genotoxic effect as mentioned by el-Ashmawy et al.
(2011). Varó et al. (2010) showed significant changes in
the expression of 36 proteins in gilthead sea bream liver
protein profile following oral administration of IVM by
difference Gel Electrophoresis Technology (DIGE). While
IVM and ivomec did not modified SCE frequencies, they
induced DNA-strand break revealed by single cell gel
electrophoresis (SCGE) as indicated by Molinari et al.
(2010).
The present communication is dealing with the hazardous
effect of IVM in germ cells through chromosomal
aberration test. Electrophoresis analysis of serum protein
was also done by using SDS-PAGE.
MATERIALS AND METHODS
Chemicals and solutions
Ivermectin was used in the form of Bomectin injection
(Bomac-Laboratories LTD). The recommended dose is
200ug/kg b.w. (Chouela et al., 2002).
Experimental animals
The Swiss albino male mice (Mus musculus) aged 9-12
weeks were used in all experiments. They were supplied
by Abbasia Farm of the Egyptian Organization for
Vaccine and Biological Preparations. They were supplied
with standard laboratory chow and tap water. Mice were
allowed to acclimate for at least one week prior to the
study.
Animals were divided into main groups. The first group,
which acted as control, contained 6 animals injected with
sterile distilled water. The second group of animals (6
mice) was injected intraperitoneally with single doses of
200ug/kg ivermectin. Meiotic chromosomes were
prepared after 12 days to cover preleptotene stage as
recommended by Ciranni and Adler (1991).
Preparation of mammalian meiotic chromosomes
Chromosomal preparations were made according to the
method described by Evans et al. (1964). Male albino
mice were injected i.p. with 0.1 ml of colchicine solution,
2 hours before sacrificing them by cervical dislocation.
Testes were removed and placed in isotonic sodium citrate
solution (2.2% wt/vol) at room temperature. Tunica was
pierced and testes were swirled in the solution to remove
adherent fat. Then testes were transferred into fresh 2.2%
sodium citrate solution and the tubules were gently pulled
out. The mass of tubules was held with fine, straight
forceps and their contents were teased out with similarly
fine, but curved forceps. When the tubules appeared flat
and opaque they have been allowed to settle, then the
supernatant fluid was transferred into centrifuge tubes.
After centrifugation at 500 r.p.m. for 5 minutes, the
supernatant fluid was discarded and the sedimented cells
were resuspended in approximately 3 ml of 1% sodium
citrate solution (hypotonic), and left for 12 minutes at
room temperature.
After centrifugation as much as possible, the supernatant
fluid was removed. Cells were resuspended in the
remainder by flicking the tube with a thumb till a thin
film of suspension was formed. Cells were fixed by
adding about 0.25 ml of fixative (3 methanol: 1 acetic
acid) rapidly, and directly on to the suspended cells. More
fixative was added, until the tube was about one-third full.
After 5 minutes, cells were sedimented again by
centrifugation and were resuspended in fresh fixative. The
change of fixative was repeated after further 10 minutes.
The final suspension was prepared in a 0.5 ml of fixative.
Few drops of this suspension were allowed to fall on a
grease free slide at room temperature. After drying, slides
were stained for 5 minutes in Giemsa.
Scoring criteria
One hundred MI spermatocytes were scored per animal.
Metaphase spreads were selected at low magnification (20
x) on the basis of morphological criteria. Numerical or
structural abnormalities were verified at high
magnification (100x).
For structural aberration analysis (MI): Only MI with 20
bivalents was scored. Reciprocal translocations as a chain
or ring multivalents were classified. The presence of
univalents (autosomal univalents and/or sex univalents) or
chromosome breaks and fragment were recorded.
Serum protein electrophoresis
Eighteen male mice were injected with single dose of
200ug/kg IVM. After two weeks, nine of the animals
received another dose of IVM. Blood were collected from
each group after 1, 7, and 14 days of the last injection.
Blood collection for protein electrophoresis
Blood serum of the treated animals and accompanied
control (three for each duration) were prepared. The
electrophoretic patterns of serum protein were studied by
using SDS-PAGE which proposed by Laemmli (1970).
Blood samples were collected from eye plexus by
nonheparinized haematocrit tubes into eppendorf tubes.
