Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(2): 37-43, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-256 Citation: I. Sarac, E. Bonciu, M. But- nariu, I. Petrescu, E. Madosa (2019) Evaluation of the cytotoxic and geno- toxic potential of some heavy metals by use of Allium test. Caryologia 72(2): 37-43. doi: 10.13128/cayologia-256 Published: December 5, 2019 Copyright: © 2019 I. Sarac, E. Bon- ciu, M. Butnariu, I. Petrescu, E. Madosa. 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 Com- mons Attribution License, which per- mits 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. Evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of Allium test Ioan Sarac1, Elena Bonciu2,*, Monica Butnariu1, Irina Petrescu1, Emilian Madosa1 1 Banat’s University of Agricultural Science and Veterinary Medicine “King Michael I of Romania” Timisoara, Romania 2 Department of Agricultural and Forestry Technology, University of Craiova, Craiova, Romania *Corresponding author: elena.agro@gmail.com Abstract. The present study aimed to evaluate the cytogenetic effects induced by heavy metals nickel (Ni) and lead (Pb) to crop plants, using the Allium sativum (garlic) as a test plant. For this purpose, were used solutions of nickel nitrate - Ni(NO3)2 - and lead nitrate - Pb(NO3)2 - at concentrations of 50, 150 and 450 ppm for 72 hours, along with an untreated control variant immersed in plain water. The biological material was immersed from the beginning in the tested solutions. The results obtained showed a strong inhibitory effect of these heavy metals on the process of rhizogenesis, as well as a significant mitodepresive effect in the meristematic cells, both phenomena being cor- related with increasing concentration of the tested solutions. At the same time, several types of chromosomal aberrations (c-mitosis, vagrants, star-anaphase, star-telophase, fragments, clumping, stickiness, bridges) have been recorded in all treatment variants. The presence of these chromosomal aberrations in all treatment variants indicates the aneugenic effects of nickel nitrate and lead nitrate in the meristematic cells of A. sati- vum. The results suggest the ecotoxicity potential of nickel and lead on plants even at low concentrations and confirm the suitability of A. sativum as a test plant for assess- ing the cytotoxicity and genotoxicity of heavy metals to plants. Keywords. Genotoxicity, chromosomal aberrations, lead, mitotic index, nickel. INTRODUCTION The concept of heavy metals has refers to any metallic chemical element that has a relatively high density and is toxic or poisonous in low concen- trations. As natural water pollutants, heavy metals are among the most toxic pollutants due to their prolonged persistence in solutions and the difficulty of being converted into insoluble compounds in surface waters (Bilal et al. 2014). The contamination of soil by heavy metals is also a major environ- mental problem (Lassoued et al. 2014) and have a toxic action on the aquatic organisms, meanwhile inhibiting the self-purification processes (Nemcsók 38 Ioan Sarac et al. et al. 2010). Heavy metals are dangerous because they tend to bioaccumulate (Coroian et al. 2017; Hariri et al. 2018; Marinova et al. 2018). Bioaccumulation means the increase in time in biological organisms of the concen- tration of the substance in an amount compared to the concentration of this substance in the environment. The presence of heavy metals in soil, is one impor- tant factor that can cause altered physiological and met- abolic processes to plants or disturbing the metabolism of essential elements (Dong et al. 2006; Mohanpuria et al. 2007; Wójcik and Tukiendorf 2014; Petrescu et al. 2015; Sarac et al. 2015; Georgieva et al. 2018; Nikolova and Georgieva 2018). Symptoms of heav y metal toxicity are the result of harmful effects of metals on physiological process- es including: inhibiting respiration and photosynthe- sis, altering the plant-water relationship that causes stress, decreased plasma membrane permeability in the root cells, adverse effects on the metabolic activities of enzymes (Arduini 1994). Lead (Pb) is one of the ubiquitously distributed most abundant toxic elements in the soil. Pb inhibits the activity of enzymes at cellular level by reacting with their sulfhy- dril groups (Yadav 2010). High lead exposure is harmful, particularly for children; its effects include damage to the nervous system, liver and kidney damage and develop- mental delays. Also, the lead exposure is associated with an increased risk of several cancers, in particular, menin- gioma, brain cancer, and kidney cancer (Liao et al. 2016). Nickel (Ni) is considered to be an essential micro- nutrient for plants (Eskew et al. 1983) but at excess concentrations this metal becomes toxic for major- ity of plant species and triggers oxidative damage (Zor- nova et al. 1999; Nakazawa et al. 2004; Gajewska et al. 2006; Sachan and Lal 