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Environmental Pollution 237 (2018) 93e102
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Environmental Pollution

journal homepage: www.elsevier .com/locate/envpol
Effects of benzo[a]pyrene on the blood and liver of Physalaemus cuvieri
and Leptodactylus fuscus (Anura: Leptodactylidae)

Lara Z�acari Fanali a, *, Lilian Franco-Belussi b, c, Cl�audia Regina Bonini-Domingos b,
Classius de Oliveira b

a Graduate Program in Animal Biology, Universidade Estadual Paulista (UNESP), S~ao Jos�e do Rio Preto, S~ao Paulo, 15054-000, Brazil
b Department of Biology, Universidade Estadual Paulista (UNESP), S~ao Jos�e do Rio Preto, S~ao Paulo, 15054-000, Brazil
c Graduate Program in Biotechnology and Environmental monitoring, CCTS, Federal University of S~ao Carlos, 18052-780, Sorocaba, S~ao Paulo, Brazil
a r t i c l e i n f o

Article history:
Received 15 September 2017
Received in revised form
13 January 2018
Accepted 9 February 2018
Available online 22 February 2018

Keywords:
Anuran
Liver melanomacrophage
Leukocytes
Erythrocytes
Micronuclei
* Corresponding author. Department of Biology, Ins
Ciências Exatas, Universidade Estadual Paulista, S~ao
Brazil.

E-mail address: lara_zacari@hotmail.com (L.Z. Fan

https://doi.org/10.1016/j.envpol.2018.02.030
0269-7491/© 2018 Elsevier Ltd. All rights reserved.
a b s t r a c t

Benzo[a]pyrene (BaP) is a bio-accumulative toxic compound found in the atmosphere, water, and soil
that may affect the life cycle of amphibians. In this study, a few contamination biomarkers, such as
hepatic melanomacrophages (MMs), mast cells, erythrocyte micronuclei (MN) and white blood cells
were used to determine how BaP acts in these cells in the anurans Physalaemus cuvieri and Leptodactylus
fuscus. Animals of both species were divided into three treatment groups: 1 day, 7 days and 13 days,
subcutaneously injected 2mg/kg BaP diluted in mineral oil and control group with only mineral oil. After
7 days, BaP caused the frequency of MN to increase in both species while reducing melanin area. The
micronucleus frequency increased due to the genotoxicity of BaP, while the decreasing melanin area may
be related to the inhibition of tyrosinase activity, an enzyme responsible for regulating melanogenesis,
decreasing the synthesis of melanin. The mast cell density increased in all groups and in both species as a
response to the inflammatory action of BaP. These cells respond to nonspecific inflammatory effects
leading, therefore, to this response in all treatments. The percentage of leukocytes remained unchanged
probably due to great intraspecific variability. Additionally, the leukocyte profiles of both species were
characterized and the differences were attributed to extrinsic factors. In short, BaP can affect the integrity
of several organs and tissues, and cell functions leading to the conclusion that this compound is hepa-
totoxic, genotoxic and immunotoxic for anurans.

© 2018 Elsevier Ltd. All rights reserved.
1. Introduction

In recent years, the number of ecotoxicological studies on am-
phibians has grown rapidly (Burlibaşa and Gavril�a, 2011), justified
by a few peculiar characteristics of this taxon. These animals are
very sensitive to pollutants, due to characteristics such as skin
permeability, water-dependent reproduction, exposed embryo-
genesis, and a life-cycle that is dependent on both aquatic and
terrestrial environments, thus maximizing their exposure to con-
taminants (Conrad, 2010; Burlibaşa and Gavril�a, 2011; Blaustein
et al., 2011; P�erez-Iglesias et al., 2016; De Oliveira et al., 2017).

Contaminants such as chemical pollutants are distributed in the
tituto de Biociências, Letras e
Jos�e do Rio Preto, S~ao Paulo,

ali).
organisms through blood flow (WHO, 1995) and exposure can,
therefore, induce nuclear abnormalities such as the formation of
micronuclei, which consists of chromosomal fragments or whole
chromosomes in anaphase (Fortin et al., 2015). Micronuclei were
detected in mature erythrocytes of different fish species (Pastore
et al., 2014) and amphibians when exposed to BaP or pesticides,
for example (Grinfeld et al., 1986; Candioti et al., 2010; P�erez-
Iglesias et al., 2014). Hematological parameters can also be
affected, due to the exposure to contaminants (Winkaler et al.,
2008). These changes of the physiological status provide informa-
tion on the level of damage, indicating the healthy status of anurans
and other animals (Sayed, 2015). In this context, differential
leukocyte count also provides information on the immunological
status (Newman et al., 1997).

