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Fipronil causes toxicity in mouse preimplantation embryos
Toxicology xxx (xxxx) xxx–xxx
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Fipronil causes toxicity in mouse preimplantation embryos ⁎
Šefčíková Zuzana ,1, Babeľová Janka1, Čikoš Štefan, Kovaříková Veronika, Burkuš Ján, Špirková Alexandra, Koppel Juraj, Fabian Dušan Institute of Animal Physiology, Centre of Biosciences, Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice, Slovak Republic
A R T I C LE I N FO
A B S T R A C T
Keywords: Preimplantation embryo Mouse Fipronil FIPRON spot-on In vitro In vivo
In this study the possible toxicity of phenylpyrazole fipronil, the related commercial product FIPRON spot-on as well as FIPRON spot-on secondary ingredients on the developmental capacities and quality of mouse preimplantation embryos was evaluated. During in vitro tests, isolated two-cell stage embryos were cultured in media with addition of the listed chemicals until blastocyst formation. Stereomicroscopic evaluation of in vitro produced embryos showed that fipronil at 1 μM and higher concentration negatively affected embryonic development. Fluorescence staining revealed that the obtained blastocysts displayed lower numbers of blastomeres at 10 μM concentrations and elevated incidence of cell death from 1 μM concentration. The presence of FIPRON spot-on at a concentration equivalent to 10 μM of fipronil caused massive degeneration of all embryos. Secondary ingredients (butylhydroxyanisolum, butylhydroxytoluenum) at corresponding concentrations negatively impacted the development and quality of preimplantation embryos as well. During in vivo tests (daily oral administration of fipronil during the preimplantation period) in embryos collected from treated mouse females, significantly elevated incidence of cell death was observed even at the acute reference dose. Fipronil impaired the development and quality of mouse preimplantation embryos in both in vitro and in vivo tests. Embryotoxicity of the commercial product FIPRON spot-on was potentiated by the presence of secondary ingredients.
1. Introduction The insecticide fipronil is a widespread active compound, commonly used to control pests in agriculture, household and health care. Fipronil belongs in the phenylpyrazoles class. It acts by blocking the chloride channels of insect gamma-aminobutyric acid (GABAA) receptors, resulting in uncontrolled central nervous system activity and subsequent death (Cole et al., 1993). Fipronil is much more toxic to insects than to mammals because of GABAA receptor affinity differences (Hainzl et al., 1998). Despite its declared safety for vertebrates, adverse health effects including reproductive disorders are well documented in various experiments. For instance, fipronil altered the reproductive cycle (lengthening the estrous phase) and changed the concentration of progesterone and estradiol levels in female rats (Ohi et al., 2004). Exposure to fipronil during the prenatal period from day 6 to 20 of gestation impaired maternal behavior in rats, while in the offspring from exposed dams it influenced the development of some reflexes
(Magalhães et al., 2015; Udo et al., 2014). On the other hand, no fetal toxicity or teratogenicity was observed after oral administration of fipronil to rats or rabbits during gestation (Marrs and Ballantyne, 2004). The reproductive toxicity of fipronil (induced DNA damage and apoptosis of spermatozoa) was also recorded in male rats after four weeks of oral fipronil ingestion (Khan et al., 2015). Adverse effects of fipronil have been confirmed in birds as well. For example, 10 days ingestion of fipronil-treated seed during the breeding period resulted in red-legged partridges in altered sexual hormone levels and reduced egg fecundation rate (Lopez-Antia et al., 2015). Other studies have found lower hatching ability in hens intoxicated with fipronil (Kitulagodage et al., 2011; Russ, 2005). Fipronil injection on day 12 of incubation caused behavioural and developmental abnormalities in hens' eggs (Kitulagodage et al., 2011). Reversible hormonal disturbances (decreased testosterone level, elevated estrogenic activity) followed by increased incidence of infertility were also reported in male Japanese quail after 15 days of fipronil application (Khalil et al., 2017).
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Corresponding author. E-mail address: [email protected] (Z. Šefčíková). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.tox.2018.08.008 Received 27 March 2018; Received in revised form 2 July 2018; Accepted 16 August 2018 0300-483X/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Šefcíková, Z., Toxicology (2018), https://doi.org/10.1016/j.tox.2018.08.008
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In general, the preimplantation period of development (i.e. the period before implantation of embryo in the uterus) is one of the most sensitive phases in mammalian ontogeny. Disturbances caused by various environmental factors during this developmental stage can affect the developmental potential of oocytes and early embryos, and consequently increase preimplantation embryo losses. Embryo losses are believed to be one of the major reasons for fertility decline in human and animal populations. Unfortunately, to date information about the potential adverse effects of fipronil on mammalian preimplantation embryos are lacking. Since humans and animals are exposed to this environmental pollutant either with a high single dose (e.g. veterinary pests control) or low doses repeated for a long time (f.e. consumption of contaminated vegetables, fruits or crops), the possible negative effects on reproduction and development need to be fully evaluated. For this reason, the purpose of the present study was to find out whether different doses of fipronil applied during the preimplantation period poses a risk to developing mouse embryos. The potential embryotoxic effect of fipronil was evaluated using a mouse model in both in vitro and in vivo conditions.
Fig. 1. Scheme of in vitro experimental design.
dissolved in DMSO (Dimethyl sulfoxide), then in culture medium. The highest final concentration of DMSO in KSOMaa was 0.1%. This concentration was also added to the control culture medium. Pilot experiments showed that the addition of DMSO at concentrations lower than 1% had no effect on in vitro embryo development or obtained blastocyst quality (data not shown).
2. Material and methods 2.1. Animals
2.3. In vivo experimental design Experiments were performed on mice of the outbred ICR (CD-1 IGS) strain (Velaz, Prague, Czech Republic) kept under standard conditions (temperature 22 ± 2 °C, humidity 55 ± 10%, 12:12-h light-dark cycle with lights on at 06:00, and free access to food and water). All animal experiments were carried out in accordance with the ethical principles approved by the Ethical Committee for Animal Experimentation at the Institute of Animal Physiology, and were performed in accordance with Slovak legislation based on EU Directive 2010/63/EU on the protection of animals used for experimental and other scientific purposes. All efforts were made to reduce animal suffering and distress and to minimize the number of animals needed to produce reliable results.
Spontaneously-ovulating female mice (5–6 weeks old) were mated with males of the same strain for one or more nights. Three days before the first overnight mating, estrus was synchronized by exposing the females to bedding contaminated with male urine and preputial gland secretions (Ma et al., 1999). Successful mating was confirmed by the presence of a vaginal plug on examination every morning at 8:00 AM, and this was designated Day 1 of pregnancy. Females in the experimental groups were fed with two different doses of fipronil (0.009 and 0.9 mg/kg BW) diluted in DMSO and water during the whole preimplantation period (starting on Day 1 and finishing on Day 3 of pregnancy). In total, three oral applications using a syringe and feeding needle were performed. Females in the control group were fed the same way with pure water and 0.01% DMSO. On Day 4 of pregnancy (approx. 80 h after supposed ovulation), the mouse females were killed by cervical dislocation and subjected to isolation of embryos by flushing the uterus and the oviduct using a flushing-holding medium containing 1% bovine serum albumin (BSA; Sigma-Aldrich). This period preceding the start of implantation was chosen to avoid embryo loss. Immediately after isolation, the collected embryos underwent stereomicroscopic classification (Nikon SMZ 745 T). The number of embryos per dam and the number of embryos isolated from the oviduct and uterus were evaluated as well.
