Cellular morphological biomarker in Oreochromis niloticus
119
ABSTRACT
e objective of this study was to evaluate a cellular morphological biomarker based on micronucleus count and nuclear
abnormalities in the tilapia Oreochromis niloticus (Linnaeus, 1758). e tilapias were collected from a sh farm nursery in
the city of Imperatriz, Maranhão, Brazil. O. niloticus blood was collected by caudal puncture, then micronucleus counting
was performed, erythrocytic abnormalities were detected, and nally it was stained with Giemsa. e micronucleus count
was determined from 2000 erythrocytes. Observations were made in three repetitions per sh (n=10). e frequency of
nuclear morphological anomalies in the investigated sh presented an average of 27.39 ± 3 micronuclei per slide. us,
the presence of micronuclei is an indicator to identify possible environmental contamination.
Keywords: micronucleus count – nuclear alteration – pollution
RESUMEN
El objetivo de este estudio fue evaluar un biomarcador morfológico celular basado en el recuento de micronúcleos y
anomalías nucleares en la tilapia Oreochromis niloticus (Linnaeus, 1758). Las tilapias se recolectaron de un vivero de una
ISSN Versión impresa: 1992-2159; ISSN Versión electrónica: 2519-5697
Biotempo, 2021, 18(1), jan-jul.: 119-124.
RESEARCH NOTE / NOTA CIENTÍFICA
CELLULAR MORPHOLOGICAL BIOMARKER IN OREOCHROMIS
NILOTICUS (LINNAEUS, 1758) (PERCIFORMES: CICHLIDAE) OF WATER
RESOURCES IN THE TOCANTINA REGION OF MARANHÃO, BRAZIL
BIOMARCADOR MORFOLÓGICO CELULAR EN OREOCHROMIS
NILOTICUS (LINNAEUS, 1758) (PERCIFORMES: CICHLIDAE) DE
RECURSOS HÍDRICOS EN LA REGIÓN TOCANTINA
DE MARANHÃO, BRASIL
Eliana da Silva-Sousa¹; Carine Almeida Miranda-Bezerra²; Gabriel Rodrigues-Oliveira¹
& Diego Carvalho Viana1,2*
1 University in the Tocantina Region of Maranhão (UEMASUL), Imperatriz, Maranhão, Brazil.
2 State University in the Tocantina Region of Maranhão (UEMASUL), Imperatriz, MA, Brazil; Postgraduate Program in
Animal Science, State University Maranhão (UEMA), Brazil.
* Corresponding author: dieob@bol.com.br
Eliana da Silva Sousa: https://orcid.org/0000-0001-7986-9849
Carine Almeida Miranda Bezerra: https://orcid.org/0000-0003-4842-1595
Gabriel Rodrigues Oliveira: https://orcid.org/0000-0003-2021-5300
Diego Carvalho Viana: http://orcid.org/0000-0002-3302-9892.
Biotempo (Lima)
doi:10.31381/biotempo.v18i1.3873
https://revistas.urp.edu.pe/index.php/Biotempo
Revista Biotempo: ISSN Versión Impresa: 1992-2159; ISSN Versión electrónica: 2519-5697 Silva-Sousa et al.
120
piscigranjas en la ciudad de Imperatriz, Maranhão, Brasil. La sangre de O. niloticus se recogió mediante punción caudal,
luego se realizó el recuento de micronúcleos, se detectaron las anomalías eritrocíticas y nalmente se tiñó con Giemsa. El
recuento de micronúcleos se determinó a partir de 2000 eritrocitos. Se realizaron las observaciones en tres repeticiones
por pez (n=10). La frecuencia de anomalías morfológicas nucleares en los peces investigados, presentó un promedio
de 27,39±3 micronúcleos por portaobjetos. Así, la presencia de micronúcleos es un indicador para identicar posibles
contaminaciones ambientales.
Palabras clave: alteración nuclear – contaminación – recuento de micronúcleos
INTRODUCTION
e micronucleus test has been considered promising
in investigations to detect the potential to cause gene
mutations and chromosomal changes (Kus et al.,
2017). e micronucleus assay is described by the
Economic Co-operation and Development (OECD)
and is recommended by the International Conference on
Harmonization (1997) among other regulatory agencies
to investigate the genotoxic action of dierent agents
(Davoren & Schiestl, 2018). e piscine micronucleus
test (MNP) has already been investigated in dierent
types of sh cells (Seriani et al., 2015) and used in eld
monitoring studies covering several species including
Nile tilapia (Nascimento-Monteiro et al., 2018).