The eppendorf tubes were put in the refrigerator for 1
hour of clotting then centrifuged at about 3000 r.p.m. for
10 minutes. The supernatant serum was transferred into a
Karima Mohammad Sweify et al
Pak. J. Pharm. Sci., Vol.32, No.2, March 2019, pp.637-646
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new clean eppendorf tube using Pasteur pipette. The tubes
were kept in the deep freezer at -20oC till analysis
Polyacrylamide gel electrophoresis (SDS-PAGE) for
serum protein analysis
Serum protein analysis of control and treated mice was
done using SDS-PAGE under reducing conditions by
using of B-mercaptoethanol according to the method of
Laemmli (1970) with some modifications.
Sample preparation and the separation process
Serum was diluted with distilled water (1:3 v/v,
respectively), i.e. 120ul distilled water was added to 40 ul
serum. Then, the diluted serum was combined with an
equal volume of treatment buffer (160ul) in eppendorf
tubes. Eppendorf tubes were put in boiling water for (5-
10) min, the samples were cooled at room temperature
and 10ul from each sample was applied to the gel wells.
Samples were stored frozen for more runs.
Once samples had been placed in the wells, the apparatus
was connected to a power supply and a current was
applied. The run was carried out with constant voltage of
100 volts at the stacking gel and 150v at the separating
gel. When the tracking dye band was within 2-3 mm of
the bottom of the gel, migration was stopped by turning of
the electrical field.
Processing the gel (staining, destaining and preserving):
Gel was removed carefully from the glass sandwich. It
was necessary to loosen the gel by spraying water around
the edges of the gel with a plastic pipette. Gel was
covered with staining solution, sealed in plastic box and
left in stain for 30 minutes at 60oC.
Destaining
After staining gel was transferred into destaining solution,
the solution was changed several times till the background
appeared clear. Then it placed in final destaining solution.
Preserving
After destaining, gel was transferred into plastic box
contained preserving solution and left for 15 minutes at
50oC. This solution helps to keep the gels flexible and
resistant to cracking.
Analysis of gel lanes
Analysis of gel lanes was carried out using computer
software program (Gel-Pro Analyzer version 3.1).
Ethical approval
All experiments were conducted according to the protocol
approved by Faculty of Medicine Ethics Committee
(MFEC), Ain Shams University, Egypt.
STATISTICAL ANALYSIS
Chromosome aberration frequencies and polyploidy
frequencies in treated and control animals were compared
by Student-t-test.
RESULTS
Chromosomal studies in germ cells
Control samples
Most of the counted dividing spermatocytes showed 20
bivalents constituent (19 II + XY) (fig.1). This fig.
represents the normal structure of diakinesis metaphase I
cell of the mouse spermatocytes. Such structure could be
speculated in about 94.17% of the examined cells (table
1).
Fig. 1: 1ry spermatocytes at diakinesis-metaphase I
showing
a-The twenty bivalents of control samples arrows showing the
attached sex chromosomes
b-17 bivalents with univalent formation of auto somal
chromosomes and the quadrivalents tanslocation.
c-18 bivalents with CIII + I of auto somes
d-19 bivalents with univalent formation of sex chromosomes (x-
y). e-Multivalent configuration (15 bivalents + CVI + A-U + x-y
u) configuration
f- 17 bivalents with univalent formation of auto somes and CIV
The mutagenic effects of ivermectin in germinal cells and serum protein of the mouse
Pak. J. Pharm. Sci., Vol.32, No.2, March 2019, pp.637-646
640
This union results by the homologous joined to each other
largely at their terminal ends. Each metaphase spread
consists of 20 pairs of chromosomes. Each pair of
homologous chromosomes known as bivalents. The
autosomes pairs regularly forming 19 identical bivalents;
the sex chromosomes associated together with the
noncentromeric ends. The auto some bivalents acquire a
more rounded shape of termination of chiasmats as the
chromosomes shorten.
A= Hypoploid metaphase with 19 chromosomes
B= Hyperploid metaphase with 25 chromosomes
C= polyploidy.
Fig. 2: a-c Primary spermatocytes at metaphase I
illustrating
On the other hand, the abnormal metaphases in the control
samples constitute 5.83% of the examined cells. The
observed aberrations are almost translocations in addition
to low frequency of autosome and sex univalent.
