The mitigating effects of Tribulus terrestris extract on the cytotoxic, genotoxic, biochemical and phytotoxic effects of omethoate in roots of Allium cepa bulbs

Abstract

The goal of this study was to investigate the protective potential of Tribulus terrestris L. extract (TTE) against omethoate induced toxicity in Allium cepa L. For this purpose, six groups of A. cepa bulbs were established. While tap water was applied to the control group, 25 µg/mL TTE, 50 µg/mL TTE, 5.7 mg/L omethoate, 5.7 mg/L omethoate + 25 µg/mL TTE, and 5.7 mg/L omethoate + 50 µg/mL TTE were applied to the experimental groups, respectively. Omethoate exposure decreased germination percentage, root elongation, weight gain, MI and chlorophyll a and chlorophyll b concentrations compared to control. MN, CAs, DNA tail percentage, proline and MDA levels as well as SOD and CAT activities of the omethoate treated group were higher than the control group. The abnormalities in the root meristem cells were epidermis cell damage, flattened nucleus, cortex cell damage, thickening of the cortex cell wall and transmission tissue thickening. TTE applied in mixture with omethoate alleviated all damages caused by omethoate. The protective effect of TTE was stronger at 50 µg/mL TTE than at 25 µg/mL TTE. TTE at a concentration of 50 µg/mL applied as a mixture with omethoate reduced MN frequency by 46% and DNA tail percentage by approximately 64% compared to omethoate alone. In the omethoate-treated group, the higher concentration of TTE decreased MDA content by 47.5%, proline levels by 38.5%, and SOD and CAT activities by 27.9% and 37.5%, respectively. The most dominant phenolic compounds in TTE were rutin, p-coumaric acid and 4_OH benzoic acid. This study offers a novel, multifaceted assessment demonstrating that TTE can mitigate omethoate-induced physiological, cytogenetic, biochemical, and anatomical damage in A. cepa, highlighting its potential as a plant-derived protective agent. In conclusion, our findings reveal that omethoate is toxic for A. cepa, and TTE mitigates this toxicity, a response related to its bioactive components.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-33131-2.

Introduction

Pesticides are extremely crucial substances for agricultural production, used to control groups of organisms such as insects, weeds, fungi, and microorganisms as well as plant diseases¹. They are persistent substances that can accumulate in grains, fruits, vegetables, water and soil². Although pesticide usage is crucial for the expansion of agricultural trade, it is also a serious threat to organisms in environmental, dietary, and occupational forms of exposure³. Other significant disadvantages associated with the utilization of insecticides include toxic residues in food, water, air, and soil and pest resistance⁴. The underlying cause of the inefficiency of many insecticides can be attributed to the fact that the chemical compound is often unable to target the pest and instead exerts its effect on non-target species⁵.

Organophosphate pesticides have been used extensively and continuously in modern agriculture, due to their considerable impact on crop protection and yield⁶. They are esters made from phosphoric acid that affect the central nervous system by inhibiting the acetylcholine esterase enzyme. The working routine of this enzyme is to control the amount of the neurotransmitter acetylcholine, which interrupts the nerve impulse by phosphorylating the OH group in the active site of the enzyme⁷. This hydrolysis of acetylcholine causes its biological activity to cease within microseconds. Organophosphate insecticides, classified both as anticholinesterases according to their principle of action and as broad-spectrum insecticides, are used globally in veterinary, household, horticultural and agricultural fields⁸. In addition to their persistence in low light, low temperature, acidic and high organic matter environments, these chemicals are also known for their ability to migrate from the roots to the green parts of plants⁹. It is well known that organophosphates can adversely affect living organisms at the cellular and molecular levels, depending on the dose and duration of exposure¹⁰. Furthermore, numerous physiological processes in plants, such as mineral nutrition, photochemical reactions, carbon and nitrogen metabolism, chlorophyll production, amino acid and fatty acid synthesis, and oxidative balance, can be interfered with by organophosphate pesticides¹¹.

Omethoate (C₅H₁₂NO₄PS) is an organophosphate insecticide with a molecular weight of 213.19 g/mol. Although it is classified as an acute insecticide, it also has notable acaricidal capabilities. It is commonly utilized as a systemic chemical on fruit trees, vegetables, ornamentals, and cereals, as well as in home gardens¹². The toxicity class of Omethoate is Ib according to the classification system established by the World Health Organization¹³. Because of its great capacity for environmental mobility and resistance to degradation, omethoate presents health and ecological pollution issues¹⁴. Omethoate residues, which can be found in fruits, vegetables, cereals, and tea, have been reported to be a major contributor to toxicity to both insects and mammals¹⁵. Since 2012, Türkiye has prohibited the use of omethoate, which has been linked to cancer, infertility, and abnormalities in neurological and neurobehavioral functioning in humans^16,17.

The utilization of natural products derived from plants for the treatment of illnesses has been a long-standing interest of humanity. Medicinal plants were initially used instinctively and based on experience, but advances in science changed this empirical framework. Over time, scientific foundations supported by modern evidence explaining the bioactive compounds and mechanisms demonstrating the effects of plants began to be used. Plants function as complex biochemical systems, synthesizing a wide array of secondary metabolites. These bioactive molecules, whether in their pure form or as constituents of natural extracts, serve as the foundation for numerous commercially utilized pharmaceutical drugs¹⁸.

Tribulus terrestris L., an annual plant from the Zygophyllaceae family, is commonly used in traditional medicine, with its fruits and roots applied to treat a variety of health issues¹⁹. The extract of the plant has excellent pharmacological activities. It is utilized as an analgesic, aphrodisiac, antioxidant, antibacterial and diuretic substance. Furthermore, it protects the immune system, central nervous system, heart, liver, and teeth and is used as a preventive against tumors, cancer, diabetes, cramps, and hypolipidemia^20,21. The bioactive properties of T. terrestris extract are related to the various constituents of the extract such as alkaloids, flavonoids, glycosides, steroidal saponins, tannins, terpenoids and polyphenolic carboxylic acids^19,22.

Testing systems using plants as model organisms are not only highly correlated with microorganism and animal testing systems but also more sensitive, cheaper and more reliable than these tests^23,24. In this setting, the Allium test, allows for easy monitoring of phytotoxicity, cytotoxicity, genotoxicity, chromosomal aberrations (CAs) and mutagenicity resulting from exposure to various pollutants without ethical approval²⁴. Despite the growing body of research on organophosphate toxicity and the diverse pharmacological properties of T. terrestris, no study has yet examined whether T. terrestris extract can alleviate omethoate-induced cytogenotoxicity, oxidative damage, or anatomical disruptions in a plant-based model such as A. cepa. In this study, the plant model organism A. cepa was used in line with the established Allium testing approach.

The aim of this study was to evaluate the detrimental effects of omethoate insecticide as well as the mitigation potential of T. terrestris against omethoate toxicity on A. cepa L. For this investigation, mitotic index (MI), micronucleus (MN) formation, CAs frequency, and DNA damage were selected as molecular markers. The activities of superoxide dismutase (SOD) and catalase (CAT) enzymes as well as malondialdehyde (MDA), free proline and chlorophyll pigments (a and b) levels were investigated to determine biochemical toxicity. Meristematic integrity of roots directly treated with the solutions was also examined. Furthermore, the phenolic content of the extract was analyzed to elucidate the protective potential of T. terrestris.

Materials and methods

Preparation of materials and solutions

In the present study, the insecticide omethoate (Merck-CA No. 1113-02−6) was applied as a toxicant to commercially purchased A. cepabulbs from the Giresun Province, which served as bioindicators. The application dose of omethoate was determined based on its EC50 value. To this end, the inhibition of the root growth of Allium bulbs that had been germinated in omethoate solutions prepared at concentrations ranging from 0 to 25 mg/L was examined. The dose at which root growth was inhibited by 50% was accepted as the EC₅₀ value. Within the tested concentrations, 5.7 mg/L caused a 50% inhibition of root growth and was therefore selected as a sublethal EC₅₀ dose. This concentration is consistent with the MI-reducing threshold reported in study of Karagol et al²⁵., where exposure to 5.7 mg/L omethoate resulted in about 42% suppression of MI, confirming that 5.7 mg/L represents a biologically relevant sublethal genotoxic dose. Karagol et al.²⁵ reported that they used the accepted tolerance limit (2 mg/L), an intermediate concentration (3.1 mg/L), and the maximum residue level (5.7 mg/L) specified for agricultural products by the Australian Pesticides and Veterinary Medicines Authority²⁶.

