Genotoxic effects of heavy metals in Trachyspermum ammi (L.) Sprague

Transcription

1 Chromosome Botany (2013) 8: GENOTOXIC EFFECTS OF HEAVY METALS IN TRACHYSPERMUM AMMI Copyright 2013 by the International Society of Chromosome Botany 81 Genotoxic effects of heavy metals in Trachyspermum ammi (L.) Sprague G. Kumar and Harshita Dwivedi 1 Plant Genetics Laboratory, Department of Botany, University of Allahabad , India 1 Author for Correspondence: harshitadwivedi88@gmail.com Received August 17, 2013; accepted October 5, 2013 ABSTRACT. Heavy metals are ubiquitous in nature. Toxicity of metals depends upon their optimum level in environment. Chromosomal plant assay is an important tool for the estimation of genome damaging property of the pollutants above the optimum level. In the present investigation on ajwain (Trachyspermum ammi (L.) Sprague), cyto-chromotoxicty was evaluated in terms of reduced mitotic activity and various mitotic irregularities comprising scattering, stickiness, precocious movement, laggards, bridges, c-mitosis, etc. Five concentrations (50, 75,100,150 and 200 ppm) of both the metal salts (Pb(NO 3 ) 2 and Cr 2 O 3 ) were used. Dose dependent increase in the chromosomal aberrations and decrease in mitotic indieces were observed. On comparing mitotoxicity of both the metals, Pb was more growth inhibitory and mitotoxic than Cr. KEYWORDS: Chromosomal aberrations, Chromosomal plant assay, Cr 2 O 3, Cyto-chromotoxicity, Mitotoxicity, Pb(NO 3 ) 2 Naturally, our environment contains metals as constituents of earth s crust. Their stability depends upon the fact that they cannot be degraded or destroyed but accumulate and persist continuously in the ecosystem. Contamination of the environment with these pollutants started way before the industrial revolution of pollution originating from Roman mining and smelters in 500 B.C.(Nriagu 1996).The previous studies suggested that low level of these metals does not give any harmful effect in the ecosystem, but as the level of these metals increases they become among the most important sorts of contamination (Tangahu et al. 2011).The primary cause of increasing percentage of these metals is anthropogenic activities. Effluents of different types of industries, mining, agricultural chemicals, combustion by-product and fuels are some sources of metal contamination. Previous studies suggested that 13 metals are considered as heavy metals. Among these heavy metals lead and chromium are highly toxic and at high level they can be carcinogenic toxicant but the pattern of toxicity is quite different. Cr toxicity is directly related with the DNA degradation whereas Pb causes genotoxicity by indirect interactions such as it interferes with DNA repairing mechanism causing genomic instability. Metal toxicity requires constant vigilance and precise and unbiased evaluation for both the environmental and human health concern (Rodriguez 2011). The toxicity of metals and their compounds largely depend upon their bioavailability, i.e. the mechanisms of uptake through the cell s membrane, intracellular distribution and binding to cellular macromolecules (Beyersmann and Hartwig 2008). Among the different models, plants are essential components of ecosystem and play vital role in food chain. Food resources from plants such as vegetables, fruits, grains, etc can be contaminated by accumulating metals from surrounding soil and water which eventually reflects in their final consumers. In case of plants, the permeability of cell membrane, the biochemical activities at the macromolecular level and the regular growth and reproduction of cells are negatively affected (Tuna et al. 2001). Ajwain Trachyspermum ammi (L.) Sprague is an Egyptian herb that is also recognized in India due to its medicinal use and also of great importance as a spice. The use of aromatic medicinal herbs to relieve and treat many human diseases has been increased in world because of their mild feature and low side effects.(abu-darwish 2009).Medicinal herbs can be easily contaminated with heavy metals from the environment(soil, water or air) during growth and manufacturing process when the readymade products are produced (Al-Eisawi 1982). Many of the previous physiochemical studies show the toxic effects of these metals. As per these available data alone should not be considered sufficient enough to determine the exact impact of toxicity of metals in the growth pattern of the plants. Plant roots are very useful in this testing because the root tips are often the first to be exposed to chemicals spread in nature, in the soil and water (Odeigah et al. 2009). The present research work aims to compile the genotoxic effect of lead nitrate Pb(NO 3 ) 2 and Chromium oxide (Cr 2 O 3 ) on the somatic cells of ajwain Trachyspermum ammi (L.) Sprague and its outcome. MATERIAL AND METHODS The seeds of ajwain var. AA-1 were collected from National Research Centre for Seed Spices, Ajmer, Rajasthan, India. For mitotic studies, germinated seeds were treated with lead nitrate and chromium oxide in aqueous solution for 3 hrs. at different concentrations viz. 50,75,100,150 and 200 ppm. One set of germinated seeds was also maintained as control by treating with distilled water only. The metal treated seeds were washed with water thoroughly and fixed in Carnoy s fixative (1:3 glacial acetic acid and alcohol solution) for 24 hrs. The root tips were preserved in absolute alcohol. Slides were prepared by using chromosome squash technique with 2% acetocarmine.active mitotic index was calculated by using the following formula:

