Genotoxic and cytotoxic effects of flumetralin in human peripheral blood lymphocytes in vitro

Abstract

Flumetralin, a synthetic plant growth regulator with herbicidal activity belonging to the 2,6-dinitroaniline class of chemicals, has been evaluated for its ability to induce genotoxicity in human peripheral blood lymphocytes (PBLs). The potential genotoxic and cytotoxic effects of flumetralin were investigated in vitro by chromosome aberration (CA) and cytokinesis-block micronucleus assays. Human PBLs were treated with 125, 250, 500, and 1000 µg/mL flumetralin for 24 and 48 h. Flumetralin statistically significantly increased the frequency of structural CAs at the three highest concentrations (250, 500, and 1000 µg/mL) for both treatment periods (24 and 48 h) when compared with both the negative and solvent controls. In addition, micronucleus formation was significantly induced at higher concentrations (250, 500, and 1000 µg/mL) for 24 h and at 125 and 500 µg/mL of flumetralin for the 48-h treatment period compared with the controls. Because of the excessive cytostatic effects of flumetralin, binuclear cells could not be detected sufficiently at the highest two concentrations (500 and 1000 µg/mL) for the 48-h treatment period. Furthermore, flumetralin significantly decreased the mitotic index and nuclear division index for all concentrations and treatment times compared with the control groups. The present results indicate that flumetralin was clastogenic and cytotoxic/cytostatic to human PBLs. This study presents the first report of the genotoxic and cytotoxic properties of flumetralin.

Keywords

Chromosome aberrations cytotoxicity flumetralin genotoxicity micronucleus

Introduction

Pesticides, including insecticides, herbicides, fungicides, and synthetic plant growth regulators, are used extensively to increase crop productivity in modern-day agriculture. However, the indiscriminate and high rate of use of these agrochemicals may be harmful to the environment and human health. It was found that exposure to some of these pesticides may lead to adverse effects, such as carcinogenicity, teratogenicity, mutagenicity, and immunotoxicity among other side effects (Dearfield et al., 1999).

Flumetralin, which is a synthetic plant growth regulator with herbicidal activity belonging to the 2,6-dinitroaniline class of chemicals, has been widely used to control axillary bud (sucker) growth on tobacco plants (Hermann et al., 2000). Occupational exposure to flumetralin may occur through dermal contact at workplaces where flumetralin is produced or used. In addition, because flumetralin products are used outdoors on tobacco crops, there is the potential for flumetralin to contaminate drinking water sources. Hence, nonoccupational exposure to flumetralin can occur through drinking water and the use of tobacco products (e.g. cigarette smoking) (USEPA, 2007).

Although flumetralin was classified as “not likely to be carcinogenic” (EFSA, 2014; USEPA, 2014) and also as “not likely to be genotoxic” to humans (EFSA, 2014), it was added to the Pesticide Action Network (PAN) International Highly Hazardous Pesticides list in June 2014 (PAN, 2014). If these different classifications related to the toxicity of flumetralin are taken into consideration, it becomes important to investigate the possible genotoxic effects of this compound in humans.

In addition, a literature survey showed that some chemicals of the 2,6-dinitroaniline class have been found to be genotoxic, mutagenic, and cytotoxic in various test systems (Bozari and Aksakal, 2013; Dimitrov et al., 2006; Könen and Çavaş, 2008; Pan et al., 2004; Patel et al., 2007). However, to date, there have not been any published studies on the possible genotoxic potential of flumetralin. The aim of this study was, therefore, to evaluate the genotoxic and cytotoxic effects of flumetralin on human peripheral blood lymphocytes (PBLs) using the in vitro chromosomal aberration (CA) and cytokinesis-block micronucleus (CBMN) assays.

The frequency of CAs and micronuclei (MNi) in human PBLs are among the most frequently used biomarkers for the early detection of biological effects induced by DNA-damaging compounds (Bonassi et al., 2008; Murgia et al., 2008). Furthermore, recent cohort and case–control studies demonstrated that increased frequencies of CAs and/or MNi in PBLs are predictors of cancer risk (Bonassi et al., 2008, 2011).