In addition to micronuclei formation and quantitative changes
of leukocytes, melanomacrophages and mast cells are also
responsive to the action of xenobiotics (Ma et al., 2011; Franco-

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L.Z. Fanali et al. / Environmental Pollution 237 (2018) 93e10294
Belussi et al., 2013, 2016; M€oller et al., 2014; Passantino et al., 2014;
Çakıcı, 2015; Regnault et al., 2014, 2016). For this reason, these cells
are used, respectively, as contamination biomarkers and inflam-
matory response indicators in several tissues (Paetow et al., 2012;
Franco-Belussi et al., 2013; Santos et al., 2014).

Melanomacrophages are macrophages that produce and store
pigments such as melanin, lipofuscin, and hemosiderin (Agius and
Roberts, 2003; Ribeiro et al., 2011). Melanin, the major pigment of
this phagocytic cell absorbs and neutralizes free radicals, cations
and other potentially toxic agents derived from the degradation of
phagocytized cellular material (Zuasti et al., 1989). These cells are
found in hematopoietic organs such as liver and spleen (Agius,
1980; Cesarini, 1996) of ectothermic animals (fish, amphibians,
and reptiles) (Wolke, 1992; Fournie et al., 2001; Loumbourdis and
Vogiatzis, 2002; Fishelson, 2006) while having detoxification,
bactericidal and innate immunity as main functions (Agius and
Roberts, 2003; Passantino et al., 2014; Franco-Belussi et al., 2013).
Mast cells specialize in secreting mediators of lipids, leukotrienes,
proteases, and histamine, which contribute to the inflammatory
process (Shakoory et al., 2004) while playing an important role in
inflammatory response mechanisms associated with a wide range
of stress conditions, such as exposure to xenobiotics (Lauriano et al.,
2012).

Polycyclic Aromatic Hydrocarbons (PAHs) are highly studied
toxic compounds due to its toxicity at low and moderate concen-
trations, and its persistence in the environment (Brandt et al.,
2002). Benzo[a]pyrene (BaP) is considered a high-risk contami-
nant due to genotoxicity (Mouchet et al., 2005) and ability to induce
micronucleus formation in erythrocytes (Fortin et al., 2015), hep-
atotoxicity (Latif et al., 2010; Regnault et al., 2014, 2016; Pastore
et al., 2014), immunotoxicity (Phalen et al., 2014) and also change
the patterns of the leukocyte cells (Sorensen, 1991; Sayed, 2015).
Because it results from the incomplete combustion of organic
matter, it can be present naturally in fires (Zakaria et al., 2002; Ou
et al., 2004; Luo et al., 2008; Jiao et al., 2009) or result from
anthropogenic action, such as the use of oil products and fossil fuel
combustion (Zakaria et al., 2002; Kim et al., 2008; Morillo et al.,
2008; Stogiannidis and Laane, 2015).

BaP is adsorbed by particles present in water, suspended in the
air (WHO, 1998; Srogi, 2007), in sediments and in the soil (Stark
et al., 2003; Morillo et al., 2008; Harris et al., 2011). Because it is
practically everywhere, anurans may be exposed to it via inhala-
tion, feeding and through the skin (Collins et al., 1991; Kanaly and
Harayama, 2000). In addition, this compound may bioaccumulate
in anurans (Brandt et al., 2002), becoming quite harmful since after
its absorption, BaP is biotransformed in the liver by the cytochrome
P450 1A1 (CYP1A1) (Caruso and Alaburda, 2008;Wakx et al., 2016).
Throughout its metabolism, toxic byproducts are formed, especially
the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetra (a) pyrene (BPDE)
(Madureira et al., 2014).

