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Comprehensive evaluation of the genotoxic effects induced by organophosphorus insecticides widely employed in agricultural practicesJosefinaCortés-Eslava1
SandraGómez-Arroyo1,3✉Emailslga@atmosfera.unam.mx
AnaRosaFlores-Márquez1
Ma.ElenaCalderón-Segura1
CésarGuerreroGuerra1
RafaelValencia-Quintana2
1Laboratorio de Genotoxicología Ambiental y 2 Bioaerosoles Atmosféricos, Instituto de Ciencias de la Atmósfera y Cambio ClimáticoUniversidad Nacional Autónoma de MéxicoCiudad Universitaria04510Coyoacán, Ciudad de MéxicoMéxico
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Laboratorio Rafael Villalobos Pietrini de Toxicología Genómica y Química Ambiental, Facultad de AgrobiologíaUniversidad Autónoma de TlaxcalaAutopista San Martin-Tlaxcala Km. 10.5 s/n, Ixtacuixtla TlaxC.P. 901203Laboratorio de Genotoxicologia Ambiental, Instituto de Ciencias de la Atmósfera y Cambio ClimaticoUniversidad Nacional Autónoma de MéxicoCiudad Universitaria04510Coyoacán, Ciudad de MéxicoMéxico
Josefina Cortés-Eslava1, Sandra Gómez-Arroyo1*, Ana Rosa Flores-Márquez1, Ma. Elena Calderón-Segura1, César Guerrero Guerra2, Rafael Valencia-Quintana3.
1Laboratorio de Genotoxicología Ambiental y 2Bioaerosoles Atmosféricos, Instituto de Ciencias de la Atmósfera y Cambio Climático, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán 04510, Ciudad de México, México.
3Laboratorio Rafael Villalobos Pietrini de Toxicología Genómica y Química Ambiental, Facultad de Agrobiología, Universidad Autónoma de Tlaxcala, Autopista San Martin-Tlaxcala Km. 10.5 s/n, Ixtacuixtla Tlax., C.P. 90120.
ABSTRACT
The risk of increasing pesticide contamination is a global hazard to ecosystems and human health. A significant proportion can reach soil and/or surface waters through leaching or wind, affecting agroecosystems and non-target plants, which can store and transfer them to higher trophic levels. The broad bean Vicia faba is an ideal model for assessing insecticide genotoxicity. This study aimed to evaluate the genotoxic effect of five organophosphate agricultural insecticides: methamidophos, parathion methyl, phoxim, azinphos methyl, and oxydemeton methyl, on the root of V. faba using the comet assay and the micronucleus tests. The results indicated that all insecticides except methamidophos caused DNA damage and micronuclei in a concentration-dependent manner. The genotoxicity induced by the agrochemicals, from lowest to highest, was: methamidophos < parathion methyl < phoxim < azinphos methyl < oxydemeton methyl. The observations suggest that the % tail intensity (% tail DNA) and tail moment are optimal parameters for assessing DNA damage. Furthermore, the mitotic index decreased with increasing insecticide concentrations. These findings underline the potential risks associated with this type of agent highlighting the need for further research and regulatory measures and support the relevance of using V. faba as a reliable system for environmental risk assessment.
Keywords:
Comet assay
Cytogenetic damage
Micronucleus
Mitotic index
Plant bioindicators
*Correspondence author: Sandra Gómez-Arroyo slga@atmosfera.unam.mx
Laboratorio de Genotoxicologia Ambiental, Instituto de Ciencias de la Atmósfera y Cambio Climatico, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán 04510, Ciudad de México, México.
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1. Introduction
Global pesticide pollution has risen due to the increasing demand for food products. Organophosphorus insecticides (OPIs) are among the most widely used worldwide due to their effectiveness in controlling a wide range of pests. These insecticides possess various chemical, physical, and biological characteristics and are applied in commercial, residential, and agricultural settings. While pesticides are designed to eliminate pests and control weeds using chemical ingredients, they can also be toxic to non-target organisms, including beneficial insects, birds, fish, and plants, as well as to the air, water, soil, and crops (Tudi et al. 2021; Dhiman et al. 2024). For instance, OPIs have been linked to the decline of bee and other insect populations, inducing deleterious effects through direct contact, dietary exposure, or contact with pollen and nectar from flowers sprayed during field application (Cutler et al. 2014; Christen 2023), which has a serious challenge worldwide for pollination and food production (Kortsch et al. 2024).
