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Acute exposure to environmentally relevant concentrations of benzophenone-3 induced genotoxicity in Poecilia reticulata
Aquatic Toxicology 216 (2019) 105293
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Acute exposure to environmentally relevant concentrations of benzophenone-3 induced genotoxicity in Poecilia reticulata
T
Sara dos Santos Almeidaa, Thiago Lopes Rochab, Gabriel Qualhatoc, Leandra de Almeida Ribeiro Oliveiraa,d, Cátia Lira do Amarala, Edemilson Cardoso da Conceiçãod, ⁎ Simone Maria Teixeira de Sabóia-Moraisc, Elisa Flávia Luiz Cardoso Bailãoa, a
Laboratório de Biotecnologia, Câmpus Henrique Santillo, Universidade Estadual de Goiás, Anápolis, Goiás, Brazil Laboratory of Environmental Biotechnology and Ecotoxicology, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, Goiás, Brazil c Laboratório de Comportamento Celular, Departamento de Morfologia, Instituto de Ciências Biológicos, Universidade Federal de Goiás, Goiânia, Goiás, Brazil d Laboratório de PD&I de Bioprodutos, Universidade Federal de Goiás, Faculdade de Farmácia, Goiânia, Goiás, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Biomarkers Comet assay DNA damage Ecotoxicology Guppy Micronuclei
The organic UV filter benzophenone-3 (BP-3), widely used in the commercial formulations of sunscreens and personal care products, is considered an emerging pollutant and has been associated with several human and environmental health concerns. However, knowledge about their mode of action and ecotoxicity on aquatic biota is scarce. In this scenario, the objective of this work was to evaluate the genotoxic, mutagenic, and erythrotoxicity effects of BP-3 in the guppy Poecilia reticulata after acute exposure. Adult females of P. reticulata were exposed to three non-lethal and environmentally relevant concentrations of BP-3 (10, 100, and 1000 ng L−1) during 96 h of exposure, and the somatic parameter [Fulton condition factor (K)], genotoxicity (comet assay), mutagenicity [micronucleus (MN) and erythrocyte nuclear abnormalities (ENA) tests] and erythrotoxicity parameters (such as total cell area and nucleus-cytoplasmic ratio) were analyzed. Results showed that the general physiological condition (K value) of fish was not affected by acute exposure to BP-3. However, BP-3 induced DNA damage at 100 and 1000 ng L−1 and increased the frequency of total ENA at 1000 ng L−1, specially lobed nucleus, when compared to control group, indicating its genotoxic and mutagenic effects. Furthermore, the BP-3 did not induce significant changes in the total cell area and nucleus-cytoplasmic ratio. In summary, results showed that the BP-3 at environmentally relevant concentration was genotoxic to freshwater fish P. reticulata, confirming its environmental risk.
1. Introduction Designed initially to protect human skin from UVA and UVB radiations, UV filters are included in a wide range of products to protect humans and materials from damage by UV irradiation and to ensure a longer shelf life to products (Brooke et al., 2008; Gackowska et al., 2014; Kim and Choi, 2014). In this way, the constant presence of UV filters in environmental matrices and in biota lead them to be considered as emerging pollutants. In addition to being frequently detected in the environment, emerging pollutants are not included in environmental monitoring programs, have no regulatory legislation and their removal by wastewater treatment plants is ineffective and may represent a risk to human health and to the environment even in concentrations in order of μg L−1 or even ng L−1 (Silva and Collins, 2011; Montagner et al., 2017).
⁎
Benzophenone-3 (BP-3) is one of the organic UV filters most used by industry (Mao et al., 2019). The occurrence of BP-3 in the aquatic environment has been widely reported (Ramos et al., 2015), such as in rivers (2031 ng L−1) (Díaz-Cruz et al., 2019), untreated residual waters (7800 ng L−1) and treated ones (700 ng L−1) (Balmer et al., 2005), drinking water (115 ng L−1) (Silva et al., 2015), and groundwater (19.2 ng L−1) (Jurado et al., 2014). The BP-3 was detected in high concentrations in lakes (up to 550 ng L−1) (Grabicova et al., 2013) and beaches in Japan (up to 5429 ng L−1) (Tsui et al., 2014). The ecotoxicological effects of BP-3 on aquatic species have been reported at different trophic levels. BP-3 induced high acute toxicity (growth inhibition or mortality) to Chlorella vulgaris, Daphnia magna, and Danio rerio after 48 or 96 h exposure (Du et al., 2017). In vitro and in vivo studies showed that BP-3 is able to interfere with reproduction, hormonal signaling, and oxidative stress in fish (Schlumpf et al., 2001;
Corresponding author. E-mail address: elisafl[email protected] (E.F.L.C. Bailão).
https://doi.org/10.1016/j.aquatox.2019.105293 Received 26 June 2019; Received in revised form 3 September 2019; Accepted 3 September 2019 Available online 04 September 2019 0166-445X/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Experimental design of this work. After the acclimation period, 45 fish were placed randomly in 15 tanks of 7 L capacity filled with 6 L of reconstituted water. Five experimental groups were established: negative control (NC), containing only reconstituted water; solvent control (SC), containing reconstituted water and the same amount of ethanol (2.5 ppm) used in the dilution of benzophenone-3 (BP-3); and three groups with environmentally relevant concentrations of BP-3 (10, 100 or 1000 ng L−1). Each group consisted of 3 tanks containing 3 fish each, so 9 fish in total for each group were tested independently (n = 9). All groups were tested in a static system without the renewal of water and BP-3 for 96 h.
2. Material and methods
Kunz et al., 2006; Coronado et al., 2008; Blüthgen et al., 2012; Liu et al., 2015), indicating its potential as an endocrine disruptor in fish. BP-3 also interferes with the embryonic development and reproduction of aquatic invertebrates, such as Chironomus riparius (Ozáez et al., 2014; Campos et al., 2019), and is reported as a threat to coral reefs worldwide (He et al., 2019; Schneider and Lim, 2019). However, the genotoxic and mutagenic potential of BP-3 to aquatic organisms has not been addressed. The comet assay is a suitable and widely used technique for the identification of genotoxicity biomarkers, allowing the verification of breaks in the DNA chain induced by exogenous agents (Bolognesi and Hayashi, 2011; Qualhato et al., 2017). Micronucleus (MN) and erythrocyte nuclear abnormalities (ENA) have been identified as an effective test to measure chromosomal damage in fish after exposure to contaminants (Qualhato et al., 2017, 2018). Currently, the association between these techniques is seen as the best test battery to evaluate the genotoxicity and mutagenicity in aquatic organisms (Rocha et al., 2014; Araldi et al., 2015). Fish have been shown to be good biomonitor, since they are relatively sensitive to changes in the environment and to effects of pollutants (Wepener et al., 2011). In addition, fish have a fundamental role in food networks and are of great commercial importance (van der Oost et al., 2003). The guppy Poecilia reticulata, an ovoviviparous freshwater fish, is a biomonitor recommended by the American Public Health Association (APHA) and the Organization for Economic Cooperation and Development (OECD) (OECD, 1992; Baird et al., 2017). It has been used as a model testing organism for the study of a wide range of substances with toxic potential (Sharbidre et al., 2011; Souza-Filho et al., 2013; Rocha et al., 2015; Qualhato et al., 2017, 2018). However, there are no studies about the effects of BP-3 on this species. In this sense, the objective of this work was to evaluate the genotoxicity, mutagenicity and erythrotoxicity of the organic UV filter BP-3 in the guppy P. reticulata during acute exposure to environmentally relevant concentrations. We evaluated both genotoxic (DNA damage), mutagenic (MN and ENA tests) and morphometric (such as total cell area and nucleus-cytoplasmic ratio) parameters on P. reticulata exposed to BP-3.