2 82 KUMAR AND DWIVEDI Fig. 1. Effects of heavy metals in chromosomes of Trachyspermum ammi (L.) Sprague. A. Prophase., B. Normal metaphase (2n=18). C. Normal anaphase (18:18). D. Precocious movement of chromosome at metaphase. E. Scattering of chromosomes. F. Stickiness at metaphase. G. C-mitosis at metaphase. H. Arrow indicating laggard chromosome at metaphase. I. Stickiness at anaphase. J.Arrow indicating incomplete bridge at anaphase. K. Arrow indicating curved bridge at anaphase. L. Arrow indicating forward movement at anaphase. Scale bar = 10μm.

3 GENOTOXIC EFFECTS OF HEAVY METALS IN TRACHYSPERMUM AMMI 83 Active mitotic index= Total no.of dividing cells Total no.of cells observed 100 OBSERVATIONS In ajwain plant, the haploid chromosome number has been found to be n=9. The cytological observation revealed that the mitotic index was recorded to be 11.96% for the control set, with standard chromosomal behaviour, showing regular array of chromosomes at metaphase plate (Fig. 1B) and regular separation (18:18) at anaphase as shown in (Fig. 1C). However, after the treatment of the root tip cells with Pb(NO 3 ) 2 and Cr 2 O 3, changes in normal chromosomal behaviour and rate of dividing cells was observed. The percentage of both the mitotic index and chromosomal abnormalities are inversely related with each other. The gradient of AMI and chromosomal aberrations with respect to the dosage of both heavy metals has been summerized in Tables 1 and 2, respectively. Figure 2 shows a sharp decline in the value of active mitotic index along with the increasing concentration of both the metals. In case of Pb treated set, the percentage of active mitotic index reduced from to 4.31% whereas in case of Cr, it was reduced from to 8.12%. Both the metals induces various types of chromosomal abnormalities (Fig.1) such as precocious movement (Fig.1D), scattering (Fig.1E), stickiness (Fig:1-F), c-metaphase (Fig.1G), laggards (Fig. 1H), bridges (Fig. 1 J-K), forward movement (Fig. 1L), etc. The gradation of abnormality percentage with increasing doses of heavy metal concentration has been shown in Fig. 3. The abnormality percentage increased from 3.94% at 50 ppm concentration to 13.51% at 00 ppm concentration of Pb whereas in case of Cr, from 2.63% at 50 ppm concentration to 200 ppm concentration of Pb whereas in case of Cr, from 2.63% at 50 ppm concentration to 11.23% at 200 ppm concentration. In Pb treated set, out of the observed abnormality percentage (13.51%), the major portion (5.95%) was covered by stickiness. Hence, it is evident from the data that most prominent abnormality in case of Pb was stickiness. On the contrary, in Cr, out of the observed abnormality percentage (11.23%), scattering (4.48%) was predominent. DISCUSSION The mitotic index reflects the frequency of cell division and it is regarded as important parameter while determining the rate of root growth (Liu et al. 1992).The result of present study demonstrates that there is a gradual reduction in mitotic index, as the concentration of metals increases. This reduction in the mitotic index and a wide range of chromosomal aberrations clearly indicate that heavy metals ars clastogenic. Chemicals that induce Table 1. Effect of Pb(NO 3 ) 2 and Cr 2 O 3 on mitotic index of Trachyspermum ammi (L.)Sprague. Treatment Concentration (ppm) Mitotic Index (%) Control Pb(NO 3 ) Cr 2 O Table 2. Effect of Pb(NO 3 ) 2 and Cr 2 O 3 in the somatic cells of ajwain (Trachyspermum ammi (L.) Sprague) Treatment Dose(ppm) Metaphasic abnormalities Anaphasic abnormalities Oth Tab(%) Sc St Pr Lg Bg Fw St Pb(NO 3 ) Cr 2 O Sc-Scattering, St-Stickiness, Pr-precocious, Lg-laggards, Bg-Bridges, Fw-Forward movement, Oth-Other abnormalities, Tab-Total abnomality