Furthermore, pesticide contamination extends beyond the target organisms, resulting in pollution. Such chemical residues harm human health through environmental and food contamination (Tudi et al. 2021). For example, studies have found traces of OPIs in fruits and vegetables, which can pose a risk to consumers. Several OPIs have demostrated their ability to damage DNA and cause cellular dysfunction, including the formation of DNA adducts, single- and double-strand DNA breaks, as well as inter- and intra-cross-links between DNA and proteins (Prathiksha et al. 2023). In addition, its indiscriminate use contributes to an imbalance in the ecosystems.
With approximately 20,000 commercial products currently available worldwide for plant and crop protection, including various pesticides and their formulations, understanding their impact on human health and environmental conservation is relevant (Landrigan et al. 2018). México, a leading pesticide importer in Latin America, presents a significant vulnerability to those who apply these substances in the field. Diverse studies report various health effects due to exposure to pesticides, such as dermatological, gastrointestinal, respiratory, reproductive, endocrine, child development, neurological, and carcinogenic effects (Scorsa et al. 2023; Thapa et al. 2024; Tucker et al. 2024; Song et al. 2025; Kamaruldzaman et al. 2023; Muñoz-Quezada et al. 2025) Furthermore, since pesticides can be ingested through residues in food and water, they have been shown to alter the intestinal microbiota, causing several diseases in humans (Giambo et al. 2021; Utembe and Kamng’ona 2021). This scenario highlights the need for additional experimental studies to investigate the impact of pesticide exposure on human health and the ecosystem. Plants offer several advantages over other biological tests that evaluate the genetic effects of these compounds.
Plant-based bioassays are widely used in toxicological and ecotoxicological assessment procedures due to their sensitivity, low cost, and ease of handling, allowing reliable methods for testing phytotoxicity, cytotoxicity, and genotoxicity. The use of plant tissue for these studies is one of the oldest, simplest, and most reliable methods available, both in vitro and in vivo. Thus, the effects of pollutants may be assessed by observing the modifications in plants and their life cycle. Researchers have utilized root tip meristem cells to assess the toxicity of various compounds and environmental agents. They believe these cells are dependable systems for environmental risk assessment. Among these methods, V. faba root tips are particularly notable for genotoxicity testing because they are sensitive to cytotoxic and genotoxic agents. This model constitutes a valuable bioassay for environmental studies due to its possession of six pairs of large chromosomes and easy of growth and handling. The unique characteristics of V. faba make it an ideal model for genotoxicity assessment, offering prospects for obtaining accurate and reliable results comparable to those from biological and ecological systems.
V. faba has long been widely used to detect the genotoxicity of pollutants in both aquatic and terrestrial systems by measuring chromosome aberration, micronucleus frequency (Gómez-Arroyo and Villalobos-Pietrini 1995; Ma 1999; Ma et al. 2005), and DNA damage using the comet assay (Koppen and Vershaeve 1996; Patlolla et al. 2012; Iqbal 2016; Iqbal et al. 2019; Zhang et al. 2020). The V. faba model is significant in environmental research due to its ability to detect genetic damage quantitatively. Results obtained from this model can be extrapolated to other biological and ecological systems, making it a crucial tool for researchers (Iqbal 2016).
The comet assay is one of the most widely used methods to assess DNA damage, which is commonly applied in genetic toxicology and environmental biomonitoring (Collins 2015; Lanier et al. 2015; Gómez-Arroyo et al. 2018; Al-Hamadany et al. 2023; Ladeira et al. 2024). This fluorescent method allows the visualization and quantitative evaluation of DNA fragments at a single-cell level. The basic principle of the comet assay is the migration of DNA through an agarose matrix under an electric field. When observed under a microscope, the nuclei resemble a comet, with a head and a tail containing migrating DNA fragments that move toward the anode.
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It is a fast, easy, and sensitive assay to detect single or double DNA strand breaks, alkali-labile sites, and DNA-protein cross-links with high reproducibility. Additionally, it does not require proliferating cells, further enhancing its reliability and making it a trusted tool for researchers in the field (Moller 2018; Walsh and Kato 2023; Tyutereva et al. 2024). Additionally, late cellular outcomes of single- or double-strand DNA breaks in dividing cells can result in micronucleus (MN) formation when cells are unable to repair DNA lesions through distinct repair pathways (Jiang et al. 2019). Moreover, chromosome breakage can result in acentric fragments and spindle disturbances in lagging chromosomes, which can be excluded from the nucleus during mitosis, generating MN, which can be observed in interphase cells and is increasingly recognized as an indicator of genotoxic potential across various species and contexts (Jiang et al. 2019). The Vicia faba-MN assay was standardized according to an international protocol, approved by the International Organization for Standardization (ISO 29200) in 2013 (Cotelle et al. 2015).As mentioned previously, higher plants occupy the first link in the food chain. Considering the risks associated with using organophosphate insecticides, this work assesses their genotoxic effects using V. faba as a model. Therefore, this study aimed to evaluate the DNA damage using two complementary methodologies: the comet assay to measure initial DNA breaks and the micronucleus in dividing cells using V. faba roots treated with the organophosphorus insecticides methamidophos (ME), parathion methyl (PM), phoxim (PH), azinphos methyl (AM), and oxidemeton methyl (OM), and to demonstrate the usefulness of both methodologies to integrate the analysis of DNA damage.