Although toxic compounds are produced during BaP meta-
bolism and disrupt homeostasis (Chovatiya and Medzhitov, 2014),
anurans have defense cells such as melanomacrophages, mast cells,
and leukocytes, which are capable of attenuating toxicity, due to
their functions. The consequence of the protective response of
these cells is the homeostasis restoration (Chovatiya and
Medzhitov, 2014), enabling the survival and adaptation of the an-
imals in anthropic environments (Blaustein et al., 2011). In general,
the effects of pollutants on the internal morphology of adult an-
urans are poorly known (Fanali et al., 2017). After undergoing
biotransformation in the liver, the metabolites of BaP reach target
organs through circulation, causing toxicity (Van Veld et al., 1988;
Buhler and Williams, 1988; Costa et al., 2011; Fanali et al., 2017).
Therefore, the purpose of the study was to determine the effects of
BaP on hepatic melanomacrophages, hepatic mast cells, leukocyte
composition and erythrocytes integrity of the of P. cuvieri and
L. fuscus.

During its reproductive period, P. cuvieri performs mating
vocalization in newly flooded environments, usually inside animal
footprints, when deep and filled with water (Uetanabaro et al.,
2008), while L. fuscus vocalizes on the ground, on the banks of
temporary ponds or depressions subject to flooding (Uetanabaro
et al., 2008). Both species coexist, have similar living habits, and
are phylogenetically close and abundant in the study region.
Because these two species share the same habitat and expose
themselves to similar environmental conditions, they could be used
as sentinel species to evaluate the cause-effect relationships of
chemical contaminants in the environment.

2. Material and methods

2.1. Animal sampling

Seventy-two adult males, 36 P. cuvieri and 36 L. fuscus, were
used in the experiment. This project was approved by the Ethics
Committee on Animal Use, protocol 094 and RAN/IBAMA/MMA
18573-1. The animals were collected in northwestern S~ao Paulo,
Brazil, from December to February, period that includes the species
reproductive season. The animals were acclimated for one week, in
28� 21� 15 cm boxes containing 2 cm of soil, which was kept
moist. Room temperature was kept at 27 �C, with natural photo-
period. During the experimental period, all animals were fed with
Drosophila shortly after the injections to avoid further stress.

2.2. Experiments with benzo[a]pyrene

The studied parameters were evaluated at 1, 7 and 13 days after
all animals were subcutaneously injected 2mg/kg benzo[a]pyrene
(B1760; purchase from Sigma-Aldrich), diluted in 0.02ml of Nujol
mineral oil, while the control consisted of mineral oil only. The
solution was injected every 48 h on the animal back, near the hind
limb. The solution concentration was adapted and based on the
work of Padr�os et al. (2003). The animals (N¼ 6, per group) were
exposed for 1, 7 and 13 days and each group had its respective
control with the same experimental period. This experimental
model was replicated for each species.

After the experiment, the hind limb was anesthetized with
xylocaine (with the excess removed with cotton to avoid smear
interference) and blood was drawn from the femoral vein using a
heparinized needle and a syringe. Smear was performed immedi-
ately after collection, due to the small blood sample and rapid
coagulation.

The animals were then euthanized with benzocaine solution
(1 g/L) and had their liver sampled, which was weighed in an
analytical balance (0.05 g precision) and analyzed as the following
procedures.

2.3. Micronucleus analysis

After drying, slides were fixed in methanol at 4 �C for 20min,
stained with Giemsa 7.5% for 15min and observed under a light
microscope (Leica DM4000 B). A total of 1000 erythrocytes were
counted for each animal.

2.4. Leukocyte analysis

For differential leukocyte count, the blood smear was stained
with Panoptic (Hematocor-Biol�ogica®) to identify leukocytes vari-
ety and establish their relative proportions. For each animal, 100
leukocytes were counted under a light microscope (Leica DM4000



L.Z. Fanali et al. / Environmental Pollution 237 (2018) 93e102 95
B). The percentage of each leukocyte type in the peripheral circu-
lating blood was determined, and knowing the total circulating
leukocytes, the percentage of each cell type was obtained.

Themeans of controls (1, 7 and 13 days) was calculated and used
to determine the leukocyte profile and compare it in the two
species.