4 84 KUMAR AND DWIVEDI 14 Actve Mitotic Index (AMI) % Cr Pb 2 0 Control Concentration (ppm) Fig. 2. Comparative account of active mitotic index values induced with different treatment doses of lead nitrate and chromium oxide. chromosomal breakage are called as clastogens and their action on chromosomes is generally regarded to involve an action on DNA (Grant 1978; Chauhan et al. 1990). In our investigation, stickiness and scattering were found to be the most frequent abnormality after treating the root tip cells with Pb and Cr, respectively. Sticky behavior of chromosomes clearly indicates the genomic loss because chromosomes lose their physical identity. Stickiness may be defined as the physical adhesion involving mainly the proteinaceous matrix of chromatin material (Patil and Bhat 1992). The complexes formed by heavy metals are highly reactive, thus it may also be possible that these complexes either directly interact with DNA or histone or non-histone proteins which bring about the change in surface property of chromosomes and led them to be sticky.(kumar and Rai 2007). The sticky chromosomes may result from defective functioning of one or two types of specific non-histone proteins involved in chromosome organization that are needed for chromatid seperation and segregation (Gaulden 1987). Stickiness may induce many of the other abnormalities such as laggards, bridges and fragments which led to the cell death. Normal spindle activity is very important for chromosomal alignment at equatorial position at metaphase and for normal cell division. In Cr treated set scattering is found to be the most prominent abnormality. This may be due to inhibition of polymerization of microtubule. Cr 3+ can reduce the free Ca 2+ level in the cytoplasm (Liu et al. 1992) that s why calmoduline (Cheung et al ) does not activate the Ca-ATPase (Xu 1985). According to Means and Deadman (1980) calmoduline was found in the mitotic spindle and regulate the polymerization and depolymerization of microtubule due to which the movement of chromosome occurs.(li and Sun 1991). Cr also binds with the carboxyl group of the peptide chains and forms complexes with the proteins and by this mechanism Cr binds with tubulin molecules and it may change their structure leading to spindle dysfunction. (Mishra et al. 2012). Apart from causing scattering, spindle dysfunction also correlated with precocious movement, c-metaphase and laggards type of chromosomal aberrations. Another reason of precocious separation of chromosome is possible due to chemical breaking of the protein moiety of nucleoprotein backbone (Patnaik 1984). Bridges might be formed due to the sticky behaviour of chromosomes which could not move towards he poles at anaphase (Kumar and Rai 2007). The breaks at the same locus and their lateral fusion might have led to the formation of dicentric chromosome (Rai and Kumar 2010). The dicentric and acentric chromosomes formed by inversion of chromosomes. These dicentric chromosomes were involved in the formation of bridges. The dicentric chromosomes were pulled equally towards both poles at anaphase and bridges were formed (Anis et al. 1998). Lagged chromosomes might depend upon the moving speed and process of an individual chromosome differing from normal ones (Qian 2004), which may led to the formation of micronuclei (Kumar and Rai 2007). In normal condition, damaged DNA was repaired at various checkpoints of cell cycle. If the process of repairing is inhibited by some agents, the damaged DNA replicates and enters into the M phase and causes various abnormal consequences. According to Rodriguez (2011), Pb can induce blockage of cell cycle at G 2 /M checkpoint