2.1. Benzophenone-3 Powder BP-3 was donated by Volta Flora Manipulation Pharmacy (Anápolis, Goiás) and was submitted to a qualitative analysis using a high efficiency liquid chromatography (HPLC) method developed by Agha et al. (2013). The BP-3 sample (10 μL) was injected into a HPLCPDA equipment (Waters Alliance e2695 chromatograph; Milford, MA, USA) in the concentration of 0.1 mg mL−1. The chromatographic separation was achieved using a Zorbax C18 column (250 x 4.6 mm, 5 μm) coupled with a guard column of the same stationary phase. Data processing was performed by Empower 2.0. The mobile phase was composed of methanol and Milli-Q water (85:15 v/v) and the flow rate was 1 mL min−1. The mobile phase was filtered through a 0.45 μm filter membrane (Millipore, MA, United States) and degassed in an ultrasonic bath for 20 min before use. The samples were solubilized in methanol and filtered through a 0.45 μm Millex® membrane (Millipore, MA, USA). The wavelength used for the BP-3 detection was 288 nm. The column oven was set at 30 °C. The qualitative analysis of BP-3 was performed by comparison of its UV spectra (190–400 nm) with the literature values. 2.2. Ethical approval for animal use The experimental procedures and animal management were approved by Animal Use Ethics Committees from Universidade Estadual de Goiás (UEG; protocol n° 009/2016) and from Universidade Federal de Goiás (UFG; protocol n° 046/2017). The experiment followed the guidelines for testing chemicals for acute toxicity established in the OECD 203 guideline (OECD et al., 1992). 2.3. Experimental design Adult females of P. reticulata were collected in the Water Treatment 2
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refrigeration (10 °C) and in the dark. The electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) was added and the slides remained immersed for 30 min. The electrophoresis run was performed at 1 V/cm and 300 mA for 25 min. After the run, the slides were neutralized with Tris buffer (Tris 0.4, pH 7.5) for 5 min. The slides were washed with distilled water and dried at room temperature. After drying, slides were fixed in absolute ethanol for 10 min and stored. The analysis was performed with Diamond™ Nucleic Acid Dye (Promega®, NSW, Australia) fluorescence dye, 495 nm emission and 558 nm excitation using a light microscope (Olympus BX60, Japan) under 20x objective and the images were scanned using Olympus CellSens software (Olympus, Japan). For image analysis, the Comet Image Analysis system (Comet Score-software; TriTek, Sumerduck, VA, EUA) was used. A hundred nucleoids were randomly selected (50 in each slide, two slides per specimen), totaling 900 nucleoids per experimental group (n = 9 fish per experimental group). The degree of DNA damage was evaluated using the following parameters: percentage of DNA in the tail, tail length, and Olive tail moment.
Station (Saneamento de Goiás - SANEAGO - 16° 37′ 59″ S and 49° 15′ 44″ W, Goiânia, Goiás, Brazil) and acclimated during 10 days in static tanks (60 L) containing reconstituted water (ISO, 1996) at 27 ± 2 °C, pH 7.1 ± 2, ammonia 0 - 0.01 μL/L, and photoperiod (12:12 h light/ dark), as recommended by Rocha et al. (2015). Fish were fed ad libitum three times per day with the commercial fish food Cardume 36% (VB Alimentos Ltd). After acclimation period, 45 fish (total weight of 0.14 ± 0.02 g; total length of 2.3 ± 0.1 cm; standard length of 1.9 ± 0.1 cm) were placed randomly in 15 tanks (Fig. 1) of 7 L capacity filled with 6 L of reconstituted water (ISO, 1996), as recommended by Rocha et al. (2015). Before exposure, BP-3 was diluted with ethanol P.A. (96%) and the final concentration of ethanol in the exposure medium was adjusted to 2.5 ppm. Five experimental groups (n = 9 fish per group) were established: negative control (NC), containing only reconstituted water; solvent control (SC), containing reconstituted water and the same amount of ethanol used in the dilution of BP-3 (2.5 ppm); and three groups with environmentally relevant concentrations of BP-3 (10, 100 or 1000 ng L−1). The concentrations used for the exposure are in agreement with the concentrations found in several studies that investigated the presence of UV filters in environmental matrices (Balmer et al., 2005; Grabicova et al., 2013; Jurado et al., 2014; Tsui et al., 2014; Silva et al., 2015; Díaz-Cruz et al., 2019). All groups were tested in a static system without the renewal of water and BP-3 for 96 h. After the exposure period, the animals were euthanized by hypothermia and blood samples were collected using the tail artery as recommended by Qualhato et al. (2017).
2.6. Mutagenicity
Where, W is the weight of the individual and L is the total length.
The mutagenicity was analyzed by micronucleus (MN) and erythrocyte nuclear abnormalities (ENA) tests, such as described by Carrasco et al. (1990) and Fenech et al. (2003) with modifications. Blood was collected from the caudal artery in a microtube containing 250 μL of 0.01 M PBS buffer and pH 7.2 and homogenized. Two slides per fish (n = 9 fish per experimental group) were prepared by the blood distension technique. The material was stained with quick panoptic dye (Instant Prov, New Prov®, Pinhais, Brazil). A total of 1000 erythrocytes were analyzed per animal (totalizing 9000 erythrocytes per experimental group; n = 9 fish per experimental group) using a Primo Star optical microscope (Zeiss, Oberkochen, Germany) under immersion objective (100 ×). The erythrocytes were classified according to the nuclear morphologies proposed by Fenech (2000) and Fenech et al. (2003): MN and nuclei of the type notched, lobed, broken eggs, and blebbed. The total frequency of abnormalities represents the sum of all the nuclear alterations (Qualhato et al., 2017).
2.5. Genotoxicity
2.7. Erythrotoxicity
The genotoxic effects (DNA damage) were assessed by the comet assay according to Singh et al. (1988), with modifications proposed by Qualhato et al. (2017). Peripheral blood was obtained through the caudal artery and collected in microtubes containing 250 μL of 0.1 M PBS buffer pH 7.2. Then 15 μL of buffer solution + blood was diluted in 120 μL low melting point (LMP) agarose, previously prepared and heated (37 °C). The samples were homogenized with a micropipette, scattered with coverslips on microscope slides coated with normal agarose (1.5%) and kept at 10 °C for 3 min for the solidification of the LMP agarose. The glass slides were carefully removed and the slides were immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 0.2 M NaOH, 0.03 M sodium lauryl sarcosine, 10% DMSO, 1% Triton X-100 and pH 10) for 2 h without illumination. After the lysis time, the slides were transferred to the electrophoresis, kept under
Erythrotoxicity was analyzed in terms of several morphometric parameters of erythrocytes (Table 1), such as described by Carella et al. (2017) with modifications. Blood was collected from the caudal artery in a microtube containing 250 μL of 0.01 M PBS buffer at pH 7.2 and homogenized. Two slides per fish (n = 9 fish per experimental group) were prepared by the blood distension technique. The material was stained with quick panoptic (Instant Prov, New Prov®, Pinhais, Brazil) and the analysis was performed according to Carella et al. (2017) with modifications, using a Primo Star optical microscope (Zeiss, Oberkochen, Germany) under immersion objective (100 ×). The slides were photographed using the Axiocam 105 image capture system (Zeiss, Oberkochen, Germany) and the images were processed using Fiji version 1.0 free software based on ImageJ. For the analysis were considered non-overlapping erythrocytes that were completely inserted
2.4. Somatic biomarkers After the exposure period (96 h), weight (g), total length (mm), and standard length (mm) were collected from all individuals, and the Fulton Condition Factor (K) was estimated by the following formula (Fulton, 1904):
K=
W L³
Table 1 List of morphological parameters of erythrocytes used in this study. Parameter Total cell area (TA) Total nucleus area (NA) Area of cytoplasm (AC) Nucleus Perimeter (PN) Circularity NA/TA NA/AC
Unity
Description 2
The area within the polygon delineated by the perimeter of the cell The area within the polygon delineated by the perimeter of the nucleus Obtained by the difference TA - NA Perimeter calculated from the centers of boundary pixels 4*π*Area/Perimeter2, also called a form factor Ratio between the core area and the total cell area Ratio between the nucleus area and the cytoplasm area
Pixels Pixels2 Pixels2 Pixels – – –
3
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into the capture field, totalizing 2000 erythrocytes per animal and 18,000 per experimental group (n = 9 fish per experimental group). 2.8. Statistical analyses Statistical analysis of the data was performed using the free software Paleontological statistics - PAST version 3.18 (Hammer et al., 2001). The assumptions of data normality and homogeneity of variances were verified by the Shapiro-Wilk test and the Levene test, respectively. Once observed the violation of the assumptions, the data transformation was then attempted through logarithmic transformation. For the data on which the transformation was successful, the results were compared using parametric tests (one-way ANOVA followed by the Tukey's test). When the transformation was not possible, the Kruskal-Wallis nonparametric test was used. All results are expressed as mean ± standard deviation of the mean and were considered significant when p < 0.05.
