InvestigationintothecytotoxicityandmutagenicityoftheMarajó Archipelagowatersusing Plagioscion squamosissimus (Perciformes: Sciaenidae) asabioindicator

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Ecotoxicology and Environmental Safety 132 (2016) 111–115
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Investigation into the cytotoxicity and mutagenicity of the Marajó
Archipelago waters using Plagioscion squamosissimus (Perciformes:
Sciaenidae) as a bioindicator
Carlos Alberto Machado da Rocha a,n, Carla Mariana Ferreira Pessoa b,
Claudia Antonia Campos Rodrigues c, Raul Henrique da Silva Pinheiro c,
Edmar Tavares da Costa d, Adriana Costa Guimarães e, Rommel Rodríguez Burbano e
a Fishery and Agribusiness Resource Coordination (Coordenação de Recursos Pesqueiros e Agronegócio), Federal Institute of Education, Science and Tech-
nology of Pará (Instituto Federal de Educação, Ciência e Tecnologia do Pará), Belém, Pará, Brazil
b Coordination of Biological Sciences (Coordenação de Ciências Biológicas), Federal Institute of Education, Science and Technology of Pará (Instituto Federal
de Educação, Ciência e Tecnologia do Pará), Belém, Pará, Brazil
c Social, Environmental and Water Resources Institute (Instituto Sócio Ambiental e Recursos Hídricos), Federal Rural University of Amazonia (Universidade
Federal Rural da Amazônia), Belém, Pará, Brazil
d Experimental Neuropathology Laboratory (Laboratório de Neuropatologia Experimental - LaNEx), Hospital Universitário Barros Barreto, Belém, Pará, Brazil
e Laboratory of Human Cytogenetics and Genetic Toxicology (Laboratório de Citogenética Humana e Genética Toxicológica), Federal University of Pará
(Universidade Federal do Pará), Belém, Pará, Brazil
a r t i c l e i n f o
Article history:
Received 23 November 2015
Received in revised form
23 May 2016
Accepted 24 May 2016
Keywords:
Marajó archipelago
Flow cytometry
Fish erythrocytes
Micronucleus test
x.doi.org/10.1016/j.ecoenv.2016.05.020
13/& 2016 Elsevier Inc. All rights reserved.
esponding author.
ail addresses: carlos.rocha@ifpa.edu.br,
a@hotmail.com (C.A.M.d. Rocha),
ssoa35@yahoo.com.br (C.M.F. Pessoa),
engel@yahoo.com.br (C.A.C. Rodrigues),
ryque@yahoo.com.br (R.H.d.S. Pinheiro), etco
esac@gmail.com (A.C. Guimarães), rommel@u
a b s t r a c t
Maintaining water quality within tolerable limits is a basic need of the riverside communities in the
Amazon. Using endemic aquatic organisms as biological models is useful for monitoring the environ-
ment. In this study, potential cytotoxic and genotoxic damages in Plagioscion squamosissimus (commonly
known as silver croaker) from the Marajó Archipelago were evaluated using a flow cytometry assay and a
survey of micronuclei (MN) frequency as well as other nuclear abnormalities (NA). P. squamosissimus
specimens were collected at four locations in the Marajó Archipelago. Blood samples from these fish
were used in the flow cytometry assay and piscine micronucleus test, and the resulting data were
analyzed using analysis of variance (ANOVA). We did not observe a difference in the erythrocyte cell cycle
distribution among the samples (P¼0.9992), which suggests the absence of cytotoxic agent-induced
apoptosis. The piscine micronucleus test exhibited differences in the samples from São Sebastião da Boa
Vista (SSBV), and those from Anajás produced the highest mutagenicity indices. The MN frequencies
were low for all groups, but the groups exhibited significantly different frequencies (P¼0.0033). Reni-
form nuclei, nuclei with extensions, and lobed nuclei were combined and considered NA. The frequency
differences for these NAwere significant among sampling sites (P o0.0001). This report is the first to use
flow cytometry in fish to evaluate cytotoxic agent-induced apoptosis. The micronucleus test results in-
dicate the presence of pollutants that can change the genetic material of the fish studied. We also de-
monstrate that the Amazonian fish P. squamosissimus is important not only as a comestible species but
also as an adequate model for biomonitoring in aquatic environments.