2.2. In vitro experimental design Adult female mice (5–6 weeks old) were synchronized with eCG (5 IU ip; Folligon, Intervet International, Boxmeer, Holland) and hCG (4 IU ip; Pregnyl, Organon, Oss, Holland; 47 h later) and mated with males of the same strain overnight. Successful mating was confirmed by identification of a vaginal plug next morning (Day 1 of pregnancy). Fertilized dams were killed by cervical dislocation and subjected to isolation of embryos at the 2-cell stage at 45 h post hCG administration on Day 2 of pregnancy. Embryos were recovered by flushing the oviduct using a flushing–holding medium (Lawitts and Biggers, 1993) containing 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and classified under stereomicroscope (Nikon SMZ 745 T). Isolated 2-cell mouse embryos were pooled, randomly divided into several sub-groups and cultured in vitro under standard conditions (a humidified atmosphere with 5% CO2 and 37 °C) for 72 h with the active component fipronil (Sigma-Aldrich, Germany) at 0, 0.1, 1, 10 and 100 μM concentrations or commercial product FIPRON spot-on at 0.0004 and 0.0437 μL/mL (Bioveta, Czech Republic, containing 0.1 μM and 10 μM of fipronil) or known additional chemicals present in FIPRON spoton: butylhydroxyanisolum (BHA) at concentration 0.087 μg and butylhydroxytoluenum (BHT) at concentration 0.0430 μg (both corresponding to 0.0437 μL of FIPRON spot-on containing 10 μM fipronil). The scheme of the in vitro experimental design is shown in Fig. 1. A culture of each sub-group of mouse embryos was performed in 400 μl of synthetic oviductal medium KSOMaa Evolve supplemented with half-strength Eagle non-essential amino acid mixture (Zenith Biotech, Canada) and 1% bovine serum albumin (BSA, Sigma-Aldrich). Each experiment was repeated two or three times. In accord with the producer’s instructions, substances were first
2.4. Evaluation of in vitro and in vivo derived embryos For the analysis of in vitro developmental capacities, the numbers of embryos reaching the blastocyst, morula or cleavage stages of development (3 to 16-cell), and those arrested at the 2-cell stage or degraded (i.e. undergoing extensive cytoplasmic fragmentation) were assessed using stereomicroscopy (Nikon SMZ 745 T). For the analysis of in vivo developmental capacities, the numbers of embryos reaching the blastocyst, morula or cleavage stages of development (2 to 16-cell), those arrested at the zygotic stage or degraded, were assessed using stereomicroscopy. In in vitro and in vivo obtained blastocysts, cell numbers and cell death incidence were evaluated using immunochemistry followed by fluorescence microscopy. Blastocysts were first stained with propidium iodide (PI, 10 μg/ml; Sigma-Aldrich; stains dead cells only), then washed in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA, SigmaAldrich), fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) 2
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fipronil at 10 and 1 μM concentration negatively affected the qualitative parameters of mouse blastocysts obtained in vitro. In blastocysts derived from 2-cell embryos cultured with fipronil at 10 μM concentration, significantly decreased cell numbers in blastocysts (51.63 ± 3.19 vs 92.33 ± 4.98; P < 0.01) were recorded. The presence of active component fipronil at 10 and 1 μM significantly increased the average percentage of dead cells (7.10 ± 0.93 and 7.50 ± 0.82 vs 5.01 ± 0.40; P < 0.05 in both cases). In blastocysts derived in the presence of commercial product FIPRON spot-on at concentration 0.0004 μL/mL (containing 0.1 μM of fipronil), significantly decreased cell numbers were observed (85.62 ± 4.15 vs 101.70 ± 4.73; P < 0.05, Fig. 3(B)). There was no significant difference in average percentage of dead cells between blastocysts derived from embryos cultured in the presence of FIPRON spot-on and blastocysts derived from the control group (Fig. 3 (C)). As mentioned above, no blastocysts had developed in the 0.0437 μL/mL FIPRON spot-on group. The presence of FIPRON spot-on additives (at a concentration equivalent to 0.0437 μL/mL FIPRON spot-on) negatively affected embryo development as well as the quality of obtained blastocysts in our in vitro experiment. Significant decrease in the number of embryos reaching blastocyst stage was recorded (chi-square tests, 50.00% in the case of BHA and 58.33% in the case of BHT compared to control 88.00%; P < 0.01 in both) (Fig. 4A). Decreased cell numbers per blastocyst in blastocysts derived from 2-cell embryos cultured in the presence of BHA were observed (72.35 ± 3.61 vs. 85.49 ± 4.21; P < 0.05) (Fig. 4B). There were significant differences in the average percentage of dead cells between blastocysts derived from embryos cultured in the presence of BHA as well (6.66 ± 0.73 vs. 4.64 ± 0.49; P < 0.05) (Fig. 4C). All blastocysts produced in in vitro experiments contained at least one dead cell. Morphological analysis showed that the majority of them were of apoptotic origin, i.e. showed both nuclear fragmentation and TUNEL positive labeling (data not shown).
in PBS at room temperature for 1 h and optionally stored in 1% paraformaldehyde in PBS at 4 °C. Fixed blastocysts were washed three times for 5 min in PBS containing 0.1% BSA and transferred into PBS with 0.5% Triton X-100 (Sigma–Aldrich, Germany) for 1 h. After permeabilization, the blastocysts were washed twice for 15 min in PBS with BSA and incubated in the TUNEL assay reagents (TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling, in situ Cell Death Detection Kit; Promega Corporation, Madison, USA, visualizes specific DNA fragmentation) for 1 h at 37 °C in the dark. Then they were washed once in PBS containing 0.1% BSA. Finally, the blastocysts were counterstained with Hoechst 33342 DNA staining (10 μl/ml in PBS; Sigma Aldrich) for 5 min at room temperature, mounted on glass slides with Vectashield (Vector Laboratories, Burlingname, CA, USA) and observed using a fluorescence microscope at magnification x 400 (BX51; Olympus, Tokyo, Japan). According to their nuclear morphology, the presence of specific DNA fragmentation in the nucleoplasm and PI positivity/negativity, embryonic cells were classified as: normal (without morphological changes in nuclei, without TUNEL labeling, and without PI inclusion in the nucleoplasm) or dead (showing at least one of the following features: fragmented or condensed nucleus, positive TUNEL labeling or positive PI staining). Dead cells showing nuclear fragmentation or condensation and TUNEL positive labeling were classified as apoptotic. In each blastocyst, the percentage of dead cells was calculated as the number of dead cells relative to the total number of blastomeres in the blastocyst. 2.5. Statistical analysis Statistical analysis was performed using GraphPad Prism Software 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). The results are expressed as the mean values ± SEM. The differences between data showing normal Gaussian distribution were assessed with ANOVA followed by Tukey´s post-hoc test. This was used to analyze the mean numbers of blastomeres per blastocyst. The differences between data which did not pass normality tests were assessed with the Kruskal-Wallis test, followed by Dunn’s post hoc test. This was used to analyze cell death incidence per blastocyst. For the assessment of differences between score-type data, standard chi-square tests with one degree (oviduct – uterus transition, percentage of blastocysts with dead cells) and four degrees of freedom (overall developmental abilities of embryos in vitro or in vivo) were used. Differences with P < 0.05 were considered statistically significant.
3.2. In vivo experiment Stereomicroscopic evaluation of oviduct/uterus flushes obtained from treated and untreated dams showed that fipronil at concentration 0.9 mg/kg significantly affected the average number of isolated embryos per dam. There was a tendency to decrease the average embryo number per mouse in the group treated with concentration 0.009 mg/ kg of fipronil as well, however it did not reach statistical significance (9.44 ± 1.00 vs 11.67 ± 0.65; P = 0.06) (Table 1). Significant differences were also recorded in oviduct/uterus transition in mouse females exposed to fipronil at dose 0.9 mg/kg. The transition of embryos from oviduct to uterus was significantly slowed down (80.67% embryos isolated from uterus and 19.33% isolated from oviduct compared to control with 93.15% embryos isolated from uterus and 6.85% from oviduct; P < 0.01) (Fig. 5). As shown in Fig. 6A, stereomicroscopic evaluation of embryos obtained from treated and untreated dams showed that fipronil at 0.9 mg/ kg affected the development of mouse embryos produced in in vivo conditions (P < 0.001). Increased numbers of degraded embryos and decreased numbers of blastocysts were isolated from the exposed dams. Oral administration of lower dose (0.009 mg/kg) did not affect ability of embryos to reach the blastocysts stage. Morphological assessment of isolated blastocysts did not reveal significant differences in cell numbers per blastocyst (Fig. 6B). However, there were significant differences in the incidence of dead cell counts found between blastocysts isolated from treated and non-treated dams (2.94 ± 0.35% and 3.86 ± 0.69 vs. 1.64 ± 0.18; P < 0.001 and P < 0.01, respectively for 0.009 and 0.9 mg/kg dose) (Fig. 6C). The proportion of blastocysts with dead cells was 54.81% in the untreated group, 69.42% in the group treated by 0.009 mg/kg and 64.21% in the group treated by 0.9 mg/kg BW of fipronil (P < 0.05; P > 0.05).
3. Results 3.1. In vitro experiment As shown in Fig. 2 (A), the presence of active component fipronil at concentrations of 100, 10 and 1 μM had negative effects on mouse embryonic development (chi-square tests, P < 0.001, P < 0.001, P < 0.05; respectively). In the case of 100 μM concentration all embryos were degraded, while at the other concentrations (10 and 1 μM), significant decrease in the number of embryos reaching blastocyst stage was recorded: 10 μM – 20.41%, 1 μM – 70.00% compared with control group – 84.00% of blastocysts. The rest of the embryos arrested at the early stages of development or degraded. The presence of fipronil at concentration 0.1 μM had no effect on embryo development. The presence of commercial product (FIPRON spot-on) at concentration 0.0437 μL/mL (containing 10 μM of fipronil) had a negative effect on mouse embryonic development as well (Fig. 3(A)). In this case, all embryos were degraded (P < 0.001). The presence of FIPRON spoton at concentration 0.0004 μL/mL (equivalent to 0.1 μM of active compound) had no effect on embryonic development. As shown in Fig. 2 (B, C), the presence of active component – 3
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Fig. 2. Analysis of developmental abilities (A) and qualitative parameters (B, C) of mouse embryos derived from 72 h in vitro cultures of 2-cell embryos in KSOMaa medium supplemented with various concentrations (0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM) of fipronil. (A) Percentages of arrested embryos (degraded, 2-cell stage) and embryos at higher developmental stages (3–16-cell stage, morulas and blastocysts). Differences from control group were analyzed using chi-square test with four degrees of freedom, *P < 0.05; ***P < 0.001. (B) Mean number of cells per blastocyst. Differences from control group were determined using ANOVA followed by Tukey post hoc test, **P < 0.01. (C) Average percentage of dead cells in blastocysts. Differences from control group were analyzed using Kruskal-Wallis test, followed by Dunn’s post hoc test, *P < 0.05.