Fig. 3. Fulton condition factor (K) of Poecilia reticulata after acute exposure (96 h) to benzophenone-3 (BP-3). The negative control (NC) group was not exposed to BP-3, the solvent control (SC) group was exposed just to the BP-3 solvent (ethanol) and the other groups were exposed to crescent concentrations of BP-3: 10, 100 or 1000 ng L−1 for 96 h. Results are presented as mean ± standard deviation of each group (n = 9). The ANOVA one-way test demonstrated no significant differences between the groups (p > 0.05).
3. Results 3.1. Qualitative analysis of benzophenone-3 The HPLC results showed that the retention time for BP-3 was 5.2 min and its UV spectra (Fig. 2) is in accordance with those reported in the literature (Ahmedova et al., 2002), indicating that the compound used in this work is pure and could be considered as BP-3.
(Fig. 4). 3.4. Mutagenicity
3.2. Somatic biomarker
P. reticulata erythrocytes containing MN or other nuclear abnormalities, such as notched, broken eggs, lobed, and blebbed nucleus, were observed after acute exposure to BP-3 (Fig. 6). Total nuclear alterations frequency increased in fish exposed to BP-3 at 1000 ng L−1 when compared to the negative and solvent control groups (Fig. 6). Individually, nuclei with lobed-type changes were more prevalent than other nuclear changes (Fig. 6B). The fish exposed to BP-3 at 1000 ng L−1 showed high frequency of this abnormality (Fig. 6B) when compared to the control groups. We observed that was a tendency to decrease the frequencies of erythrocytes nuclear alterations at the concentration of 100 ng L−1 BP-3, except for the presence of MN, when compared to the other concentrations tested (Fig. 6).
To verify if the acute exposure to BP-3 was able to interfere in the general fish health condition, the K value was determined based on the biometric data. No significant differences were found between the animals exposed to BP-3 and those from negative control and solvent control (Fig. 3), indicating that the guppies were in similar physiological conditions during the acute exposure to BP-3. No fish mortality was observed in this study. 3.3. Genotoxicity The DNA damage in P. reticulata peripheral erythrocytes was analyzed by Comet assay and expressed as % of tail DNA, Olive tail moment (OTM), and comet tail length (Fig. 4). As expected, the negative control group showed a significantly lower DNA damage when compared to all the other groups (Fig. 5). The group exposed to 10 ng L−1 of BP-3 did not differ from the solvent control group. An increase in DNA damage was observed in fish exposed to BP-3 at concentrations of 100 and 1000 ng L−1. These concentrations did not differ significantly from each other, but had significant differences with respect to the solvent control group for tail length and OTM parameters. When the tail DNA% was evaluated, only the concentration of 100 ng L−1 presented a significant increase (˜ 40%) when compared to the solvent control group
3.5. Erythrotoxicity The peripheral blood erythrocyte morphometry of P. reticulata was analyzed using seven parameters: total area of the cell, area of the nucleus, area of the cytoplasm, perimeter of the nucleus, circularity, area of the nucleus/ total area of the cell ratio, and area of the nucleus/ area of the cytoplasm ratio. The results showed that BP-3 did not alter cell morphometry, because none of these measurements presented significant differences when compared to negative and solvent controls (Table 2).
Fig. 2. Qualitative analysis of benzophenone-3 by HPLC-DAD. (A) Chromatogram of benzophenone-3 (0.1 mg/ml) at 288 nm and (B) UV spectra (190–400 nm) of featured peak. 4
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Fig. 4. DNA damage in Poecilia reticulata erythrocytes after acute exposure (96 h) to benzophenone-3 (BP-3). The parameters evaluated in the Comet assay were (A) Percentage of DNA in the tail, (B) Olive tail moment, and (C) tail length. The results are expressed as mean ± standard deviation. Different letters represent statistically significant differences between groups (n = 9) according to ANOVA with Tukey post-test (p < 0.05). NC: negative control group; SC: solvent control group.
4. Discussion
reticulata erythrocytes. This exposition did not cause alterations in the general health of animals, as we can see by the K value. According to Vazzoler (1996), K is an indicator of the degree of the healthiness of an individual. This value reflects recent nutritional aspects and/or reserve expenditure on cyclical activities, providing an indirect estimate of energy storage by animals (Le Cren, 1951; Mozsár et al., 2015). In ecotoxicological studies, measures of weight, length and K are often used to assess the effects of exposure to toxic substances. However, variations of these measurements are best characterized in chronic exposures using larvae or juvenile specimens (Smolders et al., 2002). So, we decided to use a classical approach: comet assay associated with the evaluation of MN and ENA induction. The analysis of DNA alterations in aquatic organisms is a suitable method for evaluating
BP-3 is lipophilic, photostable, and was detected in fish tissues (Balmer et al., 2005; Kim and Choi, 2014). An ecological risk assessment study pointed that the 96 h-EC50 of BP-3 on the green algae C. vulgaris was 2980 ng L−1. The 48 h-LC50 of BP-3 on the planktonic crustacean D. magna was 1090 ng L−1. And the 96 h-LC50 of BP-3 on the fish D. rerio was 3890 ng L-1 (Du et al., 2017). This study revealed the acute toxicity of BP-3 in low concentrations and pointed that more studies should be performed to enrich the toxicity test database and assessment of chemicals used as UV filters. So, considering limited ecotoxicological information, we tested the acute toxicity of environmentally relevant concentrations of BP-3 in P.
Fig. 5. DNA damage induced after acute exposure (96 h) to benzophenone-3 (BP-3) on Poecilia reticulata erythrocytes. Representative images of the DNA migration pattern of erythrocytes submitted to Comet assay after exposure to BP-3. The cells were stained with Diamond™ Nucleic Acid Dye (Promega®, NSW, Australia) and the images were captured using an Olympus BX60 light microscope (Olympus, Japan) with an Olympus CellSens software (Olympus, Japan) under 20× objective. (A) Negative control group (n = 9); (B) Solvent control group (n = 9); (C) group exposed to 10 ng L−1 of BP-3 (n = 9); (D) group exposed to 100 ng L−1 of BP-3 (n = 9); and (E) group exposed to 1000 ng L−1 of BP-3 (n = 9). 5
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Fig. 6. Frequencies of nuclear abnormalities and micronucleus in Poecilia reticulata erythrocytes after acute exposure (96 h) to benzophenone -3 (BP-3). The parameters analyzed were total nuclear abnormalities (A) and frequency of erythrocytes with lobed nuclei (B), micronuclei (C), notched nuclei (D), blebbed nuclei (E), and broken eggs nuclei (F). The results are expressed as mean ± standard deviation. Only charts with significant differences between groups are flagged with letters. Different letters represent statistically significant differences between groups (n = 9) according to ANOVA with Tukey post-test (p < 0.05).
