Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(2): 29-35, 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-257 Citation: Ö.S. Aslantürk (2019) Cytogenetic effects of Fulvic acid on Allium cepa L. root tip meristem cells. Caryologia 72(2): 29-35. doi: 10.13128/ cayologia-257 Published: December 5, 2019 Copyright: © 2019 Ö.S. Aslantürk. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) 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. Cytogenetic effects of Fulvic acid on Allium cepa L. root tip meristem cells Özlem Sultan Aslantürk Adnan Menderes University, Faculty of Art and Science, Department of Biology, 09010 Aydın, Turkey E-mail: osaslanturk@adu.edu.tr Abstract. Fulvic acid is a class of compounds of humic substances and is found in a significant proportion of the substances in the environment. It has been used for many years in industry, agriculture, and complementary medicine. In this study, cytogenetic effects of fulvic acid purified from Muğla Milas Hüsamlar leonardite (TURKEY) on Allium cepa root tip meristem cells were investigated using the Allium test. For this purpose, 10 mg/ml stock solution of fulvic acid was prepared by dissolving in citric acid and it was diluted with distilled water to 10, 20, 40, 80 and 100 µg/mL concentra- tions. Onion bulbs were exposed to these concentrations of the fulvic acid for macro- scopic and microscopic analysis. Tap water was used as a negative control, 40 µg/mL citric acid was used as solvent control (fulvic acid solvent), and 0.02M Ethyl methane sulfonate (EMS) (a mutagenic, teratogenic, and possibly carcinogenic organic com- pound) was used as a positive control. There has been statistically significant stimu- lation of root growth depending on fulvic acid concentration in comparison with the control groups (p<0.05). Furthermore, in fulvic acid treatment groups, breaks, sticki- ness and polar deviations appeared at very low rates, and total chromosome aberration ratios were insignificant compared to the control groups. These results suggest that ful- vic acid does not have cytotoxic and genotoxic effects on A. cepa. Keywords. Allium test, Chromosome aberrations, Fulvic acid, Mitotic index. 1. INTRODUCTION Fulvic acid is a class of compounds of humic substances, and it is a mix- ture of polyphenolic compounds formed through the degradation of organic substances such as plants, microbes and animals by chemical and biological processes (Motojima et al. 2011). It is a type of humic acid. Compared to oth- er humic acid types, the fulvic acid is soluble in both acid and alkaline solu- tions, is lower in molecular weight, and has a greater biological activity (Ste- venson, 1994; Bai et al. 2013; Yong 2001; Zhang et al. 2011). Piccolo (2002) redefined fulvic acid as associations of small hydrophilic molecules in which there are enough acid functional groups to keep the fulvic clusters dispersed in solution at any pH. Because, while humic acids precipitate when the pH is adjusted to 1-2, fulvic acids remain in solution after the alkaline extracts are acidified (Canellas et al. 2015). 30 Özlem Sultan Aslantürk Recently, it has been reported that fulvic acid has nutraceutical, neuroprotective (Cornejo et al. 2011; Guzmán-Martinez et al. 2013), antimicrobial, antioxi- dant, and anti-inflammatory properties (Van Rensburg et al. 2001; Yamada et al. 2007; Sherry et al. 2013). Fulvic acid has also been used as a medicine by people in Chi- na, Mexico, India, South America and Russia for centu- ries. Fulvic acid has a large capacity to retain transition metals, forming metalorganic complexes, which cause these metals to be more or less available for plants which include them into the food chain. The food industry also uses it as an ion exchanger, because it holds heavy met- als very well (Pena-Mendez et al. 2005). Over these last decades, more than 200 short-term bioassay utilizing plants, microorganisms, and insects have been developed and used to evaluate the environ- mental risks (Marcato-Romain et al. 2009). Plant assays are highly sensitive, easy to use in an experiment, inex- pensive, and good predictors of genotoxicity and carci- nogenicity (Ennever et al. 1988). The Allium test has been used by many researchers as a bioindicator of envi- ronmental pollution (Bagatini et al. 2009; Leme and Marin-Morales 2009) and genotoxicity of various agents (Aşkın Çelik and Aslantürk 2007, 2009, 2010) for a long time. With this test, mutagenic effects of substances may be analyzed by monitoring macroscopic