Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 103-109, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1230 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Sazada Siddqiui, Saad Abdurahamn Muhammad Al Amri, Huda Ahmed Al Ghamdy, Wadha Saad Saeed Alqahtani, Sarah Mohammed Alquyr, Habab Merghani Yassin (2021) Impact of Bisphenol A on seed germi- nation, radicle length and cytogenetic alterations in Pisum sativum L.. Caryo- logia 74(2): 103-109. doi: 10.36253/cary- ologia-1230 Received: March 02, 2021 Accepted: June 11, 2021 Published: October 08, 2021 Copyright: © 2021 Sazada Siddqiui, Saad Abdurahamn Muhammad Al Amri, Huda Ahmed Al Ghamdy, Wadha Saad Saeed Alqahtani, Sarah Mohammed Alquyr, Habab Merghani Yassin. 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, distri- bution, and reproduction in any medi- um, 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 Impact of Bisphenol A on seed germination, radicle length and cytogenetic alterations in Pisum sativum L. Sazada Siddiqiui*, Saad Abdurahamn Muhammad Al Amri, Huda Ahmed Al Ghamdy, Wadha Saad Saeed Alqahtani, Sarah Mohammed Alquyr, Habab Merghani Yassin Department of Biology, College of Science, King Khalid University, Abha 61413, Saudi Arabia *Corresponding author. E-mail: sasdeky@kku.edu.sa Abstract. Bisphenol A (BPA) is a global transpiring pollutant and an endocrine dis- ruptor present in the environment which has a substantial harmful effect on plants. In the present study, its effects on seed germination, radicle length and cytogenetic alterations were investigated in P. sativum root tip cells. P. sativum seeds were germi- nated after treating with various concentrations of BPA (2 mg/L, 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L and 25 mg/L) at 24±1°C for 72 hours and the cytogenetic variations were assessed. The investigation showed that BPA reduced the percentage of seed ger- mination, mitotic index, radicle length (at higher concentrations) and instigated a rise in chromosomal anomalies in a dose-related manner. In total, there is an enhanced occurrence of c-mitosis, stickiness, bridges, fragments and laggards in the BPA treated root tip cells of P. sativum seeds. Keywords: BPA, Seed germination, Mitotic index, Chromosomal anomalies, Pisum sativum L. INTRODUCTION Bisphenol A (BPA, 2,2-bis-(4- hydroxyphenyl) propane) is an impor- tant transpiring pollutant (Clarke and Smith 2011). BPA is an abundantly mass-produced industrial chemical widely used in the manufacture of vari- ous domestic and daily use items like baby feeding plastic bottles, protect- ing coverings, packing of drinks, food items and in the linings of metal cans used for storing beverages and food products. Globally every year BPA is manufactured industrially in huge quantities approximately 0.0037 billion metric tonnes (Mihaich et al. 2009). It is constantly released in marine envi- ronment by municipal, agriculture and industrial effluents (Gatidou et al. 2007; Pothitou and Voutsa 2008; Fu and Kawamura 2010). With leaching of BPA by plastics and containers used for keeping food, drinks and beverag- es, human beings are exposed to it by consuming food and drinks stored in 104 Sazada Siddiqiui et al. these containers (Huang et al. 2012) and it poses a risk for the health of all human beings (Le et al. 2008; Wag- ner and Oehlmann 2009; Cooper et al. 2011). Human beings are also at risk by eating fish found in aquatic waters polluted by BPA (Mita et al. 2011). Agrarian soils usually get polluted by biosolids containing BPA found in sewage sludge (Gatidou et al. 2007; Stasinakis et al. 2008). Through extensive research work on BPA, it has been found that it is an endocrine disruptor (Staples et al. 1998; Le et al. 2008; Clarke and Smith 2011). Small organisms living in soils and plants could come in con- tact with soils polluted by BPA (Yamamoto et al. 2001; Staples et al. 2010). Moreover, not many studies have analyzed the toxicologic effects of BPA in plants which absorbs and accumulates it (Ferrara et al. 2006). Though, it has been established that plants can form BPA-gly- cosides by metabolizing BPA (Noureddin et al. 2004), clastogenic as well as phytotoxic influence of BPA were defined (Ferrara et al. 2006). Due to the impact of BPA on the pollen