Materials and methods

Chemicals

Flumetralin (Fluka 45501, purity: ≥98.0%) was obtained from Sigma-Aldrich (St Louis, Missouri, USA). The chemical structure of flumetralin is shown in Figure 1, and its properties are shown as follows:

IUPAC name: N-(2-chloro-6-fluorobenzyl)-N-ethyl-2,6-dinitro-4-(trifluoromethyl)aniline

CAS name: 2-chloro-N-[2,6-dinitro-4-(trifluoromethyl)phenyl]-N-ethyl-6-fluorobenzenemethanamine

CAS registry number: 62924-70-3

Chemical formula: C₁₆H₁₂ClF₄N₃O₄

Molecular weight: 421.74 mol

The test substance was dissolved in dimethyl sulfoxide (DMSO, purity 99%, CAS no: 67-68-5) supplied by Sigma-Aldrich. Mitomycin-C (MMC, Sigma-Aldrich, M-05030, CAS no: 50-07-7) was used as the positive control and was dissolved in sterile distilled water. Colchicine (C-9754) and cytochalasin B (C-6762, CAS no: 14930-96-2) were supplied by Sigma-Aldrich. Giemsa stain and all other chemicals were purchased from Merck (Darmstadt, Germany).

Donors and collection of blood samples

This study was conducted using venous blood samples from four (n = 4) healthy, nonsmoking volunteer donors (two males and two females) aged 24–26 years. The project was approved by the Institutional Ethics Committee of Mersin University, Turkey, and all volunteers gave informed consent to participate in the study and signed consent forms. All blood samples were obtained on the same day of the initiation of the experiment between 9.00 a.m. and 9.30 a.m. to minimize possible confounding effects of dietary factors. All donors had no known recent exposure to high concentrations of genotoxic agents (e.g. chemicals, ionizing radiation, pesticides, dust, and fibers).

Peripheral venous blood samples were taken using heparinized sterile syringes and stored at 4°C and processed within 2 h after collection to prevent damage associated with storage.

Concentration selection

Test concentrations of flumetralin were chosen based on the highest concentration that resulted in an approximately half-maximal effective concentration (EC₅₀) reduction in the mitotic index (MI; 1000 µg/mL) when compared with that of the negative control. Therefore, the concentration range of the test substance (125, 250, 500, and 1000 µg/mL) corresponded to 1/8, 1/4, and 1/2 of the EC₅₀ and EC₅₀. Serial dilutions of flumetralin were made in DMSO under sterile conditions.

CA assay

The method of Evans (1984), with minor modifications, was applied in the preparation of the CA assay. This study was also organized according to the International Programme on Chemical Safety (IPCS) guidelines (Albertini et al., 2000). Lymphocyte cultures were set up by adding 0.2 mL of heparinized whole blood to 2.5 mL of chromosome medium B (Biochrom F-5023; Berlin, Germany). The cultures were incubated at 37°C for 72 h. The cells were treated with 125, 250, 500, and 1000 µg/mL concentrations of flumetralin for 24 h (flumetralin was added 48 h after initiating the culture) and 48 h (flumetralin was added 24 h after initiating the culture). A negative control (untreated cultures), a solvent control (8 µL/mL DMSO), and a positive control (0.2 µg/mL MMC) were also employed in every experiment. Colchicine, at a final concentration of 0.06 µg/mL, was added to the cultures 2 h before harvesting. The cells were collected by centrifugation (2000 r/min, 5 min), resuspended in a hypotonic potassium chloride (KCl) solution (0.4%) for 15 min at 37°C and then fixed in cold Carnoy’s fixative (methanol:glacial acetic acid, 3:1 v/v) for 20 min at room temperature (22 ± 1°C). The fixative treatments were repeated three times with intermittent centrifugation. Finally, metaphase spreads were prepared by dropping the concentrated cell suspension onto cold glass slides. The air-dried slides were stained following standard methods (5% Giemsa in Sorensen buffer, pH = 6.8, 15–20 min).