The application doses of T. terrestris extract, which were used as a protective product against omethoate toxicity in the study, were determined based on the IC50 values it exhibited in antioxidant activity. The IC50 value was defined as the concentration required to inhibit 50% of free radicals. In the literature, IC50 values of T. terrestris extracts prepared with different solvents are reported in the range of 10–75 µg/mL^22,27. The IC50 value of T. terrestris was determined as 25 µg/mL in accordance with the literature, as its superoxide radical scavenging activity was investigated to determine the extract dose. Thus, the genotoxicity and protective role of T. terrestris extract against omethoate toxicity were investigated at 25 and 50 µg/mL doses. Therefore, 25 µg/mL was used as the base dose representing the IC₅₀ level. Additionally, 50 µg/mL was used as the upper concentration, which was above the base dose but not cytotoxic. The T. terrestris fruits were harvested in the same year, used in the study was obtained commercially from Aşçı Spices (Türkiye) and identified by the Department of Botany at Giresun University. Experimental research and field studies on plant and plant parts (A. cepa bulbs, T. terrestris fruits), including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation. T. terrestris samples were identified by Prof. Dr. Zafer Türkmen (Giresun University, Department of Biology, Botany Division) and deposited in the herbarium of the Department of Biology at Giresun University under the voucher number TTR-2024. The T. terrestris fruits were air-dried and ground in a grinder, then extracted using the maceration method in ethanol solvent in a shaking incubator. Following a 24-hour maceration process conducted in a shaking incubator, the solvent phase was filtered using Whatman No. 4 filter paper. The filtrate was subjected to a centrifugal process at 5,000 rpm for a duration of 10 min. The supernatant was then evaporated to yield the desired extract. Then, the residues were collected and utilized for further analysis. The yield of the crude extract was calculated using the formula (mass of dry extract/mass of plant material) x 100, represented as a percentage yield (w/w). Extract stored at + 4 °C until use. Its phenolic composition was analyzed and is fully reported in the manuscript. All procedures involving plants were conducted in accordance with national, international, and institutional guidelines and legislation. In this study, the crude extract yield was approximately 10% (w/w).

Examination of phenolic constituents

LC-MS/MS chromatography was employed to determine the phenolic constituents of T. terrestris extract. 1 g plant sample was extracted using a methanol: dichloromethane solvent mixture in a 4:1 ratio. The extract was filtered through a 0.45 μm syringe filter before analysis, which was performed using a Thermo Scientific LC-MS/MS system with an ODS Hypersil column. The optimal oven temperature for the column was set at 30 °C, and the solvent flow rate was maintained at 0.7 mL/min. The analysis duration was 34 min. The LC-MS/MS analysis was supported by Hitit University Scientific, Technical Application and Research Centre (HUBTUAM)^28,29. The detailed information about analysis profile is given in Supplementary Material.

Study design: experimental groups and procedure

A. cepa bulbs were divided into six groups of fifty individuals, each of similar size. One group represented the control group and the others were the experimental groups. After removing the old adventitious roots on the bulbs with a razor blade, the bulbs were placed in test tubes filled with the prepared solutions. Stem discs were in contact with the solutions throughout the application. While the control group was treated with tap water, the experimental groups were treated with 25 µg/mL T. terrestris extract, 50 µg/mL T. terrestris extract, 5.7 mg/L omethoate, 5.7 mg/L omethoate + 25 µg/mL T. terrestris extract and 5.7 mg/L omethoate + 50 µg/mL T. terrestris extract, respectively (Table 1). All solutions were prepared fresh daily to ensure constant concentrations. Treatments were maintained at room temperature for 72 h. At the end of the application period, root samples were collected for analyses. Meanwhile, some bulbs were left unharvested and these individuals continued to be treated with the relevant solutions until the treatment period reached 144 h. Thus, samples for chlorophyll analyses were collected from onions with growing green parts. All samples were washed with distilled water and dried before further analyses.

Table 1.

Experimental groups and treatments.

Groups	Treatments
Control	Tap water
TT 1	25 µg/mL T. terrestris extract
TT 2	50 µg/mL T. terrestris extract
OT	5.7 mg/L omethoate
OTTT 1	5.7 mg/L omethoate + 25 µg/mL T. terrestris extract
OTTT 2	5.7 mg/L omethoate + 50 µg/mL T. terrestris extract

Evaluation of physiological variables

Germination percentage (%), total weight gain (g), and root length (cm) were the parameters chosen to evaluate the toxicity of omethoate on physiological functions and the potential antitoxic effects of T. terrestris extract. All onion bulbs were initially weighed (g) using a precision balance before being exposed to the solutions. At the conclusion of the treatment period, the bulbs were taken out of the solutions, dried, and reweighed (g). The total weight gain for each group was determined by subtracting the initial weight from the final weight.

The germination percentage was calculated using Eq. 1:

1

The bulbs were classified as “germinated” if adventitious roots emerged from the disk stem.

The length of the newly emerged roots was measured from the point of emergence to the root tip using a ruler in order to evaluate root elongation.

For the germination percentage analysis, a total of 50 bulbs were used, whereas 10 bulbs were selected as a representative subsample for measuring root length (ten adventitious roots measured in each bulb) and determining total weight.

Evaluation of cytogenetic variables

To determine the effect of omethoate insecticide and T. terrestris extract on cytogenetic parameters, MI, presence of MN, frequency of CAs, and DNA damage analyses were performed. For easy monitoring of mitosis and chromosomal abnormalities, squash preparations were prepared from 1 cm long root segments³⁰. Clarke’s fixative and 96% ethyl alcohol were used for fixation and washing of root tissues, respectively. Fixation was performed 2 h and washing lasted 15 min. The root tissues were then transferred to glass tubes containing 1 N HCl and hydrolyzed in a water bath for 16 min. Following this process, the root tissues were kept in 45% glacial acetic acid for half an hour. Acetocarmine was used for the final staining of the root tips. After 20 h of staining, the roots were crushed between the slide and coverslip. Random areas of 10 slides from each group were examined and photographed under an Irmeco IM-450 TI model light microscope (400X magnification) for MI, MN, and CAs analyses³¹. For the determination of MI, 100 cells per slide were scanned, and 1,000 cells per slide were scanned for the identification of MN and CAs. The total number of cells analyzed was 1,000 for MI (100 cells X 10 slides) and 10,000 for MN and CAs (1,000 cells X 10 slides). To calculate the MI, the number of cells visualized in mitosis was divided by the total number of cells. The identification of MN was based on the criteria described by Fenech et al.³². The diameter of the MN was approximately one-third of the diameter of the nucleus; the MN was stained in the same color shade as the main nucleus, and the MN was separated from the cell nucleus by a clear boundary. To minimize observer bias and increase the reliability of cytogenetic and genotoxicity analyses, all microscopic evaluations were performed blinded. Preparations were coded by an independent investigator, and the examiner was not informed of the groups used.

Comet method was used to identify the damage in DNA isolated³³ from root tip cells³⁴. Slides were kept in ethanol for 24 h and then dried in an oven. Normal melting point agarose was dissolved to a concentration of 1% in distilled water heated to 50℃. A layer of 100 µl agarose was placed on the slide and frozen in the refrigerator for 5 min. A 1% concentration of low melting point agarose solution prepared with 40 °C distilled water was mixed 1/8 with freshly isolated cell suspension. This mixture was spread over the previously frozen normal melting point agarose layer. The amount of mixture needed to form this second layer was 100 µl. Following the application of coverslips to these layers, the slides were stored in the refrigerator for 5 min. After the freezing of the agarose layers, the coverslip was removed from the slide. The slides were incubated in an electrophoresis tank filled with alkaline buffer solution for 40 min to allow the DNA to dissolve. This was followed by 20 min of electrophoresis at 4 °C and 0.7 V/cm. The neutralization process was conducted by subjecting the sample to 0.4 M Tris-HCl. Fluorescence staining of the slides was achieved by dropping 100 µL of ethidium bromide on the slides. After covering with coverslips, the slides were examined under a fluorescence microscope. Comet analysis was performed on 1,000 cells from each group. “TriTek 2.0.0.0.38 Automatic Comet Assay” software program was used as the analysis program. The DNA percentage of DNA segments distinguished as head and tail was analyzed, and an increase in the percentage of tail DNA was considered a sign of DNA damage. Based on this criterion, the DNA damage of the groups was categorized³⁵. The categories were no or very little damage (≤ 5%), low damage (5.1–20%), moderate damage (20.1–40%), high damage (40.1–75%), severe damage (≥ 75.1%). DNA tail percentages exceeding 75% in omethoate groups indicate severe fragmentation, potentially linked to apoptosis.