of kiwifruit in a dose-related manner, there is a substantial inhibition of tube development and its elongation (Speranza et al. 2011). Lately, the mitotic and chromosomal anomalies were found in cells of root meristem of Allium cepa L treated with 50, 100, 150 and 200 mg/L BPA concentration for five days (Jadhav et al. 2012). BPA treatment with 0.044-0.44 mM concentration inhibited the segregation of chromosomes, obstructed the cytokinesis completion, disrupted mitotic MT arrays and interphase and stimulated microtubules creation in P. sativum (Adamakis et al. 2013). Moreover, BPA treat- ment influences leaf blade differentiation in Arabidopsis thaliana significantly (Pan et al. 2013) and in BPA treat- ed seedlings of soybean, it reduced the photosynthetic constraints and growth indexes (Qiu et al. 2013). In animals, Bisphenol A has revealed to put forth xenoestrogenic action (Wang et al. 2021). However, the influence of BPA on plants are not clearly understood. Though BPA is consumed regularly and disposed, it may persist in the soil and can potentially cause detrimental effects on the plants. Further, there is not sufficient stud- ies available in the literature about its genotoxic effects on plants (Palani and Panneerselvam 2007). In the pre- sent study, we have evaluated the adverse effects of BPA on seed  germination, radicle length, mitotic index and chromosomal anomalies in cells of P. sativum root tips. MATERIAL AND METHODS Purchase of BPA and seeds From a seed shop in Saudi Arabia, pea seeds (P. sativum variety ARKIL, 2n = 14) were bought. Through Sigma–Aldrich Merck (Darmstadt, Germany) Bisphenol A (BPA) (BPA, 2,2-bis-(4- hydroxyphenyl) propane is procured from Bayouni Trading Co. Ltd., Jeddah, Saudi Arabia. Bisphenol A, CAS number is 80-05-7. Its melt- ing point is 158-159 °C and its solubility in water at 25ºC is 123–300 mg/L. The molecular weight of BPA is 228.29 and its chemical formula is C16H18O2. Seed treatment with BPA For 5 minutes, seeds were sterilized in 0.1% HgCl2 solution and they were washed in distilled water 2-3 times. Thirty seeds were soaked in BPA solutions of each concentrations (2 mg/L, 5 mg/ L, 10 mg/L, 15 mg/L, 20 mg/L and 25 mg/L) for 3 hours. For control group, a group of thirty seeds was soaked in distilled water. Seeds were repeatedly shaken for sufficient air supply. Thirty sterilized seeds were then spread over three Whatman filter papers, grade one and then kept in Petri-dishes (150 mm x 15 mm diameter). For more readings, these Petri dishes were kept in a Biological Oxygen Demand incubator (BOD) at a temperature of 24±20C. As per the procedure defined by Rank (2003), root elongation tox- icity and seed germination tests were performed. Radi- cle length were measured and germination of seeds were recorded, each day on an interval of 24 hours for 3 consecutive days. In similar settings, this test was done thrice. Toxicity was stated as compared with control, the difference of germination of seeds and root elongation. Cytotoxicity and genotoxicity evaluations To assess the cytotoxicity and genotoxicity evaluations caused by BPA in P. sativum plant, the root tips of germi- nated seeds were used as a source of mitotic cells. The root tips were washed in water. In a blend of ethanol and ace- tic acid (3:1–v/v, Merck), roots in length 2 cm were fixed (approximately 2 days). Staining of fixed roots were done with Schiff ’s reagent, as defined by Feulgen and Rossen- beck (Mello and Vidal 1978) and the slides were made by applying the meristematic region as per the protocol stated by Siddiqui et al. (2007). By documenting the variations in the meristematic cells mitotic index (MI), cytotoxicity was evaluated. By means of scoring various kinds of chromo- somal anomalies (CAs), genotoxicity was assessed. Each slide was observed and coded blind. By using light microscope under oil immersion, chromosomal anomalies and mitotic index in metaphase and anaphase plates were examined. At least 250 cells were scored from every single slide and mitotic index was computed. Chromosomal anomalies such as sticky chromosome, 105Impact of Bisphenol A on seed germination, radicle length and cytogenetic alterations in Pisum sativum L. c-mitosis, laggards, bridges and fragments were exam- ined