Assessments on the potential genotoxic effects of fipronil insecticide on Allium cepa somatic cells

Abstract

The potential genotoxic influences of fipronil insecticide were analyzed by studying mitotic index and phases, chromosomal abnormalities, and micronucleus percentage on the somatic cells of Allium cepa L. The roots were treated with 1, 2.5, 5 and 10 ppm of fipronil insecticide within 6, 12 and 24 h. Mitotic index was clearly diminished by fipronil in each treatment group in comparison with control. The percentages of mitotic stages have been significantly affected. Fipronil markedly enhanced the abnormality cell percentage in almost all of the concentrations and treatment times compared to the control. Chromosomal abnormalities were recorded as disturbed prophase, sticky, chromatid bridges, c-mitosis, and laggards. The micronucleus was found at interphase and its frequency was calculated at each concentration and in the control. Generally, micronucleus formation augmented with increasing concentration of fipronil as compared to control. Consequently, the genotoxic potency of fipronil insecticide with different assessments was evaluated by using the somatic chromosomes of A. cepa, and the use of a defined non-toxic dose was suggested.

Keywords:

• Fipronil
• insecticide
• chromosome
• micronucleus
• Allium test
• genotoxicity

Introduction

Pesticides are used in agricultural areas to avoid losses resulting from pests (Karaismailoglu et al. 2013; Karaismailoglu 2015). Pesticides and products contaminated with them permanently damage living systems with the risk of mutagenicity and teratogenicity (Bag 2000; Gupta 2004; Liman et al. 2011; Karaismailoglu 2015; Kuchy et al. 2015). The genotoxic potential of the different pesticides and environmental pollutants have been widely assessed with various plant and animal bioassays (Grant et al. 1960; Sta et al. 2012; Karaismailoglu 2014a; Kuchy et al. 2015). Pesticides can be highly sensitive complexes, which can link covalent bonds with nucleophilic midpoints of cellular molecules such as DNA (Crosby 1982). The use of pesticides may cause adverse effects on human health (Ji et al. 2001).

Fipronil is a selective phenylpyrazole insecticide, used in Turkey to control pests such as Agriotes sp. in corn and sunflower agricultural areas at 2.5–5 ppm concentrations ([MARA] Ministry of Agricultural & Rural Affairs (Turkey) 2009; EPA 2011). Fipronil is a highly active molecule and has a detrimental effect on the nervous system of invertebrates, blocking the uptake of ion in cells and causing hyperexcitation and cell death (Rhone-Poulene 1995; Çelik et al. 2014). Some previous studies showed that fipronil leads to production of reactive oxygen species in cells, which can cause enlarged lipid peroxidation and oxidative stress in living organisms (Möhler et al. 2004; Ki et al. 2012; Stefani Margarido et al. 2013; Gripp et al. 2017). Investigations in rats indicate that fipronil is rapidly metabolized, and is harmful to tissue (Hugnet et al. 1999). In addition, it can lead to hyperactivity, convulsions and death in fish (Beggel et al. 2012). It is lethal for crustaceans and insects (Yildirim & Agar 2016). Several studies including various toxicological analyses have indicated that fipronil insecticide has genotoxic and mutagenic effects (Tisch et al. 2007; Patricia et al. 2012; Qureshi et al. 2016; Yildirim & Agar 2016; Gripp et al. 2017).

Some plant assays can be used to study the genotoxic effects of chemicals in living systems (Singh et al. 2008), e.g. by investigating mitotic index and phase, chromosomal abnormalities, and micronucleus percentage in the somatic cells. The most widely used plants are Allium cepa (Turkoglu 2009; Karaismailoglu 2015; Verma et al. 2016; de Souza et al. 2017), Tradescantia pallida (de Souza et al. 2017), Vicia faba (Khadra et al. 2012; Yildirim & Agar 2016) and Helianthus annuus (Karaismailoglu et al. 2013). In particular, A. cepa is one of the most widely used plant materials in genotoxicity experiments, because this plant is very susceptible and dependable in comparison with the other plant systems (Grant 1994; Karaismailoglu 2015). Thus, it has been accepted as an indicator organism for bio-monitoring of environmental pollutants (Liu et al. 1992; Ateeq et al. 2002; Kuchy et al. 2015).