Evaluation of biochemical variables

Extraction of the enzymes

SOD and CAT enzymes were extracted from root tip cells by the same method³⁶. A 0.4 g piece of root tip was mechanically ground in 4 mL, 7.8 pH, 50 mM sodium phosphate buffer in a mortar previously cooled with a drop of liquid nitrogen. The supernatant separated from the residual phase by centrifugation at 10,500 g for 20 min was stored at 4+℃ until activity assays.

Determination of SOD activity

A reaction medium containing sodium phosphate buffer (1.5 mL; 0.05 M), nitroblue tetrazolium chloride (0.3 mL; 750 µM), riboflavin (0.3 mL; 20 µM), EDTA-Na₂ (0.3 mL; 0.1 mM), polyvinylpyrrolidone (0.01 mL; 4%) and methionine (0.3 mL; 130 mM) was prepared. The final volume of the medium was brought to 3 mL by adding 0.28 mL distilled water and 0.01 mL enzyme extract. The mixture containing the SOD enzyme was allowed to stand at 25 ℃ in front of 15 W fluorescent light. After 10 min of illumination, the cuvettes containing the reaction mixture were kept in the dark for 10 min to terminate the reaction. The outcome of the reaction was determined by measuring the absorption at a wavelength of 560 nm³⁷. The amount of enzyme required to inhibit the depletion of NBT by half was calculated in units and presented as activity U/mg FW (FW: fresh weight).

Determination of CAT activity

A reaction medium containing sodium phosphate buffer (1.5 mL; 200 mM) and hydrogen peroxide (0.3 mL; 0.1 M) was prepared. The final volume of the medium was brought to 3 mL by adding 1 mL distilled water and 0.2 mL enzyme extract. Enzymatic reduction of hydrogen peroxide was monitored at a wavelength of 240 nm for 1 min³⁸. The final unit of CAT activity was calculated as OD₂₄₀ nm min/g.

Determination of MDA accumulation

The effect of T. terrestris extract, omethoate, and omethoate with T. terrestris extract on MDA accumulation was investigated using 1.5 g root tip segments³⁹. The homogenization was achieved utilizing 3 mL of 5% trichloroacetic acid. The supernatant containing MDA was separated from the remaining phase by centrifugation at 12,000 g for 10 min. The supernatant (1.5 mL), 0.5% thiobarbituric acid (1.5 mL), and 20% trichloroacetic acid (1.5 mL) were mixed. The mixture was kept in a water bath at 90 °C for 35 min. The reaction was then stopped using an ice bath for 10 min. The absorbance of the supernatant separated by centrifugation at 10,000 g for 5 min was read at 532 nm. MDA levels are presented as µM/g FW (FW: fresh weight).

Determination of free proline levels

The effect of T. terrestris extract, omethoate, and T. terrestris extract applied simultaneously with omethoate on proline levels was analyzed using 0.5 g root tips⁴⁰. Roots were homogenized in 10 mL of 3% aqueous sulfosalicylic acid. The homogenate (2 mL), glacial acetic acid (2 mL), and acid-ninhydrin (2 mL) were mixed. The mixture was kept in a water bath at 100℃ for 60 min. After 10 min in the ice bath to cool abruptly, the mixture was mixed with 4 mL of toluene and vortexed for 15 s. The resulting phases were separated, and the absorbance of the chromophore phase was measured at a wavelength of 520 nm. Proline concentrations in the groups were calculated as µmol proline/g FW (FW: fresh weight) using a freshly prepared proline standard.

Determination of chlorophyll a and chlorophyll b levels

The effect of T. terrestris extract, omethoate, and T. terrestris extract applied simultaneously with omethoate on chlorophyll a and chlorophyll b levels was analyzed using 0.2 g leaf segments⁴¹. The leaf pieces were stored in glass tubes, which were covered with aluminum foil and containing 5 mL of 80% acetone, for seven days under refrigerated conditions. At the end of this process, the leaves were quickly compressed with a glass baguette to facilitate the transfer of pigments that acetone could not dissolve into the solution. Centrifugation at 3000 rpm was performed to separate the supernatant containing pigments. Thereafter, acetone (5 mL, 80%) was added to the obtained supernatant. The absorbance of this final solution was read at two different wavelengths (645 nm and 663 nm). The chlorophyll a and chlorophyll b concentrations contained in the groups were calculated as mg/g FW (FW: fresh weight) according to Witham et al.⁴².

Determination of meristematic integrity

After removing the root cap, cross-sections were taken from the roots with a steel razor blade. A volume of 1 mL of 5% methylene blue was used to stain the tissues. After waiting for 2 min to allow the stain to permeate the tissues, the slides containing the sections were covered with coverslips. The slides were photographed under x200 magnification of an Irmeco IM-450 TI model microscope⁴³.

Statistical analysis

Tables were arranged to include mean ± standard deviation data. The significance of the mean differences was examined using the Duncan and one-way ANOVA tests in the SPSS Statistics 22 (IBM SPSS/Türkiye) software. The difference between the means is statistically significant at the p < 0.05. The data showed normal distribution according to both the Shapiro-Wilk Test and the Kolmogorov-Smirnov Test (p > 0.05). Furthermore, the Skewness-Kurtosis values of the data were also within the normal distribution range (−1, + 1). Therefore, parametric tests were used for statistical analysis. Since the Levene test demonstrated homogeneous distribution of variances during data analysis, the Duncan test was applied to determine statistical differences between the groups.

Results and discussion

The effects of omethoate and T. terrestris extract treatments on physiological parameters are given in Table 2. The initial mean bulb weight of the groups ranged from 10.3 to 10.5 g, and the germination percentages of the TT 1 and TT 2 groups treated with T. terrestris extracts were similar to the control group. Furthermore, no statistical difference was observed between the root length and weight gain values of these groups and the control group (p > 0.05). This finding suggests that the selected doses of T. terrestris extract did not induce growth inhibition in A. cepa bulbs. Conversely, the OT group that received omethoate exhibited a significantly reduced germination percentage compared to the control group (p < 0.05). The mean values for root length and weight gain decreased by 50.06% and 39.7%, respectively, in the OT group when compared to the control group. In contrast, the OTTT 1 and OTTT 2 groups, where T. terrestris extract was applied in a mixture with omethoate, exhibited a higher germination percentage compared to the OT group, although this did not reach the levels observed in the control group. The 50 µg/mL extract resulted in a higher germination percentage than the 25 µg/mL extract (Table 2). The root length of the OTTT 1 and OTTT 2 groups was 1.32 and 1.64 times the OT group values, respectively. Additionally, the weight gain of the OTTT 1 and OTTT 2 groups was 1.75 and 2.65 times that of the OT group, respectively. In this case, T. terrestris extract applied concurrently with omethoate positively influenced both root length and weight gain in comparison to omethoate administered alone. The ameliorative effect of the extract was more pronounced at 50 µg/mL than at 25 µg/mL.

Table 2.

Effects of T. terrestris extract, omethoate and T. terrestris + omethoate treatments on physiological variables.

Groups	Germination (%) (n = 50)	Root elongation (cm) (n = 10)	Initial weight (g)	Final weight (g)	Weight gain (g) (n = 10)
Control	98	7.31 ± 0.19a	10.50 ± 0.41	20.40 ± 0.59	+ 9.90a
TT 1	100	7.33 ± 0.14a	10.40 ± 0.39	20.60 ± 0.55	+ 10.20a
TT 2	99	7.32 ± 0.20a	10.30 ± 0.33	20.80 ± 0.63	+ 10.50a
OT	70	3.65 ± 0.12d	10.30 ± 0.48	12.30 ± 0.99	+ 2.00d
OTTT 1	83	4.82 ± 0.22c	10.30 ± 0.43	13.80 ± 0.77	+ 3.50c
OTTT 2	92	6.02 ± 0.21b	10.30 ± 0.54	15.60 ± 0.54	+ 5.30b

Control: Tap water, TT1: 25 µg/mL T. terrestris extract, TT2: 50 µg/mL T. terrestris extract, OT: 5.7 mg/L omethoate, OTTT 1: 5.7 mg/L omethoate + 25 µg/mL T. terrestris extract, OTTT 2: 5.7 mg/L omethoate + 50 µg/mL T. terrestris extract. The means marked with different letters (a-d) in the same column are statistically different (p < 0.05).