4. Discussion
When the toxicity of insecticides is tested, the majority of studies focus on the main component (active substance) of the commercial product. However, pesticides often contain additional chemicals with harming potential as well (Cox and Surgan, 2006; Fabian et al., 2011). Based on the available information, commercial FIPRON spot-on contains fipronil at 10% concentration, butylhydroxyanisolum (BHA) at 0.02%, and butylhydroxytoluenum (BHT) at 0.01% concentration, as well as other compounds which are unknown. Getting data on the other compounds is difficult, due to the protection of confidential commercial information. To assess the relevance of the negative effects of available secondary compounds, an in vitro experiment with addition of BHA and BHT alone was performed in our study. It was found that BHA and BHT (at concentrations equivalent to FIPRON spot-on containing 10 μM fipronil) in culture medium decreased the capacity of two-cell stage mouse embryos to reach higher stages of development. In the case of BHA, the quality of in vitro obtained blastocysts was poor as well, as shown by significantly decreased average number of cells and elevated incidence of dead cells per blastocyst. It is known that butylhydroxyanisolum (E 320) and butylhydroxytoluenum (E 321) are widely used as antioxidants for long-term preservation of food, cosmetics and pharmaceuticals. Despite their antioxidant activity, negative impact on the reproductive physiology of rodents has been shown in both in vitro (Iannaccone, 1986) and in vivo studies (Kang et al., 2005; Pop et al., 2013). For example, at a concentration of 0.025 mg/mL, BHT was toxic to blastocyst-stage embryos, and a concentration of 0.075 mg/mL killed all of the embryos after a 2hour incubation period (Iannaccone, 1986). A high dose of BHA was
Although it has been established that the phenylpyrazole group of insecticides have greater affinity to invertebrate than to vertebrates (Hainzl et al., 1998; Narahashi et al., 2010), previous study has indicated that fipronil can bind to mammalian GABAA and GABAC receptors and may have potential to affect mammalian health (Tingle et al., 2003). Despite the high probability that GABAA receptors might already be expressed in embryos at the preimplantation developmental stages (Andäng et al., 2008; suggestion based on their observations in stem cells), up to the present the toxicity of fipronil on mammalian preimplantation embryos has not been evaluated. Our results show that fipronil at concentrations of 1 μM and higher in culture medium negatively affected the in vitro development of twocell mouse embryos. Furthermore, it negatively affected their quality, as shown by significantly decreased total cell counts and increased percentage of dead cells in obtained blastocysts. When two-cell stage mouse embryos were cultured in the presence of commercial product FIPRON spot-on containing 10 μM concentration of main compound fipronil, significant negative effect on embryo development characterized by overall developmental arrest of 100% of obtained embryos and their degeneration was observed. Moreover, lower tested FIPRON spot-on concentration (corresponding to 0.1 μM of fipronil) decreased the quality of obtained blastocysts, as shown by decreased average numbers of blastomeres in blastocysts. Observed differences between main compound fipronil and commercial FIPRON spot-on might be explained by the presence of secondary ingredients in the commercial preparation, which potentiated its negative effect. 4
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Fig. 3. Analysis of developmental abilities (A) and qualitative parameters (B, C) of mouse embryos derived from 72 h in vitro cultures of 2-cell embryos in KSOMaa medium supplemented with/without FIPRON spot-on at final concentration 0.0004 μL/mL corresponding to 0.1 μM concentration of active compound fipronil and 0.0437 μL/mL corresponding to 10 μM concentration of fipronil. (A) Percentages of arrested embryos (degraded, 2-cell stage) and embryos at higher developmental stages (3–16-cell stage, morulas and blastocysts). Differences from control group were analyzed using chi-square test with four degrees of freedom, ***P < 0.001. (B) Mean number of cells per blastocyst. Differences from control group were determined using ANOVA followed by Tukey post hoc test, *P < 0.05. (C) Average percentage of dead cells in blastocysts. Differences from control group were analyzed using Kruskal-Wallis test, followed by Dunn’s post hoc test, P > 0.05.
It has been shown that after a single dose of radiolabeled fipronil C- fipronil (4 or 40 mg/kg B.W.) administered to rats the maximum blood concentration was reached after approximately 6 h. Significant amounts of residues in individual tissues were found for 1 week after treatment, predominantly in adipose tissue (22 μg per gram for 4 mg/kg in females). Intermediate concentrations were found in adrenals, liver, kidney, pancreas and thyroids. In ovaries and uterus persisted amounts of fipronil were 4.6 μg and 2.5 μg, respectively for 4 mg/kg 1 week post treatment (FAO, 2001). Moreover, substantial amounts of fipronil metabolite were found in the brain of male rats after single dose of 14Cfipronil, demonstrating that even highly selective brain membrane are permeable for this compound (Cravedi et al., 2013). According to accessible information on pharmacokinetics of fipronil in mouse [peak serum concentration: 0.58 μg per gram at 5 mg/kg B.W. dose (FAO, 2001)] we might hypothesize that after repeated administration of fipronil at 0.9 mg/kg to female dams (current in vivo experiment), its blood/oviductal fluid concentration reached approximately 1μM concentration. This is in accordance with our results showing correspondence between outcomes of in vivo and in vitro experiments: in both cases, retarded embryonic development, increased incidence of apoptosis and no differences in cells numbers in blastocysts were observed. However, such extrapolation implies partial inconsistency between the effect of fipronil at 0.009 mg/kg dose in vivo and the effect of fipronil at 0.1 μM concentration in vitro: whilst increase in cell death documented after the treatment with 0.009 mg/kg dose in vivo reached statistical significancy (2.94% vs. 1.64% in controls), slight increase in cell death
reported to decrease serum testosterone in F0 male rats, whereas in F1 offspring it delayed sexual maturation, changed quality of sperm, shortened the estrous cycle and also decreased the weight of sex organs (BHA at 100 or 500 mg/kg for 13 weeks) (Jeong et al., 2005). On the other hand, there is no evidence that BHA or BHT affected reproduction in female pigs (dose up to 400 mg/kg) or in female rats (dose up to 1000 mg/kg), or that they impaired the development of fetuses (Hansen et al., 1982; McFarlane et al., 1997). To evaluate the relevance of the negative impact of fipronil on preimplantation embryos recorded in vitro, an in vivo experiment was performed as well. The results obtained from the in vivo experiment show that fipronil can impair their physiological development. Similarly as in in vitro experiments (concentrations up to 10 μM), no effect on average cell numbers was recorded in mouse embryos developing in intoxicated dams. On the other hand, mouse embryos developing in dams treated by 0.9 mg/kg of fipronil showed significantly decreased ability to reach the blastocyst stage. Moreover, as shown by morphological evaluation, even at the acute reference dose (daily oral exposure without adverse health effect 0.009 mg/kg), blastocysts collected on Day 4 of pregnancy displayed higher incidence of dead cells than the embryos from control dams. The most importantly, treatment with fipronil decreased the average embryo number obtained from individual dams: a numerical tendency at the acute reference dose (0.009 mg/kg B.W.) and significant impact at 0.9 mg/kg B.W. were observed. This finding suggests that fipronil has the potential to directly cause embryo loss.
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Fig. 4. Analysis of developmental abilities (A) and qualitative parameters (B, C) of mouse embryos derived from 72 h in vitro cultures of 2-cell embryos in KSOMaa medium supplemented with/without secondary compounds: butylhydroxyanisolum (BHA) at concentration 0.087 μg and butylhydroxytoluenum (BHT) at concentration 0.0430 μg (both corresponding to 0.0437 μL of FIPRON spot-on containing 10 μM fipronil). (A) Percentages of arrested embryos (degraded, 2-cell stage) and embryos at higher developmental stages (3–16-cell stage, morulas and blastocysts). Differences from control group were analyzed using chi-square test with four degrees of freedom, **P < 0.01. (B) Mean number of cells per blastocyst. Differences from control group were determined using ANOVA followed by Tukey post hoc test, *P < 0.05. (C) Average percentage of dead cells in blastocysts. Differences from control group were analyzed using Kruskal-Wallis test, followed by Dunn’s post hoc test, *P < 0.05). Table 1 Average number of embryos isolated from untreated and fipronil-treated mice. Parameters
0 mg/kg
0.009 mg/kg
0.9 mg/kg
Donor mice, N Isolated embryos, N Average, N ± SEM
15 175 11.67 ± 0.65
16 151 9.44 ± 1.00
18 150 8.33 ± 0.52**
Significantly different from untreated group: **P < 0.01.