For several years around the world, aquatic ecosystems
have been suering from anthropic impacts, where the
nal destination of various pollutants are the rivers,
tributaries and seas themselves (Liu et al., 2021). e
practice of shing remains a simple activity performed
in the region of the middle Rio Tocantins, Imperatriz,
Maranhão, Brazil, as it is characterized by being a protable
and familiar practice represented by professional and/
or subsistence shermen who explore multispecic and
multi-device shing (Barbosa et al., 2020).
In turn, Tilapia (Oreochromis niloticus (Linnaeus, 1758)),
represents a species of sh intensively cultivated in sh
farming worldwide and is among the most suitable species
for intensive cultivation in tropical regions. is species
has important characteristics for sh production, such as
the short reproduction cycle and rapid growth (Vaseem
& Banerjee, 2016). In addition, these sh are widely
used in studies of toxicological genetics associated with
environmental biomonitoring (Manzano et al., 2015).
In view of the above, there is no information on the
quality of the sh caught in the Tocantins River and
in controlled environments such as psicultures. us,
there is a need for environmental monitoring capable of
detecting xenobian eects using, for example, biomarker
detection techniques (Alimba et al., 2019). In general, sh
are the best bioindicators currently used by researchers to
assess changes in the aquatic ecosystem, in addition to
being considered capable of responding more accurately
to adverse eects more appropriately (Cruz et al., 2019).
For the analysis of biomarkers, the micronucleus test is
the most used by researchers, as it is a simple, low-cost
and accurate test and has been used as an experimental
model for the evaluation of contamination and the
genotoxic eects of pollutants in ecosystems aquatic (Al-
Sabti & Metcalfe, 1995; Gomes et al., 2019). us, the
increase in the frequency of micronuclei is the result of
DNA damage potentially caused by mutagens, such as
metals and organic compounds (Fenech, 2000; Ribeiro et
al., 2003; Yokoi et al., 2019).
is research is justied by the need to diagnose the
health of Tilapia (O. niloticus) through the micronucleus
test in peripheral erythrocytes and it is a research with
the generation of partial and pioneering data for the
Tocantina region of Maranhão, Brazil.
MATERIALS AND METHODS
e research was conducted captive environment
Imperatriz, Maranhão, Brasil. During the collection, ten
samples were total, four from the Fish Farm collected in
May 2017 and six in November of that year belonging to
the Tilapia species (O. niloticus). e sh was captured
and transported alive in a plastic container (bucket) to
the anatomy laboratory of the State University in the
Tocantina Region of Maranhão (UEMASUL), Brazil.
Cellular morphological biomarker in Oreochromis niloticus
121
Blood samples were collected via caudal puncture with
heparinized and disposable syringes. A drop of blood
from each specimen was used to make the smear. After the
slides were dried for about 30 minutes, the staining was
continued through the giemsa for six minutes and then
washed with distilled water for four minutes and allowed
to dry at room temperature. In each blade, a total of 2000
cells were analyzed (Dittmar et al., 2010). e analysis of
sh erythrocytes was performed according to Ditmar et al.
(2010) for determining the frequency of micronuclei and
nuclear morphological changes (AMN). After making
the blades, they were taken to the microscope for viewing
in a 100-degree objective, using emersion oil for better
visibility of the cells. Carrasco et al. (1990) described
and photographed the morphological changes found in
sh erythrocyte nuclei, in addition to micronuclei, and
classied as: bebbled; lobed; notched and vacuolated.
Fenech (2000), added the technique describing binucleus
as another morphological alteration that detects the
presence of xenobiotics in the environment. All of these
characteristics were taken into account when viewing the
erythrocytes from blood smears performed on specimens
captured for the current research. e data were analyzed
using mean and standard deviation.
Ethic aspects
e research was approved with the Ethics Committee
on Animal Experimentation at the State University of
Maranhão (UEMA / CCA), being in accordance with by
the ethical principles of animal experimentation, adopted
by the mentioned protocol committee N° 21/2017.
RESULTS
For the species of sh studied, alterations of the blebbed,
lobed, binucleus and vacuolated type were not observed
in any of the slides analyzed (Table 1). e analysis using
the micronucleus test showed an average of 27.39±3.
