Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(3): 91-99, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1895 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Sazada Siddiqui, Sulaiman A. Alrumman (2022). Methomyl has clas- togenic and aneugenic effects and alters the mitotic kinetics in Pisum sativum L. Caryologia 75(3): 91-99. doi: 10.36253/caryologia-1895 Received: October 12, 2022 Accepted: December 02, 2022 Published: April 5, 2023 Copyright: © 2022 Sazada Siddiqui, Sulaiman A. Alrumman. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID SS: 0000-0001-5448-7617 Methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in Pisum sativum L. Sazada Siddiqui*, Sulaiman A. Alrumman Department of Biology, College of Science, King Khalid University, Abha 61413, Saudi Arabia *Corresponding author. E-mail: sasdeky@kku.edu.sa; kalasaz@yahoo.co.in Abstract. Methomyl is a carbamate pesticide that is frequently applied to crops all over the world. This research aims to evaluate the Pisum sativum L mitotic process and potential genotoxicity. The Cell Proliferation Kinetics (CPK) frequencies demonstrated changes in kinetics of mitotic process, and study of Mitotic Index (MI) demonstrated that methomyl had cytotoxic properties. In fact, the telophases ratio dropped at 0.1% to 0.5% methomyl treatment, while there was an increase in prophases, metaphases, and anaphases from 0.1% to 0.5% in a dose dependent manner. In terms of genotoxic- ity, methomyl cause an increase in the frequency of clastogenic and aneugenic chromo- somal abnormalities at metaphase-anaphase at 0.1% to 0.5%. The effects on the mitotic spindle were further confirmed by an increase in the frequencies of c-mitosis from 0.1 to 0.5% methomyl treatment. The outcome of the current analysis indicates that regu- larly used insecticides methomyl has a considerable cytotoxic effect on mitotic cells of Pisum sativum L. Keywords: methomyl, mitotic index, clastogenic, aneugenic, C- mitosis Pisum sati- vum L. INTRODUCTION In public areas, agricultural lands and gardens, pesticides are extensively used to eradicate weeds, undesirable pests, and diseases transmitted by vec- tors. Nevertheless, the prolonged usage of pesticides may leave behind toxic remains that, through surface drains, spray drift, runoff, spray leftovers, and leaching may pollute nearby surface water and ground natural water bodies (Mojiri et al. 2020; Chandra et al. 2021). The accumulation of residual pesti- cides in aquatic and marine organisms food chains can pose a risk to human health and have a detrimental effect on ecological systems (Lukaszewicz et al. 2019; Jing et al. 2022a; Abdel-Wahab et al. 2021). From many decades, pesticides have been a crucial component in reduc- ing crop loss and increasing output. Due to these advantages, farmers are spraying pesticides on crops more frequently and using modern techniques like drones (Nie et al. 2020). However, the propensity of pesticides to bio accumulate in edible goods may have an undesirable impact on human 92 Sazada Siddiqui, Sulaiman A. Alrumman health (Yu et al. 2016; Ramadan et al. 2020). Beyond their maximum residue limits (MRLs), pesticides in water and agricultural products have the potential to cause both acute and chronic illnesses in people (Li and Jennings 2018; Amaç and Liman 2021). Carbamates are a diverse group of chemicals that are used as insecticides. The acetylcholinesterase (AChE) enzyme is selectively affected by carbamates, which results in a buildup of acetylcholine and overstimulation of the nervous system in both target and non-target spe- cies, including human beings (Eddleston et al. 2004). In the areas where onions, cucumbers, cabbage, and chili peppers are grown, methomyl, a carbamate insecticide, is frequently used (Ramadan et al. 2020). Acute poison- ing may result from methomyl consumption by using contaminated agrifoods and water via occupational or non-occupational ways (Jing et al. 2022 b). Due to its highly effective