CBMN assay

The CBMN assay was conducted using the methods of Fenech (2000) and Kirsch-Volders et al. (2003). To establish the cultures, 0.2 mL of heparinized whole blood was added to 2.5 mL of chromosome medium B. The cultures were incubated at 37°C for 68 h. The cells were exposed to flumetralin at concentrations of 125, 250, 500, and 1000 µg/mL for 24 and 48 h (flumetralin was added 44 and 20 h after initiating the culture, respectively). Cytochalasin B (final concentration of 6 µg/mL) was added after 44 h of incubation to block cytokinesis and obtain binucleated (BN) cells. After an additional 24-h incubation at 37°C, the cells were harvested by centrifugation, and the pellets were treated with a hypotonic solution (0.4% KCl) for 5 min at 37°C. The cells were fixed in a cold fixative (methanol:glacial acetic acid:0.9% sodium chloride, 5:1:6 v/v/v) and, after centrifugation, fixed two times with methanol:glacial acetic acid (5:1 v/v). Finally, the fluid-containing centrifuged cells were dropped onto clean slides. The air-dried slides were stained with a 5% Giemsa stain solution for 15 min.

Slide analysis

Slides were examined using a Nikon E100 light microscope at 1000× magnification (Japan). The CAs were classified according to the International System for Human Cytogenetic Nomenclature (Paz-y-Miño et al., 2002) and evaluated as structural (chromatid type: breaks, exchanges, and sister unions; chromosome type: breaks, dicentrics, rings, fragments, and translocations) and numerical (polyploid cells) aberrations. Chromatid and chromosome gaps were not considered as CAs, as recommended by Preston et al. (1987). For the analysis of CA, 100 well-spread, intact metaphases were investigated per culture for each treatment and donor (a total of 400 metaphases). The percentage of cells with structural CAs, as well as the total CAs/cell, was calculated and summarized. To determine cytotoxicity, the MI was calculated as the number of metaphases in 3000 cells (a total of 12,000 cells per concentration) and analyzed per culture for each treatment and donor (MI = 100 × cells in metaphase/3000).

The criteria used for the BN cells and MNi evaluations were in accordance with the recommendation of Fenech (2007 ). For the MN analysis, the number of MNi in 1000 BN cells was scored for each treatment and donor (a total of 4000 BN cells per concentration). To determine the nuclear division index (NDI), the numbers of cells with well-preserved cytoplasm-containing 1–4 nuclei were determined in 1000 cells (a total of 4000 cells per concentration). The NDI was calculated using the formula: NDI = 1 × M1 + 2 × M2 + 3 × M3 + 4 × M4/N, where M1–M4 represent the number of cells with 1–4 nuclei, and N is the total number of cells scored (Eastmond and Tucker, 1989).

In this study, all slides were scored by only one individual (SB).

Statistical analysis

Statistical analysis was performed using one-way analysis of variance. Comparisons between groups were made using a post hoc analysis, least significant difference test. Concentration–response relationships were determined from the correlation coefficients (r). The values of p < 0.05 were considered to be significant.

Results

The results corresponding to the induction of CA formation in human PBLs after treatment with flumetralin are shown in Table 1. In general, flumetralin statistically significantly increased the percentage of structural CAs and also the levels of the total CAs/cell at the three higher concentrations (250, 500, and 1000 µg/mL) for both treatment periods (24 and 48 h) when compared with both the negative and solvent controls; however, the aberrations were significantly lower in comparison with the respective positive control, MMC. The increases in structural CA formation were concentration dependent only for the 48-h treatment period (r ² = 0.919, p < 0.05).

Table 1.

Effects of flumetralin on CAs in human peripheral blood lymphocytes for 24- and 48-h treatment periods.

Test substance	Treatment		Structural CAs		Polyploid cells	% Cells with structural CAs ± SD	Total CAs/cell ± SD
Test substance	Time (h)	Concentration (µg/mL)	Chromatid type	Chromosome type	Polyploid cells	% Cells with structural CAs ± SD	Total CAs/cell ± SD
Negative control	–	–	17	4	1	5.00 ± 1.41	0.06 ± 0.01
DMSO	24	8 µL	18	6	0	6.00 ± 0.81	0.06 ± 0.01
MMC	24	0.2	77	62	4	29.25 ± 3.86^a3,b3	0.35 ± 0.05^a3,b3
Flumetralin	24	125	12	17	0	6.50 ± 0.57^c3	0.07 ± 0.01^c3
		250	20	17	0	8.75 ± 0.95^a2,b1,c3	0.09 ± 0.01^a1,b1,c3
		500	20	17	1	8.50 ± 1.29^a2,c3	0.10 ± 0.02^a1,b1,c3
		1000	31	13	0	10.50 ± 1.91^a3,b2,c3	0.11 ± 0.01^a2,b2,c3
DMSO	48	8 µL	24	6	0	7.25 ± 1.25	0.08 ± 0.01
MMC	48	0.2	155	116	2	49.25 ± 6.60^a3,b3	0.68 ± 0.09^a3,b3
Flumetralin	48	125	25	16	1	9.00 ± 2.16^c3	0.11 ± 0.02^c3
		250	27	17	0	10.25 ± 1.50^a1,c3	0.11 ± 0.01^a1,c3
		500	36	19	1	12.50 ± 1.73^a3,b1,c3	0.14 ± 0.03^a2,b1,c3
		1000	47	13	0	14.00 ± 0.81^a3,b2,c3	0.15 ± 0.01^a2,b2,c3