Pesticides, which have mitodepressive effects, can cause a decrease in root growth⁴⁴, germination⁴⁵, and weight gain⁴⁶. Our results confirmed the results of Zhang et al.⁴⁶, who demonstrated the dose-dependent inhibitory effect of omethoate application on plant growth through fresh and dry weight reduction of wheat plants. Increasing doses of dimethoate, a close relative of omethoate, have also been shown to retard growth of wheat seedlings⁴⁷. Similarly, growth parameters such as root and shoot elongation, dry and fresh weight, and leaf area were observed to be reduced in chickpea treated with high doses of the organophosphorus pesticide monocrotophos⁴⁸.

The analysis of germination rate and root growth is a reliable, inexpensive, and efficient method for assessing the toxicological potential of substances. According to Camilo-Cotrim et al.⁴⁹, the most common effects of active ingredients tested for their actions through the A. cepa test include reduced germination and growth. Graña⁵⁰ asserted that inhibition and delays in cell division are implicated in the disruption of normal plant development and growth, and established a connection between growth inhibition and MI reduction. Furthermore, Sidhu et al.¹¹ reported that organophosphorus pesticides reduce growth by affecting not only cell division but also cell elongation. Growth inhibition due to insecticide toxicity may also have occurred due to severe cellular dehydration caused by omethoate stress. This finding corroborates the observations made by Zhang et al.⁴⁶, who reported that wheat leaves exposed to omethoate exhibited signs of wilting within a single day. In addition, organophosphorus pesticides, including omethoate, have also been reported to disrupt the growth-promoting mechanism by inhibiting various enzymes, transcuticular diffusion and permeability necessary for plant growth¹¹. The conversion of indole-3-acetic acid to various photooxidative products, or excessive production of reactive oxygen species (ROS), may also be responsible for such inhibition⁴⁷. Macar et al.⁵¹ stated that cell membrane disruption, genetic damage, and heavy metals in pesticide formulation may play a role in pesticide-induced growth inhibition. The enhancement of germination and growth-related values in the OTTT 1 and OTTT 2 groups in comparison to the OT group signifies that T. terrestris extract alleviated the inhibitory impact of omethoate on the growth (Table 2). Interestingly, our results are not in agreement with the study of Bouzekri et al.⁵², who reported that T. terrestris extract applied alone reduced root elongation in A. cepa. However, Yeganeh et al.⁵³ pointed out that growth rates increased significantly in Cichlasoma nigrofasciatum fed with T. terrestris extract. In a similar manner, Oreochromis niloticus exhibited substantial enhancement in growth following dietary supplementation with T. terrestris extract⁵⁴. There is no study showing that T. terrestris extract reduces stress-induced growth inhibition in plants, but various plant extracts can counteract the effect of growth suppressors in A. cepa^55,56. The protective effect of T. terrestris against pesticides is largely attributable to the bioactive compounds contained in the extract. These antioxidant compounds have capacity to reduce free radical formation and thus protect membrane integrity, which plays an important role in water and nutrient uptake, against lipid peroxidation. Antioxidants may also provide a protective barrier against the deleterious effects of oxidative stress, which can impede crucial processes such as cell division, DNA, RNA, and ATP synthesis, all of which are indispensable for optimal growth⁵⁷.

As root and/or radicle tissues are responsible for the absorption and transport of nutrients, they are also the entry points into the cells of pesticides applied to the root zone. Furthermore, meristematic cells in plants undergo intense mitotic activity, which allows for the analysis of genetic material and, consequently, the identification of genotoxic effects. Consequently, root tip meristem tissues represent significant monitoring points for pesticide toxicity⁴⁹. The parameters monitored to determine the genotoxic effects of omethoate and T. terrestris extract on the treatment groups included MI, MN formation, and CAs frequency (Table 3). There was no statistically significant difference between the MI% values of the control, TT 1, and TT 2 groups (p > 0.05). Therefore, the selected concentrations of T. terrestris extract were not cytotoxic for A. cepa root tip cells. In contrast, the MI% value of 9.22% in the control group decreased to 5.61% in the omethoate-treated OT group, and the difference was statistically significant (p < 0.05). The MI% values of the OTTT 1 and OTTT 2 groups were 6.19% and 7.17%, respectively. This finding suggests that T. terrestris administered in a mixture with omethoate may have the potential to improve the omethoate-induced MI decrease in the OT group, with the 50 µg/mL extract providing greater improvement than the 25 µg/mL extract. To the best of our knowledge, this study was the first to demonstrate that omethoate decreased MI in A. cepa. However, dimethoate, which has structural similarity with omethoate, has been previously mentioned to reduce MI in Allium sativum⁵⁸. Similarly, acephate⁵⁹, chlorfenvinphos⁶⁰, malathion⁶¹ and chlorpyrifos⁶², which are among the organophosphorus insecticides, have also been shown to reduce MI in various organisms.

Table 3.

Effects of T. terrestris extract, omethoate and T. terrestris + omethoate treatments on genotoxicity variables.

	Control	TT 1	TT 2	OT	OTTT 1	OTTT 2
MCN	922 ± 12.30a	928 ± 9.87a	925 ± 10.20a	561 ± 11.80d	619 ± 13.00c	717 ± 13.80b
MI (%)	9.22	9.28	9.25	5.61	6.19	7.17
MN	0.40 ± 0.52d	0.30 ± 0.48d	0.20 ± 0.42d	50.9 ± 4.36a	40.2 ± 3.12b	27.4 ± 3.53c
SC	0.20 ± 0.42d	0.30 ± 0.48d	0.10 ± 0.32d	43.4 ± 4.06a	32.3 ± 3.47b	22.0 ± 3.30c
VC	0.20 ± 0.42d	0.00 ± 0.00d	0.10 ± 0.32d	38.1 ± 4.53a	29.9 ± 3.21b	20.0 ± 3.56c
FRG	0.00 ± 0.00d	0.00 ± 0.00d	0.00 ± 0.00d	33.7 ± 3.33a	23.1 ± 3.54b	16.5 ± 1.96c
BR	0.30 ± 0.48d	0.40 ± 0.52d	0.20 ± 0.42d	26.9 ± 2.88a	19.9 ± 3.21b	11.4 ± 2.27c
UDC	0.00 ± 0.00d	0.00 ± 0.00d	0.00 ± 0.00d	15.2 ± 2.62a	10.4 ± 2.07b	5.90 ± 1.60c
Head diameter (px)	36.000	40.000	34.000	42.000	42.000	36.000
Head density	296.536	280.285	233.472	285.822	280.738	295.750
Head DNA (%)	95.1 ± 1.37b	98.4 ± 1.71a	99.0 ± 1.05a	43.5 ± 1.72e	67.3 ± 2.31d	79.9 ± 1.60c
Tail length (px)	4.000	1.000	3.000	87.000	62.000	26.000
Tail density	15.283	4.657	2.277	371.521	136.474	74.439
Tail DNA (%)	4.90 ± 1.37d	1.60 ± 1.71e	1.00 ± 1.05e	56.5 ± 1.72a	32.7 ± 2.310b	20.1 ± 1.60c
Tail moment	0.196050	0.016344	0.028976	49.171	20.280	5.228

Control: Tap water, TT1: 25 µg/mL T. terrestris extract, TT2: 50 µg/mL T. terrestris extract, OT: 5.7 mg/L omethoate, OTTT 1: 5.7 mg/L omethoate + 25 µg/mL T. terrestris extract, OTTT 2: 5.7 mg/L omethoate + 50 µg/mL T. terrestris extract. The means marked with different letters (a-d) in the same line are statistically different (p < 0.05) (n = 10). MCN: Mitotic cell number per 10,000 cells, MI: mitotic index, MN: micronucleus, SC: sticky chromosome, VC: vagrant chromosome, FRG: fragment, BR: bridge, UDC: unequal distribution of chromosomes.