individual eukaryotic cells by physical and chemical pollutants (Dhawan et al., 2009; Lapuente et al., 2015). But it is important to highlight that the comet assay measures transient genetic damage that is not a fixed change to the DNA because the strand breaks may be repaired (Beedanagari et al., 2014). Because we can not evaluate mutagenicity using the comet assay we
genotoxicants (Monteiro et al., 2011). Using the comet assay, we observed an induction of DNA breaks in fish exposed to BP-3 at concentrations above 10 ng L−1. The comet assay has been used as the most reliable, sensitive and fast technique for assessment of environmental genotoxicants on fish (Dhawan et al., 2009). This assay could assess single/double-strand breakage and alkali-labile sites induced in 6
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Table 2 Morphometric measurements (mean ± standard deviation) of Poecilia reticulata erythrocytes after acute exposure to benzophenone-3 (BP-3). Parameter
NC
Total area (TA) Nucleus area (NA) Cytoplasm area (CA) Nucleus perimeter Nucleus circularity NA/TA CA/TA
8.03 1.95 6.07 6.53 0.52 0.24 0.34
SC ± ± ± ± ± ± ±
0.53 0.18 0.63 1.74 0.11 0.03 0.06
7.63 2.01 5.61 6.97 0.56 0.26 0.37
± ± ± ± ± ± ±
0.45 0.15 0.37 0.53 0.06 0.01 0.04
BP-3 10 ng L−1
BP-3 100 ng L−1
BP-3 1000 ng L−1
8.25 2.25 5.99 8.01 0.50 0.27 0.40
7.72 2.03 5.68 7.65 0.53 0.26 0.37
7.12 2.01 5.10 7.74 0.56 0.29 0.42
± ± ± ± ± ± ±
0.99 0.34 0.78 1.70 0.15 0.02 0.05
± ± ± ± ± ± ±
0.43 0.11 0.46 1.65 0.18 0.02 0.04
± ± ± ± ± ± ±
0.59 0.09 0.61 1.35 0.13 0.02 0.06
In summary, BP-3 at environmentally relevant concentrations induced genotoxic effects in P. reticulata erythrocytes after acute exposure. It draws attention for a chronic exposition of the aquatic biota to BP-3 that could be occurring all around the word, because of the BP-3 widespread usage in commercial and industrial products, what result in a continuous release into aquatic systems (Mao et al., 2019). BP-3 is one of the dominant UV filters in the environment and was detected in all samples of a study consisting of eight cities in four countries (China, the United States, Japan, and Thailand) and in the North American Arctic (Tsui et al., 2014; Mao et al., 2019). This broad distribution stimulates studies to investigate chronic BP-3 exposure, the next step in our work. Since this compound is an emerging pollutant, the toxicological risk assessment of BP-3 is important to stimulate the governmental policies to establish a maximum safe dose for this compound.
also used another well-established approach that has proven useful in assessing the mutagenic effect of a wide range of compounds in fish: the evaluation of MN induction (Udroiu, 2006). MNs are extra-nuclear bodies originating from acentric chromosome fragments or whole chromosomes that lag behind during cell division process (Luzhna et al., 2013). The analysis of ENA, a variant of the standard MN test, has also been widely used in fish toxicology (Ergene et al., 2007). In this assay, a number of alterations in red blood cell nuclei that may lead to their fragmentation and/or to MN formation are recorded instead of counting just the MN (Carrasco et al., 1990; Costa et al., 2008; Monteiro et al., 2011). In this work, the frequency of MN and ENA of fish not exposed to BP-3 agreed with the previously reported basal levels of alterations for P. reticulata (Souza-Filho et al., 2013; Qualhato et al., 2017). However, total nuclear changes (ENA and MN) and the frequency of erythrocytes with lobed nuclei increased in fish exposed to BP-3 at the concentration of 1000 ng L−1, indicating that the DNA breaks are not fixed from this concentration on. The lobed nuclei may result from problems segregating tangled and attached chromosomes (Çavaş and Ergene-Gözükara, 2005). It has been widely accepted in toxicology that the dose-response relationship follows a sigmoidal curve of response that is the lower and the upper doses approach zero and 100% response, respectively. Despite its long term acceptance, studies developed in the last decades suggest the possibility that alternative dose-response models may better account for observed dose responses in the low dose zone, like hormesis (Calabrese, 2015). Hormesis is a dose-response phenomenon characterized by a biphasic behavior (Calabrese and Baldwin, 2002). Thinking on biological plasticity, hormesis is a coordinated response of cells and organisms that involves multiple integrative cellular functions, each of which is quantitatively hormetic, to coordinate a final holistic response against a challenge (Calabrese and Mattson, 2017). The hormesis stimulatory response in the low dose zone (below the traditional toxic threshold) can result from an overcompensation following an initial disruption in homeostasis (Calabrese, 2015). Considering the overcompensation hypothesis, several theoretical explanations of hormesis have been proposed, including the stimulation of DNA damage repair, oxidative stress mechanism, immune function, and alteration of gene expression pattern (Shi et al., 2016). Interestingly, in this study we observed a tendency to decrease the frequencies of erythrocytes nuclear alterations at the tested intermediate concentration (100 ng L−1) of BP-3, which could indicate a possible hormetic behavior. Since we observed some ENAs and MN in this work, we decided to use some morphometric descriptors, like a total area of the cell, area of the nucleus, and perimeter of the nucleus, to correlate morphological parameters to the toxicological response. Cell and nuclei shape have been considered important indicators of the events occurring in the cellular micro-environment (Lobo et al., 2016). However, we did not succeed in this approach, maybe because the alterations observed in this work were not robust. Moreover, this shape–function paradigm may work better with biological processes such as proliferation, differentiation, cell migration, behavior, motility, cellular communication, malignancy, and growth dynamics (Lobo et al., 2016; Carella et al., 2017).
Funding This work was supported by Universidade Estadual de Goiás in Brazil(UEG; No. 201600020010791) and Fundação de Amparo à Pesquisa do Estado de Goiás in Brazil (FAPEG; No. PPP/ 201610267001019). The author SSA received a scholarship from FAPEG and EFLCB was supported by UEG with a fellowship at the program PROBIP (Scientific Production Support Program). Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by SSA, TLR, GQ, LARO, CLA, and EFLCB. The first draft of the manuscript was written by SSA, TLR, and EFLCB and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest None. References Agha, N.Y., Haidar, S., Al-Khayat, M.A., 2013. Development and validation of RP-HPLC method for analysis of four UV filters in sunscreen products. Int. J. Pharm. Sci. Rev. Res. 23, 254–258. Ahmedova, A., Mantareva, V., Enchev, V., Mitewa, M., 2002. 2-Acetylindan-1,3-dione and its Cu2+ and Zn2+ complexes as promising sunscreen agents. Int. J. Cosmet. Sci. 24, 103–110. Araldi, R.P., Melo, T.C., Mendes, T.B., Sá-Júnior, P.L., Nozima, B.H.N., Ito, E.T., Carvalho, R.F., Souza, E.B., Stocco, R.C., 2015. Using the comet and micronucleus assays for genotoxicity studies: a review. Biomed. Pharmacother. 72, 74–82. Baird, R.B., Eaton, A.D., Rice, E.W., Bridgewater, L.L., Water Environment, F, American Public Health, A, American Water Works, A, 2017. Standard Methods for the Examination of Water and Wastewater, 23rd ed. American Public Health Association, New York. Balmer, M.E., Buser, H.-R., Müller, M.D., Poiger, T., 2005. Occurrence of some organic UV filters in wastewater, in surface waters, and in fish from Swiss Lakes. Environ. Sci. Technol. 39, 953–962. Beedanagari, S., Vulimiri, S.V., Bhatia, S., Mahadevan, B., 2014. Chapter 43 - genotoxicity biomarkers: molecular basis of genetic variability and susceptibility. In: Gupta, R.C. (Ed.), Biomarkers in Toxicology. Academic Press, Boston, pp. 729–742.