& 2016 Elsevier Inc. All rights reserved.
1. Introduction
Aquatic environments accumulate many contaminants that can
sta@globo.com (E.T.d. Costa),
fpa.br (R.R. Burbano).
cause damage at the molecular, biochemical, cellular, and phy-
siological levels (Kirschbaum et al., 2009). In fact, among different
ecosystems, aquatic ecosystems, either by direct or indirect release
(loading), are the final repository for most xenobiotics discharged
therein. Fish have successfully been used in cytogenetic analyses
because they are easy to handle and maintain in a laboratory, and
they are relatively inexpensive to analyze (Hayashi et al., 1998).
Using fish red blood cells provides rapid results with little suffer-
ing by the organisms used in biomonitoring (Minissi et al., 1996).
These tests can detect clastogenic (induced chromosome breaks)
and aneugenic (induced aneuploidies or abnormal chromosome
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mailto:carlos.rocha@ifpa.edu.br
mailto:camrocha@hotmail.com
mailto:carla.pessoa35@yahoo.com.br
mailto:claudia.engel@yahoo.com.br
mailto:eng_henryque@yahoo.com.br
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http://dx.doi.org/10.1016/j.ecoenv.2016.05.020
Table 1.
Summary of physicochemical parameters of the river waters of the Marajó Archi-
pelago. The minimum, maximum, and average of each parameter are displayed.
Parameters Minimum Maximum Average
Transparency (cm) 40.00 118.89 68.12
Temperature (°C) 26.37 32.10 28.72
pH 3.30 7.88 6.14
Conductivity (mS.cm�1) 34 11002 1772.8
Total solids (mg.L�1) 10 5505 583.19
Dissolved oxygen(mg.L�1) 2.60 6.52 4.36
Total nitrogen (mg.L�1) 2.01 6.36 4.42
Total phosphorus (mg.L�1) 0.001 2.31 0.14
C.A.M.d. Rocha et al. / Ecotoxicology and Environmental Safety 132 (2016) 111–115112
segregation) agents and have been validated in vivo and in vitro
(Al-Sabti and Metcalfe, 1995). Many substances have been tested in
various organisms, such as mollusca, fish, amphibians, reptiles,
birds, and mammals (Zúñiga-González et al., 2000; Venier and
Zampieron, 2005).
Measuring the rates of apoptosis using flow cytometry facil-
itates a more complete quantitation of these rates. Using this
technique, programmed cell death can be detected using nuclear
staining and quantified by identifying cells with fragmented DNA
(Telford et al., 1992) or externalized phosphatidylserine at the cell
membrane.
The micronucleus test, developed by Schmid in 1975 using
bone marrow cells from mammals, has been extensively used to
test chemical compound genotoxicity (Campana et al., 2003). Mi-
cronuclei (MN) are small corpuscles composed of chromosomal
material. After chromatid separation during mitosis, two nuclei are
reconstituted, one at each pole. The nuclear membrane is re-
constructed around these two sets of chromosomes. However, if
an entire chromosome or acentric chromosome fragment does not
integrate into the new nucleus (because it is not attached to the
mitotic spindle), this chromosome is also considered a small in-
dividual nucleus that is referred to as a micronucleus (Villela et al.,
2003). Compared with other techniques that detect DNA damage,
the micronucleus test features certain advantages: (a) it can be
performed rapidly; (b) it is simple; (c) it is inexpensive; and
(d) preparation and analysis are easier and faster compared with
chromosome abnormality studies (Rocha et al., 2009).