MI has been employed as a biomarker to evaluate the potential of chemicals or extracts to induce cellular toxicity⁶³. A decline or an increase in MI signifies that an agent exerts a cytotoxic effect at a level that impacts mitosis mechanisms. An increase in MI in the studied samples in comparison to the control group indicates a stimulatory effect of the agent on cell division (and even tumor development), whereas a decrease signifies an inhibitory effect. Inhibition can be interpreted as delayed cell proliferation, cellular damage or death⁶⁴. As stated by Camilo-Cotrim et al.⁴⁹, a decrease in MI value is indicative of damage to normal growth and development. The findings of this study demonstrate that omethoate treatment had a negative effect on both MI values and all growth parameters (Tables 2 and 3). The concurrent decline in root elongation and weight gain in response to diminished MI indicates that omethoate exerts its effect by diminishing the frequency of cell division. This in turn leads to a decrease in cell number. Sinha and Kumar⁶⁵ reported that a decline in MI may also be associated with the capacity of the test chemical to eliminate actively dividing cells. If MI decreases by 22% compared to the control group, the tested substance is considered to have lethal effects. Additionally, a 50% decrease in MI designates the substance as being cytotoxic⁶⁶. The findings of this study demonstrate that omethoate is cytotoxic to A. cepa and there is a strong possibility that omethoate will be cytotoxic to other non-target organisms as well. As posited by Kumar and Prasad⁶³, the marked reduction in MI may be attributable to blockade in the G2 phase of the cell cycle or inhibition of DNA synthesis in the S phase. In addition, pesticides may interact with and inhibit cell cycle proteins, including DNA polymerase, DNA gyrase, RNA polymerase, kinases, and enzymes involved in mitotic spindle production, assembly, and orientation⁴⁹. Pesticides can also directly inhibit tubulin polymerization. This problem results in the failure of mitotic spindle formation and the disruption of the cell cycle⁶⁷. Decreased ATP levels and stress in the energy production center are other possible causes of pesticide-induced MI reduction⁵⁹. Disruptions in water and mineral intake, a key factor in the process of cell division, which have a direct effect on MI values, may also have resulted in a decrease in MI⁶⁸.

There was no statistical difference between the MN and CAs frequency levels of TT 1 and TT 2 groups and those of the control group (p > 0.05) (Table 3). On the other hand, MN (Fig. 1a) and CAs levels in the OT group were significantly increased compared to the control group (p < 0.05). In the A. cepa root tip cells exposed to omethoate insecticide, the most prevalent to least prevalent CA types, in order of frequency, were sticky (Fig. 1b; Table 3), vagrant (Fig. 1c; Table 3), fragment (Fig. 1d; Table 3), bridge (Fig. 1e; Table 3) and unequal distribution of chromosomes (Fig. 1f; Table 3). In the OTTT 1 and OTTT 2 groups, MN and all other CA types were significantly decreased compared to the OT group (p < 0.05). However, the values in the OTTT 1 and OTTT 2 groups did not decrease to the control level. This finding indicated that T. terrestris extract co-administered with omethoate has the potential to alleviate omethoate-induced MN and CAs formation to a great extent, if not entirely. Furthermore, the group treated with 50 µg/mL T. terrestris (OTTT 2) exhibited a greater recovery level than the group treated with 25 µg/mL (OTTT 1) (Table 3).

Fig. 1.

Omethoate-induced CAs.

MN is formed by a new membrane that develops around chromatin material that cannot move to the two poles during anaphase of mitosis. Such chromatin material results either from abnormal segregation of chromosomes due to spindle abnormalities or from chromosome breakage resulting in the formation of acentric fragments, dicentric chromosomes, and chromosome bridges. Pesticides that are spindle inhibitors or clastogens cause MN formation⁶⁹. Additionally, MN formation is an indirect indicator of numerical and structural CAs⁷⁰. Thus, MN induction in root meristems of A. cepa is a manifestation of both chromosome damage and disruption of the mitotic process⁶⁹.

CAs are considered a consequence of unrepaired single-stranded or double-stranded DNA breaks⁷¹. Chemical substances can produce genotoxic effects directly or indirectly by inhibiting the DNA repair system. While abnormalities caused by a direct genotoxic effect can be repaired by DNA repair mechanisms to maintain genome integrity, indirect genotoxic effects often become exponentially toxic⁶⁶. Fragment and bridge are indicators of the activity of clastogenic agents, while vagrant chromosome and sticky chromosome are indicators of aneugenic effect. Clastogenic agents can cause disruption, deletions, breakage, duplications and translocations of chromosomes⁷². On the other hand, aneugenic substances interfere with the machinery of cell division, like the spindle fibers, leading to an incorrect number of chromosomes in daughter cells. According to the results of this study, it can be stated that omethoate insecticide is both an aneugenic and clastogenic chemical for A. cepa. Chromosomal bridges are the result of structural changes between sister chromatids, but they can also be the result of structural exchanges between different chromosomes due to breaks, terminal deletions or incorrect rejoining. At the conclusion of cell division, particularly during telophase, these bridges are susceptible to rupture, resulting in the formation of fragments that have the potential to become MNs within the daughter cells⁷³. The appearance of chromosomal bridges highlights the effects of toxic agents on DNA⁷⁴. Sticky chromosomes are considered clastogenic aberrations that cause cell death and are associated with the formation of chromosomal bridges⁶⁶. Chromosomes can become sticky either through contraction or condensation or through DNA depolymerization and partial dissolution of nucleoproteins. Chromosomal stickiness can also result from changes in non-histone proteins associated with chromosomes⁷³. Yildiz and Arikan⁷⁵ stated that the induction of vagrant chromosomes would result in the segregation of unequal numbers of chromosomes in daughter nuclei and the subsequent formation of daughter cells with unequally sized or irregularly shaped nuclei in interphase. Vagrant, defined as the movement of a chromosome from its chromosomal set to the poles, is an error caused by a spindle thread defect in cells⁷⁶. Correct orientation of the spindle apparatus is imperative for chromosome alignment during metaphase and for proper segregation of chromosomes during anaphase. Impaired spindle apparatus orientation can lead to chromosome disorganization and disorientation, which may be responsible for delays in chromosomal segregation⁷⁷. The combined observation of vagrant chromosomes and unequal distribution of chromosomes provides clear evidence of spindle apparatus disruption in A. cepa cells.

The Comet assay is a test that is used to detect single-strand breaks or structural DNA alterations⁷⁸. The assay is distinguished by its high sensitivity, rapidity, and versatility, as well as its cost-effectiveness, making it possible to work with small numbers of cells. These features have made it much more popular than other genotoxicity tests such as sister chromatid exchanges, MN tests, and CAs⁷⁹. Nevertheless, the application of the Comet system in conjunction with the MN and CAs tests inA. cepa enables a substantial analysis of the genotoxic profile of the tested chemical. In order to determine the extent of DNA damage in A. cepa root tip cells treated with omethoate and T. terrestris extract, the percentage DNA amounts of the DNA segments in the head and tail parts were determined utilizing the Comet assay (Table 3). The categorization of DNA damage in cells was based on the percentage of tail DNA.DNA damage in the control, TT 1 and TT 2 groups was classified as “no or very little damage”. On the other hand, the percentage of tail DNA in the OT group exposed to omethoate increased to 56.5 and this damage was classified as “high damage”. DNA damage in the OT group was significantly higher than the results of the control group (p < 0.05). The tail DNA percentages of the OTTT 1 and OTTT 2 groups exposed to T. terrestris extract in a mixture with omethoate were 32.7 and 20.1, respectively (Table 3). According to these findings, T. terrestris extract applied simultaneously with omethoate reduced DNA damage to the “moderate damage” level. The damage in the OTTT 2 group was significantly lower than in the OTTT 1 group (p < 0.05).

To the best of the author’s knowledge, there has been no study to date that has utilized all MN, CAs and Comet tests for the purpose of investigating the genotoxic potential of omethoate insecticide. However, similar to the results of this study, it was revealed that the organophosphorus insecticide profenofos decreased MI and induced an increase in MN and CAs in A. cepa L. root meristem cells⁶³. Additionally, malathion was found to decrease root growth in A. cepa L. and increase MN and chromosomal disorders, including stickiness and bridge⁸⁰. It has been demonstrated that experimental mice exposed to increasing doses of dimethoate pesticide exhibited a gradual increase in MN and CAs levels and DNA damage, as revealed by Comet assay⁸¹. In the same study, it was stated that the genotoxicity induced by dimethoate could be attributed to the parent compound itself and its oxygen analog omethoate, which is much more toxic than dimethoate. Similarly, Knapik and Ramsdorf⁸² utilized Comet analysis to show that malathion, another organophosphate pesticide, induces DNA damage in Daphnia magna. In a separate study, Çeppioğlu et al.⁸³ reported that DNA damage developed in Maden-Darby Canine Kidney (MDCK) cell lines following application of the organophosphate chlorpyrifos methyl, again using the Comet assay.

DNA is highly vulnerable to changes induced by chemical agents. According to Odetti et al.⁸⁴, the interaction of diverse pesticides with DNA can occur in a direct or indirect manner, resulting in genetic instability and subsequent disruption to normal cellular processes. Sule et al.⁸⁵ stated that pesticide-induced oxidative damage may result not only from ROS production but also from accumulation of reactive nitrogen species, downregulation of antioxidant enzyme activities and mitochondrial damage. It has also been reported that ROS accumulation resulting from organophosphorus pesticide exposure can damage biological macromolecules such as DNA repair proteins and RNA, as well as DNA, and alter antioxidant defense mechanisms⁸⁶. Furthermore, radical molecules such as superoxide, hydroxyl and hydrogen peroxide, the intracellular levels of which are increased by pesticides, can cause clastogenic chromosomal damages^87,88. Cytogenetic damage of pesticides can also occur when certain metals present in their composition inhibit DNA polymerase activity by competing with various ions. These pesticides disrupt mitotic cycle and cell cycle control points, leading to abnormal cell division and CAs production⁸⁹.