in at least 150 metaphase and anaphase plates for each slide and stated in percentage. Statistical analysis By using Graph Pad software (San Diego, CA, USA), statistical analysis (ANOVA with Dunnett’s multiple- comparison test) having significance at P<0.05 was car- ried out. Data were exhibited in the form of mean ± standard error (SE). RESULTS Effect of BPA treatment on seed germination At 24 h interval, in control group 77.33% of seeds ger- minated which increased to 85% and 99% at 48 h and 72 h respectively (Table 1). In seeds treated with lower con- centration of BPA (2 and 5 mg/L), percentage of seed ger- mination decreased (p<0.001 and p<0.05) at 24 h. Simi- larly, a significant decrease was observed at 48 h (p>0.05) and 72 h (p<0.01 and p<0.001) compared to control. In all time periods, on and above a concentration of 10 mg/L treatment with BPA caused a very significant decrease in germination percentage of seed in a dose-related manner, as compared to control. Lowest percentage of seed germi- nation was reported at 20 mg/L (65% at 72 h) and at 25 mg/L (50.22% at 24 h, 60% at 48 h) in BPA treated seeds. Effect of BPA treatment on radicle length In control group, the radicle length increased with increase in time which was 4.0 ± 0.05 at 72 h (Fig. 1). At 24 h interval, significant decrease in radicle length was observed in seeds exposed to BPA in a dose-related manner. Furthermore, there was no statistically signifi- cant difference reported from 2 mg/L to 25 mg/L BPA treatment at 48 h as compared to control. In 2 mg/L, 10 mg/L and 15 mg/L BPA treated seeds no statisti- cally significant difference was noticed but in 5 mg/L and 20 mg/L significant decrease in radicle length was reported and in 25 mg/L very significant decrease was observed at 72 h. In BPA treated seeds lowest root length was recorded in 20 mg/L at 48 h (0.85 ± 0.04) and in 25 mg/L at 24 h (0.35 ± 0.02) and at 72 h (1.5 ± 0.33). Maxi- mum root length was recorded in 2 mg/L (0.77 ± 0.04) at 24 h, 10 mg/L (1.5 ± 0.7) at 48 h and at 2 mg/L (3.0 ± 0.03) at 72 h in BPA treated seeds. Effect of BPA treatment on mitotic index The control presented a mitotic index of 17.78 ± 5.66 (Fig. 2). However, further increase in BPA concentration caused a decline in the mitotic index in a dose-related manner. As compared to control, at a lesser concentra- tion of BPA (2 and 5 mg/L), the mitotic index was non- significantly lower. When compared with control, in seeds treated with 10 mg/L BPA, the mitotic index was significantly less (p<0.05), in 15 mg/L the mitotic index was found to be very significantly lower (p <0.01) and in seeds treated with 20 and 25 mg/L BPA, the mitotic index was highly significantly lower (p<0.001). In seeds treated with 25 mg/L BPA, the lowest mitotic index (5.45 ± 2.05) was determined. Table 1. Germination rates of P. sativum treated with different con- centrations of BPA. Concentrations of BPA Seed germination (%) 24 h 48 h 72 h 00.00 77.33 ± 0.33 85.0 ± 0.88 99.0 ± 3.11 2 mg/L 72.77 ± 0.88a 84.0 ± 0.68 88.0 ± 2.09b 5 mg/L 74.33 ± 0.77c 78.0 ± 3.20 82.2 ± 0.33a 10 mg/L 66.66 ± 0.15a 70.0 ± 1.15a 75.0 ± 0.88a 15 mg/L 61.22 ± 0.03a 68.0 ± 1.15a 70.0 ± 1.33a 20 mg/L 55.33 ± 0.66a 61.0 ± 0.88a 65.0 ± 0.77a 25 mg/L 50.22 ± 0.42a 60.0 ± 0.55a 65.6 ± 0.66a ap<0.001 compared to control; bp<0.01 compared to control; cp<0.05 compared to control. Data are mean of three replicates ± SEM; 00.00 = Control group. Figure 1. Effect of different concentrations of BPA on the radicle length of P. sativum. ap<0.001 compared to control; bp<0.01 com- pared to control; cp<0.05 compared to control. Yp≤0.001v/s 15, 20, 25 mg/L; Xp≤0.01 v/s 25 mg /L; pp≤0.05 v/s 20 mg/L. Data are mean of three replicates ± SEM; 0.0 = Control group. 