2. Material and methods
2.1. Experimental plants and chemicals
Vicia faba seeds were acquired in a regional market, ensuring they were “organic” that is, they were not treated with any pesticides during planting, cultivation, transportation, or storage. Bayer of Mexico kindly donated commercial formulation of insecticides with the following purity: ME: 48.3%, PM: 62.8%, PH: 50.0%, AM: 70%, OM: 20.0%. All reagents used for the comet assay were those previously described by Cortés-Eslava et al. (2018), and regarding the evaluation of micronuclei induction, as described by Salazar-Mercado and Correa (2024).
2.2. Germination of Vicia faba seeds
The health seeds of the broad bean selected were washed, and subsequently, they were germinated between two wet cotton layers at 19–20ºC in darkness for approximately five days.
2.3. In vivo exposure conditions
For the comet assay, Vicia faba plants with roots of 3 cm length were immersed in glass vials containing 50 mL of the five insecticide solutions (0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 10 mg/mL) in 1% dimethyl sulfoxide (DMSO) solution in distilled water. The seedlings were treated in darkness at room temperature for two hours. Parallel negative (distilled water and 1.0% DMSO) and positive (0.029 mg/mL potassium dichromate) controls were utilized. After treatment, the seedling roots were rinsed in tap water, and cuts of about 2 cm were maintained in cold phosphate-buffered saline (PBS) at pH = 7.4 until the isolation of nuclei (≤ 15 min).
For the micronucleus assay, when the root tips of V. faba reached 3 cm in length, they were cut transversely with a scalpel to induce the sprouting of secondary roots. They were then placed in water for seven days until the secondary roots reached 2 to 3 cm in length and immersed in the corresponding concentrations of the five insecticides (0.005, 0.025, and 0.05 mg/mL) in 1.0% DMSO and 0.0015 mg/mL of potassium dichromate for the positive control, the negative control was immersed in distilled water. The treatment was applied for four hours, after which the seedlings remained in a recovery bath with aeration and constant temperature (20°C) for 24 hours, after which the meristems were fixed and stained. Later, they were observed under a light microscope by Carl Zeiss Mod. Axiostar Plus at 400X.
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Seed germination and treatment conditions were according to the protocol recommended by the International Programme of Chemical Safety (Kanaya et al. 1994).For both assays, the concentrations were determined through preliminary experiments to be sufficient to cause an effect but without inducing cell death, except for the highest concentration of methamidophos (10 mg/mL), which caused nuclei to be pulverized in the comet assay.
2.4. Comet assay
2.4.1. Isolation of nuclei from roots
The standard protocol was performed according to Gichner et al. (2000), with some modifications. Root sections of 2.5 cm were placed in a 60 mm glass Petri dish tilted on ice with 250 µL of cold PBS. Nuclei were mechanically isolated by cutting the roots transversely with a scalpel blade, under dim light at room temperature (± 20°C). In a 0.6 mL microtube with 50 µL of 1.0% low melting point agarose (LMPA) at 40°C, 50 µL of nuclei suspension was added, by gentle pipetting using a cut tip and mixed. The mixture (80 µL) was placed on a slide previously covered with 1.0% normal melting point agarose (NMPA). The slide was placed on a cold aluminum plate for 5 min, the coverslip was removed, and a final layer of 80 µL of 0.5% LMPA at 37°C was added and placed on a cold surface for 5 min.
2.4.2. Lisis
The coverslips were removed, and all slides were immersed in a box with a flat bottom containing cold (4ºC) lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, NaOH, 10% DMSO, and 1.0% Triton X-100 in deionized water at pH = 10) during at least one h.