The target of this investigation was to determine the potential genotoxic effects of the insecticide fipronil, by investigating mitotic index and phases, chromosomal abnormalities, and micronucleus percentage in the somatic cells of Allium cepa L.

Materials and methods

Onions (2n = 16) were obtained from a commercial market. Fipronil [(RS)-5-amino-1-(2,6-dichloro-a,a,a-trifluoro-p-toly)-4-trifluoro methylsulfinylpyrazole-3-carbonitrile; molecular weight: 437.2 g/mol CAS No: 20068–37-3] (EPA 2011) was obtained from a retail outlet and utilized as stock solution for experimental tests.

Bulbs were treated with only distilled water until reaching 1 cm in length. After, they were treated with 1, 2.5, 5, and 10 ppm fipronil concentrations, at 6, 12 and 24 h application periods. Five bulbs were used for each treatment and 25 roots were evaluated per bulb. Tests were made at 22°C in the dark in order to minimize fluctuations in cell division. In preparation of the application concentration were based on the used dosage of fipronil in agriculture, and its multiples ([MARA] Ministry of Agricultural & Rural Affairs (Turkey) 2009). Distilled water was used as a control. Root tips were fixed in ethanol:glacial acetic acid (3:1) and stored at 4°C overnight. Afterwards, they were hydrolyzed in 5 N HCl at 25°C for 30 min and dyed by Schiff’s reagent for 2 h. Five slides were determined randomly from each treatment group for the genotoxic analysis and mitotic index (MI), micronucleus (MN) in interphase, and chromosome aberrations such as disturbed prophase, chromatid bridge, stickiness, c-mitosis, and laggards were investigated in the dividing cells (Karaismailoglu et al. 2013; Karaismailoglu 2014a, 2014b).

The obtained data are analyzed using analysis of variance (ANOVA) with the SPSS Inc. (2008) computer program and a significance level of p < 0.05. Dunnett’s multiple range test was used for the definition of statistical importance among the monitored differences. The outcomes of statistical analyses are given in Tables 1–3.

Table 1. Effects of the applied fipronil concentrations and control on mitotic cell division in A. cepa.

Time (h)	Concentrations (ppm)	Number of analyzed cells	MI ± SD	Prophase (%)		Metaphase (%)		Ana-telophase (%)
				T	AN	T	AN	T	AN
6	C	5000	14.51 ± 0.19	36.49	0.11	34.13	0.17	30.38	0.04
	1	5000	14.53 ± 0.08	37.15	0.11	28.45*	0.16	34.40*	–
	2.5	5000	13.84 ± 0.22*	31.49*	1.33*	27.16	0.98*	41.35*	0.39*
	5	5000	13.02 ± 0.14*	32.99*	2.63*	34.12	1.75*	34.26*	1.58*
	10	5000	10.99 ± 0.06*	49.75*	3.18*	34.16	1.21*	16.09*	3.35*
12	C	5000	16.12 ± 0.15	30.75	–	33.66	–	35.59	0.06
	1	5000	15.97 ± 0.12	29.02*	–	33.59	–	37.39*	0.29*
	2,5	5000	13.75 ± 0.21*	27.56*	2.42*	30.55*	1.47*	41.89*	1.01*
	5	5000	12.75 ± 0.33*	20.19*	2.77*	31.17*	0.91*	48.64*	1.32*
	10	5000	10.12 ± 0.44*	25.17*	3.96*	29.55*	3.44*	45.28*	2.05*
24	C	5000	16.59 ± 0.12	39.55	–	32.06	–	28.39	–
	1	5000	16.45 ± 0.15	36.41*	0.22*	32.05	0.25*	31.54*	0.47*
	2,5	5000	14.48 ± 0.19*	33.15*	3.46*	30.01*	3.21*	36.84*	1.99*
	5	5000	11.99 ± 0.25*	30.43*	3.05*	28.19*	4.08*	41.38*	2.74*
	10	5000	9.78 ± 0.11*	31.22*	5.11*	25.40*	5.76*	43.38*	4.11*

* Different from the control p < 0.05; MI: mitotic index; SD: standard deviation; C: control, T: total, AN: abnormal.