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Acute exposure to environmentally relevant concentrations of benzophenone-3 induced genotoxicity in Poecilia reticulata
T
Sara dos Santos Almeidaa, Thiago Lopes Rochab, Gabriel Qualhatoc, Leandra de Almeida Ribeiro Oliveiraa,d, Cátia Lira do Amarala, Edemilson Cardoso da Conceiçãod, ⁎ Simone Maria Teixeira de Sabóia-Moraisc, Elisa Flávia Luiz Cardoso Bailãoa, a
Laboratório de Biotecnologia, Câmpus Henrique Santillo, Universidade Estadual de Goiás, Anápolis, Goiás, Brazil Laboratory of Environmental Biotechnology and Ecotoxicology, Institute of Tropical Pathology and Public Health, Federal University of Goiás, Goiânia, Goiás, Brazil c Laboratório de Comportamento Celular, Departamento de Morfologia, Instituto de Ciências Biológicos, Universidade Federal de Goiás, Goiânia, Goiás, Brazil d Laboratório de PD&I de Bioprodutos, Universidade Federal de Goiás, Faculdade de Farmácia, Goiânia, Goiás, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Biomarkers Comet assay DNA damage Ecotoxicology Guppy Micronuclei
The organic UV filter benzophenone-3 (BP-3), widely used in the commercial formulations of sunscreens and personal care products, is considered an emerging pollutant and has been associated with several human and environmental health concerns. However, knowledge about their mode of action and ecotoxicity on aquatic biota is scarce. In this scenario, the objective of this work was to evaluate the genotoxic, mutagenic, and erythrotoxicity effects of BP-3 in the guppy Poecilia reticulata after acute exposure. Adult females of P. reticulata were exposed to three non-lethal and environmentally relevant concentrations of BP-3 (10, 100, and 1000 ng L−1) during 96 h of exposure, and the somatic parameter [Fulton condition factor (K)], genotoxicity (comet assay), mutagenicity [micronucleus (MN) and erythrocyte nuclear abnormalities (ENA) tests] and erythrotoxicity parameters (such as total cell area and nucleus-cytoplasmic ratio) were analyzed. Results showed that the general physiological condition (K value) of fish was not affected by acute exposure to BP-3. However, BP-3 induced DNA damage at 100 and 1000 ng L−1 and increased the frequency of total ENA at 1000 ng L−1, specially lobed nucleus, when compared to control group, indicating its genotoxic and mutagenic effects. Furthermore, the BP-3 did not induce significant changes in the total cell area and nucleus-cytoplasmic ratio. In summary, results showed that the BP-3 at environmentally relevant concentration was genotoxic to freshwater fish P. reticulata, confirming its environmental risk.
1. Introduction Designed initially to protect human skin from UVA and UVB radiations, UV filters are included in a wide range of products to protect humans and materials from damage by UV irradiation and to ensure a longer shelf life to products (Brooke et al., 2008; Gackowska et al., 2014; Kim and Choi, 2014). In this way, the constant presence of UV filters in environmental matrices and in biota lead them to be considered as emerging pollutants. In addition to being frequently detected in the environment, emerging pollutants are not included in environmental monitoring programs, have no regulatory legislation and their removal by wastewater treatment plants is ineffective and may represent a risk to human health and to the environment even in concentrations in order of μg L−1 or even ng L−1 (Silva and Collins, 2011; Montagner et al., 2017).
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Benzophenone-3 (BP-3) is one of the organic UV filters most used by industry (Mao et al., 2019). The occurrence of BP-3 in the aquatic environment has been widely reported (Ramos et al., 2015), such as in rivers (2031 ng L−1) (Díaz-Cruz et al., 2019), untreated residual waters (7800 ng L−1) and treated ones (700 ng L−1) (Balmer et al., 2005), drinking water (115 ng L−1) (Silva et al., 2015), and groundwater (19.2 ng L−1) (Jurado et al., 2014). The BP-3 was detected in high concentrations in lakes (up to 550 ng L−1) (Grabicova et al., 2013) and beaches in Japan (up to 5429 ng L−1) (Tsui et al., 2014). The ecotoxicological effects of BP-3 on aquatic species have been reported at different trophic levels. BP-3 induced high acute toxicity (growth inhibition or mortality) to Chlorella vulgaris, Daphnia magna, and Danio rerio after 48 or 96 h exposure (Du et al., 2017). In vitro and in vivo studies showed that BP-3 is able to interfere with reproduction, hormonal signaling, and oxidative stress in fish (Schlumpf et al., 2001;
Corresponding author. E-mail address: elisafl[email protected] (E.F.L.C. Bailão).
https://doi.org/10.1016/j.aquatox.2019.105293 Received 26 June 2019; Received in revised form 3 September 2019; Accepted 3 September 2019 Available online 04 September 2019 0166-445X/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Experimental design of this work. After the acclimation period, 45 fish were placed randomly in 15 tanks of 7 L capacity filled with 6 L of reconstituted water. Five experimental groups were established: negative control (NC), containing only reconstituted water; solvent control (SC), containing reconstituted water and the same amount of ethanol (2.5 ppm) used in the dilution of benzophenone-3 (BP-3); and three groups with environmentally relevant concentrations of BP-3 (10, 100 or 1000 ng L−1). Each group consisted of 3 tanks containing 3 fish each, so 9 fish in total for each group were tested independently (n = 9). All groups were tested in a static system without the renewal of water and BP-3 for 96 h.
2. Material and methods
Kunz et al., 2006; Coronado et al., 2008; Blüthgen et al., 2012; Liu et al., 2015), indicating its potential as an endocrine disruptor in fish. BP-3 also interferes with the embryonic development and reproduction of aquatic invertebrates, such as Chironomus riparius (Ozáez et al., 2014; Campos et al., 2019), and is reported as a threat to coral reefs worldwide (He et al., 2019; Schneider and Lim, 2019). However, the genotoxic and mutagenic potential of BP-3 to aquatic organisms has not been addressed. The comet assay is a suitable and widely used technique for the identification of genotoxicity biomarkers, allowing the verification of breaks in the DNA chain induced by exogenous agents (Bolognesi and Hayashi, 2011; Qualhato et al., 2017). Micronucleus (MN) and erythrocyte nuclear abnormalities (ENA) have been identified as an effective test to measure chromosomal damage in fish after exposure to contaminants (Qualhato et al., 2017, 2018). Currently, the association between these techniques is seen as the best test battery to evaluate the genotoxicity and mutagenicity in aquatic organisms (Rocha et al., 2014; Araldi et al., 2015). Fish have been shown to be good biomonitor, since they are relatively sensitive to changes in the environment and to effects of pollutants (Wepener et al., 2011). In addition, fish have a fundamental role in food networks and are of great commercial importance (van der Oost et al., 2003). The guppy Poecilia reticulata, an ovoviviparous freshwater fish, is a biomonitor recommended by the American Public Health Association (APHA) and the Organization for Economic Cooperation and Development (OECD) (OECD, 1992; Baird et al., 2017). It has been used as a model testing organism for the study of a wide range of substances with toxic potential (Sharbidre et al., 2011; Souza-Filho et al., 2013; Rocha et al., 2015; Qualhato et al., 2017, 2018). However, there are no studies about the effects of BP-3 on this species. In this sense, the objective of this work was to evaluate the genotoxicity, mutagenicity and erythrotoxicity of the organic UV filter BP-3 in the guppy P. reticulata during acute exposure to environmentally relevant concentrations. We evaluated both genotoxic (DNA damage), mutagenic (MN and ENA tests) and morphometric (such as total cell area and nucleus-cytoplasmic ratio) parameters on P. reticulata exposed to BP-3.