documented after the exposure of 0.1 μM fipronil in vitro was not statistically significant (5.50% vs. 5.01% in controls). More apparent effect of fipronil on in vivo developed blastocysts might be explained in two ways: 1. Overall increase in apoptotic incidence in both untreated and treated embryos (induced by the imperfectness of artificial culture conditions) would overlap the negative effect of 0.1 μM fipronil in vitro. 2. Increased incidence of apoptosis in in vivo developed embryos would be correlated to damaging effect of fipronil to other sensitive tissues in maternal body (Badgujar et al., 2015; Montanha et al., 2018), secondarily causing disturbance of hormonal homeostasis or elevation of apoptosis-inducing mediators in reproductive organs. Apoptosis is a physiological process occurring spontaneously in both in vivo and in vitro developed preimplantation embryos (Brison and Schultz, 1997; Fabian et al., 2005; Gjørret et al., 2003; Pomar et al., 2005). It is usually triggered to fulfill a reparatory function through elimination of damaged cells. Increased incidence of apoptosis in
Fig. 5. Oviduct – uterus transition. Oviduct-to-uterus transition of in vivo derived embryos obtained from untreated and fipronil-treated (0.009 mg/kg, 0.9 mg/kg) mouse females. Statistical differences between groups were determined using standard chi-square test, ** P < 0.01.
preimplantation embryos is considered to be an indicator of inadequate developmental environment and is used as a sensitive marker of embryo quality in both experimental and clinical conditions (Betts and King, 2001; Huppertz and Herrler, 2005). Furthermore, it has been suggested that when apoptosis incidence reaches a certain threshold, it might be detrimental to further development of conceptus (Hardy, 1997; Huang and Chan, 2016; Jurisicova et al., 1998). However, the values of such 6
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Fig. 6. Analysis of developmental abilities (A) and qualitative parameters (B, C) of in vivo derived embryos obtained from untreated and fipronil-treated (0.009 mg/ kg, 0.9 mg/ kg) mouse females. (A) Percentage of degraded embryos, zygotes (1-cell stage), slowly developing (2–16- cell stage) embryos, morulas and blastocysts. Differences from untreated group were analyzed using chi-square test with four degrees of freedom, ***P < 0.001. (B) Mean number of cells per blastocyst. Differences from untreated group were determined using ANOVA followed by Tukey post hoc test, P > 0.05. (C) Average percentage of dead cells in blastocysts. Differences from untreated group were analyzed using Kruskal-Wallis test, followed by Dunn’s post hoc test, ***P < 0.001; **P < 0.01.
for fipronil, since it was found that fipronil treatment completely inhibited the function of all human glycine receptor subtypes (Islam and Lynch, 2012). In the last decade, evidence that fipronil is involved in the induction of oxidative stress has increased. Prolonged exposure to low doses of fipronil contributed to significant induction of oxidative stress by reducing the activity of antioxidant enzymes in the maternal rat organism and also in the offspring (Tukhtaev et al., 2013). Imbalance between pro-oxidants and anti-oxidants during pregnancy contributes to various complications in the maternal organism and in the offspring (Agarwal et al., 2012; Tukhtaev et al., 2013; Wang et al., 2016). The high sensitivity of embryonic cells to oxidative stress has been documented as well (Kurzawa et al., 2004). Because of fipronil's widespread application and its relatively slow degradation with a half-life up to 7.3 months in soil and water, it poses a serious risk for the surrounding environment and consequently for animal and human health (Simon-Delso et al., 2015; Tingle et al., 2003). Any contamination, especially during the early gestation period, should not be underestimated. Since fipronil residues are transferable from the pets for 30 days after treatment with Frontline, they present possible health risk to humans as well (Jennings et al., 2002). In conclusion, the results of our in vitro tests indicate that phenylpyrazole fipronil and its commercial product FIPRON spot-on can negatively influence the development and qualitative parameters of mouse preimplantation embryos. The sensitivity of mouse embryonic
threshold have not been defined yet in any mammalian species. Still, the elevation in the percentage of apoptotic cells in blastocysts directly reflects the increase in irreversible intracellular damage or malfunction of essential cellular systems (Levy et al., 2001; Penaloza et al., 2006) induced by fipronil. Evidence from earlier toxicity studies suggested that oral exposure to 0.3 mg/kg of fipronil during the perinatal period induced reproductive disorders such as shorter estrus cycle in young female rats and decreased quality of sperm in male rats (de Barros et al., 2016, 2017). Treatment during the time of organogenesis led to decreased somatic parameters and increased incidence of skeletal malformations in rat fetuses (Eisa et al., 2017). Approximately doubled length of the estrous cycle was found in female rats treated with Frontline Top Spot (commercial product containing fipronil) (Ohi et al., 2004). Higher doses of Frontline (fipronil at concentration 280 mg/kg) reduced the pregnancy index in female rats (Ohi et al., 2004). Taken together, our results indicate that mouse embryonic cells are sensitive to fipronil. Whether its negative effect is mediated via GABAA receptors depends on the unresolved question of their potential expression in preimplantation embryos. However, the presence of several receptors of neurotransmitters and biogenic monoamines has already been documented in preimplantation embryos, and in vitro tests have proved their function (reviewed in [Čikoš et al.,2010,2011]Čikoš et al. (2010, 2011). Moreover, another recent study has suggested that glycine receptors could potentially be the novel vertebrate toxicity targets
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cells to the tested secondary compounds of FIPRON spot-on (BHA, BHT) was also confirmed, as shown by decreased capacity of two-cell stage mouse embryos to reach higher stages of development, decreased cell proliferation and increased apoptosis incidence in the obtained blastocysts. The results of our in vivo experiment show that even the acute reference dose (0.009 mg/kg) of orally-administered fipronil might significantly increase apoptosis incidence in developed blastocyst. Fipronil-induced changes during the preimplantation period of embryonic development could be considered as a potential risk factor in mammalian reproduction.
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Evaluation of the possible endocrine disruptive effect of butylated hydroxyanisole, butylated hydroxytoluene and propyl gallate in immature female rats. Farmacia 61, 202–211. Russ, M., 2005. An Investigation of the Effects Locust Pesticides, Fenitrothion and Fipronil, on Avian Development Using an in Ovo Model. Masters Thesis. University of Wollongong. Simon-Delso, N., Amaral-Rogers, V., Belzunces, L.P., Bonmatin, J.M., Chagnon, M., Downs, C., Furlan, L., Gibbons, D.W., Giorio, C., Girolami, V., Goulson, D., Kreutzweiser, D.P., et al., 2015. Systemic insecticides (neonicotinoids and fipronil): trends, uses, mode of action and metabolites. Environ. Sci. Pollut. Res. 22, 5–34. Tingle, C.C., Rother, J.A., Dewhurst, C.F., Lauer, S., King, W.J., 2003. Fipronil: environmental fate, ecotoxicology, and human health concerns. Rev. Environ. Contam. Toxicol. 176, 1–66. Tukhtaev, K.R., Tulemetov, S.K., Zokirova, N.B., Tukhtaev, N.K., Tillabaev, M.R., Amirullaev, O.K., Yarieva, O.O., Otajonova, A.N., 2013. Prolonged exposure of low doses of fipronil causes oxidative stress in pregnant rats and their offspring. Internet J. Toxicol. 10, 1–8. Udo, M.S., Sandini, T.M., Reis, T.M., Bernardi, M.M., Spinosa, H.S., 2014. Prenatal exposure to a low fipronil dose disturbs maternal behavior and reflex development in rats. Neurotoxicol. Teratol. 45, 27–33. Wang, X., Martínez, M.A., Wu, Q., Ares, I., Martínez-Larrañaga, M.R., Anadón, A., Yuan, Z., 2016. Fipronil insecticide toxicology: oxidative stress and metabolism. Crit. Rev. Toxicol. 46, 876–899.