The different types of chromosomes damage which may
find in diakinesis metaphase I could be mentioned
according the description of Leonard (1975): First of all,
the term of reciprocal translocation creates exchanges the
terminal segments between non-homologous
chromosomes. At meiotic synapsis heterozygosity for a
reciprocal translocation results from production of A
quadrivalent configuration in form of a ring (RIV). This
occurs when the noncentromeric ends of the
chromosomes maintain association with terminalyzing
chiasmata.
A chain of four fig. (CIV) by failure of association with
chromosomes in one arm. A chain of three plus a
univalent fig. (CIII + 1), by failure of association of two
adjacent arms. In addition, more complex configurations
can be observed. It was found that sex univalent and the
chain of four figs. (CIV) were frequently observed in the
control samples. Autosome univalent and chain of
hexavalents (CVI) or a quadrivalents ring (RIV) were
rather rare.
Effects of ivermectin on spermatocyte chromosomes:
Structural aberrations: Cytological analysis of diakinesis-
metaphase I spermatocytes of the treated samples
revealed significant increase in chromosome aberrations
over the control values. Single i.p. injections of 200ug/kg
b.w. ivermectin caused 33.83% of abnormal metaphases.
The detected types of aberrations can be arranged in a
descending manner as, sex univalents, CIV, and autosome
univalents. Sex univalents observed in 66 metaphases out
of 600, with a percentage of11, fig. (1). Chain of four
(CIV) occurred in 56 of 11 cells that is equal 9.3%.
Autosome univalents found in 6% of the scored cells.
Other types of aberrations, autosome plus sex univalents,
ring of four (RIV), chain of six (CVI) and fragments were
rather rare (table 1).
Fig. 3: Gel represent protein pattern of Mus musculus
1day after treatment with single dose of ivermectin. Lanes
from left to right respectively: Lane 1: High molecular
weight marker. Lane 2: Control. Lanes (3-8): Treated
animals. Lane 9: Control.
Fig. 4: Gel represent protein pattern of Mus musculus 7
days after treatment with single dose of ivermectin. Lanes
from left to right respectively: Lane 1: Low molecular
weight marker. Lane 2: High molecular weight marker.
Lane 3: Control Lanes (4-9): Treated animals.
It is of interest to mention that, some of the examined
metaphases were found to have multiple reciprocal
translocations (fig. 1). In addition, polyploidy was also
observed in 3% of the counted cells (fig. 2).
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Pak. J. Pharm. Sci., Vol.32, No.2, March 2019, pp.637-646
641
Electrophoretic patterns of serum protein in Mus
musculus
In the present work, the effect of ivermectin was observed
as changes in the number of bands of various serum
proteins.
Scoring of SDS-PAGE gel of mice sera proteins in control
series revealed the presence of 26 to 32 bands while they
were 28: 33 in samples obtained from the treated mice.
The majority of the control bands matched that of the
treated. However, some newly formed bands were
observed in the experimental samples (table 2). From the
table, it is clear that the number of the newly formed
bands are 1, 6, 1 in mice received single dose of
ivermectin; they are 3, 1, 2 in samples obtained after two
injections.
Fig. 5: Gel represent protein pattern of Mus musculus 14
days after treatment with single dose of ivermectin. Lanes
from left to right respectively: Lane 1: Low molecular
weight marker. Lane 2: High molecular weight marker.
Lane 3: Control. Lanes (4-9): Treated animals.
Fig. 6: Gel represent protein pattern of Mus musculus 1
day after treatment with repeated dose of ivermectin.
Lanes from left to right respectively: Lane 1: Low
molecular weight marker. Lane 2: Control. Lanes (3-9):
Treated animals.
Concerning the relative mobility (RF), no significant
alterations
were recorded in almost all of the detected fractions
comparing to the control values (figs. 3-8). The % area
showed significant increase and / or decrease due IVM
therapy while most of the protein showed non-significant
alteration (tables 3), eg: after one day of single IVM
dosing, significant increase was observed in the % area of
5 bands (peak number (PK)) 16, 18, 19, 22 and 29 ( data
not shown). Whereas 6 bands showed significant decrease
in their % areas, PK numbers, 7, 8, 9, 10, 14 and 25. Non-
significant variations were observed in a number of 18
bands. Generally, single injection of IVM induced much
variation in % areas of the separated proteins than that
produced by the double treatment. In samples prepared
after 7 days of single dosing, significant decrease in the
area percentage was appeared in 7 bands. The average
area percentage of serum belonging to animals received
single dose of IVM was statistically increased from one
bands after 14 days comparing to that of the accompanied
control (P<0.01). Remarkable reduction in the average
area percentage was occurred after 1 day of double
injections (7 bands). Significant increase in area
percentages was recorded in three bands after 14 days of
repeated injections. In samples studied after 1, 7, & 14
days of repeated dosing, 20, 23 & 25 bands were found.