2.1. Benzophenone-3 Powder BP-3 was donated by Volta Flora Manipulation Pharmacy (Anápolis, Goiás) and was submitted to a qualitative analysis using a high efficiency liquid chromatography (HPLC) method developed by Agha et al. (2013). The BP-3 sample (10 μL) was injected into a HPLCPDA equipment (Waters Alliance e2695 chromatograph; Milford, MA, USA) in the concentration of 0.1 mg mL−1. The chromatographic separation was achieved using a Zorbax C18 column (250 x 4.6 mm, 5 μm) coupled with a guard column of the same stationary phase. Data processing was performed by Empower 2.0. The mobile phase was composed of methanol and Milli-Q water (85:15 v/v) and the flow rate was 1 mL min−1. The mobile phase was filtered through a 0.45 μm filter membrane (Millipore, MA, United States) and degassed in an ultrasonic bath for 20 min before use. The samples were solubilized in methanol and filtered through a 0.45 μm Millex® membrane (Millipore, MA, USA). The wavelength used for the BP-3 detection was 288 nm. The column oven was set at 30 °C. The qualitative analysis of BP-3 was performed by comparison of its UV spectra (190–400 nm) with the literature values. 2.2. Ethical approval for animal use The experimental procedures and animal management were approved by Animal Use Ethics Committees from Universidade Estadual de Goiás (UEG; protocol n° 009/2016) and from Universidade Federal de Goiás (UFG; protocol n° 046/2017). The experiment followed the guidelines for testing chemicals for acute toxicity established in the OECD 203 guideline (OECD et al., 1992). 2.3. Experimental design Adult females of P. reticulata were collected in the Water Treatment 2
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refrigeration (10 °C) and in the dark. The electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) was added and the slides remained immersed for 30 min. The electrophoresis run was performed at 1 V/cm and 300 mA for 25 min. After the run, the slides were neutralized with Tris buffer (Tris 0.4, pH 7.5) for 5 min. The slides were washed with distilled water and dried at room temperature. After drying, slides were fixed in absolute ethanol for 10 min and stored. The analysis was performed with Diamond™ Nucleic Acid Dye (Promega®, NSW, Australia) fluorescence dye, 495 nm emission and 558 nm excitation using a light microscope (Olympus BX60, Japan) under 20x objective and the images were scanned using Olympus CellSens software (Olympus, Japan). For image analysis, the Comet Image Analysis system (Comet Score-software; TriTek, Sumerduck, VA, EUA) was used. A hundred nucleoids were randomly selected (50 in each slide, two slides per specimen), totaling 900 nucleoids per experimental group (n = 9 fish per experimental group). The degree of DNA damage was evaluated using the following parameters: percentage of DNA in the tail, tail length, and Olive tail moment.
Station (Saneamento de Goiás - SANEAGO - 16° 37′ 59″ S and 49° 15′ 44″ W, Goiânia, Goiás, Brazil) and acclimated during 10 days in static tanks (60 L) containing reconstituted water (ISO, 1996) at 27 ± 2 °C, pH 7.1 ± 2, ammonia 0 - 0.01 μL/L, and photoperiod (12:12 h light/ dark), as recommended by Rocha et al. (2015). Fish were fed ad libitum three times per day with the commercial fish food Cardume 36% (VB Alimentos Ltd). After acclimation period, 45 fish (total weight of 0.14 ± 0.02 g; total length of 2.3 ± 0.1 cm; standard length of 1.9 ± 0.1 cm) were placed randomly in 15 tanks (Fig. 1) of 7 L capacity filled with 6 L of reconstituted water (ISO, 1996), as recommended by Rocha et al. (2015). Before exposure, BP-3 was diluted with ethanol P.A. (96%) and the final concentration of ethanol in the exposure medium was adjusted to 2.5 ppm. Five experimental groups (n = 9 fish per group) were established: negative control (NC), containing only reconstituted water; solvent control (SC), containing reconstituted water and the same amount of ethanol used in the dilution of BP-3 (2.5 ppm); and three groups with environmentally relevant concentrations of BP-3 (10, 100 or 1000 ng L−1). The concentrations used for the exposure are in agreement with the concentrations found in several studies that investigated the presence of UV filters in environmental matrices (Balmer et al., 2005; Grabicova et al., 2013; Jurado et al., 2014; Tsui et al., 2014; Silva et al., 2015; Díaz-Cruz et al., 2019). All groups were tested in a static system without the renewal of water and BP-3 for 96 h. After the exposure period, the animals were euthanized by hypothermia and blood samples were collected using the tail artery as recommended by Qualhato et al. (2017).
2.6. Mutagenicity
Where, W is the weight of the individual and L is the total length.
The mutagenicity was analyzed by micronucleus (MN) and erythrocyte nuclear abnormalities (ENA) tests, such as described by Carrasco et al. (1990) and Fenech et al. (2003) with modifications. Blood was collected from the caudal artery in a microtube containing 250 μL of 0.01 M PBS buffer and pH 7.2 and homogenized. Two slides per fish (n = 9 fish per experimental group) were prepared by the blood distension technique. The material was stained with quick panoptic dye (Instant Prov, New Prov®, Pinhais, Brazil). A total of 1000 erythrocytes were analyzed per animal (totalizing 9000 erythrocytes per experimental group; n = 9 fish per experimental group) using a Primo Star optical microscope (Zeiss, Oberkochen, Germany) under immersion objective (100 ×). The erythrocytes were classified according to the nuclear morphologies proposed by Fenech (2000) and Fenech et al. (2003): MN and nuclei of the type notched, lobed, broken eggs, and blebbed. The total frequency of abnormalities represents the sum of all the nuclear alterations (Qualhato et al., 2017).
2.5. Genotoxicity
2.7. Erythrotoxicity
The genotoxic effects (DNA damage) were assessed by the comet assay according to Singh et al. (1988), with modifications proposed by Qualhato et al. (2017). Peripheral blood was obtained through the caudal artery and collected in microtubes containing 250 μL of 0.1 M PBS buffer pH 7.2. Then 15 μL of buffer solution + blood was diluted in 120 μL low melting point (LMP) agarose, previously prepared and heated (37 °C). The samples were homogenized with a micropipette, scattered with coverslips on microscope slides coated with normal agarose (1.5%) and kept at 10 °C for 3 min for the solidification of the LMP agarose. The glass slides were carefully removed and the slides were immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 0.2 M NaOH, 0.03 M sodium lauryl sarcosine, 10% DMSO, 1% Triton X-100 and pH 10) for 2 h without illumination. After the lysis time, the slides were transferred to the electrophoresis, kept under
Erythrotoxicity was analyzed in terms of several morphometric parameters of erythrocytes (Table 1), such as described by Carella et al. (2017) with modifications. Blood was collected from the caudal artery in a microtube containing 250 μL of 0.01 M PBS buffer at pH 7.2 and homogenized. Two slides per fish (n = 9 fish per experimental group) were prepared by the blood distension technique. The material was stained with quick panoptic (Instant Prov, New Prov®, Pinhais, Brazil) and the analysis was performed according to Carella et al. (2017) with modifications, using a Primo Star optical microscope (Zeiss, Oberkochen, Germany) under immersion objective (100 ×). The slides were photographed using the Axiocam 105 image capture system (Zeiss, Oberkochen, Germany) and the images were processed using Fiji version 1.0 free software based on ImageJ. For the analysis were considered non-overlapping erythrocytes that were completely inserted
2.4. Somatic biomarkers After the exposure period (96 h), weight (g), total length (mm), and standard length (mm) were collected from all individuals, and the Fulton Condition Factor (K) was estimated by the following formula (Fulton, 1904):
K=
W L³
Table 1 List of morphological parameters of erythrocytes used in this study. Parameter Total cell area (TA) Total nucleus area (NA) Area of cytoplasm (AC) Nucleus Perimeter (PN) Circularity NA/TA NA/AC
Unity
Description 2
The area within the polygon delineated by the perimeter of the cell The area within the polygon delineated by the perimeter of the nucleus Obtained by the difference TA - NA Perimeter calculated from the centers of boundary pixels 4*π*Area/Perimeter2, also called a form factor Ratio between the core area and the total cell area Ratio between the nucleus area and the cytoplasm area
Pixels Pixels2 Pixels2 Pixels – – –
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into the capture field, totalizing 2000 erythrocytes per animal and 18,000 per experimental group (n = 9 fish per experimental group). 2.8. Statistical analyses Statistical analysis of the data was performed using the free software Paleontological statistics - PAST version 3.18 (Hammer et al., 2001). The assumptions of data normality and homogeneity of variances were verified by the Shapiro-Wilk test and the Levene test, respectively. Once observed the violation of the assumptions, the data transformation was then attempted through logarithmic transformation. For the data on which the transformation was successful, the results were compared using parametric tests (one-way ANOVA followed by the Tukey's test). When the transformation was not possible, the Kruskal-Wallis nonparametric test was used. All results are expressed as mean ± standard deviation of the mean and were considered significant when p < 0.05.