2.5. Histological processing

P. cuvieri and L. fuscus liver fragments were fixed in Metacarn
(60% methanol, 30% chloroform, and 10% acetic acid) for the his-
tological analyses. Subsequently, samples were washed in water,
dehydrated in alcoholic series and placed in historesin (Leica-his-
toresin embedding kit). The 2 mm sections were obtained with the
aid of a microtome (RM 2265, Leica, Switzerland), stained and
observed under a light microscope (Leica DM4000 B) coupled with
an image capture system (Leica DFC 280).

2.6. Quantitative analyses

The sections were stained with Hematoxylin-eosin and
pigmentation was quantified using Image Pro-Plus program,
Media-Cybernetics Inc. (version 6.0) based on the different color
intensity according to Santos et al. (2014). For each animal, a slide
with 20 sections was prepared, 25 random fields were photo-
graphed and the melanomacrophage melanin area was analyzed.

A solution containing 0.025 g toluidine blue with borax (pH 4.0)
diluted in 50ml distilled water was used to detect the presence of
mast cells in the liver. The sections were washed in distilled water
for 1min, stained with toluidine blue for 30 s, washed in distilled
water, allowed to dry for assembly with Canada's balsam. Ten
randomized histological sections of each animal were selected for
mast cell count. The section area was quantified using the Image
Pro-Plus program, Media-Cybernetics Inc. (version 6.0).

2.7. Statistical analysis

We used a 2� 3 factorial design with two treatments (one BaP
concentration and the control), crossed with three exposure times:
1 d, 7 d, and 13 d. We used six animals per treatment to quantify
melanomacrophage area, micronucleus frequency, mast cell den-
sity and leukocyte percentage.

A Generalized Linear Model (GLM) with binomial distribution
and log link function including treatment and exposure time, along
with their interactions were used to model the frequency of
micronucleus and leukocyte types (lymphocytes, basophils, eosin-
ophils, monocytes, and neutrophils) (counting data). We tested the
model assumptions using diagnostic plots from the R (Team Core,
2016) sjPlot package (Lüdecke, 2016). Residuals showed homoge-
neity of variance and normal distribution. The leukocyte profiles
were compared between both species using the animals of the
control groups. For this, a GLM was performed for each leukocyte
type and modulated by the species.

To model melanomacrophage area, our continuous response
variable, we fitted a Linear Mixed-Effects Model (package lme4;
Bates et al., 2015) (Zuur et al., 2009) with restricted maximum
likelihood (REML; Bolker et al., 2009) including treatment and time
of exposure as fixed factors, along with their interaction. The
sampling units (pictures), in which we estimated our response
variables, were nested within each animal (true replicate). To
control for dependency among the 25 pictures from the same an-
imal (Crawley, 2012: 703), we included a random intercept for
animal (categorical with 6 levels; see Moen et al., 2016). We tested
model assumptions using diagnostic plots with the R package sjPlot
(Lüdecke, 2016). Residuals showed homogeneity of variance and
normal distribution. We used least-squares means of the R package
lsmeans (Lenth, 2016) to test for differences between treatment
levels and exposure time. Models were summarized and the P-
values estimated based on conditional F-tests with Kenward and
Roger (1997) approximation for the degrees of freedom using sjPlot.

A Generalized Linear Mixed Model (GLMM) with binomial dis-
tribution including treatment and exposure time as fixed effects,
along with their interaction, was used to model mast cell density in
the hepatic tissue. A random animal intercept was included to
control the dependence between the 10 sections of the same ani-
mal (categorical with 6 levels; Crawley, 2012: 703; Moen et al.,
2016). The analysis was conducted in the glmmADMB package
(Fournier et al., 2012). We used the least squares means in the
lsmeans package (Lenth, 2016) to test differences between levels of
treatment factors and exposure time. All analyses were performed
using software R v. 3.3.2 (R Core Team, 2016).

3. Results

3.1. Benzo[a]pyrene effect on micronucleus frequency

Benzo[a]pyrene displayed a genotoxic effect on P. cuvieri and
L. fuscus after 7 days of exposure to the contaminant, which
resulted in an increased frequency of micronucleus from 0.0± 0.0
to 0.001± 0.0008 (100%) in P. cuvieri and from 0.00016± 0.00016 to
0.0023± 0.0012 (1356%) in L. fuscus (Fig. 1; P< 0.0001).