Conflict of interest The authors declare no conflict of interest. Acknowledgments The authors thank Andrew Billingham for his revision of the English text, and Anna Olšavská and Dana Čigašová for their technical assistance. This study was supported by the Slovak Research and Development Agency under contract APVV 14-0763. References Agarwal, A., Aponte-Mellado, A., Premkumar, B.J., Shaman, A., Gupta, S., 2012. The effects of oxidative stress on female reproduction: a review. Reprod. Biol. Endocrinol. 10, 49. Andäng, M., Hjerling-Leffler, J., Moliner, A., Lundgren, T.K., Castelo-Branco, G., Nanou, E., Pozas, E., Bryja, V., Halliez, S., Nishimaru, H., Wilbertz, J., Arenas, E., Koltzenburg, M., Charnay, P., El Manira, A., Ibañez, C.F., Ernfors, P., 2008. Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation. Nature 451, 460–464. Badgujar, P.C., Pawar, N.N., Chandratre, G.A., Telang, A.G., Sharma, A.K., 2015. Fipronil induced oxidative stress in kidney and brain of mice: Protective effect of vitamin E and vitamin C. Pestic. Biochem. Physiol. 118, 10–18. Betts, D.H., King, W.A., 2001. Genetic regulation of embryo death and senescence. Theriogenology 55, 171–191. Brison, D.R., Schultz, R.M., 1997. Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor alpha. Biol. Reprod. 56, 1088–1096. Čikoš, Š., Burkuš, J., Bukovská, A., Fabian, D., Rehák, P., Koppel, J., 2010. Expression of adiponectin receptors and effects of adiponectin isoforms in mouse preimplantation embryos. Hum. Reprod. 25, 2247–2255. Čikoš, Š., Fabian, D., Makarevich, A.V., Chrenek, P., Koppel, J., 2011. Biogenic monoamines in preimplantation development. Hum. Reprod. 26, 2296–2305. Cole, L.M., Nicholson, R.A., Casida, J.E., 1993. Action of phenylpyrazole insecticides at the GABA-gated chloride channel. Pestic. Biochem. Physiol. 46, 47–54. Cox, C., Surgan, M., 2006. Unidentified inert ingredients in pesticides: implications for human and environmental health. Environ. Health Perspect. 114, 1803–1806. Cravedi, J.P., Delous, G., Zalko, D., Viguié, C., Debrauwer, L., 2013. Disposition of fipronil in rats. Chemosphere 93, 2276–2283. de Barros, A.L., Rosa, J.L., Cavariani, M.M., Borges, C.S., Villela e Silva, P., Bae, J.H., AnselmoFranci, J.A., Cristina Arena, A., 2016. In utero and lactational exposure to fipronil in female rats: Pregnancy outcomes and sexual development. J. Toxicol. Environ. Health A 79, 266–273. de Barros, A.L., Bae, J.H., Borges, C.S., Rosa, J.L., Cavariani, M.M., Silva, P.V., Pinheiro, P.F.F., Anselmo-Franci, J.A., Arena, A.C., 2017. Perinatal exposure to insecticide fipronil: effects on the reproductive system in male rats. Reprod. Fertil. Dev. 29, 1130–1143. Eisa, A.A., Abo-Elghar, G.E., Ammar, I.M., Metwally, H.G., Arafa, S.S., 2017. Embryotoxicity and teratogenicity of fipronil in rats (Rattus norvegicus). Zagazig J. Agric. Res. 44, 1851–1861. Fabian, D., Koppel, J., Maddox-Hyttel, P., 2005. Apoptotic processes during mammalian preimplantation development. Theriogenology 64, 221–231. Fabian, D., Bystriansky, J., Burkuš, J., Rehák, P., Legáth, J., Koppel, J., 2011. The effect of herbicide BASTA 15 on the development of mouse preimplantation embryos in vivo and in vitro. Toxicol. In Vitro 25, 73–79. FAO, 2001. Pesticide residues in food – 2001. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticide Residues. Geneva, Switzerland. 17-26 September 2001. Gjørret, J.O., Knijn, H.M., Dieleman, S.J., Avery, B., Larsson, L.-I., Maddox-Hyttel, P., 2003. Chronology of apoptosis in bovine embryos produced in vivo and in vitro. Biol. Reprod. 69, 1193–1200. Hainzl, D., Cole, L.M., Casida, J.E., 1998. Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct. Chem. Res. Toxicol. 11, 1529–1535. Hansen, E.V., Meyer, O., Olsen, P., 1982. Study of toxicity of butylated hydroxyanisole (BHA) in pregnant gilts and their foetuses. Toxicology 23, 79–83. Hardy, K., 1997. Cell death in the mammalian blastocyst. Mol. Hum. Reprod. 3, 919–925.
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Fipronil causes toxicity in mouse preimplantation embryos ⁎
Šefčíková Zuzana ,1, Babeľová Janka1, Čikoš Štefan, Kovaříková Veronika, Burkuš Ján, Špirková Alexandra, Koppel Juraj, Fabian Dušan Institute of Animal Physiology, Centre of Biosciences, Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice, Slovak Republic
A R T I C LE I N FO
A B S T R A C T
Keywords: Preimplantation embryo Mouse Fipronil FIPRON spot-on In vitro In vivo
In this study the possible toxicity of phenylpyrazole fipronil, the related commercial product FIPRON spot-on as well as FIPRON spot-on secondary ingredients on the developmental capacities and quality of mouse preimplantation embryos was evaluated. During in vitro tests, isolated two-cell stage embryos were cultured in media with addition of the listed chemicals until blastocyst formation. Stereomicroscopic evaluation of in vitro produced embryos showed that fipronil at 1 μM and higher concentration negatively affected embryonic development. Fluorescence staining revealed that the obtained blastocysts displayed lower numbers of blastomeres at 10 μM concentrations and elevated incidence of cell death from 1 μM concentration. The presence of FIPRON spot-on at a concentration equivalent to 10 μM of fipronil caused massive degeneration of all embryos. Secondary ingredients (butylhydroxyanisolum, butylhydroxytoluenum) at corresponding concentrations negatively impacted the development and quality of preimplantation embryos as well. During in vivo tests (daily oral administration of fipronil during the preimplantation period) in embryos collected from treated mouse females, significantly elevated incidence of cell death was observed even at the acute reference dose. Fipronil impaired the development and quality of mouse preimplantation embryos in both in vitro and in vivo tests. Embryotoxicity of the commercial product FIPRON spot-on was potentiated by the presence of secondary ingredients.
1. Introduction The insecticide fipronil is a widespread active compound, commonly used to control pests in agriculture, household and health care. Fipronil belongs in the phenylpyrazoles class. It acts by blocking the chloride channels of insect gamma-aminobutyric acid (GABAA) receptors, resulting in uncontrolled central nervous system activity and subsequent death (Cole et al., 1993). Fipronil is much more toxic to insects than to mammals because of GABAA receptor affinity differences (Hainzl et al., 1998). Despite its declared safety for vertebrates, adverse health effects including reproductive disorders are well documented in various experiments. For instance, fipronil altered the reproductive cycle (lengthening the estrous phase) and changed the concentration of progesterone and estradiol levels in female rats (Ohi et al., 2004). Exposure to fipronil during the prenatal period from day 6 to 20 of gestation impaired maternal behavior in rats, while in the offspring from exposed dams it influenced the development of some reflexes
(Magalhães et al., 2015; Udo et al., 2014). On the other hand, no fetal toxicity or teratogenicity was observed after oral administration of fipronil to rats or rabbits during gestation (Marrs and Ballantyne, 2004). The reproductive toxicity of fipronil (induced DNA damage and apoptosis of spermatozoa) was also recorded in male rats after four weeks of oral fipronil ingestion (Khan et al., 2015). Adverse effects of fipronil have been confirmed in birds as well. For example, 10 days ingestion of fipronil-treated seed during the breeding period resulted in red-legged partridges in altered sexual hormone levels and reduced egg fecundation rate (Lopez-Antia et al., 2015). Other studies have found lower hatching ability in hens intoxicated with fipronil (Kitulagodage et al., 2011; Russ, 2005). Fipronil injection on day 12 of incubation caused behavioural and developmental abnormalities in hens' eggs (Kitulagodage et al., 2011). Reversible hormonal disturbances (decreased testosterone level, elevated estrogenic activity) followed by increased incidence of infertility were also reported in male Japanese quail after 15 days of fipronil application (Khalil et al., 2017).
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Corresponding author. E-mail address: [email protected] (Z. Šefčíková). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.tox.2018.08.008 Received 27 March 2018; Received in revised form 2 July 2018; Accepted 16 August 2018 0300-483X/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Šefcíková, Z., Toxicology (2018), https://doi.org/10.1016/j.tox.2018.08.008
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In general, the preimplantation period of development (i.e. the period before implantation of embryo in the uterus) is one of the most sensitive phases in mammalian ontogeny. Disturbances caused by various environmental factors during this developmental stage can affect the developmental potential of oocytes and early embryos, and consequently increase preimplantation embryo losses. Embryo losses are believed to be one of the major reasons for fertility decline in human and animal populations. Unfortunately, to date information about the potential adverse effects of fipronil on mammalian preimplantation embryos are lacking. Since humans and animals are exposed to this environmental pollutant either with a high single dose (e.g. veterinary pests control) or low doses repeated for a long time (f.e. consumption of contaminated vegetables, fruits or crops), the possible negative effects on reproduction and development need to be fully evaluated. For this reason, the purpose of the present study was to find out whether different doses of fipronil applied during the preimplantation period poses a risk to developing mouse embryos. The potential embryotoxic effect of fipronil was evaluated using a mouse model in both in vitro and in vivo conditions.
Fig. 1. Scheme of in vitro experimental design.
dissolved in DMSO (Dimethyl sulfoxide), then in culture medium. The highest final concentration of DMSO in KSOMaa was 0.1%. This concentration was also added to the control culture medium. Pilot experiments showed that the addition of DMSO at concentrations lower than 1% had no effect on in vitro embryo development or obtained blastocyst quality (data not shown).