parameters, like the appearance and growth of the roots or by genotox- ic parameters, like type and frequency of chromosome aberrations, and abnormal cell division. Another advan- tage of this test is the presence of an oxidase enzyme system, which is essential for promutagen evaluations (Fiskesjö, 1985; Nielsen and Rank,1994 ). The Allium test is important, since it is an excellent model in vivo, where roots grow in direct contact with the test substance ena- bling possible damage to DNA of eukaryotes to be pre- dicted. Therefore, results from this test can be extrapo- lated for all animal and plant biodiversity (Tedesco and Laughinghouse IV 2012). Although fulvic acid is found in a significant pro- portion of the substances in the environment, and has been used for many years in industry, agriculture and complementary medicine, there is still minimal scien- tific evidence of its biological properties. In this study, cytogenetic effects of fulvic acid on Allium cepa root tip meristem cells were investigated using the Allium test. 2. MATERIALS AND METHODS 2.1. Supply of Fulvic acid Fulvic acid purified from Muğla Milas Hüsamlar leonardite (TURKEY) in Chemical Engineering Labora- tory of Gazi University in Ankara (TURKEY) was used in this research (Sönmez 2011). This study was conduct- ed between March and December 2017. 2.2. Preparation of the Fulvic acid solution The 10 mg/ml stock solution of fulvic acid was pre- pared by dissolving in citric acid, as it has a structure, which is soluble in weak acid. Stock fulvic acid solution was diluted with distilled water to 10, 20, 40, 80 and 100 µg/mL concentrations. Fresh solution was prepared just before the experiment. 2.3. Allium Test Small bulbs (1.5–2.0 cm in diameter) of the common onion, A. cepa, (2n = 16) were purchased at a local super- market in Aydın, Turkey. Prior to initiating the test, the outer scales of the bulbs and the dry bottom plate were removed without damaging the root primordia. For each treatment, seven onion bulbs were placed on top of test tubes filled with tap water (pH 7.3) for 48 h. The test tubes were kept in an incubator at 22±1ºC. After 48 h, two unhealthy onions with the most poor- ly growing roots were removed and the other healthy onion bulbs in water were treated with 10, 20, 40, 80 and 100 µg/mL fulvic acid for 24 hours. 0,02M Ethyl meth- ane sulfonate was used as positive control for 3 h, 40 µg/ mL citric acid was used as solvent control, and tap water was used as negative control. Citric acid is a  weak  organic acid  that has the chemical formula  C6H8O7. It occurs naturally in  citrus fruits. In  biochemistry, it is an intermediate product in the  citric acid cycle, which occurs in the  metabolism  of all  aerobic organisms (Berovic and Legisa, 2007). Ethyl methanesulfonate  (EMS) used as positive control in experiment is a mutagenic, teratogenic, and possibly car- cinogenic  organic compound  and its chemical  formula C3H8SO3. EMS is often used in  genetics  as a muta- gen. Mutations induced by EMS can then be studied in genetic screens or other assays (Merck Index, 1989). After the completion of treatment the roots were counted and their lengths were measured for each onion. To determine mean root length in a root bundle for each bulb, root lengths of experimental and con- trol bulbs were measured with ruler at the end of treat- ment time. After then root tips were removed from the bulbs, fixed in 3:1 (v/v) ethanol:glacial acetic acid and stored overnight at 4ºC. The next day they were placed in 70% (v/v) aqueous alcohol and refrigerated until use. An average of five slides was made for each bulb using 31Cytogenetic effects of Fulvic acid on Allium cepa L. root tip meristem cells five root tips which hydrolyzed in 1N hydrochloric acid (HCl) for 3 min, and microscope slides were prepared by squashing the stained root tips in 2% (w/v) acetic orcein. Each slide was examined using Olympus BX51 at a total magnification of 40×10. Chromosomal aberrations were determined by scoring cells with bridges, fragments, sticky chromosomes, and polar deviations in 1000 cells per slide. Also micronucleus formation was determined in 1000 cells per slide. 5000 cells scored in total for each bulb (Fiskesjö 1993, 1997; Pavlica et al. 2000). 