biological action in con- trolling pests and safeguarding the crops, methomyl (C5H10N2O2S), S-methyl-1-N- [(methyl carbamoyl)- oxy]-thioacetimidate, belongs to carbamate pesticide group that is commonly applied in various countries (Laicher et al. 2022; Pietrini et al. 2022). Several pesti- cides are designed to strike a particular group of targets, although their noxious constituents will affect the whole organism, both target and non-target (Castellanos et al. 2022). According to a study, methomyl causes genotoxic effects in fish (Afaf et al. 2022). Fish and aquatic crea- tures including Danio rerio, coastal aquatic system and water spinach have also shown toxicity to methomyl (Jablonski et al. 2022; Camilo-Cotrim et al. 2022). DNA damage is a preliminary biotic phenomenon which could disrupt biological developments and struc- tures and produce genotoxic disorders associated with carcinogenic complications (Acar et al. 2022; Siddiqui and Sulaiman 2022 a and b; El-Houseiny et al. 2022). As per a recent report, numerous species undergo carcino- genic progressions due to various causes, such as DNA damage instigated by chemical contaminants (Pesaven- to et al. 2018; Velázquez et al. 2022; Liman et al. 2022). This study aims to analyze the potential adverse effects of methomyl on mitotic processes and DNA integrity in the terrestrial plant Pisum sativum L. MATERIAL AND METHODS Purchasing of chemicals and seeds Methomyl insecticide were bought from Sigma Chemicals Ltd., United States (CAS No. 16752-77-5). Pisum sativum L (Pea) seeds were procured from a licensed trader at a community market in Abha, Saudi Arabia. Exposure conditions Even sized P. sativum L seeds were chosen, pre- soaked for 12 hours in distilled water and then divided into various groups of 30 seeds each. After that, the seeds were exposed to various methomyl concentrations (0.1, 0.2, 0.3, 0.4, and 0.5%) for 1 h by soaking in 250 mL solutions of methomyl. Double-distilled water was used to soak the seeds in the control group. Throughout the treatment time, the containers were shaken repeatedly to make available ample aeration to the seeds. Following treatment, seeds were extensively rinsed with double dis- tilled water (DDW) to eliminate any remaining traces of adhering methomyl and were placed in Petri dishes on moisturized Whatman Filter Paper. For the following 72 hours, the Petri dishes were kept in dark in a plant growth cabinet at 25±2°C. The experiment was conduct- ed on newly emerging roots that were 1-2 cm long. The complete experiment was conducted thrice in identical conditions. Evaluation of mitotic kinetics and genotoxicity One to two cm long roots were collected between 8 to 10 am, soaked for 24 h in a fixation solution (ethanol: glacial acetic acid, 3:1), then transferred to 70% ethanol, maintained at 5°C till microscopic examination. For each sample, 10 roots were hydrolyzed in 1N HCl for 10 minutes, and with 2% acetocarmine solution, root tips were dyed for 10 minutes for preparing each slide. Chro- mosome preparation was done from root tips as stated by Qian et al. 1998 with minor modifications (Siddiqui and Suleiman 2022b). To calculate the MI, which is a proportion of dividing cells, 1000 cells from each sample were evaluated. The no. of cells in each division phase to all mitotic cells was used to compute CPK frequen- cies. All mitotic cells were studied in a light microscope under oil immersion (100 x). All slides were examined blind and coded. Ratio of aberrant cells over 500 metaphase/anaphase cells per root tips were used to calculate the frequency of chromosomal aberrations. Chromosomal aberra- tions were categorized as per their origin in clastogenic (resulting in chromosomal breakage) or aneugenic (dis- rupting spindle function and leading to asynchronic chromosomal migration). Laggards and vagrants chro- mosomes have been scored with regards to aneugenic abnormalities. Single bridges, fragments, double bridges, and sticky chromosomes were taken