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2.4.3. Unwinding and electrophoresisA
All slides were drained vertically; the lysis solution was removed from the back and placed in an electrophoresis chamber with the electrophoresis buffer (300 mM NaOH, 1.0 mM EDTA at pH ˃ 13) in a refrigerator at 4ºC in darkness. Nuclei were incubated for 18 min to allow DNA unwinding. Electrophoresis was performed at 0.74 V/cm (25 V, 300 mA) for 20 minutes. Subsequently, the slides were neutralized 3X with 0.4 M Tris buffer at pH 7.5, stained or fixed for 15 min in ethanol, and left to dry overnight after being stored in slide boxes until registration.2.4.4. Staining and comet scoring
Each slide was stained with 50 µL of ethidium bromide (20 µg/mL), placed in a wet camera between two paper towel layers soaked in filtered water, and protected from light to avoid evaporation and fluorescence decay. Fifty nuclei per slide and three slides by treatment were analyzed in an epifluorescence Axiostar Plus, Carl Zeiss microscope with an exciting 515–560 nm and a barrier filter of 590 nm. The computerized image analysis system Comet Assay IV (Perceptive Instruments) was used to measure the DNA damage through the tail length (µm), tail intensity (%), and tail moment, and the photographs were obtained with a Stingray monochrome camera Mod. F046B 1RF (Fig. 1).
All slides, including treatments and controls, were relabeled before microscopic analysis and scored without knowledge of the code to avoid bias.
2.5. Micronucleus assay
2.5.1. Fixation of meristems
After treatment, the roots were washed with running water and carefully dried. One cm of the lateral roots was cut with a scalpel, placed in a mixture of ethyl alcohol-acetic acid (3:1) in labeled glass jars, wrapped with aluminum foil to protect from light, and left in the freezer for 24 h.
2.5.2. Histological preparations
Root sections were rehydrated with fresh 70% ethanol for 15 min. They were hydrolyzed with 5 N HCl in a water bath at 28°C for 20 to 25 min, stirring continuously. HCl was removed, and the sections were washed three times with distilled water. Immediately, they were placed on an excavated slide, excess water was dried, and stained with Schiff’s reagent for 15 min. Finally, the meristems were transferred to a labeled flat slide, 45% acetic acid was added, and the tissue was crushed to form a monolayer (“squash”) using a cover slip for handling. Permanent slides were obtained using the dry ice technique and analyzed under a light microscope at 400X magnification.
2.5.3. Mitotic index (MI) determination and observation of micronucleus (MN)
The secondary root meristems of 5 individual seedlings exposed to insecticide treatments for four hours were analyzed. One thousand cells were quantified randomly for all treatment and control groups. The MI was calculated as a percentage of the ratio between dividing cells and the total number of cells scored, following the formula:
MI = number of cells in mitosis x 100
total number of cells
The MN media was expressed as the number of interphase cells with micronuclei per 1000 scored cells per slide, and five slides per treatment of three independent experiments.
2.6. Statistical analysis
At least two independent experiments were carried out. The comet assay results, including the parameters length, intensity and moment of the tail, were analyzed using the Anderson-Darling test. The data did not follow a normal distribution. As a result, the nonparametric Kruskal-Wallis test and Dunn’s post-hoc analysis were performed.
In the MN test, the results of at least three independent experiments were considered, and a one-way analysis of variance (ANOVA) was performed. Comparisons were estimated to be significant in cases where a significant F value was obtained with a p < 0.05. The Newman-Keuls multiple comparison test was performed to determine significant differences between the treated and negative control groups, ensuring a comprehensive analysis.
3. Results
The comet assay results are detailed in Figs. 2A, 2B, and 2C. The values of the negative and positive controls remained consistent with our previous experiments, indicating the reliability of our findings. A significant difference (*p < 0.05, **p < 0.01, and ***p < 0.001) in genotoxic effect was observed, with methamidophos showing the minor effect and oxidemethon methyl the highest, in the order of increasing genotoxicity: methamidophos < parathion methyl < phoxim < azinphos methyl < oxidemethon methyl. A dose concentration-response behavior was observed with all insecticides except methamidophos, confirming the genotoxic effect at all tested concentrations. Notably, the highest concentrations exceeded the genotoxicity of the positive control (0.029 mg/µL potassium dichromate).
Figure 2 depicts the genotoxicity induced in the root of V. faba by the organophosphate insecticides metamidophos, parathion methyl, phoxim, azinfos methyl, and oxydemeton, as evidenced by the three criteria used. Figure 2A shows the tail length induced by the different pesticides. The Fig. 2B refers to the porcentage tail intensity, which corresponds to the percentage of concentrated DNA in the tail of the comet. This graph effectively reflects the toxicity of the analyzed pesticides more clearly. This parameter, often overlooked in other publications, provides rapid and general information on genotoxicity, demonstrating the efficiency of our study. Finally, Fig. 2C shows the effect of insecticides through the tail moment. It integrates the intensity of the tail, with its length. In each case, an increase in genotoxicity is observed as the concentration of the insecticides increases, indicating a concentration-response relationship, except for the genotoxicity induced by ME. The same concentrations induced increasing damage in the following order: ME < PM < PH < AM < OM. The highest concentration tested was 10 mg/mL.