5 GENOTOXIC EFFECTS OF HEAVY METALS IN TRACHYSPERMUM AMMI Total Abnormality Percentage(%) Cr Pb Control Concentration(ppm) Fig. 3. Comparative account of total abnormality percentage induced by lead nitrate and chromium oxide. due to severe DNA degradation, and giving the cells extra time to either repair the damage(őconell and Cimprich, 2005) or activate an apoptosis (Santos and Rodriguez 2012), which may led to the chromosomal aberrations. Pb has the ability to replace the Ca 2+ /Zn 2+ in enzymes involved in DNA processing and repairing and enhancing the genotoxicity when combined with other DNA damaging agents (Garcia-Leston et al. 2010). On the basis of these results, it can be concluded that although both the studied heavy metals are capable of inducing wide range of chromosomal abnormalities but Pb(NO 3 ) 2 was proved to be highly genotoxic in comparison to Cr 2 O 3. Mutagenesis is the important tool for identifying the deleterious effect of heavy metals at genome level in organisms. This type of mutagenic data from chromosome assay in plants may be useful as it reports the deleterious effect of these pollutants at the genome level, which may help for maintaining a stable ecosystem. LITERATURE CITED Abu-Darwish, M. S., Essential oils and heavy metals content of some aromatic medicinal plants grown in Ash- Shoubak region,south of Jordan. Daptogen interregional centre, Journal of food, Agriculture and Environment Vol. 7 Issue 3-4: Al-Eisawi, D. M, List of Jordan vascular plants.mitt. Bot. Staat. M unchen 18: Anis, M., Shiran, B. and Wani, A. A Genotoxic effect of aldrin and malathion on the root meristem of Vicia faba. Jour. Cytol. Genet. 33: Beyersmann, D., Hartwig, A Carcinogenic metal compounds: recent inside into molecular and cellular mechanisms. Archives of Toxicology 82: Chauhan, L. K. S. and Sundararaman, V Effect of substituted urea on plants.i. Cytological effects of isopruturon on the root meristem cells of A. cepa. Cytologia 55: Cheung, W. Y Calcium and cell division, Vol Acad Press, London, New York. García-Lestón, J., Méndez, J., Pásaro, E. and Laffon, B Genotoxic effects of Lead: An updated review. Environment International, 36: Gaulden, M. E. and Hypothesis Some mutagens directly alter specific chromosomal proteins (DNA topoisomerase II and peripheral proteins) to produce chromosome stickiness, which causes chromosome aberrations. Mutagenesis 2: Grant, W. F Chromosome aberrations in plants as monitoring system. Environ. Health perspect. 27: Kumar, G. and Rai, P Genotoxic potential of mercury and cadmium in soybean. Turk Journ. Biol. 31: Li, X. and Sun, D. Y A study on CaM distribution in cells of living things. Chin. Journ. Cell Biol. 13(1): 1-6. Liu et al Effects of trivalent and hexavalent chromium on root growth and cell division of Allium cepa Hereditas 11 7: Means, A. R. and Deadman, J. R Calmodulin-an intracellular calcium receptor. Nature 285: Mishra, S.N. et al., 2012.Genotoxicity testing of chromium trioxide-a study using Vicia bioassay.jour. Phytol.,4(5): Nriagu, J. O A History of Global Metal Pollution. Science 272: 223. O Connell, M. J. and Cimprich, K. A G2 damage checkpoints: what is the turn-on? Journ. Cell Sci. 118: 1-6. Odeigah et al., Cytogenotoxicity evaluation of two industrial effluents using Allium cepa assay. Afr. Journ. Environm. Sci. Techn. 4: Patil, B. C. and Bhat, G. I A comparative study of MH and EMS in the induction of chromosomal aberrations on lateral root meristem in Clitoria ternatea L Cytologia 50: Patnaik, S., Saran, B. L. and Patnik, S. M Effect of zarda (processed tobacco leaf) extract on the chromosomes of Allium cepa. Cytologia 49: Qian, X. W Mutagenic effects of chromium trioxide on root tip cells of Vicia faba, Journ. Zhejiang Univ. Sci, 5(12): Rai, P.K. and Kumar 2010.The genotoxic potential of two heavy metals in inbred lines of maize (Zea mays L.). Turk. Journ. Bot. 34: Santos and Rodriguez, Review on some emerging endpoints of chromium (IV) and lead phytotoxicity. Univ. Aveiro, Aveiro, Portugal. Ecotoxicol.:

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