3.2. Benzo[a]pyrene effect on melanomacrophage

BaP changed the melanin area present in the melanomacro-
phage in both species after 7 days of exposure. In P. cuvieri, the
melanin area decreased approximately 42%, from 2123.6± 483.8 to
1221.5± 233.1 (P ¼ 0.042) whereas in L. fuscus it decreased by
4125.1± 934.7 to 1474.2± 236.3 (P ¼ 0.042), corresponding to 64%
(Figs. 2e4).

3.3. Benzo[a]pyrene effect on mast cell density

BaP changed the mast cell density of P. cuvieri in all groups
treated with the contaminant. The densities increased from
1.8± 0.7 to 4.3± 1.1 after 1 day, from 3.7± 1.0 to 6.6± 1.6 after 7
days, and from 5.1± 1.6 to 10.6± 3.3 after 13 days of exposure
(P<0.0001). The densities doubled approximately for each experi-
mental time.

The mast cell density in L. fuscus also increased in all treated
groups from 0.02± 0.02 to 0.2± 0.1 after 1 day, 0.2± 0.1 to 0.6± 0.2
after 7 days, and 0.7± 0.2 to 1.9± 0.4 after 13 days, corresponding
to 11.5, 1.28 and 1.65�, respectively (Figs. 5 and 6; P<0.0001).

3.4. Leukocyte profile and benzo[a]pyrene effect on the percentage
of leukocytes

Leukocyte percentages remained unchanged in both species
treated with BaP; however, the leukocyte profiles of the controls of
both species differed regarding lymphocytes, neutrophils and eo-
sinophils (Fig. 7).

4. Discussion

4.1. Benzo[a]pyrene effect on micronucleus frequency

After 7 days of exposure, the genotoxic effect of BaP was evident
and resulted in increased micronucleus frequency in both species.
The micronucleus test is widely used to estimate cytogenetic
damage induced by physical or chemical agents (Udroiu, 2006) and



Fig. 1. Micronuclei frequency in Physalaemus cuvieri and Leptodactylus fuscus after 1, 7 and 13 days of exposure to BaP compared to control groups administered mineral oil only for
the same time intervals. The asterisk (*) indicates significant difference (P < 0.05) between the control and the treated groups for the same time interval.

L.Z. Fanali et al. / Environmental Pollution 237 (2018) 93e10296
several studies corroborate this fact. In one of these studies, Pleu-
rodeles waltl specimens of the Urodela order were experimentally
contaminated with 0.05 ppm benzo[a]pyrene and the results
showed that the micronucleus amount was time-dependent and
peaked on the sixth day of treatment (Grinfeld et al., 1986).

P�erez-Iglesias et al. (2015) investigated Hypsiboas pulchellus
tadpoles exposed to the herbicide imazethapyr and reported that
the micronucleus frequency increased at the concentration of
1.17mg/L after 48 h and for all studied concentrations (0.39, 0.78
and 1.17mg/L) after 96 h. In addition, the herbicide caused other
nuclear alterations such as binucleate cells and DNA damage. These
responses relates to the variability of pesticide-induced toxicity in
different species of amphibians. Another study reports that tad-
poles had increased micronucleus after 16 days exposed to 248 nM
BaP. Micronucleus formation resulting from the exposure to the
contaminant confirms the disruption of the spindle apparatus in
erythrocytes. The results show that micronuclei might be useful
indicators of sub-lethal effects on amphibian. Therefore, by
applying these biomarkers in native species and wild populations
exposed to toxicants could be valuable for investigating the causes
of declining amphibian populations (Sadinski et al., 1995).

At 13 days of treatment, micronucleus frequency was lower in
comparison to 7d. As BaP is a bio-accumulative toxic compound
(Brandt et al., 2002) and anurans received a greater amount of it
after 13 days of exposure when comparing to 7d, an increased
frequency of MN would be expected. P�erez-Iglesias et al. (2015)
revealed that H. pulchellus tadpoles exposed to the herbicide
imazethapyr for 96 h (0.39, 0.78 and 1.17mg/L) displayed a lower
micronucleus frequency in comparison to the animals that were
exposed for 48 h (in the concentration of 1.17mg/L). Another
insecticide, the pirimicarb, was used by Candioti et al. (2010) in
Rhinella arenarum tadpoles, which also demonstrated a lower fre-
quency of MN in 96 h when comparing to 48 h, using concentra-
tions of 80mg/L and 250mg/L.