Their area percentages were not statistically differing
comparing to the control values.
Fig. 7: Gel represent protein pattern of Mus musculus 7
days after treatment with repeated dose of ivermectin.
Lanes from left to right respectively: Lane 1: Low
molecular weight marker. Lane 2: High molecular weight
marker. Lane 3: Control. Lanes (4-9): Treated animals.
Fig. 8: Gel represent protein pattern of Mus musculus 14
days after treatment with repeated dose of ivermectin.
Lanes from left to right respectively: Lane 1: Low
molecular weight marker. Lane 2: High molecular weight
marker. Lane 3: Control. Lanes (4-9): Treated animals.
DISCUSSION
Ivermectin is a semi-synthetic lactone drug that exhibits
broad anti-helminthic specificity (Goa et al., 1991). Over
the past several years, severe adverse reactions to
ivermectin treatment have been reported on individuals
residing in onchocerciasis endemic areas that are also
endemic for Loa loa (Boussinesq et al., 2003).
The mutagenic effects of ivermectin in germinal cells and serum protein of the mouse
Pak. J. Pharm. Sci., Vol.32, No.2, March 2019, pp.637-646
642
In this study, the aberrant metaphases in control sample
represented in 5.83% of the examined cells. Sex univalent
and chain of four (CIV) were frequently observed. Auto
some univalent and (CVI) or (RIV) were rather rare.
These observations are in a good agreement with that of
Das and Roy (1990), Benova (1992) and Ahmed and
Othman (2004). They found the aberrant metaphases in
the untreated mice were ranging between 5.5: 10%. In the
current study, chromosomes spreads were prepared for 12
days post-treatment with single dose of 200ug/kg IVM, to
cover preleptotene stage.
The latter is the most sensitive stage before meiotic
divisions, when cytogenetic analysis is carried out in MI
spermatocytes (Ciranni and Adler, 1991). Further, the
majority of chemicals act as S-dependent agents, and
premeiotic S-phase occurs in the mouse 12 days before
meiotic divisions (Adler, 1996).
IVM treatment elevated the abnormality to 33.83%, i.e. 6-
fold increase was occurred. Sex univalents, CIV,
autosome univalents represented in 11%, 9.3% and 6%,
respectively. Autosomes plus sex univalents, RIV, CVI,
fragments were rarely picked out.
Literature on cytogenetic effects of IVM is very meager.
However, many chemicals and drugs have mentioned to
inducing such univalents formation of mice (Kar and Das,
1987). Abd-EL-Baset et al. (2000) observed various signs
of chromosomal aberrations in germ cells of mice due to
exposure to sunset yellow and / or ponceu 4R as a
synthetic colors. Also, chromosomal aberrations were
significantly recorded in germ cells of mice after exposure
to pepon and prostaplex (used in treatment of prostatic
disorders (Ahmed and Othman, 2004).
Concerning the effects of IVM on pairing behaviour, both
autosomes and sex chromosomes showed susceptibility to
univalent formation. X-Y bivalents showed a higher
sensitivity than the auto somal bivalents. That is mean,
following IVM treatment, X and Y separation was highly
detected than autosomes. This observation is in
accordance with that of Hu and Zhu (1990) following the
effect of uranyl fluoride containing uranium; Imai et al.
(1981) in hybrids between wild mice and inbred
laboratory mice; Amer et al. (2002) as the effect of
malathion. According to Das and Roy (1990) such
behaviour may result either from early breakdown of
association or from a complete lack of it.