Table 2. Chromosomal abnormalities in the root tips of A. cepa exposed to concentrations of fipronil and control.

Time (h)	Concentrations (ppm)	Abnormality percentages (%)					Total Abnormality
		Disturbed prophase	Stickiness	Chromatid bridge	C-mitosis	Laggards
6	C	0.11 ± 0.04	‒	‒	0.11 ± 0.04	‒	0.22
	1	0.11 ± 0.06	0.11 ± 0.04*	‒	0.14 ± 0.04	‒	0.36*
	2.5	0.76 ± 0.11*	0.84 ± 0.15*	0.98 ± 0.06*	0.27 ± 0.15	0.97 ± 0.06*	3.82*
	5	1.19 ± 0.17*	1.86 ± 0.11*	0.87 ± 0.10*	1.01 ± 0.26*	1.54 ± 0.08*	6.47*
	10	2.02 ± 0.28*	3.21 ± 0.33*	1.70 ± 0.27*	1.84 ± 0.08*	1.71 ± 0.44*	10.48*
12	C	‒	‒	‒	‒	0.22 ± 0.12	0.22
	1	‒	0.22 ± 0.04*	‒	‒	0.11 ± 0.08	0.33*
	2.5	0.76 ± 0.15*	1.55 ± 0.08*	0.47 ± 0.09*	0.33 ± 0.08*	0.77 ± 0.11*	3.88*
	5	1.45 ± 0.21*	2.07 ± 0.11*	0.94 ± 0.11*	0.86 ± 0.05*	1.02 ± 0.25*	6.34*
	10	3.08 ± 0.55*	2.54 ± 0.17*	1.81 ± 0.06*	1.14 ± 0.18*	2.08 ± 0.12*	10.65*
24	C	‒	‒	‒	‒	‒	‒
	1	0.11 ± 0.04*	0.27 ± 0.04*	‒	‒	0.16 ± 0.06*	0.54*
	2.5	0.26 ± 0.08*	1.95 ± 0.22*	1.19 ± 0.17*	0.88 ± 0.21*	1.01 ± 0.08*	5.29*
	5	0.63 ± 0.04*	3.27 ± 0.34*	2.28 ± 0.11*	1.76 ± 0.33*	1.74 ± 0.12*	9.68*
	10	1.09 ± 0.12*	6.73 ± 0.45*	3.39 ± 0.28*	2.61 ± 0.39*	3.05 ± 0.17*	16.87*

* Different from the control p < 0.05.

Table 3. The effects of fipronil insecticide on the micronucleus assay.

Time (h)	Concentrations (ppm)	Number of analyzed cells	MN (%)
6	C	5000	‒
	1	5000	‒
	2.5	5000	0.45 ± 0.06*
	5	5000	0.36 ± 0.04*
	10	5000	0.98 ± 0.008*
12	C	5000	‒
	1	5000	‒
	2.5	5000	0.27 ± 0.04*
	5	5000	0.63 ± 0.08*
	10	5000	1.13 ± 0.12*
24	C	5000	‒
	1	5000	‒
	2.5	5000	0.49 ± 0.11*
	5	5000	0.94 ± 0.16*
	10	5000	1.21 ± 0.08*

* Different from the control p < 0.05.