This response is supported by some hypothesis, being among
them that the most damaged cells went through induced cell death
by the contaminant, with less damaged erythrocytes remaining in
the blood; severely damaged cells lost their division capacity, thus
consequently precluding the formation of micronucleus (Candioti
et al., 2010; P�erez-Iglesias et al., 2015). Both hypothesis may
explain the behavior found in our study, especially when one
considers the toxicological characteristics of BaP.
4.2. Benzo[a]pyrene effect on melanomacrophages

In both species, the melanomacrophage melanin area did not
change after 1 day of exposure to BaP. However, De Oliveira et al.
(2017) reported the effect of BaP, pesticides and other agrochemi-
cals on the hepatic MMs of some anuran species for shorter expo-
sure times. Therefore, we hypothesized that our studied time
intervals were not long enough to stimulate cell response in both
species at this level of histopathological evaluation. Size, rate of
ingestion, growth rate, membrane permeability and ventilatory
frequency are biological processes that influence PAHs absorption



Fig. 2. Melanomacrophages area in Physalaemus cuvieri and Leptodactylus fuscus after 1, 7 and 13 days of exposure to BaP compared to control groups administered mineral oil only
for the same time intervals. The asterisk (*) indicates significant difference (P < 0.05) between the control and the treated groups for the same time interval.

Fig. 3. Amount of melanin present in the melanomacrophages of Leptodactylus fuscus after 7 days of exposure to BaP (BaP 7d) compared to the control group treated with mineral
oil only (CT 7d). It is possible to observe the decrease of the pigmented area in animals treated with BaP. Coloration: Hematoxilin-eosin.

Fig. 4. Amount of melanin present in the melanomacrophages of Physalaemus cuvieri after 7 days of exposure to BaP (BaP 7d) compared to the control group treated with mineral oil
only (CT 7d). It is possible to observe the decrease of the pigmented area in animals treated with BaP. Coloration: Hematoxilin-eosin.



Fig. 5. Mast cells density in Physalaemus cuvieri and Leptodactylus fuscus after 1, 7 and 13 days of exposure to BaP compared to the control groups treated with mineral oil only for
the same time intervals. The asterisk (*) indicates significant difference (P < 0.05) between control and treated groups for the same time interval.

Fig. 6. Mast cell occurrence in the hepatic tissue of Physalaemus cuvieri. Coloration:
Toluidine blue.

L.Z. Fanali et al. / Environmental Pollution 237 (2018) 93e10298
by the organisms (Juhasz and Naidu, 2000). We believe that these
processes are influencing immediate 1-day response of the MMs in
P. cuvieri and L. fuscus.

After being absorbed by the animals, BaP is biotransformed in
the liver by an enzymatic class called cytochrome P450 (Caruso and
Alaburda, 2008; Wakx et al., 2016). BaP induces its own biotrans-
formation by activating the aromatic hydrocarbon receptor (AhR).
The AhR is a cytosolic transcription factor that responds to envi-
ronmental toxins (Sibilano et al., 2012) and, after activation, moves
to the nucleus and binds to the ARNT receptor (nuclear transporter
of the aryl hydrocarbon receptor). This complex binds to DNA ele-
ments that respond by regulating the expression of various genes,
including genes encoding several enzymes that allow biotransfor-
mation. An example is cytochrome P450 1A1 (CYP1A1) that while
metabolizing BaP, generates toxic byproducts, especially the 7,8-
dihydroxy-9,10-epoxy-7,8,9,10-tetra(a)pyrene (BPDE) (Madureira
et al., 2014).