2017). On the other hand, some authors reported cytotoxic effects even at low doses (20 to 100µM) of nickel ions as well as antioxidative enzyme changes in Allium cepa roots (Gantayat et al. 2017). Concentrations of Ni could increase by human activi- ties such as application of phosphate fertilizers and pes- ticides (Gimeno-García et al. 1996) or industrial and agricultural wastewater discharges, domestic sewage dis- charge and atmospheric deposition (Yan et al. 2018). The vegetal meristematic tissues that are used for testing the effects of chemicals on chromosomes should be easy to obtain and less expensive. From this point of view, the species A. sativum and A. cepa are well suited to cytogenetic studies because the meristematic roots appear lightly, have relatively large chromosomes in small numbers and can be easily observed by optical microscope (Doroftei et al. 2010; Bonciu 2012; Bonciu et al. 2018). MATERIALS AND METHODS Plant material The biological material consisted of garlic bulbs, clean and without traces of pests or diseases that had been spread in several bulbils. They were cleaned from the dried leaves and formed by removing any roots after which they were transferred to small glass bottles containing the heavy metal solutions: nickel nitrate - Ni(NO3)2 and lead nitrate - Pb(NO3)2 in concentrations of 50, 150 and 450 ppm for each of them. Three treatment variants with 4 repetitions were performed for each of the heavy metals experienced, along with an untreated control immersed in plain water. In each variant, four garlic bulbils were immersed directly into the treatment solutions for 72 hours, time required for the meristematic roots to be emitted. Microscopic preparations After sampling, the meristematic roots were fixed with a mixture of absolute ethyl alcohol and glacial acetic acid in a volume ratio of 3: 1 for 16 hours in the refrigerator, followed by acid hydrolysis with 1 N HCl for 5 minutes and HCl 50% consisting of equal parts of HCl and distilled water for 16 min at room temperature. Roots’ staining was performed by the Feulgen technique with Schiff’s reagent; the staining time was 90 minutes, followed by the intensi- fication of the coloration in plain water for 20 minutes. Statistical analyses After 72 hours, the meristematic roots were counted and measured at each variant. The cytogenetic effects of heavy metals were assessed by calculating the mitotic index (MI) and analysing the chromosomal aberrations observed in the various stages of mitosis. The micro- scopic preparations have been studied using a micro- scope with digital camera Kruss (Kruss manufacturer Hamburg, Germany). Five preparations for each variant and 500 cells were analysed for calculating the mitotic index and the chromosome aberration frequency. Statistical analysis was done using MS Excel 2007. The obtained data were analysed statistically with one- way analysis of variance (ANOVA). The differences between treatment means were compared using the LSD-test at a probability level of 0.05% subsequent to the ANOVA analysis. The mitotic index and chromosomal aberrations were calculated using the following formulas: 39Evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of Allium test Mitotic index (MI%) = total number of cells in division / total number of analysed cells x 100; Chromosomal aberrations (CA%) = total number of aberrant cells / total number of cells in division × 100. RESULTS The treatment of A. sativum bulbils with nickel and lead depending on the concentration negatively influ- enced the process of issuing meristematic roots. The treated roots was smaller in size and they had a smaller number than the control. Thus, their number decreased as the concentration of the heavy metal solu- tions tested in all variants increased: from 46 registered roots to the control variant, to 14-28 roots to the variant treated with Ni(NO3)2 respectively 5-18 roots to the vari- ant treated with Pb(NO3)2 (Figure 1). The results showed that both heavy metals treat- ments caused a decrease in MI at all the treatment groups (Table 1). Thus, the value of the MI decreased with the increase concentration of heavy metal solutions. The intensity of mitotic activity was decreasing in order of treatment with lead nitrate to nickel nitrate treatment. However, the strongest mitodepresive effect was seen in the treatment of Pb(NO3)2 at the concentration of 450 ppm, when MI was 5.32%, ie with 46.4% lower mitotic activity compared to the control variant. Heavy metals tested induced a high number of CA when compared with control. The increase of CA was dependent on the increasing treatment concentrations (Table 1). The types of CA identified in meristematic cells of A. sativum were the following: C-Mitosis (Figure 2A); fragments and vagrants (Figure 2B); star-anaphase; star-telophase (Figure 2C); clumping; stickiness (Figure 2D); bridges (Figure 2E). As can be seen from the data of Table 1, the