2.4. Statistical Analysis Statistical analyses were performed using the SPSS 20.0 software package program. Data on physicochemi- cal parameters, root length, root number, and mitotic index and chromosomal aberrations were compared using analysis of variance (One Way ANOVA) to con- firm the variability of the data and validity of results. Post-hoc test was used to describe the magnitude of var- iability. Differences between corresponding controls and exposure treatments were considered statistically signifi- cant at p <0 .05. 3. RESULTS 3.1. Morphological Analysis The results of the morphological analysis (root num- ber and root length) are presented in Table 1. These results show that all tested concentrations of fulvic acid caused increase in the root growth, and average root number in comparison to negative control, positive con- trol, and solvent control. The measured average root length is 2.26±1.07 cm in negative control, 1.40±0.40 cm in positive control, and 1.36 cm in solvent control. The average root length after 20 and 40 µg/ml fulvic acid treatment is found very high (4.10±0.41 and 4.22±0.69 cm, respectively) compared to controls (Table 1). The number of roots also increased in fulvic acid treatment groups compared to control groups. The highest root number is found in group treated with 80 µg/ml fulvic acid (Table 1). The root morphology in fulvic acid treat- ed groups was thinner and more fragile compared to the negative control group. 3.2. Cytogenetic Analysis With the objective of investigating the possible mechanism involved in root growth stimulation, cytoge- netic analysis was performed. Fulvic acid was found to stimulate mitotic index. A statistically significant differ- ence in the mitotic index of root meristems was found in negative, positive and solvent control. The increase in the mitotic index was found to be positively correlat- ed with the increase in concentration of the fulvic acid (Table 2). In the positive and solvent control groups, the mitotic index decreased significantly compared to the control group, and the mitotic index value approached zero in the solvent control group (Table 2). Cytogenetic alterations were investigated, and the results are described in Table 2 and Figure 1. Table 2 presents the percentage of the aberrant cells in dividing cells. Very few cells with polar deviation were observed in the negative control group. No chromosome aber- ration was observed except for polar deviation. In this group, total chromosome aberration was found very low (0.07%). The chromosome aberration rate in the posi- tive control group was found to be significantly higher than the control group (36.09%). Especially the anaphase bridge and stickiness have been observed to appear at a very high rate (p<0.05). In addition, breaks and polar deviations were observed in the positive control group. No chromosome aberration was observed in the solvent control group (citric acid), because there were only two divided cells in total and the mitotic index value was near zero in this group. The cell membrane and nucleus were deformed (Fig. 1a). In the groups treated with fulvic acid, breaks, sticki- ness and polar deviations appeared at very low rates. Total chromosome aberration percentages in these groups were insignificant compared to control and sol- vent control groups. The highest total chromosome aber- ration percentage in fulvic acid treated groups was 20 Table 1. The average root numbers and root lengths in control and treatment groups after 24h treatment (Analysis were carried by One Way ANOVA). Concentrations Average root number ± SD Average root lengths (cm ± SD) Negative control 19.2 ± 8.07 2.26 ± 1.07 Solvent control 20.2 ± 8.87 1.36 ± 0.24 EMS (positive control) 21.0 ± 7.81 1.40 ± 0.40 FA10 37.2 ± 4.43* 3.36 ± 0.54 FA20 31.8 ± 4.56 4.10 ± 0.41* FA40 37.6 ± 5.92* 4.22 ± 0.69* FA80 39.2 ± 5.17* 3.98 ± 1.29* FA100 34.8 ± 6.14* 3.68 ± 0.68 One Way ANOVA Analysis *p<0.05 is significant (EMS: 0.02M Ethyl methane sulfonate; Solvent control: 40 µg/ml citric acid; FA10: 10 µg/ml fulvic acid; FA20: 20 µg/ml fulvic acid; FA40: 40 µg/ml fulvic acid; FA80: 80 µg/ml fulvic acid; FA100: 100 µg/ml fulvic acid ). 32 Özlem Sultan Aslantürk and 40 μg/ml, respectively (Fig. 1d, e). This percentage is statistically insignificant when compared to the control and solvent control group. In addition, this percentage is very low compared to the chromosome aberration value obtained from the positive control group (EMS), and the difference is statistically significant (p <0.05). In the pos- itive control group (EMS), chromosome aberration per- centage has been found high, especially stickiness and anaphase bridge. Micronucleus formation results are also present in Table 2. Micronucleus formation was found at a very low level of 0.04 ‰ in 10 µg/ml fulvic acid treated group only which was statistically not significant. No micronu- cleus formation was found in other experimental groups (including negative, positive and solvent controls). As a result of this study, root length and mitotic index results show that fulvic acid promotes root growth by inducing division in Allium cepa root meristem cells. Chromosome aberration and micronucleus results also indicate that fulvic acid does not induce cytotoxic and genotoxic effects in the root meristem cells. 