The parameters evaluated in the comet assay—tail length, tail intensity, and tail moment—were subjected to statistical analysis for each pesticide concentration tested. Initially, the Anderson-Darling test was applied to assess the normality of the data distribution. While some parameters followed a normal distribution, others did not; therefore, the nonparametric Kruskal-Wallis test was utilized, followed by Dunn’s post-hoc analysis to determine specific group differences. For tail length, statistically significant differences were observed in comparison to the negative control at all concentrations, except for 0.25 and 0.5 mg/mL for methamidophos and parathion methyl, respectively (Fig. 2A). In the case of tail intensity, significant differences were also found for most concentrations relative to the negative control. Exceptions were noted at 0.25 mg/mL for methamidophos, and at 0.25 and 0.5 mg/mL for parathion methyl and phoxim (Fig. 2B). Analysis of tail moment revealed significant differences at all tested concentrations when compared to the control group, except at the lowest concentration (0.25 mg/mL) of parathion methyl and phoxim (Fig. 2C). Overall, a concentration-dependent increase in genotoxic damage was observed across all pesticides, except for methamidophos, for which a consistent trend was not evident.
Table 1 shows the results from the mitotic index and micronucleus tests. The root meristems exposed to treatment with five insecticides showed a significant reduction in cell division, even at the lowest concentration. The treatment with the highest concentration showed the lowest mitotic index value, indicating a reduction in cell division due to the insecticide treatments. The results were significant across all the tested concentrations of the five organophosphate insecticides (p < 0.05) compared to the negative control (Figs. 3A and 3B).
Experimental groups | aMitotic index (%) | ± S.E. |
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Negative controls average (Distilled water) (cDMSO) | 12.3 ± 0.54 | 2.00 ± 0.38* |
Positive control dK2 Cr2 O7 | 5.56 ± 1.82* | 13.25 ± 0.95* |
Treatments (mg/mL) | ||
Metamidophos 0.005 0.025 0.050 | 9.90 ± 1.77 N.S. 9.50 ± 1.06 N.S. 8.10 ± 1.02 N.S. | 1.33 ± 0.70 N.S. 2.30 ± 0.62 N.S. 3.03 ± 1.89 N.S. |
Parathion methyl 0.005 0.025 0.050 | 6.80 ± 1.69* 5.90 ± 1.61* 4.90 ± 0.43 * | 5.75 ± 1.11* 7.25 ± 1.31* 9.00 ± 1.97* |
Phoxim 0.005 0.025 0.050 | 6.02 ± 1.24* 4.11 ± 1.05* 4.90 ± 0.78* | 6.25 ± 1.11* 7.75 ± 1.31* 9.25 ± 1.04* |
Azinphos methyl 0.005 0.025 0.050 | 8.40 ± 1.15* 6.20 ± 0.94* 5.90 ± 0.67* | 4.00 ± 0.91* 7.25 ± 1.31* 9.75 ± 1.65* |
Oxidemethon methyl 0.005 0.025 0.050 | 5.50 ± 0.98* 5.00 ± 0.81* 4.70 ± 0.62* | 9.50 ± 1.55* 11.75 ± 1.31* 14.50 ± 1.75* |
| a Mitotic index was analyzed in 3000 cells from each of three independent experiments | ||
| b Mean of micronuclei obtained in 3000 cells from each of three independent experiments | ||
| c1% dimethyl sulfoxide | ||
| d 0.0015 mg/mL potassium dichromate | ||
| *Significant differences between the control groups and each treated group were obtained by ANOVA and therefore, the Newman-Keuls multiple comparison test was applied, p< 0.05. | ||
| N.S. = No significant differences | ||
All treatments with organophosphate insecticides induced MN with different frequencies (Table 1, Figs. 3C and 3D).
Discussion
Pesticides emerged to enhance agricultural production and meet the increasing demands of a growing global population. Approximately one-third of agricultural production relies on the application of pesticides; without pesticides, the loss in producing fruits, vegetables, and cereals would be 78%, 54%, and 32%, respectively (Tudi et al. 2021). Over the last decade, the world population has increased by another billion, and given current growth rates, it is expected to reach 9.9 to 10 billion by 2050, demanding more substantial food production. Therefore, the application of agrochemicals continues to rise; consequently, pesticide constituents released into the environment pose potential hazards to both humans and other non-targeted organisms, as well as to ecosystems. These agrochemicals cause environmental pollution by contaminating rivers, groundwater, and irrigation water for agricultural purposes, posing a potential risk to all living organisms and ecosystems (Kaur et al. 2024).