Results

The influence of fipronil insecticide on the MI and the frequency of mitotic phases are presented in Table 1. Generally, all concentrations of fipronil markedly decreased the MI of A. cepa somatic chromosomes compared to the control (p < 0.05). Exceptionally, the MI values between the application groups and the control group is statistically insignificant in 1 ppm fipronil applications at all treatment periods. There were clear distinctions in MI of A. cepa root tips treating concentrations of fipronil when compared to the control (p < 0.05) (Table 1). As a result of 10 ppm fipronil application, MI substantially declined at each application period (Table 1). When mitotic phase frequencies were compared with the control in application periods, there were statistically significant results as well (p < 0.05). Almost all of the treatments significantly influenced the mitotic phase frequencies (Table 1). As the percentage of anaphase-telophase rise, the frequencies of metaphase and prophase decreased in most of the analyzed cells. A significant increase in the frequencies of abnormalities in phases with increasing fipronil concentrations was determined (Table 1).

Chromosome abnormalities were studied in mitotic phases and their types and percentages are demonstrated in Table 2 and Figure 1. The used fipronil concentrations caused five types of abnormalities: disturbed prophase, c-mitosis, sticky, laggards, and chromatid bridges.

Figure 1. Chromosomal abnormalities induced with fipronil in root meristem cells of A. cepa: (a) disturbed prophase; (b) stickiness; (c) c-mitosis; (d) chromatid bridge; (e) laggards; (f) micronucleus. Scale bars = 10 μm.

The outcomes of the micronucleus test in A. cepa root tips exposed to control group and concentrations of fipronil are given in Table 3 and Figure 1. The percentage of micronucleus induction was found to be dependent on the application dose and determine to be significantly distinct in all treatments in comparison with the control (p < 0.05), with the exception of the 1 ppm treatment. It was clearly higher at 10 ppm than the other applications of fipronil in all the treatments (Table 3). However, at 6, 12 and 24 h, applications of 2.5 and 5 ppm concentrations of fipronil markedly increased the MN frequency.

Discussion

Random or uncontrolled use of chemical agents causes environmental pollution and negative effects on living systems. Therefore, evaluation of the effects of fipronil insecticide and its impact on mitotic index and chromosomes provide useful information regarding the effects of these mostly treated toxic materials (Karaismailoglu 2015).

Mitotic index can be used as a biological signal of cell increment, which calculates the ratio of cells in various mitotic phases (Yuet Ping et al. 2012). The impacts of different concentrations of fipronil on MI in A. cepa somatic cells are given in Table 1. Mitotic effect decreased clearly with increased fipronil solutions at each application periods, as compared with the controls (p < 0.05) (Table 1). Furthermore, at concentrations of 1 ppm fipronil, MI was not significantly different to the control at almost all the treatment periods (p < 0.05) (Table 1). The most genotoxic dose of fipronil is 10 ppm; this has a more mitodepressive effect than 1, 2.5 and 5 ppm applications at all application periods.

If the MI rate declines below 22% of the control, this status induces lethal impacts on living systems. Reductions under 50% are of sublethal impact and are referred to as the genotoxicity limit value (Panda & Sahu 1985; Karaismailoglu et al. 2013; Karaismailoglu 2014b, 2015). In this investigation, sublethal impact was determined at 10 ppm as compared to control in 6, 12 and 24 h treatments, and sublethal impacts values were found as 24.25, 37.22 and 41.04%, respectively. These results are compatible with outcomes obtained from prior works, which include toxic effects of various pesticides (Liman et al. 2011; Karaismailoglu 2013, 2014a).

The mitodepressive effects of fipronil insecticide can negatively affect the mitotic cycle and hence prevent cells entering prophase and stop the mitosis cycle in interphase (Badr 1986; Kuchy et al. 2015). This reduction in MI owing to environmental substances may be due to a block on DNA/protein synthesis of the living organisms (Elghamery et al. 2000). de Oliveira et al. (2012) stated that concentrations of fipronil may cause DNA damage on mice.