most common types of CA were stickiness, C-Mitosis and bridges, and the least frequent were vagrants chromo- somes. Compared with the control variant, the total CA rate recorded insignificant values for the variant treated with 50 ppm Ni(NO3)2, significant for the variant treated with 150 ppm Ni(NO3)2 and distinctly significantly posi- tive for the variant treated with 450 ppm Ni(NO3)2. On the other hand, in case of the Pb(NO3)2 treated variants compared to the control variant, the total CA recorded significantly positive values for the variant treated with 50 ppm Pb(NO3)2 and strongly positive for the variants treated with 150 and 450 ppm Pb(NO3)2 respectively. DISCUSSION We chose to study the cytotoxic effects of Ni and Pb heavy metals on A. sativum because, according to many authors (Saxena et al. 2004; Gul et al. 2006; Unyayar et al. 2006; Liu et al. 2009), the evaluation of CA in A. sati- vum meristematic roots is a reliable biotest example that can be applied to detect a wide range of genetic damage. At a macroscopic level, heavy metals induced inhi- bition of the growth of meristematic garlic roots. Gen- erally, heavy metals are known to decreasing the plant growth and ground cover (McGrath et al. 2001). Some effects of lead on growth, physiology, metabolism and yield attributes of plants are the following: inhibition in seed germination, fresh and dry biomass, leaf area, chlo- rophyll and growth to Helianthus annuus (Mahmood et al. 2013); decline in growth, chlorophyll, carotenoids and prolinecontent to Brassica juncea (John et al. 2009); decrease in plant growth, root hair to Vigna unguiculata (Kopittke et al. 2007), etc. In our experiment, reducing the number of meris- tematic root has been accentuated with increasing con- 0 50 150 450 50 150 450 46 28 21 14 18 9 5 0 50 100 150 200 250 300 350 400 450 500 Concentration (ppm) Number of meristematic roots after 72 h Fig. 1. The inhibitory effect of different concentrations of Ni(NO3)2 and Pb(NO3)2 on the rhizogenesis to A. sativum. A B C D E Fig. 2. Some chromosomal aberrations identified in meristematic cells of A. sativum exposed to Ni(NO3)2 and Pb(NO3)2: C-Mitosis (A), fragments and vagrants (B), star-telophase (C), stickiness in telophase (D), anaphase bridge (E). 40 Ioan Sarac et al. centrations of test solutions (especially to treatments with lead nitrate). Proportionately with increasing con- centrations, intensity of the mitotic division has been decreased to all variants, as an active protection reac- tion of the plants exposed to heavy metals action. These findings are in agreement with Doroftei et al. (2010) who have tested the cytogenetic effects of lead nitrate to A. cepa. The lead and nickel inhibited cell division to other plants too, like Zea mays (Kozhevnikova et al. 2007). The root growth is an integrative process depend- ing on whole-organism signalling and individual growth trajectories of cells (Beemster et al. 2003). The intensity of the mitotic division is directly related to the growth of plant roots; conceptually, mitotic division in the api- cal root meristem provides cells during longitudinal growth (Sanz et al. 2012). The number of dividing cells in the root apical meristem generates a cell flux with importance in modulating root growth (Baskin, 2013). Studies of cell length profiles have shown that the prolif- erative fraction of dividing cells in the apical root meris- tem proliferation domain is indistinguishable from one, even in response to moderate levels of stress (Ivanov and Dubrovsky 2013). The results of this study highlight the increasing of CA frequencies to A. sativum dependent on different concentrations of nickel nitrate and lead nitrate. Sticki- ness, C-mitosis and bridges were most often identified in all treatment variants but the highest frequency was recorded to variants treated with lead nitrate. According to some authors, sticky chromosomes might have result- ed from increased chromosome contraction and conden- sation or possibly from depolymerisation of DNA and partial dissolution of nucleoproteins (Kuras et al. 2005; Turkoglu 2013b). Asita and Mokhobo (2013) suggested that the induction of Allium’s sticky chromosomes under the influence of pesticides indicates abnormal DNA con- densation, abnormal chromosomal wrapping, and inac- tivation of the axes, and all these anomalies cell division can have adverse effects on the environment. C-Mitosis indicates a chemical-inhibited spindle formation similar to the effect of colchicine and induction of these aberra- tions suggests a turbogenic effect (Shahin and El-Amoo- di 1991; Turkoglu 2013b). Regarding the anaphase bridg- es, these chromosomal aberration cause structural chro- mosome mutations and may lead to loss of genetic mate- rial (George 2000; Pampalona et al. 2016). According to Turkoglu (2013b), bridges could form due to dicentric chromosome presence or due to the breakage and fusion of chromosomes and