2. Material and methods 2.1. Animals
2.3. In vivo experimental design Experiments were performed on mice of the outbred ICR (CD-1 IGS) strain (Velaz, Prague, Czech Republic) kept under standard conditions (temperature 22 ± 2 °C, humidity 55 ± 10%, 12:12-h light-dark cycle with lights on at 06:00, and free access to food and water). All animal experiments were carried out in accordance with the ethical principles approved by the Ethical Committee for Animal Experimentation at the Institute of Animal Physiology, and were performed in accordance with Slovak legislation based on EU Directive 2010/63/EU on the protection of animals used for experimental and other scientific purposes. All efforts were made to reduce animal suffering and distress and to minimize the number of animals needed to produce reliable results.
Spontaneously-ovulating female mice (5–6 weeks old) were mated with males of the same strain for one or more nights. Three days before the first overnight mating, estrus was synchronized by exposing the females to bedding contaminated with male urine and preputial gland secretions (Ma et al., 1999). Successful mating was confirmed by the presence of a vaginal plug on examination every morning at 8:00 AM, and this was designated Day 1 of pregnancy. Females in the experimental groups were fed with two different doses of fipronil (0.009 and 0.9 mg/kg BW) diluted in DMSO and water during the whole preimplantation period (starting on Day 1 and finishing on Day 3 of pregnancy). In total, three oral applications using a syringe and feeding needle were performed. Females in the control group were fed the same way with pure water and 0.01% DMSO. On Day 4 of pregnancy (approx. 80 h after supposed ovulation), the mouse females were killed by cervical dislocation and subjected to isolation of embryos by flushing the uterus and the oviduct using a flushing-holding medium containing 1% bovine serum albumin (BSA; Sigma-Aldrich). This period preceding the start of implantation was chosen to avoid embryo loss. Immediately after isolation, the collected embryos underwent stereomicroscopic classification (Nikon SMZ 745 T). The number of embryos per dam and the number of embryos isolated from the oviduct and uterus were evaluated as well.
2.2. In vitro experimental design Adult female mice (5–6 weeks old) were synchronized with eCG (5 IU ip; Folligon, Intervet International, Boxmeer, Holland) and hCG (4 IU ip; Pregnyl, Organon, Oss, Holland; 47 h later) and mated with males of the same strain overnight. Successful mating was confirmed by identification of a vaginal plug next morning (Day 1 of pregnancy). Fertilized dams were killed by cervical dislocation and subjected to isolation of embryos at the 2-cell stage at 45 h post hCG administration on Day 2 of pregnancy. Embryos were recovered by flushing the oviduct using a flushing–holding medium (Lawitts and Biggers, 1993) containing 1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) and classified under stereomicroscope (Nikon SMZ 745 T). Isolated 2-cell mouse embryos were pooled, randomly divided into several sub-groups and cultured in vitro under standard conditions (a humidified atmosphere with 5% CO2 and 37 °C) for 72 h with the active component fipronil (Sigma-Aldrich, Germany) at 0, 0.1, 1, 10 and 100 μM concentrations or commercial product FIPRON spot-on at 0.0004 and 0.0437 μL/mL (Bioveta, Czech Republic, containing 0.1 μM and 10 μM of fipronil) or known additional chemicals present in FIPRON spoton: butylhydroxyanisolum (BHA) at concentration 0.087 μg and butylhydroxytoluenum (BHT) at concentration 0.0430 μg (both corresponding to 0.0437 μL of FIPRON spot-on containing 10 μM fipronil). The scheme of the in vitro experimental design is shown in Fig. 1. A culture of each sub-group of mouse embryos was performed in 400 μl of synthetic oviductal medium KSOMaa Evolve supplemented with half-strength Eagle non-essential amino acid mixture (Zenith Biotech, Canada) and 1% bovine serum albumin (BSA, Sigma-Aldrich). Each experiment was repeated two or three times. In accord with the producer’s instructions, substances were first
2.4. Evaluation of in vitro and in vivo derived embryos For the analysis of in vitro developmental capacities, the numbers of embryos reaching the blastocyst, morula or cleavage stages of development (3 to 16-cell), and those arrested at the 2-cell stage or degraded (i.e. undergoing extensive cytoplasmic fragmentation) were assessed using stereomicroscopy (Nikon SMZ 745 T). For the analysis of in vivo developmental capacities, the numbers of embryos reaching the blastocyst, morula or cleavage stages of development (2 to 16-cell), those arrested at the zygotic stage or degraded, were assessed using stereomicroscopy. In in vitro and in vivo obtained blastocysts, cell numbers and cell death incidence were evaluated using immunochemistry followed by fluorescence microscopy. Blastocysts were first stained with propidium iodide (PI, 10 μg/ml; Sigma-Aldrich; stains dead cells only), then washed in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA, SigmaAldrich), fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) 2
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fipronil at 10 and 1 μM concentration negatively affected the qualitative parameters of mouse blastocysts obtained in vitro. In blastocysts derived from 2-cell embryos cultured with fipronil at 10 μM concentration, significantly decreased cell numbers in blastocysts (51.63 ± 3.19 vs 92.33 ± 4.98; P < 0.01) were recorded. The presence of active component fipronil at 10 and 1 μM significantly increased the average percentage of dead cells (7.10 ± 0.93 and 7.50 ± 0.82 vs 5.01 ± 0.40; P < 0.05 in both cases). In blastocysts derived in the presence of commercial product FIPRON spot-on at concentration 0.0004 μL/mL (containing 0.1 μM of fipronil), significantly decreased cell numbers were observed (85.62 ± 4.15 vs 101.70 ± 4.73; P < 0.05, Fig. 3(B)). There was no significant difference in average percentage of dead cells between blastocysts derived from embryos cultured in the presence of FIPRON spot-on and blastocysts derived from the control group (Fig. 3 (C)). As mentioned above, no blastocysts had developed in the 0.0437 μL/mL FIPRON spot-on group. The presence of FIPRON spot-on additives (at a concentration equivalent to 0.0437 μL/mL FIPRON spot-on) negatively affected embryo development as well as the quality of obtained blastocysts in our in vitro experiment. Significant decrease in the number of embryos reaching blastocyst stage was recorded (chi-square tests, 50.00% in the case of BHA and 58.33% in the case of BHT compared to control 88.00%; P < 0.01 in both) (Fig. 4A). Decreased cell numbers per blastocyst in blastocysts derived from 2-cell embryos cultured in the presence of BHA were observed (72.35 ± 3.61 vs. 85.49 ± 4.21; P < 0.05) (Fig. 4B). There were significant differences in the average percentage of dead cells between blastocysts derived from embryos cultured in the presence of BHA as well (6.66 ± 0.73 vs. 4.64 ± 0.49; P < 0.05) (Fig. 4C). All blastocysts produced in in vitro experiments contained at least one dead cell. Morphological analysis showed that the majority of them were of apoptotic origin, i.e. showed both nuclear fragmentation and TUNEL positive labeling (data not shown).
in PBS at room temperature for 1 h and optionally stored in 1% paraformaldehyde in PBS at 4 °C. Fixed blastocysts were washed three times for 5 min in PBS containing 0.1% BSA and transferred into PBS with 0.5% Triton X-100 (Sigma–Aldrich, Germany) for 1 h. After permeabilization, the blastocysts were washed twice for 15 min in PBS with BSA and incubated in the TUNEL assay reagents (TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling, in situ Cell Death Detection Kit; Promega Corporation, Madison, USA, visualizes specific DNA fragmentation) for 1 h at 37 °C in the dark. Then they were washed once in PBS containing 0.1% BSA. Finally, the blastocysts were counterstained with Hoechst 33342 DNA staining (10 μl/ml in PBS; Sigma Aldrich) for 5 min at room temperature, mounted on glass slides with Vectashield (Vector Laboratories, Burlingname, CA, USA) and observed using a fluorescence microscope at magnification x 400 (BX51; Olympus, Tokyo, Japan). According to their nuclear morphology, the presence of specific DNA fragmentation in the nucleoplasm and PI positivity/negativity, embryonic cells were classified as: normal (without morphological changes in nuclei, without TUNEL labeling, and without PI inclusion in the nucleoplasm) or dead (showing at least one of the following features: fragmented or condensed nucleus, positive TUNEL labeling or positive PI staining). Dead cells showing nuclear fragmentation or condensation and TUNEL positive labeling were classified as apoptotic. In each blastocyst, the percentage of dead cells was calculated as the number of dead cells relative to the total number of blastomeres in the blastocyst. 2.5. Statistical analysis Statistical analysis was performed using GraphPad Prism Software 5.01 (GraphPad Software, Inc., La Jolla, CA, USA). The results are expressed as the mean values ± SEM. The differences between data showing normal Gaussian distribution were assessed with ANOVA followed by Tukey´s post-hoc test. This was used to analyze the mean numbers of blastomeres per blastocyst. The differences between data which did not pass normality tests were assessed with the Kruskal-Wallis test, followed by Dunn’s post hoc test. This was used to analyze cell death incidence per blastocyst. For the assessment of differences between score-type data, standard chi-square tests with one degree (oviduct – uterus transition, percentage of blastocysts with dead cells) and four degrees of freedom (overall developmental abilities of embryos in vitro or in vivo) were used. Differences with P < 0.05 were considered statistically significant.