Our ongoing commitment to assessing the genetic effects of pesticides has been a comprehensive effort over many years, given the lack of effective regulation of their use. We have conducted numerous studies on occupationally exposed workers, as well as in vivo and in vitro research, which has identified various damages in several biological test models. Our specific focus has been on organophosphorus insecticides, which have a long history of causing chromosomal aberrations in plant and cultured animal cells (Gómez-Arroyo and Villalobos-Pietrini 1995; Cortés-Eslava et al. 2018; Siddiqui and Alrumman 2022; Hernández-Toledano et al. 2023).
The systematic study of genotoxicity induced by contaminants and other stressors is favored by establishing more reliable and robust biomarkers. Plant bioassays are a relevant and integral part of the test battery to detect environmental genotoxic pollutants.
The root of Vicia faba has proven to be an ideal model for examining the risks of environmental agents. Several researchers have reported the efficacy of the V. faba assay in assessing the risk of organic and inorganic contaminants (Gómez-Arroyo and Villalobos-Pietrini 1995; Kanaya et al. 1994; Cotelle et al. 2015; Barbafieri and Giorgetti 2016; Arya et al. 2017; Cortés-Eslava et al. 2018; Jiang et al. 2019). This assay is particularly significant because it enables the observation of genotoxic effects on the root tips of V. faba cells, which may also, pose severe risks to animal and human health with high probability. The V. faba assay wth its sensivity, reproducibility, and cost-effectiveness is a powerful tool in environmental genotoxicity studies due to its sensitivity, reproducibility, and cost-effectiveness. In addition, using plant tissue to study the induction of chromosomal aberrations is one of the oldest, simplest, most reliable, and inexpensive methods available. The use of V. faba as a genetic model in our study not only enhances the credibility of our findings but also provides a practical and accessible approach for future research in this field, instilling confidence in the reliability of our methodology. Our study’s findings contribute to the current understanding of genotoxicity and provide a solid foundation for future research in this critical area. They inspire further investigations and lead to the development of effective regulatory measures.
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As far as we know, studies on the effect of organophosphorus insecticides (OPIs) on DNA, particularly in plants, are scarce (Arya et al. 2017; Srivastava and Singh 2020; Ghisi et al. 2021; Shahid et al. 2023; Siddiqui and Alrumman 2022). The scarcity of research in this crucial area underscores the novelty and significance of the present study. Cortés-Eslava et al. (2018) reported the expression of markers of apoptotic-like programmed cell death (AL-PCD), including chromatin compaction, cytoplasmic vacuolization, nuclear shrinkage, protoplast condensation, and nuclear disintegration, resulting in the generation of apoptotic-like bodies. It also caused other features associated with PCD, such as increased active caspase-3-like protein, the release of cytochrome C (Cyt C) into the cytoplasm, and a decrease in extracellular signal-regulated protein kinase (ERK) expression. Experimental evidence suggests that OPIs induce oxidative stress, including the production of reactive oxygen species (ROS), decrease the antioxidant capacity of enzymes, and damage mitochondria (Sharma et al. 2024; Silva et al. 2021; Sule et al. 2022). This evidence is consistent with various studies showing the reversal of genotoxicity induced by different OPI via oxidant stress using different natural antioxidants in diverse experimental models: cell cultures (Karami-Mohajeri et al. 2021), germinal cells (Bakir et al. 2020), Drosophila melanogaster (Gomes et al. 2020), catfish (Mansour et al. 2022), Wistar rats (Seth et al. 2021; Hassan et al. 2022). Common phenomena seen in cells exposed to OPI include the formation of DNA adducts, single and double-stranded DNA breaks, and inter- and intra-crossovers of DNA proteins (Prathiksha et al. 2023). Genetic damage refers to any alteration in the DNA sequence that can lead to mutations, cell death, or the development of cancer. It is known that OPIs are organic esters of phosphoric acid, in which the hydroxyl groups have been replaced by alkoxide groups in their molecule. They contain pentavalent phosphorus attached to a double bond with sulfur or oxygen, two R groups, and a ‘leaving group.’ Likewise, phosphate esters are found in biological molecules such as DNA or ATP, which are responsible for providing energy to cells through various enzyme-mediated hydrolysis processes (Corbridge 2013). Therefore, it is unsurprising that living cells can be compatible with and process OPI. Their efficacy, toxicity, and danger are attributed to their biocompatibility; that is, cells have mechanisms to absorb and metabolize natural phosphate esters, which can alter their chemical structure and lead to cell malfunction (Jiang et al. 2019; Valdez-Salas et al. 2000). All these interactions can lead to genetic damage, as observed in the present study. The genetic damage induced by OPI poses a significant risk to human and animal health, as these compounds can disrupt normal cellular processes and lead to various diseases (Kaur et