4. DISCUSSION In this study, cytogenetic effects of fulvic acid were evaluated by analyzing root growth and root morphol- ogy. Fulvic acid caused an increase in root growth and number, and there was a statistically significant differ- ence between fulvic acid and control groups (negative, positive and solvent controls). Cyto- and genotoxic- ity were estimated by observing cytological parameters, such as the mitotic index and number of chromosome abnormalities, including chromosome breaks, sticki- ness, and polar deviations. The mitotic index (MI) of A. cepa meristem cells treated with the EMS and citric acid (solvent control) was significantly decreased (0.83% and 0.01%, respectively) in comparison to negative control. Although EMS and citric acid significantly decreased the Table 2. Mitotic index values, percentage of chromosomal aberrations and thousandths of micronuclei in control and treatment groups after 24h treatment. Concentrations Total cells Total dividing cells Mitotic index (MI ± SD) Breaks (%± SD) Anaphase bridge (%± SD) Stickiness (%± SD) Polar deviation (%± SD) Total aberrant cells (%± SD) Micronuclei (%o ± SD) Negative control 25000 1241 4.96 ± 0.31 0 ± 0 0 ± 0 0 ± 0 0.07 ± 0.03 0.07 ± 0.03 0 ± 0 Solvent control 25000 2 0.01 ± 0.17* 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 EMS (positive control) 25000 209 0.83 ± 0.11* 1.17± 0.26 10.47 ± 9.59*20.05 ± 6.90* 4.39 ± 6.03 36.09 ± 5.48* 0 ± 0 FA10 25000 2893 11.57 ± 1.93* 0.04 ± 0.01 0 ± 0 0± 0 0.51 ± 0.61 0.52 ± 0.60 0.04 ± 0.01 FA20 25000 3111 12.44 ±0.51* 0 ± 0 0 ± 0 0.03 ± 0.07 2.87 ± 0.53 2.90 ± 0.52 0 ± 0 FA40 25000 3559 14.23± 0.41* 0 ± 0 0 ± 0 0 ± 0 2.90 ± 0.42 2.90 ± 0.42 0 ± 0 FA80 25000 3566 14.26 ± 0.35* 0.03 ± 0.06 0 ± 0 0.03 ± 0.06 2.68 ± 0.51 2.73 ± 0.61 0 ± 0 FA100 25000 2988 11.95 ± 0.54* 0.03 ± 0.08 0 ± 0 0 ± 0 2.38 ± 0.17 2.41 ± 0.21 0 ± 0 One Way ANOVA Analysis *p<0.05 is significant (EMS: 0.02M Ethyl methane sulfonate; Solvent control: 40 µg/ml citric acid; FA10: 10 µg/ ml fulvic acid; FA20: 20 µg/ml fulvic acid; FA40: 40 µg/ml fulvic acid; FA80: 80 µg/ml fulvic acid; FA100: 100 µg/ml fulvic acid. 25000 cells/ group were evaluated for MI and CA) d a e b c Fig. 1. a: membrane and nucleus deformation in solvent control (cit- ric acid) group; b: stickiness; c: stickiness and polar deviation in pos- itive control group; d: polar deviation in 20 µg/ml fulvic acid treat- ment group; e: polar deviation 80 µg/ml fulvic acid treatment group. 33Cytogenetic effects of Fulvic acid on Allium cepa L. root tip meristem cells mitotic index, fulvic acid treatment increased the mitotic index in A. cepa meristem cells at all concentrations sig- nificantly (Table 2). The mitotic index is measure of the mitotic activity of a cell population. It measures the pro- portion of cells in the M-phase of the cell cycle (Rojas et al. 1993). Therefore, the increase of MI in groups treat- ed with fulvic acid, in comparison to negative control, suggests that fulvic acid could have proliferative effect on the meristem cells of A. cepa. The increased mitotic index in A. cepa root tip cells treated with fulvic acid is probably due to induction of DNA synthesis and pro- motion of cell cycle. The MI results of fulvic acid treat- ment groups are consistent with literature data. Previous studies suggest that humic substances including fulvic acid enhanced stimulation of seedling germination and growth of plants (Kulikova et al. 2002; Pena-Méndez et al. 2005; Van Rensburg 2015). Humic substances affect the development of organisms. Being utilized as a sub- strate (a source of organic carbon) or nutrient source (N, P, trace elements and vitamins), humic substances can serve as a moiety of the biosynthesis chains. On the other hand, beneficial effects of humic substances on the plants are often attributed to hormone-like activity of these substances (Nardi, 1994; Nardi et al. 2002; Piccolo et al. 1992; Kulikova et al. 2002). Since humic substances