into considera- 93Methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in Pisum sativum L. tion in clastogenic aberrations. C-mitosis as defined by Grant (1978) is an inactivation of spindle ensued by a haphazard scattering of chromosomes over the cell and is quantified and scored by assessing the frequency over 100 metaphases per root tips. All the aberrant and c-mitosis cells were studied in a light microscope under oil immersion (100x). All slides were examined blind and coded. Statistical analysis A one-way ANOVA test using GPIS 1.13 software (GRAPHPAD, California, USA) was applied to find sig- nificance of differences in variables. All results were articulated as mean ± standard error. RESULTS It is clear from the results that methomyl is toxic to MI, CPK, c- mitosis, aneugenic and clastogeneic aberra- tions. The observed MI, CPK, c-mitosis, aneugenic and clastogeneic aberrations are well represented in (Fig. 1, Table 1, Fig. 2, Fig. 3 and Fig. 4). The clastogenic abnor- malities observed were single bridges, fragments, double bridges, sticky chromosome and aneugenic abnormali- ties were laggards and vagrants. Effect of methomyl treatment on mitotic index of P. sati- vum L. Fig. 1 shows how methomyl affected the MI of root tip cells in P. sativum. In control group, seeds treated with DDW for 1 hour had a MI of 9.3%. From 0.1 to 0.2% methomyl treated seeds, a non-significant decline (p>0.05) in MI was observed and at 0.3% concentration, there was a significant decrease (p< 0.05) in MI and at 0.4 to 0.5%, a very significant decrease (p< 0.01) in MI was reported in comparison to control for 1 hour. Over- all, MI decreases dose dependently in all concentrations from 0.1 to 0.5%. Effect of methomyl in Cell Proliferation Kinetics (CPK) of P. sativum L Cell proliferation kinetics (CPK), assessed as the ratio of prophases, metaphases, anaphases and telo- phases revealed a rise in prophase, metaphase and ana- phase from (0.1 to 0.5%) and a decrease in telophase at 0.1 to 0.5% of methomyl treated root tips in comparison to control (Table 1). A significant increase (p<0.05) was reported in prophase at 0.4 % (58.34±1.2); metaphase at 0.2% (27.3±3.8), and anaphase at 0.2% (21.3±1.9) but a sig- nificant decrease (p<0.05) was observed in telophase at 0.3% (19.12 ±3.6) in comparison to control. Prophase at 0.5% (60.12±2.6); metaphase from 0.3 to 0.5% (27.4±3.5; 28.45±3.6; 29.40±1.2 respectively) and anaphase from 0.3 to 0.5% (23.1±1.7; 24.5±1.9; 25.5±1.6 respectively) result- ed in a very significant increase (p<0.01) and telophase from 0.4 to 0.5% (17.6±2.5; 16.80±3.6) showed a very sig- nificant decrease (p<0.01) in comparison to control. Effect of methomyl treatment on C-mitosis of P. sativum L Fig. 2 demonstrates how methomyl affects c-mitosis in P. sativum root tips cells. Seedlings treated for 1h with Figure 1. Effect of methomyl on mitotic index of P. sativum for 1 h. *p<0.05; compared to control group. Data are mean of three repli- cates ±SE, 0.0 = Control group. Table 1. Effect of methomyl on cell proliferation kinetics in P. sati- vum L. Concentration (%) Prophases Metaphases Anaphases Telophases 0.0 52.5±4.8 21.7± 2.7 18.5±3.4 23.12.8±2.3 0.1 50.7±4.6 23.5±1.7 20.4±2.4 21.5.6±3.0 0.2 50.4±2.4 27.4±3.5* 21.3±1.9* 20.9±1.30 0.3 55.7± 2.2 27.3.3±3.8** 23.1±1.7 ** 19.12±3.6* 0.4 58.34±1.2* 28.45±3.6** 24.5±1.9 ** 17.6±2.5** 0.5 60.12±2.6 ** 29.40±1.2** 25.5± 1.6 ** 16.80±3.6** *p<0.05; **p<0.01 compared to control group. Data are mean of three replicates ±SE, 0.0 = Control group. 