al. 2024; Zhu et al. 2024), highlighting the severe situation.Currently, there are few assays available to detect pesticide genotoxicity in plant species (Kontek 2007; Cortés-Eslava et al. 2018; de Morais et al. 2019; Shahid et al. 2023). The comet assay, also known as single cell gel electrophoresis, was adapted for use with plant cells to detect early induced DNA damage. This technique, initially applied to animal cells, was later incorporated into plant tissues by Koppen and Vershaeve (1996) and Gichner and Plewa (1998), significantly enhancing the utility of plants in both basic and applied studies in environmental mutagenesis. We have verified the versatility of the comet assay, which can be used with all plant species (both wild and cultivated) and in various tissues, such as roots and leaves (Gómez-Arroyo et al. 2018; Cortés-Eslava et al. 2018; 2023). The use of the comet assay has increased significantlly, Møller (2018) published a retrospective study and concluded, “The trial is ready for the next 30 years”. A critical challenge for methodology has been reaching reliable negative control standards. For years, we have conducted numerous tests with V. faba, adjusting the temperature to low levels (4°C), maintaining dark conditions, and providing rigorous care to ensure pH values, thereby ensuring the reliability of the data and the robustness of the test. The comet assay has proven to be a sensitive system for studying environmental genotoxicity and can be applied to diverse species, tissues, and cells. Unfortunately, researchers use a wide variety of parameters to present results. With the emergence of image-analyzing systems to evaluate the magnitude of DNA damage, the most used parameters have been length, intensity, and moment of the tail. These results are expressed in different units that cannot be easily compared with another. However, they can express the general damage trend induced by the chemical or physical agents under study. In the present research, these were selected, and relevant information was provided. On the one hand, length (proportional to the extent of DNA damage, measured in micrometers) denotes an increasing effect with concentration, reaching values close to 200 micrometers with 8 µg/mL Phoxim; however. It decreased by 10 µg/mL to less than 150 micrometers. When an image analyzer is unavailable, this tool provides limited information, particularly regarding the assay’s dynamic range. It has been reported that tail length increases only at the lowest levels of damage and soon reaches its maximum (Walsh and Kato 2023), as also observed in this study, particularly with oxidemethon methyl.
The tail intensity, which refers to the total fluorescence of damaged DNA migrating toward the anode and forming a tail, and is specified as a percentage, was found to have a linear relationship with insecticide concentration. This relationship was consistent except for oxidemethon methyl, where the highest concentration led to severe damage, pulverizing the nuclei and preventing their migration toward the anode, forming “clouds” that were impossible to record with the comet Assay IV program. Currently, the Organization for Economic Cooperation and Developmet (OECD) recommends tail intensity as the best descriptor of DNA break frequency, as it utilizes quantitative damage measures (ranging from 0 to 100%). However, several researchers still use the tail moment, which relates the tail length multiplied by the tail fluorescence intensity divided by 100, as it considers both the length and DNA content of the comet tail. The TM has the disadvantage of not having standard units, and given a particular TM, it is challenging to visualize the level of damage being described (Collins et al. 2023). In the present study, this value followed the same trend as the previous ones, showing lower records than intensity. However, it also increased with the concentration of the insecticide, except in the case of methamidophos, like the other parameters. Of the three parameters evaluated in the comet assay during the present investigation, we recommend the tail intensity and the tail moment based on our findings.
Our results at the cytological level showed a decrease in the mitotic index value of the root meristematic cells of V. faba parallel to the increase in concentrations, which, according to Ghisi et al. (2021) and Camilo-Cotrim et al. (2022), modifications in the mitotic index, whether an increase or decrease, may indicate cytotoxicity. An increase in the mitotic index indicates increased cell division, damaged cells, and disordered cell proliferation. Additionally, the reduced mitotic index can indicate an inhibition/delay in cell division, which may damage normal plants (Camilo-Cotrim et al. 2022).
On the other hand, the induction of micronuclei increased with the concentration. These cytogenetic studies can be crucial in rapidly assessing the genetic effects following insecticide treatment. Given their ease and short execution time, they are essential for cytogenetic analysis as part of routine laboratory tests on plant genotoxicity (Kwasniewska and Bara 2022).
The present study used the MN assay to discern the final product of DNA damage. Chromosome breakage and disturbance of the chromosome segregation machinery are two main mechanisms for MN formation, and their expression requires cells in division (Fan and Li 2024).