106 Sazada Siddiqiui et al. Effect of BPA treatment on chromosomal anomalies. As shown in Table 2 and Fig. 3 treatment with BPA caused numerous mitotic anomalies in P. sativum. In control, the occurrence of abnormal metaphase-anaphase plates was 00 ± 00. In the present study, in case of root tips of P. sativum enhanced occurrence of chromosomal anomalies such as sticky chromosomes, c-mitosis, lag- gards, bridges and fragments were observed in various doses of BPA treatment (Table 2, Fig. 3). Treatment with BPA resulted in a dose-related increase in the percentage of root tip cells with abnormal metaphase-anaphase plates. In lower concentration (2 mg/L of BPA treatment), minimum chromosomal anomalies such as fragments (0.42 ± 0.01), c-mitosis (0.52 ± 0.01), sticky chromosomes (0.61 ± 0.01), laggards (0.83 ± 0.06) and bridges (0.91 ± 0.02) were found which were non-significant (p>0.05) when compared with control. Highest percentage of bridg- es (10.72 ± 2.2), c-mitosis (8.1 ± 2.15), fragments (6.78 ± 0.56), sticky chromosomes (6.1 ± 0.77) and laggards (6.01 ± 2.56) were found in 25 mg/L BPA treated root tip cells. St ick y chromosomes were high ly signif ica nt (p<0.001) in 5 to 25 mg/L, c–mitosis was found to be sig- nificant (p<0.05) at 25 mg/L, laggards were found to be significant (p<0.05) at 20 mg/L, bridges were found to be very significant (p<0.01) at 10 mg/L and highly signifi- cant (p<0.001) at 15 to 25 mg/L (p<0.01) and fragments were found to be very significant (p<0.01) at 15 mg/L and highly significant (p<0.001) at 20 to 25 mg/L when compared with control. DISCUSSION The outcome of the present study revealed that BPA inhibits and delays the germination of seeds, mitotic index, radicle length and chromosomal anomalies in Figure 2. Effect of different concentrations of BPA on the mitotic index in root tip cells of P. sativum. ap<0.001 compared to con- trol; bp< 0.01 compared to control; cp< 0.05 compared to control. Yp≤0.001v/s 15, 20, 25 mg/L; Xp≤0.01 v/s 25 mg /L; Data are mean of three replicates ± SEM; 0.0 = Control group. Table 2. Chromosomal anomalies in metaphase–anaphase plates in root tip cells of P. sativum treated with different concentrations of BPA. Anomalies in 150 plates Concentrations of BPA 00.00 2 mg/L 5 mg/L 10 mg/L 15 mg/L 20 mg/L 25 mg/L Sticky chromosome (%) 00 ± 00 0.61 ± 0.01 2.78± 0.09a 4.99 ± 0.90a 4.25± 0.04a 7.80 ± 0.44a 6.10 ± 0.77a C-mitosis (%) 00 ± 00 0.52 ± 0.01 3.15 ± 1.12 2.25 ± 1.20 5.70 ± 1.13 6.70 ± 3.20 8.10 ± 2.15c Laggards (%) 00 ± 00 0.83 ± 0.06 1.32 ± 0.91 2.45 ± 1.01 6.75 ± 2.05 8.15 ±3.25c 6.01 ± 2.56 Bridges (%) 00 ± 00 0.91 ± 0.02 2.25 ± 1.00 4.23 ± 1.20b 5.78 ± 0.09a 7.62 ± 1.50a 10.72 ± 2.2a Fragments (%) 00 ± 00 0.42 ± 0.01 0.71 ± 0.45 1.75 ± 0.76 2.91 ± 0.66b 4.62 ± 0.78a 6.78 ± 0.56a ap<0.001 compared to control; bp<0.01 compared to control; cp<0.05 compared to control. Data are mean of three replicates ± SEM; 0.0 = Control group. Figure 3. Chromosomal anomalies induced by BPA in P. sativum root tip cells. (A) Sticky chromosome, (B) C-mitosis, (C) Laggards, (D) Bridge at anaphase (E) Fragment. 107Impact of Bisphenol A on seed germination, radicle length and cytogenetic alterations in Pisum sativum L. seeds of P. sativum in a dose-related manner. It was shown in our experimental outcome that there is a sub- stantial concentration-effect of BPA on the germination of seeds, mitotic index, radicle length and chromosom- al anomalies in seedlings of P. sativum (Table 1, 2 and Fig. 1-3). Seed germination is inhibited by BPA (Zhiyong et al. 2013; Pan et al. 2013; Dokyung et al. 2018)). Simi- lar findings have been found by the present study that BPA delays and inhibits the germination of P. sativum seeds. Seed germination is affected by various causes for example light, temperature of incubation, humidity and oxygen level (Isabelle et al. 2000). Eunkyoo et al. (2004) proved that an essential helix-loop-helix transcrip- tion feature PIF3-like 5 (PIL5) protein was a significant adverse regulator of phytochrome-mediated germination of seeds. It is known that etiolated seedlings generally have higher quantities of phytochromes A (Hanumappa et al.1999), therefore, the possible functioning of BPA on phytochromes in seeds germination phase is interesting. In the present test, it was revealed that BPA showed inhibitory effects on root length in P. sativum treated with different doses. This may be caused by the nox- ious influence of BPA in root tips mitotic cell division (Adamakis et al. 2013; 2016; Amer 2017; Dokyung et al. 2018. In P. sativum the root tip mitotic index is directly associated with decrease in root length. The same influ- ence of BPA on mitotic index was recorded (Pan et al. 2013; Jadhav et al. 2012). Primary roots elongation is facilitated by relating hormonal signal