94 Sazada Siddiqui, Sulaiman A. Alrumman DDW in the control group exhibited 0% c-mitosis. A significant increase (p<0.05) in the number of c-mitosis cells were seen in seeds treated with 0.1% methomyl for 1 hour and from 0.2 to 0.5%, there was a very significant increase (p<0.01) in c-mitosis cells in comparison to con- trol for 1 hour. Overall, c-mitosis increases dose depend- ently in all concentrations ranging from 0.1 to 0.5%. Effect of methomyl on aneugenic and clastogeneic aberra- tion cells in P. sativum L The incidence of aneugenic aberrations (laggards and vagrants) in metaphase-anaphase plates in the control group was zero. Percentage of aneugenic aberrations (lag- gards and vagrants) in the metaphase-anaphase plate dose dependently increased with methomyl treatment (Fig. 3 and Fig. 5). Seeds treatment with 0.1% methomyl resulted in a 1-fold increase and 0.2% treatment resulted in a 1.37- fold increase which was not significant and 0.3% metho- myl treated seeds resulted in a 2.4-fold increase which was significant (p<0.05) in comparison to control. Fur- ther increase in concentration from 0.4 to 0.5% methomyl treated seeds resulted in a rise in incidence of aneugenic aberrations, 5.9-fold, and 9.56-fold respectively, which was very significant (p<0.01) in comparison to control. The incidence of clastogeneic aberrations (single bridges, fragments, double bridges and sticky chromo- some) at metaphase-anaphase plates in control group was zero (Fig. 4, and Fig. 5). Percentage of root tip cells with clastogeneic aberrations (single bridges, fragments, double bridges and sticky chromosomes) at metaphase- anaphase plate increased dose dependently with metho- myl treatment (Fig. 4). Treatment of seeds with 0.1% methomyl resulted in 1.81-fold increase which was non- significant as compared to control. However, from 0.2 to 0.5 % methomyl treated seeds resulted in 6.25-fold, 12.19-fold, 18.92-fold and 21.28-fold increase in clastoge- neic aberrations respectively which was very significant (p<0.01) in comparison to control. 0.0 0.1 0.2 0.3 0.4 0.5 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 ** ** **C -m it o si s (% ) Methomyl concentration (%) * ** Figure 2. Effect of methomyl on c-mitosis in P. sativum for 1 h. *p<0.05; **p<0.01 compared to control group. Data are mean of three replicates ±SE, 0.0 = Control group. 0.0 0.1 0.2 0.3 0.4 0.5 0 2 4 6 8 10 12 ** T o ta l a n eu g en ic a b er ra ti o n s (% ) Methomyl concentration (%) * ** Figure 3. Effect of methomyl on total aneugenic aberrations in P. sativum for 1 h. *p<0.05; **p<0.01 compared to control group. Data are mean of three replicates ±SE, 0.0 = Control group. 0.0 0.1 0.2 0.3 0.4 0.5 0 5 10 15 20 25 30 ** ** * * T o ta l c la st o g en ic a b er ra ti o n s (% ) Methomyl concentration (%) * ** Figure 4. Effect of methomyl on total clastogenic aberrations in P. sativum for 1 h. *p<0.05; **p<0.01 compared to control group. Data are mean of three replicates ±SE, 0.0 = Control group. 95Methomyl has clastogenic and aneugenic effects and alters the mitotic kinetics in Pisum sativum L. DISCUSSION This study shows that reduction in cell division indi- cates that tested methomyl have a mitodepressive poten- tial. When mitotic activity is reduced, the amount of DNA also declines. This could be due to the blockage of cell cycle in the G2 phase or DNA synthesis inhibition or stopping the cell from starting mitosis (Siddiqui et al. 2007; Siddiqui et al. 2012; Siddiqui and Alrumman 2020 a and b). The significant decrease in mitotic index observed in this study might be the effect of methomyl interfering with the cell cycle by blocking G2 phase of cell cycle or DNA synthesis inhibition, or it could be the outcome of a rise in the frequency of chromosomal anomalies with analogous increase in methomyl concentration. These findings are also consistent with the outcomes of several research teams which have stated the cytotoxic effects of ethephon (Ayşe and Kılıç 2017; Bonciu et al. 2022), vari- ous synthetic plant growth regulators (Singh et al. 2022; Asif et al. 2022), and various pesticides (Lukaszewicz et al. 2019; Siddiqui and Alrumman 2022 a and b; Omeiri et al. 2022; Hafez et al. 2022; Bandopadhyay et al. 2022). In this study, Methomyl raised the percentage of metaphase, prophase and anaphase and reduced the percentage of telophase in all concentrations in a dose dependent manner, as per the outcomes of proportions of distribution