In the present study, a multivalent configurations were
occasionally observed such as (18 II + III + I), (18II +
CIV), (17II + CVI), (15II + CXI). Such phenomenon may
explaine as proposed by Lyon and Meredith (1966) who
believed that the type of aberration may be correlated
with the relative length of the chromosome segments that
are exchanges. They added, in very small segments
chiasmata will be rare, resulting in a multivalent figs. of
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Pak. J. Pharm. Sci., Vol.32, No.2, March 2019, pp.637-646
643
the chain of IV or chain III + I type. The presence of
several configurations also indicates a serial induction of
translocations. For example, fig. of CIV suggests breaks
in 2 chromosomes, the presence of two quadrivalents
indicates breaks in 4 chromosomes (Cacheiro et al.,
1974). This explanation is in accordance with that of
Matter and Generoso (1974) who proposed that the types
of genetic damage referred to chromosomal breakage and
all classes of aberrations that may arise from initial
breaks. This view is further confirmed by that of Cacheiro
et al. (1974); Pacchierotti et al. (1983).
The present observations pointed to the mutagenic effects
of IVM. The frequency of translocation is significantly
higher than that found in the control samples.
The effect of mutagens in germ cell in both mans and
mouse has been discussed in many occasions but rare for
IVM. In a number of studies in mice carrying male-
sterilizing auto somal translocations, spermatocytes loss
has been associated with the apposition of unpaired auto
somal and sex chromosome elements during pachytene
and this in turn, might interfere with the genetic activity
of sex chromosomes (Chandley et al., 1986).
In addition, the genotoxic effects of ivermectin were
reviewed by several investigators. Accordingly, Li et al.
(2004) suggested that the increasing proportion of Gpi-
AA genotype and perhaps Gpi-A allele in a population
might be useful as a potential resistant biomarker of Oxya
chinensis to pesticide avermectin. Osei-Atweneboana et
al. (2012) showed that IVM resistance had been selected
and the genotype (1183GG/1188CC/1308TT, 1545GG)
was strongly associated with the resistance phenotype.
Also, the genotoxicity of IVM was examined by El
Makawy and Mahrous (2008) through the cytokinesis
block micronucleus assay and chromosomal aberrations in
buffalo lymphocytes in vitro and Sweify et al. (2015) on
bone marrow cells of mice in vivo.
Moreover, El-Nahas and El-Ashmawy (2008) concluded
that ivermectin had slight effects on male fertility.
Molinari et al. (2009) highlighted that IVM and ivomec
exert both genotoxicity and cytotoxicity in mammalian
cells in vitro, at least in CHO(K1) cells. Although both
abamectin and ivermectin do not induce in vitro and in
vivo gene mutations in either bacterial or mammalian
cells, there is no concrete evidence of a clear clastogenic
effect exerted both in vitro and in vivo in mammalian cells
(Molinari et al., 2010). el-Ashmawy et al. (2011)
concluded that combined treatment of ivermectin and
verapamil severely affect fetal genetic material and
development and induced genotoxic effect in somatic
cells of the dams. Molinari et al. (2013) concluded that
the decrease in DNA lesions was mostly related to IVM-
induced cytotoxicity rather than attributable to a repair
process.
These studies revealed high clastogenic and genotoxic
potential of IVM
The variation in the protein fractions due to IVM
treatment was examined through SDS-PAGE. It is flexible
and powerful technique widely used for protein separation
based on their M. wt. (Laemmli et al., 1993 and Oswald,
2016). Comparing to the control samples, some newly
bands were developed in the treated samples. The
appearance of these bands may due to break down of a
high M.wt protein (Dutta et al., 1992); or may be
attributed to the formation of adducts between IVM and
DNA which in turn influenced the transcription rate of
Table 2: Summary of the electrophoretic changes in protein profile pattern o f Mus musculus following treatment with
ivermectin
Treated groups
Treatment
Control groups
New bands
Shared bands
Total bands
Total bands
1
28
29
Single injection with
(200ug/kg) ivermectin
28
6
27
33
27
1
32
33
32
3
26
29
Repeated injection with
(200 ug/kg) ivermectin
two weeks apart
26
1
27
28
27
2
29
31
29
Table 3: Effect of ivermectin on % areas of serum protein bands of the treated mice
The type of changes and the number of bands
Time intervals
Treatment
Non significant variations
Decrease
Increase
18
6
5
1day
Single injections
21
7
5
7 days
27
5
1
14 days
20
7
2
1day
Repeated injections
23
4
1
7 days
25
3
3
14 days
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Pak. J. Pharm. Sci., Vol.32, No.2, March 2019, pp.637-646
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genes encoding plasma protein (Mackiewicz et al., 1992).