To monitor the biochemical toxicity caused by omethoate insecticide and the protective role of T. terrestris extract against this toxicity, proline, MDA and chlorophyll pigment (chlorophyll a and chlorophyll b) levels and SOD and CAT enzyme activities in A. cepa root tip cells were investigated (Fig. 2). As observed in the physiological and cytogenetic parameters, the biochemical parameter values in TT 1 and TT 2 groups treated with 25 µg/mL and 50 µg/mL T. terrestris extracts did not demonstrate a significant difference when compared to the control group (p > 0.05). Conversely, the OT group exposed to omethoate exhibited elevated levels of MDA, proline, SOD activity, and CAT activity, reaching 4.1, 2.3, 1.9, and 2.9 times those observed in the control group, respectively. Additionally, chlorophyll a and chlorophyll b levels in the OT group decreased by 55.2% and 56.4% compared to the control group (Fig. 2). All biochemical parameter values in the omethoate group and the control group were significantly different from each other (p < 0.05). In both OTTT 1 and OTTT 2 groups, the administration of T. terrestris extract in combination with omethoate reduced the biochemical toxicity induced by omethoate. Between the two concentrations tested, the 50 µg/mL extract provided a stronger protective effect than the 25 µg/mL extract. The mean decrease in MDA amount, proline level, SOD activity, and CAT activity in the OTTT 2 group was 47.5%, 38.5%, 28.0%, and 37.5%, respectively, compared to the OT group (Fig. 2). Moreover, the levels of chlorophyll a and chlorophyll b in the OTTT 2 group were 1.5 and 1.6 times those observed in the OT group, respectively.

Fig. 2.

Protective role of T. terrestris extract against biochemical toxicity caused by omethoate insecticide. Cont: Tap water, TT1: 25 µg/mL T. terrestris extract, TT2: 50 µg/mL T. terrestris extract, OT: 5.7 mg/L omethoate, OTTT 1: 5.7 mg/L omethoate + 25 µg/mL T. terrestris extract, OTTT 2: 5.7 mg/L omethoate + 50 µg/mL T. terrestris extract. The means marked with different letters (a-d) are statistically different (p < 0.05) (n = 10). MDA: (µM/g FW), Proline: µmol/g FW, SOD: U/mg FW, CAT: OD_240nm min./g FW. Full biochemical values are provided in Supplementary Material.

In accordance with the findings of this study, it was previously shown by Zhang et al.⁴⁶ that omethoate exposure increased MDA and proline concentrations and SOD activity in wheat in a dose-dependent manner. Nevertheless, the aforementioned study demonstrated that a low concentration of omethoate increased activity of the CAT enzyme in wheat, while a high concentration of omethoate decreased the activity of the same enzyme in a dose-dependent manner. In this case, it can be suggested that the omethoate dosage administered to A. cepa in our study was not sufficient to induce damage to the CAT enzyme; rather, it was sufficient to stimulate CAT activity. In another study, similar to our findings, it was determined that organophosphorus chlorpyrifos pesticide negatively affected growth and positively affected the activities of antioxidant enzymes in A. cepa⁹⁰. In addition, it was revealed that the activities of enzymes such as SOD and CAT, as well as the levels of proline and MDA, increased in Solanum lycopersicum seedlings treated with emamectin, cypermethrin and imidacloprid pesticides⁹¹. Furthermore, the application of the insecticide thiamethoxam resulted in the accumulation of proline and MDA in Brassica juncea. It was also noted by Homayoonzadeh et al.⁹² that the organophosphate insecticide phosalone caused an increase in the specific activities of SOD and CAT and proline levels in Pistacia vera.

The term “ROS” is used to denote a group of oxygen-containing reactive molecules that are by-products of normal cellular metabolic processes⁸⁵. Under normal conditions, these signaling molecules play an important role in the regulation of different processes, including plant growth and development, metabolism, and responses to stressful (biotic and abiotic) conditions. However, an accumulation of these molecules can lead to oxidative stress within cells, potentially resulting in irreversible damage to DNA and subsequent cell death. Among these radicals, superoxide inactivates antioxidant enzymes such as CAT, peroxidases, and pseudocatalases⁹³, while hydrogen peroxide is known to be highly toxic at high concentrations and has the potential to produce the highly destructive and unstable hydroxyl free radical in the presence of redox metal ions such as iron and copper (Fenton reaction)⁹⁴. As a result, the hydroxyl produced in this hydrogen peroxide-mediated cascade of events, which starts with the formation of superoxide radical in the cell, is closely associated with mutations, genetic instability and DNA damage⁹⁵. The targets of oxidative imbalance in cells include not only DNA but also lipids and proteins⁹⁴. Plants have a complex and multilayered network of antioxidative systems to protect themselves from harmful ROS. SOD catalyzes the breakdown of superoxide radical into molecular oxygen and hydrogen peroxide⁹¹, while CAT enzyme is a universal oxidoreductase that decomposes hydrogen peroxide to water and molecular oxygen⁹⁰. The increase in SOD and CAT enzyme activities (Fig. 2) indicated that omethoate induced ROS accumulation in A. cepa roots and activated the enzymatic antioxidant response in the cells. Moreover, the high MDA levels in A. cepa root meristem tissues exposed to omethoate indicate that omethoate-induced oxidative stress is at a level that disrupts membrane integrity (Fig. 2). In this process known as lipid peroxidation, MDA, the primary metabolite of arachidonic acid and formed by the degradation of polyunsaturated fatty acids in membranes, serves as a reliable biomarker for oxidative stress. This three-carbon dialdehyde has been identified as a mutagenic, tumorigenic, and highly reactive molecule, indicative of endogenous DNA damage⁹⁶. Although the cooperation between CAT and SOD is known to play a crucial role in plant resistance to stressful environments⁴⁶, the increased enzyme activities observed during omethoate stress in this study were not sufficient to maintain membrane structure (Fig. 2), normal growth (Table 2), and genetic integrity (Table 3; Fig. 1). The amino acid proline (Fig. 2) is one of the metabolites whose accumulation is increased by omethoate application. In addition to being included in the composition of proteins, proline improves the stability of proteins and the integrity of membranes in plant cells, especially under abiotic stress conditions. It facilitates the activation of enzymes, maintains osmotic balance, generates signals, and serves as a redox buffer, ROS scavenger, and metal chelator in the cytoplasm⁹⁷. The increase in osmolyte content during pesticide toxicity indicates that the plant is experiencing stress⁹⁸. Similar to increased SOD and CAT activities, the elevated level of proline was not enough to shield cells from harm caused by omethoate in A. cepa.

Chlorophyll pigment levels provide direct information about the photosynthetic activity of the plant. The decrease in the levels of chlorophyll a and chlorophyll b due to omethoate application (Fig. 2) may be evidence that omethoate can be transported from the root environment to the leaf part of the plant, where it can damage the photosynthetic apparatus. The decline in chlorophyll b was more pronounced than that of chlorophyll a, suggesting that chlorophyll b is more susceptible to omethoate toxicity compared to chlorophyll a. Similar to the findings of this study, a mixture of pesticides containing omethoate was previously reported to reduce chlorophyll levels in Salix babylonica⁹⁹. In another study, phosphomidon and monocrotophos, which are among the organophosphate insecticides such as omethoate, were noted to have phytotoxic effects on wheat, maize, and mustard, reducing chlorophyll levels¹⁰⁰. In addition, acetamiprid, imidacloprid, abamectin, thiamethoxam and abamectin + chlorantraniliprole insecticides were found to reduce total chlorophyll levels in Lycopersicum esculentum¹⁰¹.

The decrease in chlorophyll content may be due to high chlorophyllase activity, decreased chlorophyll synthesis, chloroplast degradation and chlorophyll oxidation caused by ROS^102,103. It was also revealed that omethoate application caused direct damage to leaf ultrastructure, including disruption of the chloroplast envelope membrane and abnormal changes in the lamellar arrangement of the chloroplast¹⁰⁴. According to Fan et al.¹⁰⁵, there is a positive correlation between ROS production and chlorophyll degradation. In addition, organophosphates have been reported to cause Fe²⁺ deficiency in plants, leading to inhibition of the synthesis of enzymes such as catalase, δ-aminolevulinic acid, and peroxidase, which are the main components of the chlorophyll biosynthesis pathway¹⁰⁶.