Table 1. Number of micronuclei (NM) and nuclear abnormalities (NA) in peripheral erythrocytes from
specimens (N=10) collected in the local sh farm of Imperatriz, Maranhão, Brasil.
Study Specimens Total Cells Total MN Total NA
Fish farm Oreochromis niloticus 2000 73 0
DISCUSSION
According to Cantanhêde et al. (2016) the micronucleus
formation process depends on the event of cell division
after exposure to the genotoxic agent. In addition, the
time required for cell segmentation depends on the type
of tissue, species and environmental conditions (Hader &
Erzinger, 2017). In addition, dierent aquatic environ-
ments have dierent dissolved compounds, these compo-
nents have been associated with impacts on biota, howe-
ver studying their combined eects and determining the
risk of exposure is still a challenge (Shiroma et al., 2019).
Taking into account the importance of blood in the syste-
mic circulation in sh, changes in hematological indices
in response to water contamination are considered a bio-
marker sensitive to the health of these organisms (Seriani
et al., 2015). In general, changes in sh hematological
indexes occur before any morphological and degenerative
damage begins (Mazon et al., 2002). In addition, the sig-
nicant increase in NM frequencies is associated with ge-
nome instability and is strongly correlated with dierent
pathogens, reproductive disorders and cancer formation
in sh (Alimba et al., 2015; Yamamoto et al., 2017). Ac-
cording to Fenech (2007), the presence of NM serves as a
marker of genomic instability, being a typical characteris-
tic of cancer. A study by Yokoi et al. (2019) demonstrated
that micronuclei are more prevalent in cancer cells than
in healthy cells suggesting that cells with a higher NM
index have high instability.
e elevated levels of micronuclei and nuclear abnormali-
ties were observed in the erythrocytes of the genetic mod-
el, the Nile tilapia (O. niloticus) exposed to water samples
from the two sources tested when compared to a control
group indicating the presence of genotoxic and hazardous
pollutants in the water bodies of Estero de Paco and Es-
tero de Vitas, Philippines (Alam et al., 2019). Manganese
and iron were found at high concentrations (3.61 mg·L-
1 and 19.8 mg·L-1, respectively) in the water of the Rio
Doce after the dams of Fundão and Santarém broke in
Mariana/MG (Brazil). ese same metals were found in
sh (O. niloticus) and crustacean muscle (15 mg·kg-1 and
8 mg·kg-1 wet weight, respectively) in the specimens col-
Revista Biotempo: ISSN Versión Impresa: 1992-2159; ISSN Versión electrónica: 2519-5697 Silva-Sousa et al.
122
lected near the Rio Doce’s outfall. ese exposures caused
signicant erythrocyte micronucleus formation in the or-
ganisms exposed to the highest concentration, as well a
signicant increase in the DNA damage index of erythro-
cytes (Coppo et al., 2018).
According to Grisolia et al. (2009), one should be awa-
re of the dierential sensitivity of aquatic organisms to
genotoxic agents and their responses to them and their
relationships in the aquatic ecosystem. In view of these
changes, other studies point to the inuence of intraspe-
cic factors in responses to the MNP test, these factors
include: age, sex, diet, health, reproductive status and ge-
netic lineage (Al-Sabti, 1995).
Matias & Flohr (2015) developed a mathematical mo-
del (Model WTox) to assess and classify environmental
risk based on the results of the study of the relationship
between the organism and complex mixtures, addressing
parameters of global and specic toxicology including the
micronucleus test. Toxicity is classied into levels ranging
from A to E, ranging from extremely toxic to non-toxic,
respectively. According to this criterion, the value descri-
bed for the current study is highly toxic.
e age of sh is another possible variable, since it has
been reported in studies with mammals that older indi-
viduals tend to be more sensitive to cytogenetic damage
and in sh in the early stages of development than in the
adult stage due to exposure to genotoxic agents (Christie
& Costa, 1983). Diet is another factor that can inuen-
ce the metabolic activation of cell pathways by genotoxic
chemicals (De Flora et al., 1993). us, in sh, studies
are needed to better delimit the responses to the micro-
nucleus test according to sex, environment, diet and spe-
cic-species.
ACKNOWLEDGEMENTS
e authors thank the Maranhão Foundation for Su-
pport to Research and Scientic and Technological De-
velopment of Amparo à Pesquisa e ao Desenvolvimento
Cientíco e Tecnológico do Maranhão (FAPEMA), Ma-
ranhão, Brazil.
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Accepted May 30, 2021.