The Archipelago of Marajó, fully located in the State of Pará in
the Brazilian Amazon, constitutes one of the richest regions of
Brazil in terms of water and biologicalresources. The Archipelago
is formed by a group of islands, which as a whole, is the largest
fluvial-maritime island in the world, with 49,606 square kilo-
meters (BRASIL, 2007), extending from the mouth of the Amazon
River. It is located between the equator and the parallel 1,55°S
latitude and between 48°W and 51°W meridian of longitude.
The Amazon exhibits several consequences from urban growth,
including accelerated deforestation; natural resource degradation;
pressure on urban infrastructure and equipment; an absence of
adequate basic sanitation that has resulted in infectious disease
proliferation and is directly linked to child mortality; pollution of
rivers primarily due to poor sanitation; a lack of adequate loca-
tions for urban waste disposal, which is often deposited in the
open or in waterways; and illegal occupation of the territory
(Maniçoba, ,2006). In contrast, riverside communities would not
exist without an organic relationship with the river and/or lake
because their way of life is based on the principle of water-earth-
work association (Canto et al., 2009). Thus, maintaining water
quality within tolerable limits is a basic need for these
populations.
The aim of the present study was to test Plagioscion squamo-
sissimus erythrocytes from four municipalities in the Marajó Ar-
chipelago (São Sebastião da Boa Vista [SSBV], Anajás, Portel, and
Chaves), PA, Brazil, for genotoxic and cytotoxic effects from the
river water.
2. Materials and methods
The Marajó Archipelago includes sixteen municipalities that be-
long to the statistical entity called Mesoregion Marajó. Munici-
palities sampled in this study (SSBV, Anajás, Portel and Chaves) were
indicated by the Association of Municipalities of the Archipelago of
Marajó (AMAM) depending on the availability of infrastructure.
Table 1 summarizes the physicochemical parameters of the
study area. The temperature is the parameter that experiences the
least fluctuation throughout the year across the Archipelago. This
pattern of fairly regular temperature distribution shows the typical
thermal stability of low-latitude regions. Large changes in the
other parameters are more seasonal than spatial, differing mark-
edly between the rainy and less rainy periods.
We used the fish species Plagioscions quamosissimus [Heckel,
1840] (South American silver croaker, corvina or pescada in Por-
tuguese) as a bio-indicator. This species is a carnivorous fresh-
water fish restricted to South America and originally from the
Amazon basin with a wide distribution in Brazil. In total, 68P.
squamosissimus specimens were collected with a mean length of
19.2275.143 cm and a mean weight of 78.03736.603 g, including
the control group and specimens from the four locations in the
Marajó Archipelago. At each municipality included in the project in
the Marajó archipelago, 15 silver croaker specimens were collected
using cast nets and gill nets. The same fishing gear was used to
collect eight specimens from a reference location (control) in the
Baixo Rio Jarumã near Abaetetuba, PA, Brazil (coordinates: 1° 41′
13.6″ S and 48° 52′ 48.8″ W). Blood samples were collected from
each specimen using heparinized syringes. Blood samples were
used for two analyses: (I) Approximately 2 mL of blood from each
fish was removed and placed in opaque Eppendorf micro-
centrifuge tubes on ice and then transported to the Human Cy-
togenetics and Environmental Toxicology Laboratory (Laboratório
de Citogenética Humana e Toxicologia Ambiental) at UFPA for flow
cytometry analyses. (II) Blood smears were also prepared for each
fish and transported to the Aquatic Biology, Reproduction and
Larviculture Laboratory (Laboratório de Biologia Aquática, Re-
produção e Larvicultura) at the IFPA for Piscine Micronucleus
Analyses.
2.1. Flow cytometry assay
In this experiment, the cell cycle distribution was assessed
following the procedure described by Nicoletti et al. (1991). Per-
ipheral blood erythrocytes from the fish were incubated at 37° C
for 3 h in a lysis solution containing 0.1% citrate, 0.1% triton X-100,
and 50 μg/mL propidium iodide in the absence of light. Cellular
fluorescence was determined using flow cytometry (EasyCyteTM
Mini System cytometer Guava Technologies Incs., Hayward, CA, USA)
and CytoSoft 4.1 software. Five thousand events per experiment
were evaluated, and cellular debris was omitted from the analysis.