of specific mitotic stages. There is an increase at all concentrations of metaphase, prophase and anaphase phases. The outcomes are consistent with the findings of Liman et al. (2010), Priya et al. (2014), and Ozkul et al. 2016). Furthermore, the percentage of telophase stage decreased in comparison to control. These findings suggest that decline in telophase stag- es and henceforth MI might be due to arrest of one or more mitotic stages or due to a slowdown in the rate of cell development during mitosis (Ping et al. 2012). C-mitosis was found in the present study. C-mito- sis was created by unstable microtubules (Odeigah et al. 1997) or disruptions in the development of spindle fib- ers (Shimoi et al. 2019; Haliem 1990). The incidence of c-mitosis in root tip cells of Pisum sativum shows that spindle formation was harmfully affected (El-Ghamery et al. 2000). Considerable numbers of c-mitosis detected in this study implies that methomyl is a strong spindle Figure 5. Clastogenic and aneugenic aberrations in methomyl treated P. sativum L root tip cells. Clastogenic aberrations A to F: A) Bridge in anaphase; B) Single bridge in telophase; C-D) Chromosome fragment in metaphase; E) Double bridge at anaphase; F) Sticky chromo- some at metaphase. Aneugenic aberrations G to J: G-H-I) Chromosome vagrant at metaphase; J) Vagrant chromosome at anaphase; K –L) C-mitosis in metaphase; Bar - 10 μm. 96 Sazada Siddiqui, Sulaiman A. Alrumman inhibitor. C-mitosis is also an indication of spindle poi- soning, as per Rank (2003). The cause of the generation of c-mitosis might be due to disruptions in spindle for- mation, affected by methomyl. In relation to genotoxicity, methomyl enhanced the incidence of clastogenic as well as aneugenic anomalies at the metaphase-anaphase plate. Single bridges, frag- ments, double bridges and stickiness, were the clasto- genic anomalies whereas vagrants and laggards were the aneugenic anomalies observed in the present study. In treated seeds, a number of bridges were created in ana- phases I and II plate. Bridges were most likely formed by breakage and combining of chromosomal bridges, which got enhanced with methomyl treatment. Chromosome stickiness and subsequent failure of free anaphase divi- sion or irregular translocation or inversion of chromo- somal fragments can all lead to the creation of chromo- somal bridges (Jing et al. 2022a; Honles et al. 2022). The fusion of broken chromosomes was the primary cause of the formation of bridges as per Rosculet et al (2019; Honles et al. 2022). Increases in methomyl concentrations were associ- ated with stickiness. Stickiness may result from partial detachment of nuclear proteins and alterations in their association design or from partial detachment of nucle- oproteins and alterations in their association design or due to nucleic acid depolymerization activated by methomyl treatment. Disruptions in cytochemical bal- ance reaction may lead to stickness (Dewitte et al. 2010; Rosculet et al. 2019). Nucleic acid depolymerization because of herbicidal treatment or by partial detachment of nucleoproteins (Kaufman et al. 1955) or by incomplete separation of nucleoprotein variation in their organiza- tion design (Evans 1962) might cause stickness. The fragments formed from chromatid and chromo- somal break imply its mutagenic events within the cell. In a previous study, Siddiqui et al. (2020 a,b) had report- ed that pesticides cause various chromosomal anomalies. Generation of giant cells having diverse chromosomal anomalies had been reported in a previous study by food colorants (Prajitha and Thoppil 2016). The laggards observed during the current study may result from failure of chromosome movement or from deferred ending of stickiness of ends of chromosomes. At metaphase I, chromosome lagging could result from disturbances in bivalents motion to equatorial plate. Single univalent lagging was the most common inci- dence (Zeyad et al. 2019). Laggards and bridges could be created due