Fig. 3. Fulton condition factor (K) of Poecilia reticulata after acute exposure (96 h) to benzophenone-3 (BP-3). The negative control (NC) group was not exposed to BP-3, the solvent control (SC) group was exposed just to the BP-3 solvent (ethanol) and the other groups were exposed to crescent concentrations of BP-3: 10, 100 or 1000 ng L−1 for 96 h. Results are presented as mean ± standard deviation of each group (n = 9). The ANOVA one-way test demonstrated no significant differences between the groups (p > 0.05).
3. Results 3.1. Qualitative analysis of benzophenone-3 The HPLC results showed that the retention time for BP-3 was 5.2 min and its UV spectra (Fig. 2) is in accordance with those reported in the literature (Ahmedova et al., 2002), indicating that the compound used in this work is pure and could be considered as BP-3.
(Fig. 4). 3.4. Mutagenicity
3.2. Somatic biomarker
P. reticulata erythrocytes containing MN or other nuclear abnormalities, such as notched, broken eggs, lobed, and blebbed nucleus, were observed after acute exposure to BP-3 (Fig. 6). Total nuclear alterations frequency increased in fish exposed to BP-3 at 1000 ng L−1 when compared to the negative and solvent control groups (Fig. 6). Individually, nuclei with lobed-type changes were more prevalent than other nuclear changes (Fig. 6B). The fish exposed to BP-3 at 1000 ng L−1 showed high frequency of this abnormality (Fig. 6B) when compared to the control groups. We observed that was a tendency to decrease the frequencies of erythrocytes nuclear alterations at the concentration of 100 ng L−1 BP-3, except for the presence of MN, when compared to the other concentrations tested (Fig. 6).
To verify if the acute exposure to BP-3 was able to interfere in the general fish health condition, the K value was determined based on the biometric data. No significant differences were found between the animals exposed to BP-3 and those from negative control and solvent control (Fig. 3), indicating that the guppies were in similar physiological conditions during the acute exposure to BP-3. No fish mortality was observed in this study. 3.3. Genotoxicity The DNA damage in P. reticulata peripheral erythrocytes was analyzed by Comet assay and expressed as % of tail DNA, Olive tail moment (OTM), and comet tail length (Fig. 4). As expected, the negative control group showed a significantly lower DNA damage when compared to all the other groups (Fig. 5). The group exposed to 10 ng L−1 of BP-3 did not differ from the solvent control group. An increase in DNA damage was observed in fish exposed to BP-3 at concentrations of 100 and 1000 ng L−1. These concentrations did not differ significantly from each other, but had significant differences with respect to the solvent control group for tail length and OTM parameters. When the tail DNA% was evaluated, only the concentration of 100 ng L−1 presented a significant increase (˜ 40%) when compared to the solvent control group
3.5. Erythrotoxicity The peripheral blood erythrocyte morphometry of P. reticulata was analyzed using seven parameters: total area of the cell, area of the nucleus, area of the cytoplasm, perimeter of the nucleus, circularity, area of the nucleus/ total area of the cell ratio, and area of the nucleus/ area of the cytoplasm ratio. The results showed that BP-3 did not alter cell morphometry, because none of these measurements presented significant differences when compared to negative and solvent controls (Table 2).
Fig. 2. Qualitative analysis of benzophenone-3 by HPLC-DAD. (A) Chromatogram of benzophenone-3 (0.1 mg/ml) at 288 nm and (B) UV spectra (190–400 nm) of featured peak. 4
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Fig. 4. DNA damage in Poecilia reticulata erythrocytes after acute exposure (96 h) to benzophenone-3 (BP-3). The parameters evaluated in the Comet assay were (A) Percentage of DNA in the tail, (B) Olive tail moment, and (C) tail length. The results are expressed as mean ± standard deviation. Different letters represent statistically significant differences between groups (n = 9) according to ANOVA with Tukey post-test (p < 0.05). NC: negative control group; SC: solvent control group.
4. Discussion
reticulata erythrocytes. This exposition did not cause alterations in the general health of animals, as we can see by the K value. According to Vazzoler (1996), K is an indicator of the degree of the healthiness of an individual. This value reflects recent nutritional aspects and/or reserve expenditure on cyclical activities, providing an indirect estimate of energy storage by animals (Le Cren, 1951; Mozsár et al., 2015). In ecotoxicological studies, measures of weight, length and K are often used to assess the effects of exposure to toxic substances. However, variations of these measurements are best characterized in chronic exposures using larvae or juvenile specimens (Smolders et al., 2002). So, we decided to use a classical approach: comet assay associated with the evaluation of MN and ENA induction. The analysis of DNA alterations in aquatic organisms is a suitable method for evaluating
BP-3 is lipophilic, photostable, and was detected in fish tissues (Balmer et al., 2005; Kim and Choi, 2014). An ecological risk assessment study pointed that the 96 h-EC50 of BP-3 on the green algae C. vulgaris was 2980 ng L−1. The 48 h-LC50 of BP-3 on the planktonic crustacean D. magna was 1090 ng L−1. And the 96 h-LC50 of BP-3 on the fish D. rerio was 3890 ng L-1 (Du et al., 2017). This study revealed the acute toxicity of BP-3 in low concentrations and pointed that more studies should be performed to enrich the toxicity test database and assessment of chemicals used as UV filters. So, considering limited ecotoxicological information, we tested the acute toxicity of environmentally relevant concentrations of BP-3 in P.
Fig. 5. DNA damage induced after acute exposure (96 h) to benzophenone-3 (BP-3) on Poecilia reticulata erythrocytes. Representative images of the DNA migration pattern of erythrocytes submitted to Comet assay after exposure to BP-3. The cells were stained with Diamond™ Nucleic Acid Dye (Promega®, NSW, Australia) and the images were captured using an Olympus BX60 light microscope (Olympus, Japan) with an Olympus CellSens software (Olympus, Japan) under 20× objective. (A) Negative control group (n = 9); (B) Solvent control group (n = 9); (C) group exposed to 10 ng L−1 of BP-3 (n = 9); (D) group exposed to 100 ng L−1 of BP-3 (n = 9); and (E) group exposed to 1000 ng L−1 of BP-3 (n = 9). 5
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Fig. 6. Frequencies of nuclear abnormalities and micronucleus in Poecilia reticulata erythrocytes after acute exposure (96 h) to benzophenone -3 (BP-3). The parameters analyzed were total nuclear abnormalities (A) and frequency of erythrocytes with lobed nuclei (B), micronuclei (C), notched nuclei (D), blebbed nuclei (E), and broken eggs nuclei (F). The results are expressed as mean ± standard deviation. Only charts with significant differences between groups are flagged with letters. Different letters represent statistically significant differences between groups (n = 9) according to ANOVA with Tukey post-test (p < 0.05).