chromatids. In our experiment, the stickiness had a frequency of 3.62-5.42% at the nickel nitrate treatment, while for treatment with lead nitrate the frequency of these CA was at the level of 6.28-7.40% at all concentrations (50, 150, 450 ppm). Sticky chromosomes can probably lead to cell death (Singh 2015). These results suggest the strong genotoxic effect of lead nitrate even at low concentra- tions of 50 and 150 ppm. The current findings agreed well with other reports which showed cytotoxicity and genotoxicity of lead in plant cells (Choudhury and Pan- da 2004; Arya et al. 2013). Some nickel compounds have been established as human carcinogens (Coogan et al. 1989), but at the same time, some plant seeds, such as soybeans, can act as pro- tectors when introduced into diet of treated mice, by reducing the percentage of CA (Fahmy et al. 2014). Cer- tain hormones can act as agents for inducing resistance Table 1. Mitotic index, type and percentage of mitotic aberrations induced by some heavy metals on the meristematic roots to A. sativum. Treatment / Exposure time (hours) Conc. (ppm) MI ± SD (%) CA (%) Total aberrations (%)C-M V S-A S-T F CL S B Ct / 72 0 11.45±0.5 2.35 0 0 0 0 0 0 0 2.35 Ni(NO3)2 / 72 50 10.66±0.4 1.61 0.65 0.42 0 0 0 3.62 2.31 8.61 150 8.13±0.3 1.90 0.83 1.81 0.83 0 1.23 5.11 3.22 14.93* 450 6.84±0.8 3.15 1.40 2.73 1.43 2.11 3.41 5.42 3.91 23.56** Pb(NO3)2 / 72 50 8.73±0.4 4.65 1.62 3.90 1.70 2.80 3.96 6.28 4.82 29.73** 150 6.51±0.8 6.23 2.11 4.31 2.82 3.70 4.20 6.50 5.31 35.18*** 450 5.32±0.5 7.51 2.64 4.85 4.20 4.21 6.52 7.40 6.10 43.43*** Ct = Control; Conc. = Concentration; MI = Mitotic index; SD = Standard deviation; CA = Chromosomal aberrations; C-M = C-Mitosis; V = Vagrants; S-A = Star-Anaphase; S-T = Star-Telophase; F = Fragments; CL = Clump- ing; S = Stickiness; B = Bridges; The differences between treatment means were compared using the LSD-test at a probability level of 0.05%: *significant at P<0.05, **signifi- cant at P<0.01, ***significant at P<0.001 as compared to the control variant. 41Evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of Allium test of plants to heavy metal toxicity. Thus, some authors have reported that Brassica juncea plants sprayed with 28-homobrassinolide hormone, showed improved resist- ance against the some heavy metal toxicity (Hayat et al. 2007). Also, the organic acids (citrate and malate) have been reported to have a role in the plant protection against heavy metal stress (Haydon et al. 2007). CA called C-mitosis had a frequency of 1.61-3.15% at the nickel nitrate treatment, while for treatment with lead nitrate the frequency of C-mitosis was at the level of 4.65-7.51%, these results demonstrating the high geno- toxic potential of lead to plants. C-mitosis is the result of damaged mitotic apparatus due to genotoxic substances in the cells and is stimulated by many chemicals (Fiskes- jö 1993; Firbas and Amon 2014). Cytogenetic tests on A. sativum reveal a decrease in the mitotic index following heavy metal treatments. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 72, Issue 2 - 2019 Firenze University Press Karyotype analysis of a natural Lycoris double-flowered hybrid Jin-Xia Wang1, Yuan-Jin Cao1, Yu-Chun Han1, Shou-Biao Zhou1,2, Kun Liu1,* Insights on cytogenetic of the only strict African representative of genus Prunus (P. africana): first genome size assessment, heterochromatin and rDNA chromosome pattern Justine Germo Nzweundji1, Marie Florence Sandrine Ngo Ngwe2, Sonja Siljak-Yakovlev3,* Assessment of cytotoxicity and mutagenicity of insecticide Demond EC25 in Allium cepa and Ames Test Arzu Özkara Cytogenetic effects of Fulvic acid on Allium cepa L. root tip meristem cells Özlem Sultan Aslantürk Evaluation of the cytotoxic and genotoxic potential of some heavy metals by use of Allium test Ioan Sarac1, Elena Bonciu2,*, Monica Butnariu1, Irina Petrescu1, Emilian Madosa1 Fluorescence In Situ Hybridisation Study of Micronuclei in C3A Cells Following Exposure to ELF-Magnetic Fields Luc Verschaeve1,2,*, Roel Antonissen1, Ans Baeyens3, Anne Vral3, Annemarie Maes1 Phytochemical analysis and in vitro assessment of Polystichum setiferum extracts for their cytotoxic and antimicrobial activities Nicoleta Anca Şuţan1,*, Irina Fierăscu2, Radu Fierăscu2, Deliu Ionica1, Liliana Cristina Soare1 Telomeric heterochromatin and meiotic recombination in three species of Coleoptera (Dorcadion olympicum Ganglebauer, Stephanorrhina princeps Oberthür and Macraspis tristis Laporte) Anne-Marie Dutrillaux, Bernard Dutrillaux* A whole genome analysis of long-terminal-repeat retrotransposon transcription in leaves of Populus trichocarpa L. subjected to different stresses Alberto Vangelisti#, Gabriele Usai#, Flavia Mascagni#, Lucia Natali, Tommaso Giordani*, Andrea Cavallini Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and eastern Honshu Hikari Myoshu, Masahiro A. 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