Previous studies have linked the production of reactive oxygen species (ROS) to the mechanism of action of pesticides. Hernández-Toledano et al. (2023) show the role of organophosphate metabolites, specifically dialkyl phosphates, mentioning them as the main biomarkers of exposure to OPIs and whose genotoxicity has been demonstrated both in vitro and in vivo; in their analysis, they conclude that the metabolites are more genotoxic than the original organophosphates, suggesting their contribution to DNA damage associated with exposure to OPIs. Lihui et al. (2022) achieved the modulation of oxidant stress induced by the organophosphorus insecticide profenofos through the application of albicanol, a powerful antioxidant against heavy metals, which notably decreased the effects that causes oxidative stress in carp hepatocytes, such as arrest in G1-S phase, increased level of malondialdehyde, and decreased levels of superoxide dismutase, catalase, and total antioxidant capacity.
Salazar-Mercado and Correa (2024) examine the interaction between pesticides and bioindicator plants, including V. faba, confirming their effectiveness for detecting and biomonitoring agrochemicals. Given their indiscriminate application, exposure to these organophosphate pesticides can occur in both occupationally exposed individuals and the general population. Therefore, additional studies are necessary to clarify the genotoxicity of these compounds.
Conclusion
V. faba, once again, has proven to be an excellent model for genetic toxicology studies, capable of detecting DNA fragmentation and chromosomal aberrations, as evidenced by the induction of micronuclei by five organophosphorus insecticides evaluated in this work. The present approach is of great value; here, we clearly show both the early effect on DNA through the comet assay and micronucleus test as evidence of the unrepaired damage induced by the agrochemicals tested. The use of a sensitive model such as V. faba, whose advantages reside in its availability throughout the year, in addition to being a fast, easy to grow, handle, and low-cost test system as compared to other short-term tests, as well as its fast rate of cell division and large chromosomes easy to score, and it is sensitive, simple and straightforward, allowing robust results with a small number of cells. Therefore, the usefulness of higher plants in environmental genotoxicology was demonstrated.
Acknowledgments
The authors thank Omar Guadalupe Carballo Gómez for collaboration in the experimental stag and Hugo Ahuatctzi Cortés for for their technical assistance in facilitating image capture in the light microscope.
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Author contribution
Sandra Gómez-Arroyo led the project that funded the materials, reagents, and equipment, and reviewed and edited the original manuscript. Josefina Cortés-Eslava designed and performed the project’s experimental part and redacted the original manuscript. Ana Rosa Flores-Márquez developed part of the experiments and reviewed the manuscript. Ma. Elena Calderón-Segura reviewed the manuscript, César Guerrero Guerra facilitated obtaining the photographs with the light microscope and performed the statistical analysis, and Rafael Valencia-Quintana reviewed and corrected the manuscript.
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Funding
This work was supported by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-DGAPA) of the Universidad Nacional Autónoma de México through the project IN225608-2.
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Data availability statement
Not applicable
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DeclarationsEthics approval and consent to participate
Not applicable
Consent for publication
We confirm that all authors have read the manuscript and agree to its submission to Environmental Pollution.
Conflict of interest
The authors declare that they have no competing interests.
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Fig. 1
Images of Vicia faba root cell nuclei with the comet methodology captured using a fluorescence microscope (200X magnification) were obtained with a Stingray monochrome camera. Figure 1A presents control nuclei, Fig. 1B shows DNA damage induced by the insecticides, and Fig. 1C reveals pulverized nuclei (“cloud”) induced by oxidemethon methyl at 10 mg/mL.
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Fig. 2A
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Fig. 2B
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Fig. 2C
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Fig. 2
Our comprehensive study on genotoxicity induced in the root of V. faba by the organophosphate insecticides metamidophos, parathion methyl, phoxim, azinfos methyl, and oxydemeton methyl is presented. The tail length is observed in Fig. 2A, the tail intensity is evidenced in Fig. 2B and tail moment results are shown in Fig. 2C.
*The significance of the results is denoted by the p-values: *Indicates p < 0.05, **p < 0.01, and *** p < 0.001 significant difference compared with the control groups. These values were determined by the non-parametric Kruskal-Wallis test followed by Dunn’s post-hoc analysis providing a strong statistical basis for the conclusions drawn.
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Fig. 3
Detailed photomicrographs of Vicia faba-root tips were obtained by Schiff’s reagent staining and visualized under a light microscope at 400x magnification. In Fig. 3A, a control image with different stages of mitosis is observed: Interphase (I), Prophase (P), Metaphase (M), Anaphase (A), and Telophase (T). No mitotic figures are shown after insecticide treatment in Fig. 3B. The micronuclei induction (Black arrows) by insecticide treatment is shown in Figs. 3C and 3D.