originate from the chemical and biological decomposi- tion of plant and animal residues, and from metabolic activities of microorganisms, they might have character- istics of hormones. It was shown that humic substances enhanced plant growth by exhibiting auxin-like activ- ity (Kulikova et al. 2002). Some researchers reported that humic and, in particular, fulvic acids showed some auxin, gibberellin or cytokinin-like activity (Phuong and Tichy 1973; Nardi, 1994; Kulikova et al. 2002). Further- more, fulvic acid, as a plant growth regulator, is involved in plant response to several environmental stress fac- tors, and is reported to affect growth and development of plants (Heil 2005; Shahid et al. 2012). There are also studies of growth promoting effects of fulvic acid on plants as well as growth enhancing effects on animals (Nardi et al. 2002; Heil 2005; Bai et al. 2013), because of its antioxidant, antimicrobial and anti-inflammatory properties (Yamada et al. 2007; Van Rensburg et al. 2001; Sherry et al. 2013). Gao et al. (2017) have shown that when fulvic acid is used as food supplements for 60 days, it increases growth performance of Paramisgurnus dabryanus (Sauvage) and improves its intestinal health conditions (Gao et al., 2017). Also, Bai et al. (2013) reported that supplementation of diets with fulvic acid is an effective way to increase growth performance, reduce backfat thickness, and improve meat quality in growing-finishing pig (Bai et al. 2013). Chromosome aberration and micronucleus results of this study show that fulvic acid does not induce gen- otoxic effects in the root meristem cells in comparison with control groups. Although in the positive control group (EMS), chromosome aberration rate (especially stickiness and anaphase bridges) has been found high, but in the fulvic acid treatment groups, breaks, sticki- ness and polar deviations appeared at very low rates. The total chromosome aberration percentages in fulvic acid treatment groups were found insignificant compared to control and solvent control groups (Table 3). As a result of literature screening, different data on cytotoxic, genotoxic and mutagenic effects of fulvic acid have been reached. Qui et al. (2007) reported that fulvic acid has protective effect against copper toxicity to the polychaete Hydroidas elegans larvae, and such an effect is caused by the reduction in labile copper due to Cu- FA (copper-fulvic acid) complexation (Qui et al. 2007). Also, it has been suggested that humic and fulvic acids have desmutagenic effect (inactivation of mutagens out- side the cell) on Vicia faba root tip cells treated with maleic hydrazide, whereas they have no antimutagenic effect (Ferrara et al. 2000). Ferrara et al. (2004) reported anticlastogenic, antitoxic and sorption effects of humic substances (soil humic acid, peat humic acid and peat fulvic acid) on the maleic hydrazide tested in legumi- nous plants, Vicia faba and Pisum sativum L (Ferrara et al. 2004). However, no data has been found on the direct cytogenetic effects of fulvic acid in the literature. Therefore, the data obtained in this study is impor- tant in terms of its contribution to the scientific litera- ture in this regard. Furthermore, fulvic acid is currently being used in planting and growing plants, especially in agriculture, and as complementary in treatment of human and animal health. Its use is becoming increas- ingly widespread. Therefore, it is important to deter- mine whether this substance is safe for the environment, and animal and human health. The results of this study suggest that fulvic acid stimulate the root growth in A. cepa, and it does not have cytotoxic and genotoxic effects on A. cepa root meristem cells. These results are impor- tant, because it is a preliminary study on the safety of using of fulvic acid. However, in order to be able to say that the use of fulvic acid is safe, more detailed studies have to be carried out using different test systems. ACKNOWLEDGEMENT I would like to thank the Chemical Engineer Yusuf Mert SÖNMEZ who donated the pure fulvic acid for use in this study. Also I would like to thank Assoc. Prof. Dr. 34 Özlem Sultan Aslantürk Tülay AŞKIN ÇELİK for her helpful advice in this man- uscript. DISCLOSURE STATEMENT No potential conflict of interest was reported by the author. REFERENCES Aşkın Çelik T, Aslantürk ÖS. 2007. 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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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