2.2. Piscine micronucleus test
In total, 136 slides were prepared (two slides per fish). The
smears were fixed in absolute ethanol for 10 min at the collection
sites. At the Aquatic Biology, Reproduction and Larviculture La-
boratory (Laboratório de Biologia Aquática, Reprodução e Larvi-
cultura) at the IFPA, the preparations were stained with 5% Giemsa
diluted in phosphate buffer (pH 6.8) for 20 min and examined
under a microscope at 1000X magnification. The number of nor-
mal erythrocytes without MN and the number of damaged cells
Table 3.
Micronucleus test results for erythrocytes from Plagioscion squamosissimus fish in
the Marajó Archipelago.
Locations (Groups) Nuclear changes /2000 cells
MN NA
I. SSBV
μ7 SD 1.171.2 6.372.7
MED (Q1-Q3) 1.0 (0.0–2.0) 7.0 (4.0–8.5)
II. Anajás
μ7 SD 0.770.6 4.773.3
MED (Q1-Q3) 1.0 (0.0–1.0) 4.0 (2.0–5.5)
III. Portel
μ7 SD 0.170.3 2.172.7
MED (Q1-Q3) 0.0 (0.0 – 0.0) 1.0 (0.0 – 3.0)
IV. Chaves
μ7 SD 0.170.3 3.072.6
MED (Q1-Q3) 0.0 (0.0–0.0) 2.0 (1.0–3.5)
C.A.M.d. Rocha et al. / Ecotoxicology and Environmental Safety 132 (2016) 111–115 113
with MN and/or nuclear morphological abnormalities (NA) were
determined by analyzing 2000 cells per fish. Only cells with an
intact plasma membrane were analyzed. MN were defined as
rounded or oval intracytoplasmic bodies associated or not with the
main nucleus with a diameter 1/10–1/30 of the larger nucleus and
found on the same focal plane as the nucleus (Al-Sabti and Met-
calfe, 1995; Ayllon and Garcia-Vazquez, 2000). Reniform nuclei,
nuclei with extensions, and lobed nuclei were combined and
considered NA (Ayllon and Garcia-Vazquez, 2000; Bolognesi et al.,
2006).
2.3. Statistical analyses
First, the data were tested for normality using Lilliefors test.
The values from the flow cytometry assays exhibited a normal
distribution and were subjected to one-criterion analysis of var-
iance (one-way ANOVA). Since the micronucleus test data from
certain groups did not meet the normality assumptions, a non
parametric analysis of variance was performed, the Kruskal-Wallis
test, followed by the SNK test. For all analyses, BioEstat 5.0 soft-
ware (Ayres et al., 2007) was used, and the significance level was
0.05.
V. Control
μ7 SD 0.070.0 0.971.0
MED (Q1-Q3) 0.0 (0.0–0.0) 0.5 (0.0–2.0)
MN¼micronuclei; NA¼nuclear morphological alterations; μ¼mean; SD¼standard
deviation; MED¼median; Q1¼first quartile; Q3¼third quartile.
3. Results
Our flow cytometry results show no significant difference in
the erythrocyte cell cycle distribution among the groups (P[AN-
OVA]¼0.9992), which suggests an absence of cytotoxic agent-in-
duced apoptosis. Table 2 summarizes the cell cycle distribution
results, with the data presented as the means7standard deviation
for five independent experiments.
The micronucleus test results are summarized in Table 3. The
data are presented as the means7standard deviation and median
values (first and third quartiles). According to Ramsdorf et al.
(2009), when a distribution is not symmetrical, the median is a
more suitable representation than the mean.