3.2. In vivo experiment Stereomicroscopic evaluation of oviduct/uterus flushes obtained from treated and untreated dams showed that fipronil at concentration 0.9 mg/kg significantly affected the average number of isolated embryos per dam. There was a tendency to decrease the average embryo number per mouse in the group treated with concentration 0.009 mg/ kg of fipronil as well, however it did not reach statistical significance (9.44 ± 1.00 vs 11.67 ± 0.65; P = 0.06) (Table 1). Significant differences were also recorded in oviduct/uterus transition in mouse females exposed to fipronil at dose 0.9 mg/kg. The transition of embryos from oviduct to uterus was significantly slowed down (80.67% embryos isolated from uterus and 19.33% isolated from oviduct compared to control with 93.15% embryos isolated from uterus and 6.85% from oviduct; P < 0.01) (Fig. 5). As shown in Fig. 6A, stereomicroscopic evaluation of embryos obtained from treated and untreated dams showed that fipronil at 0.9 mg/ kg affected the development of mouse embryos produced in in vivo conditions (P < 0.001). Increased numbers of degraded embryos and decreased numbers of blastocysts were isolated from the exposed dams. Oral administration of lower dose (0.009 mg/kg) did not affect ability of embryos to reach the blastocysts stage. Morphological assessment of isolated blastocysts did not reveal significant differences in cell numbers per blastocyst (Fig. 6B). However, there were significant differences in the incidence of dead cell counts found between blastocysts isolated from treated and non-treated dams (2.94 ± 0.35% and 3.86 ± 0.69 vs. 1.64 ± 0.18; P < 0.001 and P < 0.01, respectively for 0.009 and 0.9 mg/kg dose) (Fig. 6C). The proportion of blastocysts with dead cells was 54.81% in the untreated group, 69.42% in the group treated by 0.009 mg/kg and 64.21% in the group treated by 0.9 mg/kg BW of fipronil (P < 0.05; P > 0.05).
3. Results 3.1. In vitro experiment As shown in Fig. 2 (A), the presence of active component fipronil at concentrations of 100, 10 and 1 μM had negative effects on mouse embryonic development (chi-square tests, P < 0.001, P < 0.001, P < 0.05; respectively). In the case of 100 μM concentration all embryos were degraded, while at the other concentrations (10 and 1 μM), significant decrease in the number of embryos reaching blastocyst stage was recorded: 10 μM – 20.41%, 1 μM – 70.00% compared with control group – 84.00% of blastocysts. The rest of the embryos arrested at the early stages of development or degraded. The presence of fipronil at concentration 0.1 μM had no effect on embryo development. The presence of commercial product (FIPRON spot-on) at concentration 0.0437 μL/mL (containing 10 μM of fipronil) had a negative effect on mouse embryonic development as well (Fig. 3(A)). In this case, all embryos were degraded (P < 0.001). The presence of FIPRON spoton at concentration 0.0004 μL/mL (equivalent to 0.1 μM of active compound) had no effect on embryonic development. As shown in Fig. 2 (B, C), the presence of active component – 3
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Fig. 2. Analysis of developmental abilities (A) and qualitative parameters (B, C) of mouse embryos derived from 72 h in vitro cultures of 2-cell embryos in KSOMaa medium supplemented with various concentrations (0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM) of fipronil. (A) Percentages of arrested embryos (degraded, 2-cell stage) and embryos at higher developmental stages (3–16-cell stage, morulas and blastocysts). Differences from control group were analyzed using chi-square test with four degrees of freedom, *P < 0.05; ***P < 0.001. (B) Mean number of cells per blastocyst. Differences from control group were determined using ANOVA followed by Tukey post hoc test, **P < 0.01. (C) Average percentage of dead cells in blastocysts. Differences from control group were analyzed using Kruskal-Wallis test, followed by Dunn’s post hoc test, *P < 0.05.
4. Discussion
When the toxicity of insecticides is tested, the majority of studies focus on the main component (active substance) of the commercial product. However, pesticides often contain additional chemicals with harming potential as well (Cox and Surgan, 2006; Fabian et al., 2011). Based on the available information, commercial FIPRON spot-on contains fipronil at 10% concentration, butylhydroxyanisolum (BHA) at 0.02%, and butylhydroxytoluenum (BHT) at 0.01% concentration, as well as other compounds which are unknown. Getting data on the other compounds is difficult, due to the protection of confidential commercial information. To assess the relevance of the negative effects of available secondary compounds, an in vitro experiment with addition of BHA and BHT alone was performed in our study. It was found that BHA and BHT (at concentrations equivalent to FIPRON spot-on containing 10 μM fipronil) in culture medium decreased the capacity of two-cell stage mouse embryos to reach higher stages of development. In the case of BHA, the quality of in vitro obtained blastocysts was poor as well, as shown by significantly decreased average number of cells and elevated incidence of dead cells per blastocyst. It is known that butylhydroxyanisolum (E 320) and butylhydroxytoluenum (E 321) are widely used as antioxidants for long-term preservation of food, cosmetics and pharmaceuticals. Despite their antioxidant activity, negative impact on the reproductive physiology of rodents has been shown in both in vitro (Iannaccone, 1986) and in vivo studies (Kang et al., 2005; Pop et al., 2013). For example, at a concentration of 0.025 mg/mL, BHT was toxic to blastocyst-stage embryos, and a concentration of 0.075 mg/mL killed all of the embryos after a 2hour incubation period (Iannaccone, 1986). A high dose of BHA was
Although it has been established that the phenylpyrazole group of insecticides have greater affinity to invertebrate than to vertebrates (Hainzl et al., 1998; Narahashi et al., 2010), previous study has indicated that fipronil can bind to mammalian GABAA and GABAC receptors and may have potential to affect mammalian health (Tingle et al., 2003). Despite the high probability that GABAA receptors might already be expressed in embryos at the preimplantation developmental stages (Andäng et al., 2008; suggestion based on their observations in stem cells), up to the present the toxicity of fipronil on mammalian preimplantation embryos has not been evaluated. Our results show that fipronil at concentrations of 1 μM and higher in culture medium negatively affected the in vitro development of twocell mouse embryos. Furthermore, it negatively affected their quality, as shown by significantly decreased total cell counts and increased percentage of dead cells in obtained blastocysts. When two-cell stage mouse embryos were cultured in the presence of commercial product FIPRON spot-on containing 10 μM concentration of main compound fipronil, significant negative effect on embryo development characterized by overall developmental arrest of 100% of obtained embryos and their degeneration was observed. Moreover, lower tested FIPRON spot-on concentration (corresponding to 0.1 μM of fipronil) decreased the quality of obtained blastocysts, as shown by decreased average numbers of blastomeres in blastocysts. Observed differences between main compound fipronil and commercial FIPRON spot-on might be explained by the presence of secondary ingredients in the commercial preparation, which potentiated its negative effect. 4
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Fig. 3. Analysis of developmental abilities (A) and qualitative parameters (B, C) of mouse embryos derived from 72 h in vitro cultures of 2-cell embryos in KSOMaa medium supplemented with/without FIPRON spot-on at final concentration 0.0004 μL/mL corresponding to 0.1 μM concentration of active compound fipronil and 0.0437 μL/mL corresponding to 10 μM concentration of fipronil. (A) Percentages of arrested embryos (degraded, 2-cell stage) and embryos at higher developmental stages (3–16-cell stage, morulas and blastocysts). Differences from control group were analyzed using chi-square test with four degrees of freedom, ***P < 0.001. (B) Mean number of cells per blastocyst. Differences from control group were determined using ANOVA followed by Tukey post hoc test, *P < 0.05. (C) Average percentage of dead cells in blastocysts. Differences from control group were analyzed using Kruskal-Wallis test, followed by Dunn’s post hoc test, P > 0.05.