In the literature, there are various studies showing that T. terrestris extract reduces the biochemical toxicity caused by pesticides. According to the study of Ali et al.¹⁰⁷, T. terrestris extract significantly reduced the hepatotoxicity induced by α-cypermethrin with its antioxidant power thanks to the phenolic and flavonoid compounds in its composition. In addition, it has been suggested that T. terrestris applied against the organophosphorus pesticide malathion has the potential to reduce free radical and MDA levels, representing the potential of scavenging ROS⁵⁷. In another study, T. terrestris extract was demonstrated to reduce lipid peroxidation in rams, increase antioxidant capacity, reduce the %DNA ratio in the Comet tail, and thus have an effect on DNA integrity¹⁰⁸.

The damage to meristematic cells induced by omethoate insecticide in A. cepa root tip tissues (Fig. 3) and the protective role of T. terrestris on the cells are presented in Table 4. Meristematic cells from the TT 1 and TT 2 groups that were exposed only to T. terrestris extract solutions exhibited the same typical appearance as the cells in the control group (Table 4; Fig. 3a, c and e, and 3h). In the OT group treated with omethoate, it was determined that the meristematic tissue lost its normal appearance and contained significant damage. The meristematic cell damage observed in the OT group included epidermis cell damage (Fig. 3b), flattened nucleus (Fig. 3d), cortex cell damage (Fig. 3f), thickening of the cortex cell wall (Fig. 3g) and transmission tissue thickening (Fig. 3i). Epidermis cell damage, flattened nucleus, and thickening of the cortex cell wall were classified as severe damage. Meanwhile, cortex cell damage and transmission tissue thickening were classified as moderate damage. T. terrestris extract applied in mixture with omethoate decreased meristematic cell damages in OTTT 1 and OTTT 2 groups compared to OT group treated with omethoate alone. Epidermis cell damage, flattened nucleus and thickening of the cortex cell wall observed in OTTT 1 group were at moderate damage level, while cortex cell damage and transmission tissue thickening were at low damage level. Furthermore, while epidermis cell damage, flattened nucleus, and cortex cell wall damage were present at a low level in the OTTT 2 group, cortex cell damage and transmission tissue thickening were not observed in this group (Table 4). There is no study in the literature showing meristematic cell damage induced by omethoate insecticide in A. cepa root tip tissues. However, in previous studies, it was observed that omethoate insecticide caused anatomical damages such as cell wall disruption of abaxial epidermis cells, inner cells and stomatal guard cells in Zea mays¹⁰⁴. Previous studies have also suggested that exposure to pesticides such as abamectin⁴⁴, tetraconazole¹⁰⁹, paraquat⁶⁸ and clethodim¹¹⁰ in A. cepa root tissues causes disorders in meristematic tissue anatomy.

Fig. 3.

Omethoate-induced meristematic cell damages.

Table 4.

Effects of T. terrestris extract, omethoate and T. terrestris + omethoate treatments on meristematic tissue integrity.

Groups	ECD	FN	CCD	CCWT	TTT
Control	0	0	0	0	0
TT 1	0	0	0	0	0
TT 2	0	0	0	0	0
OT	3	3	2	3	2
OTTT 1	2	2	1	2	2
OTTT 2	0	1	0	1	0

Control: Tap water, TT1: 25 µg/mL T. terrestris extract, TT2: 50 µg/mL T. terrestris extract, OT: 5.7 mg/L omethoate, OTTT 1: 5.7 mg/L omethoate + 25 µg/mL T. terrestris extract, OTTT 2: 5.7 mg/L omethoate + 50 µg/mL T. terrestris extract. (n = 10). ECD: epidermis cell damage, FN: flattened nucleus, CCD: cortex cell damage, CCWT: cortex cell wall thickening, TTT: transmission tissue thickening. (0): no damage, (1): low damage, (2): moderate damage, (3): severe damage.

Since roots are the main route for water and nutrient uptake, morphological and anatomical changes in root tissues exposed to pollutants can affect the entire physiological homeostasis of the plant. In plants, root meristem tissues are the first region to encounter chemicals applied to the root environment. The disruption extending from the epidermis cells to the cortex cells and then to the transmission tissues suggests that omethoate can be transported horizontally in the root tissues after uptake by roots. The abnormality in the epidermis cells was characterized by the convergence, tightening and shrinkage of the cells, and this abnormality might be associated with a resistance of the cells to take up omethoate into the root tissues. Epidermis cell damage may also be associated with membrane damage, which was confirmed by MDA accumulation⁴⁴. The thickening of the cortex cell wall can also be interpreted as a measure to prevent omethoate from reaching the transmission tissues. Belete¹¹¹ stated that cell wall thickening, which is a structural defense, occurs as a result of lignification and suberization. According to Macar et al.⁵¹, the flattened nucleus caused by omethoate, which has reached the cells, is an irreversible damage that can be observed in plants overexposed to toxic contaminants. The nucleus, which typically has an ellipsoidal or spherical appearance, can undergo changes in volume and shape in response to cellular changes such as DNA helix structure, DNA volume or nuclear protein concentration¹¹². The mechanical pressure exerted by the epidermis cells converging to resist pesticide penetration may also have played a role in the deformation of the nuclei¹¹³. Damaged transmission tissue in the roots is a serious deformation that prevents the transfer of water and nutrients to the upper parts of the plant¹⁰⁹. This finding suggests that exposure to omethoate can have a detrimental effect on the vital activities of plants.

When T. terrestris extract was applied in mixture with omethoate, it reduced the meristematic cell damage as well as growth inhibition, genotoxicity, and biochemical toxicity induced by omethoate. The alleviating effect of the extract on pesticide-induced stress was stronger at 50 µg/mL than at 25 µg/mL. Our findings were consistent with the findings of numerous studies in the literature which demonstrate that plant extracts have the capacity to alleviate meristem damage caused by a variety of pesticides^114,115.

T. terrestris extract provided significant protection against growth inhibition, genotoxic effects, biochemical toxicity and meristematic cell damage induced by omethoate in A. cepa. Extract treatment provided different levels of protection against each parameter. Although the 50 µg/mL extract dose provided a higher protection than the 25 µg/mL dose for some parameters, the difference between the two doses was less pronounced for other parameters. This indicates that T. terrestris extract provides protection not by a single mechanism but by multiple different mechanisms. T. terrestris extract exhibited a significant ameliorative role against the changes in the tested biochemical parameters. Application of 25 µg/mL extract caused a 28.01% decrease in MDA and 18.09% decrease in proline levels, whereas application of 50 µg/mL extract caused 47.47% and 38.46% decrease in the same parameters, respectively. Similarly, significant improvements were obtained in other biochemical parameters and pigment levels tested (Table 5).

Table 5.

Protective rates (%) of 25 µg/mL and 50 µg/mL of T. terrestris extract against genotoxicity and biochemical toxicity induced by omethoate.

	OTTT 1	OTTT 2
MN	21.02	46.16
SC	25.57	49.30
VC	21.52	47.50
FRG	31.45	51.03
BR	26.02	57.62
UDC	31.57	61.18
Proline	18.09	38.46
MDA	28.01	47.47
SOD	12.5	17.64
CAT	22.11	39.50
Chl a	31.6	82.29
Chl b	36.98	92.21

OTTT 1: 5.7 mg/L omethoate + 25 µg/mL T. terrestris extract, OTTT 2: 5.7 mg/L omethoate + 50 µg/mL T. terrestris extract. MN: micronucleus, SC: sticky chromosome, VC: vagrant chromosome, FRG: fragment, BR: bridge, UDC: unequal distribution of chromosomes. CAT: catalase, MDA: malondialdehyde, SOD: superoxide dismutase. CAT: catalase, MDA: malondialdehyde, SOD: superoxide dismutase. .

The findings revealed that T. terrestris also exhibited a protective effect on genotoxicity and biochemical toxicity parameters (Table 5). 25 µg/mL T. terrestris induced maximum protection against fragment and unequal distribution of chromosomes, with both abnormalities reduced by approximately 31% in the OTT1 group. The reduction detected in other CAs was in the range of 21%−26%. In the OTT2 group treated with 50 µg/mL T. terrestris, the maximum protection was 61.1% against unequal distribution of chromosomes. This was followed by bridge formation with a 57.62% protection rate. 50 µg/mL of T. terrestris treatment provided significant protection against genotoxicity by reversing the chromosomal damage induced by omethoate in the range of 46.16% to 61.18%. When all parameters were evaluated together, T. terrestris treatment induced higher performance in the improvement of biochemical parameters compared to genotoxic parameters. In particular, the decrease in chlorophyll pigment levels induced by omethoate regressed significantly, and the most significant protection was observed in chlorophyll levels in this study. In the group treated with 50 µg/mL T. terrestris, there was an 82.29% improvement in chlorophyll a level and a 92.22% improvement in chlorophyll b level.