paths and a vari- ety of enzymes for example phospholipase D, auxin and phosphatidic acid (Ohashi et al. 2003; Li et al. 2006; Saini et al. 2013). Though, the paths comprised in the molecular process related to the elongation of roots altered by BPA is not known. The decrease in the quantity of mitotic cells in root tips treated with BPA may be because of its mode of action on the progress of cell cycle. Synthesis of DNA may be inhibited by BPA (Adamakis et al. 2019; Ozge et al. 2019) or in G2 stage of cell cycle, BPA could also obstruct the cells and thus blocking them to enter into mitosis. Moreover, BPA might affect enzymes for DNA- repair, by altering the structure of proteins present in the enzymes or in mitotic cells, by decreasing the forma- tion of enzymes at transcription phase that could induce chromosomal anomalies (Ozge et al. 2019; Nasir et al. 2018). P. sativum seeds treated with BPA showed numer- ous chromosomal anomalies in root tips mitotic cells for example c-mitosis, bridges, laggards, fragments and sticky chromosomes. The occurrence of chromosomal anomalies increases with increase in BPA concentra- tion. In cell division, spindle fiber arrangement and its movement are a mechanism reliant on ATP (Can et al. 2005; Nasir et al. 2018; Adamakis et al. 2019). Because of decreased synthesis and obtainability of ATP, arrange- ment of spindle fiber in root tips treated with BPA, cells might get influenced, and it could disturb the chromo- somal organization at metaphase plate and chromosomal migration to opposite poles in anaphase. The irregularity in spindles formation and segregation of chromosomes in mitosis, will cause chromosomal anomalies like lag- gards, bridges and sticky chromosomes. In BPA treated root tips, C-mitosis is generally linked to spindle defects (Shahin and El-Amoodi, 1991). Since earlier studies have shown that BPA is a strong inhibitor of spindle microtubule organization (George et al. 2008; Adamakis et al. 2013; Xin et al. 2014; Adamakis et al. 2016) which may explain high incidence of C-mito- sis in BPA group.  The bridges found in the cells of BPA treated root tips are possibly produced by breaking and merging of chromosome bridges which got enhanced with treat- ment by BPA. Chromosome bridges may be formed because of stickiness of chromosomes and consequent collapse of freed anaphase separation or because of an uneven translocation or chromosome segment inversion (Gomurgen 2000; Siddiqui 2012; Siddiqui and Al-Rum- man 2020 a and b). Moreover, chromosome fragments may get formed because reactive oxygen species can induce double strand breaks in DNA. CONCLUSION Conclusively, the outcome of this investigation revealed that BPA has substantial repressing effects on seed germination and enhances chromosomal anomalies in P. sativum root tip cells. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 74, Issue 2 - 2021 Firenze University Press Chromosomal analysis of eight cultivars in three species of cultivated Yam (Dioscorea L.) species in Nigeria Joshua Matthew1,2,*, Julius Oloaye Faluyi1 Mutagenic and cytotoxic activity of insecticide Napoleon 4EC in Allium cepa and Ames test Dilek Akyil Assessment of antimitotic and programmed cell death stimulation potentials of Galium sinaicum (Delile ex Decne) Boiss. at the cellular level Shaimaa S. Sobieh1,*, Dina M. Fahmy2 First genome size assessments for Marshallia and Balduina (Asteraceae, Helenieae) reveal significant cytotype diversity Teresa Garnatje1,a, Jaume Pellicer1,2,a,*, Joan Vallès3, Nathan Hall4, Curtis Hansen4, Leslie Goertzen4 Comparative study and genetic diversity in Salvia (Lamiaceae) using RAPD Molecular Markers Ruonan Zheng1,*, Shuhua Zhao2, Majid Khayatnezhad3, Sayed Afzal Shah4 Identification of regions of constitutive heterochromatin and sites of ribosomal DNA (rDNA) in Rhogeessa hussoni (Genoways & Baker, 1996) (Chiroptera; Mammalia; Vespertilionidae) Adriano Silva dos Santos1, Karina de Cassia Faria2 Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris Timir Baran Jha1,*, Biplab Kumar Bhowmick2, Partha Roy1 Toxic and genotoxic effects of aqueous extracts of Polygonum weyrichii Fr. Schmidt on the Allium test taken as an example Maria V. Smirnova*, Anna V. 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