individual eukaryotic cells by physical and chemical pollutants (Dhawan et al., 2009; Lapuente et al., 2015). But it is important to highlight that the comet assay measures transient genetic damage that is not a fixed change to the DNA because the strand breaks may be repaired (Beedanagari et al., 2014). Because we can not evaluate mutagenicity using the comet assay we
genotoxicants (Monteiro et al., 2011). Using the comet assay, we observed an induction of DNA breaks in fish exposed to BP-3 at concentrations above 10 ng L−1. The comet assay has been used as the most reliable, sensitive and fast technique for assessment of environmental genotoxicants on fish (Dhawan et al., 2009). This assay could assess single/double-strand breakage and alkali-labile sites induced in 6
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Table 2 Morphometric measurements (mean ± standard deviation) of Poecilia reticulata erythrocytes after acute exposure to benzophenone-3 (BP-3). Parameter
NC
Total area (TA) Nucleus area (NA) Cytoplasm area (CA) Nucleus perimeter Nucleus circularity NA/TA CA/TA
8.03 1.95 6.07 6.53 0.52 0.24 0.34
SC ± ± ± ± ± ± ±
0.53 0.18 0.63 1.74 0.11 0.03 0.06
7.63 2.01 5.61 6.97 0.56 0.26 0.37
± ± ± ± ± ± ±
0.45 0.15 0.37 0.53 0.06 0.01 0.04
BP-3 10 ng L−1
BP-3 100 ng L−1
BP-3 1000 ng L−1
8.25 2.25 5.99 8.01 0.50 0.27 0.40
7.72 2.03 5.68 7.65 0.53 0.26 0.37
7.12 2.01 5.10 7.74 0.56 0.29 0.42
± ± ± ± ± ± ±
0.99 0.34 0.78 1.70 0.15 0.02 0.05
± ± ± ± ± ± ±
0.43 0.11 0.46 1.65 0.18 0.02 0.04
± ± ± ± ± ± ±
0.59 0.09 0.61 1.35 0.13 0.02 0.06
In summary, BP-3 at environmentally relevant concentrations induced genotoxic effects in P. reticulata erythrocytes after acute exposure. It draws attention for a chronic exposition of the aquatic biota to BP-3 that could be occurring all around the word, because of the BP-3 widespread usage in commercial and industrial products, what result in a continuous release into aquatic systems (Mao et al., 2019). BP-3 is one of the dominant UV filters in the environment and was detected in all samples of a study consisting of eight cities in four countries (China, the United States, Japan, and Thailand) and in the North American Arctic (Tsui et al., 2014; Mao et al., 2019). This broad distribution stimulates studies to investigate chronic BP-3 exposure, the next step in our work. Since this compound is an emerging pollutant, the toxicological risk assessment of BP-3 is important to stimulate the governmental policies to establish a maximum safe dose for this compound.
also used another well-established approach that has proven useful in assessing the mutagenic effect of a wide range of compounds in fish: the evaluation of MN induction (Udroiu, 2006). MNs are extra-nuclear bodies originating from acentric chromosome fragments or whole chromosomes that lag behind during cell division process (Luzhna et al., 2013). The analysis of ENA, a variant of the standard MN test, has also been widely used in fish toxicology (Ergene et al., 2007). In this assay, a number of alterations in red blood cell nuclei that may lead to their fragmentation and/or to MN formation are recorded instead of counting just the MN (Carrasco et al., 1990; Costa et al., 2008; Monteiro et al., 2011). In this work, the frequency of MN and ENA of fish not exposed to BP-3 agreed with the previously reported basal levels of alterations for P. reticulata (Souza-Filho et al., 2013; Qualhato et al., 2017). However, total nuclear changes (ENA and MN) and the frequency of erythrocytes with lobed nuclei increased in fish exposed to BP-3 at the concentration of 1000 ng L−1, indicating that the DNA breaks are not fixed from this concentration on. The lobed nuclei may result from problems segregating tangled and attached chromosomes (Çavaş and Ergene-Gözükara, 2005). It has been widely accepted in toxicology that the dose-response relationship follows a sigmoidal curve of response that is the lower and the upper doses approach zero and 100% response, respectively. Despite its long term acceptance, studies developed in the last decades suggest the possibility that alternative dose-response models may better account for observed dose responses in the low dose zone, like hormesis (Calabrese, 2015). Hormesis is a dose-response phenomenon characterized by a biphasic behavior (Calabrese and Baldwin, 2002). Thinking on biological plasticity, hormesis is a coordinated response of cells and organisms that involves multiple integrative cellular functions, each of which is quantitatively hormetic, to coordinate a final holistic response against a challenge (Calabrese and Mattson, 2017). The hormesis stimulatory response in the low dose zone (below the traditional toxic threshold) can result from an overcompensation following an initial disruption in homeostasis (Calabrese, 2015). Considering the overcompensation hypothesis, several theoretical explanations of hormesis have been proposed, including the stimulation of DNA damage repair, oxidative stress mechanism, immune function, and alteration of gene expression pattern (Shi et al., 2016). Interestingly, in this study we observed a tendency to decrease the frequencies of erythrocytes nuclear alterations at the tested intermediate concentration (100 ng L−1) of BP-3, which could indicate a possible hormetic behavior. Since we observed some ENAs and MN in this work, we decided to use some morphometric descriptors, like a total area of the cell, area of the nucleus, and perimeter of the nucleus, to correlate morphological parameters to the toxicological response. Cell and nuclei shape have been considered important indicators of the events occurring in the cellular micro-environment (Lobo et al., 2016). However, we did not succeed in this approach, maybe because the alterations observed in this work were not robust. Moreover, this shape–function paradigm may work better with biological processes such as proliferation, differentiation, cell migration, behavior, motility, cellular communication, malignancy, and growth dynamics (Lobo et al., 2016; Carella et al., 2017).
Funding This work was supported by Universidade Estadual de Goiás in Brazil(UEG; No. 201600020010791) and Fundação de Amparo à Pesquisa do Estado de Goiás in Brazil (FAPEG; No. PPP/ 201610267001019). The author SSA received a scholarship from FAPEG and EFLCB was supported by UEG with a fellowship at the program PROBIP (Scientific Production Support Program). Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by SSA, TLR, GQ, LARO, CLA, and EFLCB. The first draft of the manuscript was written by SSA, TLR, and EFLCB and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest None. References Agha, N.Y., Haidar, S., Al-Khayat, M.A., 2013. Development and validation of RP-HPLC method for analysis of four UV filters in sunscreen products. Int. J. Pharm. Sci. Rev. Res. 23, 254–258. Ahmedova, A., Mantareva, V., Enchev, V., Mitewa, M., 2002. 2-Acetylindan-1,3-dione and its Cu2+ and Zn2+ complexes as promising sunscreen agents. Int. J. Cosmet. Sci. 24, 103–110. Araldi, R.P., Melo, T.C., Mendes, T.B., Sá-Júnior, P.L., Nozima, B.H.N., Ito, E.T., Carvalho, R.F., Souza, E.B., Stocco, R.C., 2015. Using the comet and micronucleus assays for genotoxicity studies: a review. Biomed. Pharmacother. 72, 74–82. Baird, R.B., Eaton, A.D., Rice, E.W., Bridgewater, L.L., Water Environment, F, American Public Health, A, American Water Works, A, 2017. Standard Methods for the Examination of Water and Wastewater, 23rd ed. American Public Health Association, New York. Balmer, M.E., Buser, H.-R., Müller, M.D., Poiger, T., 2005. Occurrence of some organic UV filters in wastewater, in surface waters, and in fish from Swiss Lakes. Environ. Sci. Technol. 39, 953–962. Beedanagari, S., Vulimiri, S.V., Bhatia, S., Mahadevan, B., 2014. Chapter 43 - genotoxicity biomarkers: molecular basis of genetic variability and susceptibility. In: Gupta, R.C. (Ed.), Biomarkers in Toxicology. Academic Press, Boston, pp. 729–742.
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