The protective properties of T. terrestris extract against omethoate toxicity are closely related to the active components it contains. For this purpose, the antioxidant phenolic components of T. terrestris were analyzed by LC-MS/MS. The chromatogram was given in Fig. 4 and the amount of phenolics, limit of detection (LOD), limit of quantification (LOQ), percentage recovery (RSD%), and R² values for the standards were given in Table 6. The phenolic constituents of T. terrestris extract were listed as rutin, p-coumaric acid, 4-OH benzoic acid, salicylic acid, gentisic acid, vannilin, kaempferol, caffeic acid, quercetin, naringenin, syringic aldehyde, and syringic acid according to their abundance levels (Table 6; Fig. 4). The finding that T. terrestris extract, which was found to have no side effects at selected doses, reduced the toxicity caused by omethoate pesticide exposure in A. cepa is related to the phenolic compounds found in the extract. As suggested by Sasipriya and Siddhuraju¹¹⁶, the antioxidant properties of phenolic compounds can be attributed to their redox properties, which are related to a number of possible mechanisms, including the quenching of singlet and triplet oxygen, the absorption and neutralization of free radicals, metal chelating activity, and/or the degradation of peroxides. Since phenolic compounds in the extract of T. terrestris administered together with omethoate may reduce oxidative stress by contributing to antioxidant defense with these properties, SOD and CAT activity and proline level may have decreased in OTTT groups compared to the OT group. Rutin, the most abundant phenolic compound in T. terrestris extract (Table 6), exhibits high DPPH radical scavenging activity as a natural flavone derivative, chelates metals and inhibits lipid peroxidation¹¹⁷. Recently, it has been confirmed that some plant phenolics, including rutin, interact with DNA to mask the alkylation of primary amines¹¹⁸. This may be related to the fact that T. terrestris decreases the percentage of Comet tail DNA formed due to omethoate. Furthermore, Khan et al.¹¹⁹ suggested that rutin may block the entry of mutagens into the cytosol or provide a protective barrier to prevent DNA-mutagen interaction by physically interacting with DNA. Mechanistically, rutin protect redox balance by inducing Nrf2 and increasing CAT activity¹²⁰. It has been reported that p-coumaric acid, the second most abundant phenolic component of T. terrestris extract, has the capacity to reduce oxidative stress and the cytotoxic effects of various chemicals by means of its radical scavenging ability¹²¹. P-coumaric acid also suppressed H₂O₂ formation in rat intestinal cells and the effect of alkylating agents on DNA, preventing mutation and uncontrolled cell proliferation¹²². Furthermore, p-coumaric acid administered in conjunction with toluene resulted in a decline in SOD and CAT levels, as well as MDA levels, in rats when compared to the toluene-only group¹²³. p-coumaric acid exerts its antioxidant effect by donating electrons or hydrogen atoms to ROS such as O₂•⁻, H₂O₂, •OH, and ¹O₂-thereby interrupting radical chain reactions¹²⁴. In another study, pretreatment with gentisic acid, a metabolite of salicylic acid, which was highly present in T. terrestris extract, caused a significant decrease in MDA level, a restoration of antioxidant enzyme activity and reduction in MN formation, and DNA fragmentation against cyclophosphamide-induced genotoxicity and hepatotoxicity in Swiss albino mice¹²⁵. Gentisic acid also inhibited MN formation in Rattus norvegicus hepatoma cell culture, probably by chemically or enzymatically inactivating the mutagen, scavenging reactive molecules, and preventing mutagen-genetic material contact¹²⁶. Salicylic acid, a significant constituent of the T. terrestris extract, eliminated Pb²⁺ toxicity in A. cepa by thickening the root epidermis cell wall, limiting ROS accumulation, protecting membranes from ROS-mediated peroxidation, modifying antioxidant enzyme activities, and exhibiting an inductive effect on the expression of DNA repair-sensitive genes¹²⁷. Furthermore, it has been documented that 4_OH benzoic acid and caffeic acid are constituents of the phenolic composition of T. terrestris and 4_OH benzoic acid is a potent hydroxyl radical scavenger¹²⁸.

Fig. 4.

LC-MS/MS chromatogram of T. terrestris and phenolic content ratios.

Table 6.

Phenolic compound profiles of T. terrestris extract with LOD (mg/L), LOQ (mg/L) RSD%, and R² values.

Phenolic compound	Amount (µg phenolic substance/g plant)	LOD	LOQ	RSD%	R 2
rutin	190.984	0,022	0,073	1,6	0,9950
p-coumaric acid	36.242	0,069	0,109	3,5	0,9985
4_OH benzoic acid	31.432	0,243	0,809	4,1	0,9993
salicylic acid	27.483	0,030	0,099	2,3	0,9989
gentisic acid	23.344	0,013	0,043	2,3	0,9985
vannilin	16.753	0,023	0,076	3,7	0,9994
kaempferol	10.663	0,092	0,306	3,9	0,9948
caffeic acid	5.081	0,042	0,058	2,5	0,9991
quercetin	4.502	0,001	0,005	2,9	0,9983
naringenin	2.520	0,005	0,017	3,9	0,9941
syringic aldehyde	2.368	0,127	0,423	3,2	0,9989
syringic acid	0.555	0,194	0,647	4,6	0,9987
taxifolin	ND	0,001	0,005	3,8	0,9994
epicatechin	ND	0,003	0,006	2,5	0,9988
sinapic_acid	ND	0,091	0,303	2,8	0,9952
resveratrol	ND	0,019	0,062	4,1	0,9986
ellagic acid	ND	0,087	0,289	4,9	0,9962
cinnamic acid	ND	0,047	0,156	3,7	0,9954
galangin	ND	0,034	0,113	5,0	0,9954
flavone	ND	0,037	0,124	1,7	0,9995
protocatechuic acid	ND	0,049	0,162	4,9	0,9983
protocatechuic aldehyde	ND	0,026	0,087	2,2	0,9984
sesamol	ND	0,031	0,103	2,1	0,9961
catechin	ND	0,010	0,032	3,1	0,9999
pyrogallol	ND	0,074	0,247	3,8	0,9982
gallic acid	ND	0,061	0,203	4,5	0,9850
ferulic acid	ND	0,063	0,118	4,5	0,9985
rosmarinic acid	ND	0,011	0,036	2,6	0,9999
oleuropein	ND	0,003	0,005	2,0	0,9991

ND Not detected.

Conclusion

The present study revealed that the toxicity induced in A. cepa by omethoate insecticide, an acute insecticide classified as ‘toxicity class Ib’ by the World Health Organization, is multifaceted. The deleterious effects of omethoate exposure have been demonstrated to include the inhibition of growth and induction of oxidative stress in A cepa. In this study, both the toxicity caused by omethoate insecticide and the protective potential of T. terrestris extract against these damages were demonstrated simultaneously and for the first time with the A. cepa model organism. T. terrestris extract applied in a mixture with omethoate insecticide caused an amelioration in all stress and toxicity indicators associated with omethoate. In addition, the ameliorative potential of T. terrestris extract was most likely related to its rich phenolic composition. The findings of this study demonstrated that comprehensive studies investigating the mechanisms of toxicity of pesticides should be prioritized. On the other hand, this study indicated that T. terrestris extract can meaningfully lessen the harmful effects of omethoate. It is important to recognize that the work was carried out using an in vivo A. cepa model. These controlled laboratory conditions cannot fully represent the complexity of real soil–plant environments, and future field-based studies will be necessary to validate the ecological relevance and practical applicability of our findings.

Statement regarding experimental research on plants

Experimental research and field studies on plant and plant parts (A. cepa bulbs, T. terrestris fruits), including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

D. A., T. K. M., E. Y. and K. Ç. designed the experiments and performed the analyses; K. Ç. carried out the statistical analysis; D. A. and T. K. M. wrote the manuscript with the help of E. Y. and K. Ç.; T. K. M. edited the final version of the manuscript.

Data availability

This article provides all the data supporting the findings of the study. For further queries, the corresponding author was contacted.

Declarations

Competing interests

The authors declare no competing interests.