The reductions in MI may be in the consequence of cells not advancing from the synthesis stage to the mitosis phase of the cell cycle (Patlolla et al. 2012), or because of blocked DNA synthesis (Sudhakar et al. 2001; Karaismailoglu 2014b, 2015) after fipronil insecticide applications. Besides, notable reductions in the number of mitosis with the effect of some metallic insecticides have been shown in the some toxicity tests used A. cepa (Akdeniz and Özmen 2011; de Souza et al. 2017). Pyrethroid pesticides in other works were found to have genotoxic potency in rodent bone marrow (Agarwal et al. 1994), in peripheral lymphocyte cultures (Surralles et al. 1990) and in aquatic organisms (Campana et al. 1999).

The influences of the test solutions on stages of the cell cycle in A. cepa somatic cells are given in Table 1. As the stage frequencies are compared with the control in all application periods, there are statistically important results (p < 0.05). As can be seen Table 1, the rates of the mitotic stages were clearly impacted in all treatments (p < 0.05). As the rates of prophase and metaphase diminished, ana-telophase was enhanced in the examined somatic cells. The influences on mitotic stages of fipronil solutions might be connected to obstacles in prophase or retention in the mitotic stages in answer to mitotic stress (Liman et al. 2011). In addition, applied doses of fipronil generally caused a significant increment in the percentages of abnormalities in mitotic stages in onion somatic cells (p < 0.05). Similar outcomes have been found in previous works, which identified negative influences of some pesticides on cell cycles (Kaymak & Goc Rasgele 2009; Karaismailoglu 2014a, 2014b, 2015, 2016, 2017).

Chromosome abnormalities may be used to study genotoxic influences of pesticides (Caritá & Marin-Morales 2008). The impacts of fipronil solutions and the control applications on mitotic anomalies and rates of total anomalies in onion somatic cells are presented in Table 2. Chromosomal abnormalities were recorded as disturbed prophase, c-mitosis, stickiness, chromatid bridges and laggards, and demonstrated in Figure 1. The most frequently seen type of chromosomal anomaly was sticky chromosomes, which can result from deterioration in DNA (Mercykutty & Stephen 1980). Also, c-mitosis may occur with preventing spindle formation like colchicines effect (Badr 1983). Another common anomaly is chromatid bridges, which form with breaks and fusion of chromatins (Shehab & Adam 1983). Disturbed prophase forms as a result of chromatin erosion. Stickiness, chromatid bridges and disturbed prophase are genotoxic effects and irreversible (Karaismailoğlu 2015), and they may cause cellular death. However, laggards are formed by the failure of spindle fiber structure, and indicate a low genotoxic impact and can be recycled (Fiskesjö 1985). Increased concentrations of fipronil induced increased rates of total anomalies. With the 10 ppm treatment for each application period, genotoxicity was more than expected. This study showed that fipronil induces cell inhibition, and chromosomal, numerical, and structural abnormalities, ranging from chromosome destruction to the disorder of the mitotic spindle. These outcomes are in agreement with prior studies (Türkoğlu 2012; Yuet Ping et al. 2012; Karaismailoglu 2015).

Micronucleus assays have a key role in evaluation of the toxicity influences of pesticides (Gebel et al. 1997; Karaismailoglu 2013, 2014a, 2014b). MN formation and its frequencies in treated groups are presented in Table 3 and Figure 1. The MN rate clearly increased with enhanced fipronil concentrations in comparison with the control groups, with the exception of 1 ppm at all treatment periods (p < 0.05). The MN rate was clearly higher at the highest fipronil dose (10 ppm) than at others. MN forms with fractures of the microtubules and results in impairments at ploidy situations (like poly or aneu-ploidy) owing to breaks in chromosomes (Konuk et al. 2007; Karaismailoglu 2015). MN may also occur due to single-strands breaks in DNA by fipronil insecticide (Qureshi et al. 2016)

Consequently, exposure to fipronil because of reduced frequency of mitotic index, leading to the formation of chromosomal aberrations and micronuclei. While this insecticide reduces loss of product and enhances output in agricultural fields, it is not practical to hold it back fully. This paper finds that if fipronil is utilized in concentrations below 2.5 ppm, the genotoxic effects on A. cepa would be decreased.

Disclosure statement

No potential conflict of interest was reported by the author.