The groups exhibited significant differences in the Piscine Mi-
cronucleus Test. The samples from SSBV and Anajás produced the
highest mutagenicity indices for both typical MN and NA. A graph
of the typical MN frequencies is shown in Fig. 1. The frequencies
were low for all groups, but we observed significant differences
between the groups (P[Kruskal-Wallis]¼0.0033). Reniform nuclei,
nuclei with extensions, and lobed nuclei were combined and
considered NA. The different frequencies for these alterations were
significant among sampling sites(P[Kruskal-Wallis]o0.0001),
which is also shown in Fig. 1.
4. Discussion
According to Metcalfe (1989), the use of biological responses
(here, cytotoxicity and genotoxicity bioassays using
Table 2.
Cell cycle distribution of erythrocytes from Plagioscion squamosissimus fish col-
lected in the Marajó Archipelago.
Locations (Groups) DNA Content (%)
Sub-G1 G1 phase S phase G2 phase
I. SSBV 3.0970.27 51.571.91 22. 671.47 21.570.28
II. Anajás 2.9370.19 50.670.86 21.670.67 20.670.62
III. Portel 2.5371.00 49.071.50 22.371.15 20.670.36
IV. Chaves 3.5970.83 48.472.24 21.970.56 19.271.45
V. Control 2.4870.28 52.170.48 23.671.35 22.271.33
representatives of the local fish fauna) is more advantageous than
the use of physical and chemical water measurements. Physical
and chemical water measurements only record the single time
point at which they were collected, similar to a photograph of the
river, thus requiring more analyses for efficient temporal
monitoring.
Nucleic acid analysis is the second most common flow cyto-
metry application after defining cellular lineages. Cytometrically
analyzing DNA content establishes the conditions necessary to
assess apoptosis (Arden et al., 2007) and measure ploidy and
proliferative activity in tumors.
To our knowledge, the present study is the first to use flow
cytometry to examine apoptosis in fish. We did not observe dif-
ferences between collection sites, which does not exclude the
possibility of non-cytotoxic mutagens that promote DNA damage,
but that are not detected using this technique.
The basal (or spontaneous) frequency of MN in fish is typically
low (Ferraro et al., 2004); however, interesting differences be-
tween species have been reported. Thus, when 1000 erythrocytes
were considered, the observed frequencies were 0.0870.13 in
Hoplias malabaricus (Ferraro et al., 2004), 0.170.32 in Eigen-
mannia virescens (Bücker et al., 2006), 0.5671.12 in Aequidens
tetramerus (Rocha et al., 2011a) and 2.471.19 in Colossoma mac-
ropomum (Rocha et al., 2011b). In the present study, using Plagi-
oscion squamosissimus, formation of typical MN in the control
group was not observed; however, we observed NA, which was
also observed in a study by Ramsdorf et al. (2009) that examined
H. malabaricus.
Studies similar to ours are rare in the Amazon, but two inter-
esting reports have examined other fish species and are described
below. Porto et al. (2005) evaluated the genotoxic effects of mer-
cury using MN tests in three Characiformes species from different
trophic levels in the food web: Prochilodus nigricans (detritivore),
Mylossoma duriventris (omnivore), and Hoplias malabaricus (pisci-
vore). The authors observed MN in two Amazon Basin rivers stu-
died: the Madeira River (a mercury-contaminated area) and the
Solimões River (an uncontaminated area), both in the state of
Amazonas. However, the mean frequency of MN was significantly
Fig. 1. Blood cell micronuclei (MN) and other nuclear abnormalities (NA) evaluated in the Plagioscion squamosissimus groups (experimental and control). The results are
expressed as the median values/q1–q3. *Indicates significant differences compared with the control group (po0.05) using Kruskal–Wallis followed by the SNK test. The
graph exhibits boxes with a central line that corresponds to the median axis as well as first (lower line) and third (top row) quartiles. Also shown are the lowest and highest
scores through the lower and upper limits of vertical straight lines, which originate from the first and third quartiles, respectively.