CA: chromosomal aberration; MMC: mitomycin-C; DMSO: dimethyl sulfoxide.

All data are expressed as mean ± SD; n = 4. A total of 400 cells were scored per concentration in the CA assay. a: significant from negative control; b: significant from solvent control (DMSO); c: significant from positive control (MMC).

^a1,b1,c1 p < 0.05.

^a2,b2,c2 p < 0.01.

^a3,b3,c3 p < 0.001.

Table 2 shows the MN frequency in binuclear cells obtained in cultures treated with flumetralin in comparison with the control groups. The percentage of micronucleated binuclear (MNBN) cells and the percentage of MN were significantly increased at the three highest concentrations (250, 500, and 1000 µg/mL) for the 24-h treatment period, and at the 125 and 500 µg/mL concentrations for the 48-h treatment period compared with both the negative and solvent controls, although these increases were not concentration dependent (p > 0.05). Because of the excessive cytostatic effects of flumetralin, binuclear cells could not be observed sufficiently at the highest two concentrations (500 and 1000 µg/mL) for the 48-h treatment times. Additionally, except for the highest concentration (1000 µg/mL) for the 24-h treatment, flumetralin did not increase MN formation to the same level as the positive control.

Table 2.

Effects of flumetralin on MN formation in human peripheral blood lymphocytes for 24- and 48-h treatment periods.

Test substance	Treatment		Number of binuclear cells				MNBN cells (%) ± SD	MN (%) ± SD
Test substance	Time (h)	Concentration (µg/mL)	0 MN	1 MN	2 MN	3 MN	MNBN cells (%) ± SD	MN (%) ± SD
Negative control	–	–	3986	14	0	0	0.35 ± 0.05	0.35 ± 0.05
DMSO	24	8 µL	3984	16	0	0	0.40 ± 0.08	0.40 ± 0.08
MMC	24	0.2	3937	56	6	1	1.57 ± 0.29^a3,b3	1.78 ± 0.39^a3,b3
Flumetralin	24	125	3982	17	1	0	0.45 ± 0.05^c3	0.48 ± 0.09^c3
		250	3958	37	5	0	1.05 ± 0.12^a3,b3,c3	1.18 ± 0.17^a3,b3,c3
		500	3952	44	4	0	1.20 ± 0.08^a3,b3,c3	1.30 ± 0.08^a3,b3,c2
		1000	3937	57	6	0	1.58 ± 0.12^a3,b3	1.73 ± 0.17^a3,b3
DMSO	48	8 µL	3983	17	0	0	0.43 ± 0.15	0.43 ± 0.15
MMC	48	0.2	3784	199	12	5	5.40 ± 0.67^a3,b3	5.95 ± 0.61^a3,b3
Flumetralin	48	125	3958	36	6	0	1.05 ± 0.17^a2,b2,c3	1.20 ± 0.14^a3,b2,c3
		250	3350^b	42	8	0	1.47 ± 0.09^a3,b3,c3	1.70 ± 0.15^a3,b3,c3
		500	^c				^c
		1000	^c				^c

MN: micronucleus; DMSO: dimethyl sulfoxide; MMC: mitomycin-C; MNBN: micronucleated binuclear.

All data are expressed as mean ± SD; n = 4. A total of 4000 cells were scored per concentration for the MN analysis. a: significant from negative control; b: significant from solvent control (DMSO); c: significant from positive control (MMC).

Due to excessive toxicity, a total of 3400 cells were scored per concentration for the MNBN cells (%) and MN (%).

Insufficient binuclear cells.

^a1,b1,c1 p < 0.05.

^a2,b2,c2 p < 0.01.