At 7 days, the melanin area in the hepatic melanomacrophages
decreased. Regnault et al. (2014) observed a decrease in the pig-
mented area of MMs in Xenopus tropicalis anurans exposed to
10 mg.L�1 BaP, which remained constant after 12, 18 and 24 h of
exposure. This may be related to hepatic stress and hepatocytes
apoptosis (Regnault et al., 2014). More recently, Fanali et al. (2017)
also observed this same response for Hypsiboas albopunctatus an-
urans treated with 7mg/kg BaP after 3 days. Melanin from pig-
mented cells protects adjacent tissues by absorbing potentially
harmful substances and slowly releasing them at non-toxic con-
centrations, which results in great affinity between BaP and
melanin (Larsson, 1993). However, long-term exposure may cause
high levels of harmful chemicals to accumulate in the melanin, and
ultimately stimulate melanin-containing cells to degenerate and
secondary lesions in surrounding tissues (Larsson, 1993), thus
decreasingMM area. Additionally, Joo et al. (2005) showed that BaP
inhibits tyrosinase activity, the enzyme responsible for regulating
melanogenesis, induced by the melanocyte stimulating hormone



L.Z. Fanali et al. / Environmental Pollution 237 (2018) 93e102 99
(a-MSH) (Park et al., 2009; Videira et al., 2013), consequently,
decreasing melanin synthesis in melanoma cells of mouse (Joo
et al., 2015). Therefore, BaP may have affected the melanin syn-
thesis of MMs and caused the melanin area of this cell to decrease.

After 13 days, although not significant, the melanin area of the
MMs increased in both species. Regnault et al. (2016) stated that
BaP hampers hepatic metabolism due to increased lipid content.
This condition induces oxidative stress (Regnault et al., 2014, 2016),
and since melanin has anti-oxidant function, MM melanin may
have increased as a physiological response to BaP oxidative stress
(Fanali et al., 2017).
Fig. 7. Leukocytes percentage (A, C, E, G, I) in Physalaemus cuvieri and Leptodactylus fuscus
profiles (K) of Physalaemus cuvieri and Leptodactylus fuscus. The asterisk (*) indicates signific
4.3. Benzo[a]pyrene effect on mast cell density

Our study showed that mast cells increased in both species for
all treatments. Mast cells secret lipid mediators, leukotrienes,
proteases, and histamine, which contribute to the inflammatory
process (Shakoory et al., 2004). Thus, these cells can be used as
indicators of inflammatory response in various tissues (Franco-
Belussi et al., 2013).

Franco-Belussi et al. (2013) injected Eupemphix nattereri with
E. coli LPS and observed that liver mast cells increased greatly after
2 h while mast cells in the testis increased after 12 h and 48 h of LPS
after 1, 7 and 13 days of exposure to BaP. Cont¼ control; Trat¼ treatment. Leukocyte
ant difference (P < 0.05). Data are presented as mean ± SE. B, D, F, H, J scale bar: 5 mm.



L.Z. Fanali et al. / Environmental Pollution 237 (2018) 93e102100
exposure. Franco-Belussi et al. (2014) showed that the number of
mast cells in the E. nattereri testis tripled 12 h after LPS injection.
This may have happened due to the infiltration of inflammatory
and immune cell exudates in the interstitial region.

Ma et al. (2011) studied mice and showed that benzo[a]pyrene
increased lymphocytes and enzymes involved in inflammation, and
enlarged sinusoids as well. M€oller et al. (2014) reported increased
hepatic B and T lymphocytes of trout contaminated with BaP and
concluded that the compound causes inflammation. In the liver of
P. cuvieri and L. fuscus, BaP must have triggered an inflammatory
response in the hepatic tissue, which increased the density of mast
cells due to its protective function in inflammatory responses.

Leukocyte profile and benzo[a]pyrene effect on the percentage
of leukocytes.

Despite the diversity of anurans, a great majority of the studies
on their hematology are related to total cell count (erythrocytes and
leukocytes) and morphometry of red blood cells (Cabagna
Zenklusen et al., 2011), while detailed studies are lacking. The un-
derstanding of leukocyte responses of amphibians to diseases is
limited (Campbell and Ellis, 2007), but is an important parameter
for assessing organism health, demonstrating pollutant effects, and
identifying biological changes caused by xenobiotics (Neta et al.,
2015).