C.A.M.d. Rocha et al. / Ecotoxicology and Environmental Safety 132 (2016) 111–115114
greater in piscivorous species compared with detritivorous and
omnivorous species collected from the Madeira River. According to
the authors, the higher MN frequency detected in this species may
be due to three factors: (I) the higher trophic level associated with
the species; (II) the low pH and conductivity in their natural ha-
bitat, which favor methylmercury production and bioaccumula-
tion; and (III) the sedentary behavior of the fish. Melo et al. (2013)
compared the baseline frequencies of MN erythrocytes and other
NA among different fish species in the order Gymnotiformes
(Rhamphichthys marmoratus, Steatogenys elegans, Sternopygus ma-
crurus, Parapteronotus hasemani, Gymnotus mamiraua, Gymnotus
arapaima, Brachyhypopomus beebei, and Brachyhypopomus n. sp.)
sampled at various locations in the eastern Amazon. Determining
the basal MN and NA frequencies of these fish helped identify
potentially useful species for models in genotoxicity studies. Only
one impacted sample collected at one location in the Caripetuba
River (Pará State) exhibited a significant number of NA, which may
be due to the release of sewage by the neighboring mining in-
dustry and the fuel released by small boats used by a local com-
munity. Such studies can provide incentives for further research in
eastern Amazon areas affected by mining, deforestation, and other
anthropic activities.
Identifying potentially genotoxic substances was not the aim of
this study. However, these substances and the environmental
conditions that can produce detrimental effects have been re-
ported in other studies that monitored the waters of Marajó. Alves
et al. (2012) for example, studied the water quality and trophic
state of the Arari River in the Marajó Archipelago and observed
high trophic levels (eutrophication). The authors noted that the
high trophic levels were due to high nutrient levels in the water,
especially the total phosphorus. Additionally, the authors claimed
that the Arari River is undergoing a natural eutrophication process
because effluent releases and anthropogenic contamination are
incipient.
There are in the literature numerous references on genotoxic
and cytotoxic effects on aquatic organisms as consequences of
large spills of oil and oil products (Frenzilli et al., 2004; Barsiene
et al., 2006; Negreiros et al., 2011; Paul et al., 2013). Although on
much smaller scale, oil spills from small local boats occur daily in
the Marajó waters. Such events possibly have relevance in the
significant differences found in the frequency of micronuclei and
other nuclear changes compared to the negative control.
While collecting fish samples for the present study, we ob-
served accidental oil spills from different vessels. In an environ-
mental education study, conducted with traditional communities
along the banks of the Rio Acuti-Pereira in the municipality of
Portel, Garcia and Schmitz (2008) reported that among the pro-
blems noted, river contamination due to oil spills from vessels was
fairly frequent in the region. The spills killed fish and resulted in
disease among the people living near the water. River con-
tamination also increased due to rudimentary tanks maintained
along the river by the people living there. Untreated waste was
also frequently dumped into the river.
In conclusion, the present study is the first use of flow cyto-
metry in fish to evaluate cytotoxic agent-induced apoptosis. The
frequencies of MN and other NA, which were significantly higher
in fish from the Marajó Archipelago compared with the control
group, indicate the presence of pollutants that can cause changes
in the genetic material of the fish analyzed. Our results also de-
monstrate that the Amazonian fish P. squamosissimus is important
not only as a comestible species but also as an adequate model for
biomonitoring in aquatic environments.
Acknowledgements
The authors acknowledge the financial support of the Amazon
Pará Foundation for Research Support (Fundação Amazônia Para-
ense de Amparo à Pesquisa - FAPESPA, Grant nos. 014/2012 and
159/2014).
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	Investigation into the cytotoxicity and mutagenicity of the Marajó Archipelago waters using Plagioscion squamosissimus...
	Introduction
	Materials and methods
	Flow cytometry assay
	Piscine micronucleus test
	Statistical analyses
	Results
	Discussion
	Acknowledgements
	References