It has been shown that after a single dose of radiolabeled fipronil C- fipronil (4 or 40 mg/kg B.W.) administered to rats the maximum blood concentration was reached after approximately 6 h. Significant amounts of residues in individual tissues were found for 1 week after treatment, predominantly in adipose tissue (22 μg per gram for 4 mg/kg in females). Intermediate concentrations were found in adrenals, liver, kidney, pancreas and thyroids. In ovaries and uterus persisted amounts of fipronil were 4.6 μg and 2.5 μg, respectively for 4 mg/kg 1 week post treatment (FAO, 2001). Moreover, substantial amounts of fipronil metabolite were found in the brain of male rats after single dose of 14Cfipronil, demonstrating that even highly selective brain membrane are permeable for this compound (Cravedi et al., 2013). According to accessible information on pharmacokinetics of fipronil in mouse [peak serum concentration: 0.58 μg per gram at 5 mg/kg B.W. dose (FAO, 2001)] we might hypothesize that after repeated administration of fipronil at 0.9 mg/kg to female dams (current in vivo experiment), its blood/oviductal fluid concentration reached approximately 1μM concentration. This is in accordance with our results showing correspondence between outcomes of in vivo and in vitro experiments: in both cases, retarded embryonic development, increased incidence of apoptosis and no differences in cells numbers in blastocysts were observed. However, such extrapolation implies partial inconsistency between the effect of fipronil at 0.009 mg/kg dose in vivo and the effect of fipronil at 0.1 μM concentration in vitro: whilst increase in cell death documented after the treatment with 0.009 mg/kg dose in vivo reached statistical significancy (2.94% vs. 1.64% in controls), slight increase in cell death
reported to decrease serum testosterone in F0 male rats, whereas in F1 offspring it delayed sexual maturation, changed quality of sperm, shortened the estrous cycle and also decreased the weight of sex organs (BHA at 100 or 500 mg/kg for 13 weeks) (Jeong et al., 2005). On the other hand, there is no evidence that BHA or BHT affected reproduction in female pigs (dose up to 400 mg/kg) or in female rats (dose up to 1000 mg/kg), or that they impaired the development of fetuses (Hansen et al., 1982; McFarlane et al., 1997). To evaluate the relevance of the negative impact of fipronil on preimplantation embryos recorded in vitro, an in vivo experiment was performed as well. The results obtained from the in vivo experiment show that fipronil can impair their physiological development. Similarly as in in vitro experiments (concentrations up to 10 μM), no effect on average cell numbers was recorded in mouse embryos developing in intoxicated dams. On the other hand, mouse embryos developing in dams treated by 0.9 mg/kg of fipronil showed significantly decreased ability to reach the blastocyst stage. Moreover, as shown by morphological evaluation, even at the acute reference dose (daily oral exposure without adverse health effect 0.009 mg/kg), blastocysts collected on Day 4 of pregnancy displayed higher incidence of dead cells than the embryos from control dams. The most importantly, treatment with fipronil decreased the average embryo number obtained from individual dams: a numerical tendency at the acute reference dose (0.009 mg/kg B.W.) and significant impact at 0.9 mg/kg B.W. were observed. This finding suggests that fipronil has the potential to directly cause embryo loss.
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Fig. 4. Analysis of developmental abilities (A) and qualitative parameters (B, C) of mouse embryos derived from 72 h in vitro cultures of 2-cell embryos in KSOMaa medium supplemented with/without secondary compounds: butylhydroxyanisolum (BHA) at concentration 0.087 μg and butylhydroxytoluenum (BHT) at concentration 0.0430 μg (both corresponding to 0.0437 μL of FIPRON spot-on containing 10 μM fipronil). (A) Percentages of arrested embryos (degraded, 2-cell stage) and embryos at higher developmental stages (3–16-cell stage, morulas and blastocysts). Differences from control group were analyzed using chi-square test with four degrees of freedom, **P < 0.01. (B) Mean number of cells per blastocyst. Differences from control group were determined using ANOVA followed by Tukey post hoc test, *P < 0.05. (C) Average percentage of dead cells in blastocysts. Differences from control group were analyzed using Kruskal-Wallis test, followed by Dunn’s post hoc test, *P < 0.05). Table 1 Average number of embryos isolated from untreated and fipronil-treated mice. Parameters
0 mg/kg
0.009 mg/kg
0.9 mg/kg
Donor mice, N Isolated embryos, N Average, N ± SEM
15 175 11.67 ± 0.65
16 151 9.44 ± 1.00
18 150 8.33 ± 0.52**
Significantly different from untreated group: **P < 0.01.
documented after the exposure of 0.1 μM fipronil in vitro was not statistically significant (5.50% vs. 5.01% in controls). More apparent effect of fipronil on in vivo developed blastocysts might be explained in two ways: 1. Overall increase in apoptotic incidence in both untreated and treated embryos (induced by the imperfectness of artificial culture conditions) would overlap the negative effect of 0.1 μM fipronil in vitro. 2. Increased incidence of apoptosis in in vivo developed embryos would be correlated to damaging effect of fipronil to other sensitive tissues in maternal body (Badgujar et al., 2015; Montanha et al., 2018), secondarily causing disturbance of hormonal homeostasis or elevation of apoptosis-inducing mediators in reproductive organs. Apoptosis is a physiological process occurring spontaneously in both in vivo and in vitro developed preimplantation embryos (Brison and Schultz, 1997; Fabian et al., 2005; Gjørret et al., 2003; Pomar et al., 2005). It is usually triggered to fulfill a reparatory function through elimination of damaged cells. Increased incidence of apoptosis in
Fig. 5. Oviduct – uterus transition. Oviduct-to-uterus transition of in vivo derived embryos obtained from untreated and fipronil-treated (0.009 mg/kg, 0.9 mg/kg) mouse females. Statistical differences between groups were determined using standard chi-square test, ** P < 0.01.
preimplantation embryos is considered to be an indicator of inadequate developmental environment and is used as a sensitive marker of embryo quality in both experimental and clinical conditions (Betts and King, 2001; Huppertz and Herrler, 2005). Furthermore, it has been suggested that when apoptosis incidence reaches a certain threshold, it might be detrimental to further development of conceptus (Hardy, 1997; Huang and Chan, 2016; Jurisicova et al., 1998). However, the values of such 6
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Fig. 6. Analysis of developmental abilities (A) and qualitative parameters (B, C) of in vivo derived embryos obtained from untreated and fipronil-treated (0.009 mg/ kg, 0.9 mg/ kg) mouse females. (A) Percentage of degraded embryos, zygotes (1-cell stage), slowly developing (2–16- cell stage) embryos, morulas and blastocysts. Differences from untreated group were analyzed using chi-square test with four degrees of freedom, ***P < 0.001. (B) Mean number of cells per blastocyst. Differences from untreated group were determined using ANOVA followed by Tukey post hoc test, P > 0.05. (C) Average percentage of dead cells in blastocysts. Differences from untreated group were analyzed using Kruskal-Wallis test, followed by Dunn’s post hoc test, ***P < 0.001; **P < 0.01.
for fipronil, since it was found that fipronil treatment completely inhibited the function of all human glycine receptor subtypes (Islam and Lynch, 2012). In the last decade, evidence that fipronil is involved in the induction of oxidative stress has increased. Prolonged exposure to low doses of fipronil contributed to significant induction of oxidative stress by reducing the activity of antioxidant enzymes in the maternal rat organism and also in the offspring (Tukhtaev et al., 2013). Imbalance between pro-oxidants and anti-oxidants during pregnancy contributes to various complications in the maternal organism and in the offspring (Agarwal et al., 2012; Tukhtaev et al., 2013; Wang et al., 2016). The high sensitivity of embryonic cells to oxidative stress has been documented as well (Kurzawa et al., 2004). Because of fipronil's widespread application and its relatively slow degradation with a half-life up to 7.3 months in soil and water, it poses a serious risk for the surrounding environment and consequently for animal and human health (Simon-Delso et al., 2015; Tingle et al., 2003). Any contamination, especially during the early gestation period, should not be underestimated. Since fipronil residues are transferable from the pets for 30 days after treatment with Frontline, they present possible health risk to humans as well (Jennings et al., 2002). In conclusion, the results of our in vitro tests indicate that phenylpyrazole fipronil and its commercial product FIPRON spot-on can negatively influence the development and qualitative parameters of mouse preimplantation embryos. The sensitivity of mouse embryonic
threshold have not been defined yet in any mammalian species. Still, the elevation in the percentage of apoptotic cells in blastocysts directly reflects the increase in irreversible intracellular damage or malfunction of essential cellular systems (Levy et al., 2001; Penaloza et al., 2006) induced by fipronil. Evidence from earlier toxicity studies suggested that oral exposure to 0.3 mg/kg of fipronil during the perinatal period induced reproductive disorders such as shorter estrus cycle in young female rats and decreased quality of sperm in male rats (de Barros et al., 2016, 2017). Treatment during the time of organogenesis led to decreased somatic parameters and increased incidence of skeletal malformations in rat fetuses (Eisa et al., 2017). Approximately doubled length of the estrous cycle was found in female rats treated with Frontline Top Spot (commercial product containing fipronil) (Ohi et al., 2004). Higher doses of Frontline (fipronil at concentration 280 mg/kg) reduced the pregnancy index in female rats (Ohi et al., 2004). Taken together, our results indicate that mouse embryonic cells are sensitive to fipronil. Whether its negative effect is mediated via GABAA receptors depends on the unresolved question of their potential expression in preimplantation embryos. However, the presence of several receptors of neurotransmitters and biogenic monoamines has already been documented in preimplantation embryos, and in vitro tests have proved their function (reviewed in [Čikoš et al.,2010,2011]Čikoš et al. (2010, 2011). Moreover, another recent study has suggested that glycine receptors could potentially be the novel vertebrate toxicity targets
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cells to the tested secondary compounds of FIPRON spot-on (BHA, BHT) was also confirmed, as shown by decreased capacity of two-cell stage mouse embryos to reach higher stages of development, decreased cell proliferation and increased apoptosis incidence in the obtained blastocysts. The results of our in vivo experiment show that even the acute reference dose (0.009 mg/kg) of orally-administered fipronil might significantly increase apoptosis incidence in developed blastocyst. Fipronil-induced changes during the preimplantation period of embryonic development could be considered as a potential risk factor in mammalian reproduction.
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