^a3,b3,c3 p < 0.001.

Flumetralin significantly decreased the MI and the NDI for all concentrations and treatment times compared with the control groups (negative and solvent controls; Table 3). Furthermore, except for the lowest concentration (125 µg/mL) for the 24-h treatment, flumetralin decreased the NDI as much as or more than the respective positive control, MMC.

Table 3.

Effects of flumetralin on MI and NDI in human PBLs for 24- and 48-h treatment periods in number of mono, bi-, tri-, and tetranucleated cells.

Test substance	Treatment		MI ± SD	Number of mono, bi-, tri-, and tetranucleated cells				NDI ± SD
Test substance	Time (h)	Concentration (µg/mL)	MI ± SD	1N	2N	3N	4N	NDI ± SD
Negative control	–	–	5.73 ± 0.46	3010	911	41	38	1.28 ± 0.04
DMSO	24	8 µL	5.47 ± 0.34	3089	846	25	40	1.26 ± 0.01
MMC	24	0.2	2.74 ± 0.30^a3,b3	3527	471	2	0	1.12 ± 0.01^a3,b3
Flumetralin	24	125	4.80 ± 0.46^a1,c3	3196	790	7	7	1.21 ± 0.01^a3,b2,c3
		250	4.81 ± 0.60^a1,c3	3418	580	1	1	1.15 ± 0.02^a3,b3
		500	4.00 ± 0.49^a3,b3,c2	3643	353	2	2	1.09 ± 0.03^a3,b3
		1000	3.30 ± 0.75^a3,b3	3793	206	1	0	1.05 ± 0.02^a3,b3,c3
DMSO	48	8 µL	5.20 ± 0.51	3054	903	27	16	1.25 ± 0.02
MMC	48	0.2	2.36 ± 0.26^a3,b3	3611	388	1	0	1.10 ± 0.01^a3,b3
Flumetralin	48	125	4.61 ± 0.55^a2,c3	3636	360	3	1	1.10 ± 0.02^a3,b3
		250	3.34 ± 0.68 ^a3,b3,c2	3826	173	1	0	1.04 ± 0.02^a3,b3,c2
		500	2.45 ± 0.35^a3,b3	3954	46	0	0	1.01 ± 0.01^a3,b3,c3
		1000	1.85 ± 0.31^a3,b3	3947	53	0	0	1.02 ± 0.01^a3,b3,c3

DMSO: dimethyl sulfoxide; MI: mitotic index; NDI: nuclear division index; MMC: mitomycin-C.

All data are expressed as mean ± SD; n = 4. A total of 12,000 cells were scored per concentration for the MI and 4000 cells were scored for the NDI; a: significant from negative control; b: significant from solvent control (DMSO); c: significant from positive control (MMC).

^a1,b1,c1 p < 0.05.

^a2,b2,c2 p < 0.01.

^a3,b3,c3 p < 0.001.

Although the decrease in the MI during the 24-h treatment period occurred in a concentration-dependent manner (r ² = 0.956, p < 0.05), other decreases in the MI and the NDI were not concentration dependent (p > 0.05).

Discussion

In the present study, the genotoxic and cytotoxic effects of flumetralin, a plant growth regulator and/or herbicide belonging to the 2,6-dinitroaniline chemical class, were evaluated. We found that flumetralin induced a significant increase in the formation of structural CAs, as well as the frequency of total CAs/cell, compared with the controls at the three highest concentrations (250, 500, and 1000 µg/mL) in both the 24- and 48-h treatment periods. Furthermore, when compared with the control groups, flumetralin was found to significantly induce MN formation at all concentrations (125, 250, 500, and 1000 µg/mL) in the 24-h treatment period, and at 125 and 250 µg/mL in the 48-h treatment period. Most probably because the highest concentrations of flumetralin (500 and 1000 µg/mL) inhibited cell growth in the lymphocyte cultures, binuclear cells were not detected sufficiently at these concentrations for the 48-h treatment period.

Nevertheless, there are no data on the genotoxicity or mutagenicity of flumetralin. Hence, the genotoxic potential of flumetralin has been compared to that of other 2,6-dinitroaniline herbicides, such as fluchloralin, trifluralin, and pendimethalin.