Some authors evidenced that in both fish and mammals, BaP
and some of its metabolites, such as 7,8-BaP quinone (7,8-BPQ),
BaP-7,8-dihydrodiol (BD), BaP-7,8-dihydrodiol-9,10-epoxide
(BPDE), have an immunotoxic capacity (De Jong et al., 1999;
Carlson et al., 2004; Gao et al., 2005; Phalen et al., 2014). Latif
et al. (2010) studied birds and concluded that BaP has hema-
totoxic capacity due to decreasing total leukocytes, whichmay have
resulted from the increased destruction rate of circulating leuko-
cytes. The AhR expression induced P450 thus generating systemic
oxidative stress (Dalton et al., 2002), which eventually impaired
hematopoiesis (Latif et al., 2010). Nonetheless, in our study leuko-
cyte remained unchanged, which may be related to hemato and
cytotoxicity (Santacroce et al., 2015) that probably impaired the
leukocyte response. Also, Matiasovic et al. (2008) concluded that
unchanged lymphocytes, neutrophils, monocytes, and eosinophils
may be due to high variances within individual groups, possibly
explaining the unchanged leukocyte percentages in this study.

The leukocytes remained unchanged in P. cuvieri and L. fuscus,
but the leukocyte profiles were different between both species,
more specifically, lymphocytes, neutrophils, and eosinophils. The
variability of red and white blood counts in amphibians is based on
extrinsic factors such as temperature, diet, season, photoperiod and
intrinsic factors such as species, gender, and lifestyle (Heatley and
Johnson, 2009). As observed in our work, lymphocytes are the
most abundant leukocytes in both P. cuvieri and L. fuscus species of
anurans, similarly to Lithobates catesbeianus (Cathers et al., 1997)
and Polypedates teraiensis (Das and Mahapatra, 2014). However,
neutrophil is the most abundant leukocyte in Bufo marinus (Cannon
et al., 1987) and Bufo americanus (Forbes et al., 2006). Heatley and
Johnson (2009) argue that a conspecific animal or of the same
genus can be sampled for comparison to minimize extrinsic and
intrinsic variability for better hematological interpretation of
amphibians.

Our results call the need for employing species of the same sex,
feeding habits and sampling time period to minimize errors arising
from interpretation of the data given the great variability of
leucocytes.

5. Conclusion

Although there are particularities among species, BaP acts
similarly in the body of the both species, which can be observed by
the cellular alterations, like increased MN frequency and mast cell
density, and decreased MMs area. These changes may trigger sys-
temic processes in animals that are living in contaminated wet
lands, like suppressing amphibian immune defenses, rendering
them susceptible to additional chemical exposures like pesticides,
metals and microbial infections. Other implication of this finding
are that the exposure to BaP can affect the proper functioning of the
cells due to the hepatotoxic, genotoxic, and immunotoxic charac-
teristics of BaP. Then, BaP contamination can potentially impair the
anurans life.

Acknowledgements

The authors thank Bruno S.L.Valverde for the assistance during
the experiments, Ruth M.V.Kakogiannos for the manuscript trans-
lation and revision, Iv~a G.L. for the manuscript revision. Thanks are
also due to FAPESP for master fellowship (2014/07466-8) to LZF and
post-doctoral fellowship to LFB (2014/00946-4). To CNPq for
fellowship to CO (305081/2015-2).

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	Effects of benzo[a]pyrene on the blood and liver of Physalaemus cuvieri and Leptodactylus fuscus (Anura: Leptodactylidae)
	1. Introduction
	2. Material and methods
	2.1. Animal sampling
	2.2. Experiments with benzo[a]pyrene
	2.3. Micronucleus analysis
	2.4. Leukocyte analysis
	2.5. Histological processing
	2.6. Quantitative analyses
	2.7. Statistical analysis

	3. Results
	3.1. Benzo[a]pyrene effect on micronucleus frequency
	3.2. Benzo[a]pyrene effect on melanomacrophage
	3.3. Benzo[a]pyrene effect on mast cell density
	3.4. Leukocyte profile and benzo[a]pyrene effect on the percentage of leukocytes

	4. Discussion
	4.1. Benzo[a]pyrene effect on micronucleus frequency
	4.2. Benzo[a]pyrene effect on melanomacrophages
	4.3. Benzo[a]pyrene effect on mast cell density

	5. Conclusion
	Acknowledgements
	References