to deferred ending of stickiness of ends of chromosomes (Kaur and Grover 1985). Laggards are responsible for the formation of micronuclei at telophase I. Acentric fragments or laggards are liable for micro- nuclei generation at telophase II and hence it leads to the changes in size and number of pollen grains arising from mother cells. The other frequent aneugenic form of anomaly observed in dividing cells was vagrant chromosomes. As per Rank (2003), vagrant chromosomes are pointers of spindle poisoning. These aberrations might have devel- oped as a result of the disruption in spindle formation, which was affected by methomyl treatment. Genotoxic stress or genomic instability caused by DNA damage may result in illnesses, senescence, altera- tions in gene expression or cellular aging (Bonciu et al. 2018; Iturburu et al. 2018; Shabbir et al. 2021; Omeiri et al. 2022; Castellanos et al. 2022). In both plants and ani- mals, a rise in genomic instability has been advocated as a basis for the decline in population fitness. Genotoxic- ity biomarkers must be taken into consideration when assessing potential noxious effects in aquatic organisms since genotoxic substances have the potential to cause damage that extends beyond the individual and can be seen over several generations (Frenzilli et al. 2009; Fiore- si et al. 2020; Ergin et al. 2020 Amac and Liman 2021; Menzyanova et al. 2022). CONCLUSION According to the findings of the current study, methomyl can alter kinetics of mitotic cell process in root tip cells and can have genotoxic effects on P. sati- vum L through aneugenic and clastogenic processes. These findings raise concern about noxious effects of pesticides on non-target organisms. For the benefit of human welfare, additional genotoxicological and risk assessment studies needs to be conducted on various eukaryotic test systems. FUNDING We are grateful to King Abdul Aziz City for Sci- ence and Technology (KACST), Riyadh, Saudi Arabia for funding this project under grant number: 13-AGR2119- 07. ACKNOWLEDGMENTS We are grateful to King Abdul Aziz City for Sci- ence and Technology (KACST), Riyadh, Saudi Arabia for funding this project under grant number: 13-AGR2119- 07. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 75, Issue 3 - 2022 Firenze University Press Chromosome Mapping of Repetitive DNAs in the Picasso Triggerfish (Rhinecanthus aculeatus (Linnaeus, 1758)) in Family Balistidae by Classical and Molecular Cytogenetic Techniques Kamika Sribenja1, Alongklod Tanomtong1, Nuntaporn Getlekha2,* Chromosome number of some Satureja species from Turkey Esra Kavcı1, Esra Martin1, Halil Erhan Eroğlu2,*, Fatih Serdar Yıldırım3 L-Ascorbic acid modulates the cytotoxic and genotoxic effects of salinity in barley meristem cells by regulating mitotic activity and chromosomal aberrations Selma Tabur1,*, Nai̇me Büyükkaya Bayraktar2, Serkan Özmen1 Characterization of the chromosomes of sotol (Dasylirion cedrosanum Trel.) using cytogenetic banding techniques Kristel Ramírez-Matadamas1, Elva Irene Cortés-Gutiérrez2, Sergio Moreno-Limón2, Catalina García-Vielma1,* Contributions of species Rineloricaria pentamaculata (Loricariidae:Loricariinae) in a karyoevolutionary context A Cius¹, CA Lorscheider2, LM Barbosa¹, AC Prizon¹, CH Zawadzki3, LA Borin-Carvalho¹, FE Porto4, ALB Portela-Castro1,4 Cadmium induced genotoxicity and antioxidative defense system in lentil (Lens culinaris Medik.) genotype Durre Shahwar1,2,*, Zeba Khan3, Mohammad Yunus Khalil Ansari1 Biogenic synthesis of noble metal nanoparticles using Melissa officinalis L. and Salvia officinalis L. extracts and evaluation of their biosafety potential Denisa Manolescu1,2, Georgiana Uță1,2,*, Anca Șuțan3, Cătălin Ducu1, Alin Din1, Sorin Moga1, Denis Negrea1, Andrei Biță4, Ludovic Bejenaru4, Cornelia Bejenaru5, Speranța Avram2 Polyploid cytotypes and formation of unreduced male gametes in wild and cultivated fennel (Foeniculum vulgare Mill.) 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