The results of this study are in agreement with the previous reports on the in vitro clastogenic effects of some 2,6-dinitroanilines on mammalian cells. Panneerselvam et al. (1995) reported that fluchloralin significantly induced the frequency of CAs and MNBN cells in cultured human lymphocytes. Sinha et al. (1998) found the clastogenic and DNA damaging effects of this herbicide in cultured Chinese hamster ovary (CHO) cells. Similarly, Ribas et al. (1995) tested the genotoxic effects of trifluralin in human PBLs using the single gel electrophoresis assay, and they found an increase in the comet tail lengths, indicating the DNA damaging effects of this 2,6-dinitroaniline herbicide. Similar results were also reported by Patel et al. (2007) who found that pendimethalin induced DNA damage in CHO cells using the comet assay.

Our results on the clastogenicity of flumetralin are also consistent with those of Gebel et al. (1997) who reported that treatment with trifluralin significantly increased the MN frequency in mouse bone marrow cells as well as those of Dimitrov et al. (2006) who found pendimethalin significantly increased the CA and MN formation in bone marrow cells of mice.

Dinitroanilines are characterized as microtubule-depolymerizing chemicals, some of which (e.g. oryzalin and trifluralin) have well-documented anti-microtubule effects on plant tubulins (Anthony and Hussey, 1999). However, it is well known that dinitroanilines selectively inhibit the microtubules of plants and protozoa but do not act on fungal or vertebrate microtubules (Morissette et al., 2004). Accordingly, in the present study, the analysis of data from the CA test showed that flumetralin induced more structural CAs than numerical CAs. We can, therefore, suggest that flumetralin acts mainly as a clastogenic agent producing structural CAs, rather than causing aneugenicity in humans. Additionally, the total increase in CAs observed was due to the clastogenic effects of flumetralin, which can lead to the formation of CAs by breaking the phosphodiester backbone of DNA.

Considering the most common abnormality (i.e. chromatid/chromosome breaks) observed in this study, it can also be stated that the MNi produced by flumetralin in human PBLs likely resulted from the clastogenic damage caused by this compound. Moreover, there was indeed a statistically significant correlation between structural CAs and MN formation for both treatment times (r ² = 0.960 and r ² = 1, p < 0.05 for the 24- and 48-h treatment periods, respectively).

In the present study, peripheral lymphocytes exposed to all concentrations of flumetralin showed significant decreases in the MI and the NDI for both treatment times when compared with the negative and solvent controls. In addition, the test compound significantly decreased the NDI at the highest concentration for the 24-h treatment period, and at the three highest concentrations (250, 500, and 1000 µg/mL) for the 48-h treatment period when compared with the positive control, MMC. Thus, it can be said that flumetralin inhibited cell division/cell growth more than MMC, and it showed a greater cytostatic effect than MMC. As with genotoxicity, there is no published research explaining the cytotoxicity of flumetralin. In a study by Sinha et al. (1998), fluchloralin was shown to be cytotoxic to CHO cells, and they suggested that the inhibition of DNA synthesis may be due to interference with DNA replication enzymes. A cytotoxic effect was also observed in rat hepatocytes (Yamano and Morita, 1995), FTRL-5 cells (rat thyroid-derived cell line; Pan et al., 2004), and CHO cells (Patel et al., 2007) after exposure to pendimethalin.

It was reported that the CAs and DNA double-stranded breaks induced by clastogenic agents may lead to cytotoxicity (Kirkland and Müller, 2000). The decrease in the MI and the NDI also might be caused by a decreasing adenosine triphosphate level as well as the stress from the functioning of the energy production center (Jain and Andsorbhoy, 1988). In addition, the inhibition of DNA synthesis and cell proliferation might be due to the inhibition of certain cell cycle-specific proteins and/or enzymes (Hung et al., 1996). One or more of these reasons may be responsible for the observed cytotoxic effects of flumetralin.

Conclusion

In conclusion, it can be stated that flumetralin had significant clastogenic and cytotoxic/cytostatic effects at the tested concentrations (125, 250, 500, and 1000 µg/mL) for human PBLs in vitro. Flumetralin may, therefore, pose a potential risk to humans, especially for tobacco farmers and smokers. In addition to the present study, further in vivo or in vitro studies should be conducted to investigate the genotoxicity of flumetralin using different test systems.

Footnotes

Declaration of conflicting interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This study was supported by Mustafa Kemal University Research Fund (project code: 282).

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