Also El-Deeb et al.(1996) suggested that exposure to
pollutants may play a role in increasing polymorphism
ratio among treated samples by causing disappearance or
appearance of certain bands. So, our results may be
explained through the reports of Stromberg and Guillot
(1987) and Mycek et al. (1997). They postulated that
some pollutants or drugs cause misreading of the genetic
code and therefore cause abnormal protein synthesis.
On the other hand, the % areas of the protein fraction was
elevated to some treated samples, meanwhile it was
reduced in others. Increasing and / or decrease in the
relative area percentage were observed which indicate the
effect of IVM on the electrophoretic patterns of the serum
protein fraction, with this respect Abd El-Latif (2000)
found that fluoxetine (antidepressant) induce various
abnormal pictures of protein areas during the different
developmental stage of mice embryos referring to the
binding of the drug with certain specific proteins which
induced many signs of teratogenicity and genotoxicity.
The present findings are in harmony with the results
reported by Ibrahim et al. (1995) who proposed that the
formation of new bands or fluctuation of the % area of
some bands are most probably cause from irreversible
protein break down which appear in the presence of the
old bands after drug exposure. Finally, although the
remarkable side effect, of IVM, yet parenteral use of IVM
may be a safe and effective treatment for severe parasitic
infections (Turner et al., 2005 and Barrett et al., 2016). It
is of interest to mention the conclusions of Omura (2008)
that IVM has improved the lives and productivity of
billions of humans, livestock and pets around the globe,
and promises to help consign to the history books two
devastating and disfiguring diseases that have plaqued
people throughout the tropics for generations, while new
uses for it are continually being found.
The effect of avermectin on serum protein was mentioned
in the literature. Impair protein and energy metabolism,
immune system function, and performance resultant from
clinical psoroptic mange, improved substantially within
8 weeks of successful treatment for injectable ivermectin
as stated by Rehbein et al. (2016). Merola et al. (2009)
showed p-glycoprotein defects for increased susceptibility
to ivermectin toxicosis in dogs. Ivermectin interferes with
the binding of retinol to the 19.7 kDa Onchocerca protein
as indicated by Lal and James (1996). Moreover,
Interleukin-6 (IL-6) and C-reactive protein (CRP) were
elevated in 25.7% and 50.7% of onchocerciasis patients,
respectively, after ivermectin treatment (Njoo et al.,1994).
In the present study, the mobility of certain protein
fractions was found to be affected even with the lowest
dose of ivermectin. Based on the above findings, it is
clear that the formation of new bands as well as the
increase in band relative percentage is most probably
caused by irreversible protein breakdown which appears
in the presence of these odd bands after the exposure to
IVM. These results indicate the effect of IVM on the
electrophoretic patterns of the serum protein fractions.
CONCLUSION
In the present work, IVM induced significant increase in
the reciprocal translocation figs. of primary
spermatocytes. The changes in the serum protein fractions
add another warning for the mutagenicity of IVM. It is
concluded from the present work that the mutagenic
effects fortunately diminished with time. According to the
obtained results cautious use of IVM is advisable.
ACKNOWLEDGEMENTS
The helps and cooperation of College of Women, Ain
Shams University, Egypt, is gratefully acknowledged.
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Many medications are available for scabies treatment including oral and topical ivermectin. However, studies comparing these two forms as a scabies treatment are few. This study compares efficacy and safety of topical versus oral ivermectin as scabies treatment. The study included 62 confirmed uncomplicated scabies patients, divided into: Group I (32 patients, received topical ivermectin) and Group II (30 patients, received oral ivermectin). Patients were assessed, clinically and by KOH smear at 1, 2 and 4 weeks. Treatment was repeated after one week in patients with persistent infection. Adverse events were recorded. Most patients (87.5% and 73.5% in group I and group II respectively) were symptom free after a single treatment. A second treatment was required in 4 patients of group I and 8 patients of group II. However, 2 weeks after treatment symptoms and signs completely resolved in all cases with no recurrence at 4 weeks. This study suggests that both topical and oral ivermectin are safe and equally effective in treatment of uncomplicated scabies. Single treatment, whether topical or oral, is associated with high cure rate in a week post treatment. However, repeating treatment after one week may be required to achieve 100% cure.