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Effects of Deltamethrin on the Ultrastructures of the Root Meristem Cells of Allium cepa
Pesticide Biochemistry and Physiology 64, 135–147 (1999) Article ID pest.1999.2416, available online at http://www.idealibrary.com on
Effects of Deltamethrin on the Ultrastructures of the Root Meristem Cells of Allium cepa L. K. S. Chauhan, P. N. Saxena, and S. K. Gupta1 Cell Biology Section, Industrial Toxicology Research Centre, P.O. Box 80, Mahatma Gandhi Road, Lucknow 226001, India Received October 29, 1998; accepted April 28, 1999 The EC50 value of commercially formulated deltamethrin (Decis 2.8% EC) was determined to be 0.87 ppm against Allium root growth. Root meristem cells were exposed to deltamethrin (0.5–2 ppm) for 6 to 24 h and examined by transmission electron microscopy. Deltamethrin induced various morphological abnormalities in the cell wall, plasma membrane, and Golgi apparatus dictyosomes. The microfibrillar arrangement of cell wall appeared to be amorphous in treated cells. The plasma membrane showed extensive dehiscence from the cell wall. The stacked cisternal membrane of dictyosomes were seem to be degenerated and the vesicles emanating from the dictyosomes were found to be accumulated around the degenerated dictyosome remnants. Sporadic dilation of dictyosome cisternae was also observed in the treated cells. Most nuclei in treated cells were observed to contain condensed chromatin material. The number of microtubles in the preprophase band was significantly reduced in 24 h treatment. Deltamethrin frequently disturbed the parallel array of spindle microtubules in metaphase cells. Telophase cells exposed to deltamethrin revealed incomplete, if not irregular, cell wall formation. Present findings suggest that the action of deltamethrin on microtubules is to an extent similar to that of microtubule depolymerizing drugs, which is the primary cause of toxicity leading to the induction of various mitotic aberrations. However, induction of structural and functional aberrations in the Golgi apparatus dictyosomes indicates the effect of deltamethrin on intercisternal elements and microfilaments, which possibly keeps the cisternal stack intact and directs the intracellular transport of vesicles. q1999 Academic Press
INTRODUCTION
Synthetic pyrethroids are photostable, environmentally compatible, and potent insecticides for widespread use in agriculture and veterinary and public health (1). Their usage is perferred over organochlorine and organophosphate insecticides in view of their safety to nontarget organisms (plants and animals) due to rapid hydrolytic disposition (2). The toxicity of deltamethrin (CAS No. 5291863-5; proper name S-alpha-cyano-3-phenoxybenzyl-1R-cis-3-2, 2-dibromoviny 1-2, 2-dimethylethylcyclopropane carboxylate) has been elucidated in animals (3, 4) and plants (5–7), indicating similarities in its toxicokinetic profile in higher eukaryotes. Considering Allium root metristem cells as a suitable model and also 1 To whom correspondence should be addressed. Fax: 0522-228227.
a recepient of these agricultural chemicals, we extended our previous cytological observations (6) to the ultrastructural level to elucidate the subcellular targets of deltamethrin toxicity. The observations are expected to improve our understanding of the mode of action of deltamethrin and establish Allium cepa as an appropriate model for biomonitoring of environmental chemicals at cellular and subcellular levels. MATERIALS AND METHODS
Chemicals Commercial formulation of deltamethrin (Decis 2.8% EC) was obtained from Hoechst India Ltd. Fixatives and other embedding mediums used in electron microscopy were procured from LADD Research Industries, England, and all other chemicals/reagents used in this study were purchased from E. Merck/Glaxo, India.
135 0048-3575/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
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EC50 Determination Following the standard protocol of Fiskesjo (8), healthy and clean onion bulbs were mounted on glass jars filled with aqueous concentrations (containing less than 0.6% DMSO) of deltamethrin (0.25–2.84 ppm) for rooting at 22 6 18C in B.O.D. incubator. A minimum of five bulbs was rooted in each concentration. Control bulbs were maintained in concentrations of DMSO equal to those used for test concentration. Every 24 h after each concentration was renewed and 5th day, root lengths from treated and control bulbs were measured. The average root growths were plotted against the test concentrations and the point showing 50% growth compared to controls (considered as 100%) was marked as the EC50 value. Electron Microscopy Fresh and healthy onion bulbs were thoroughly washed and rooted in tap water. Roots
1–2 cm long were exposed to 0.5, 1.0, and 2.0 ppm of deltamethrin for 6 or 24 h at 22 6 18C. Control bulbs were maintained in distilled water mixed with a quantity of DMSO (.0.1%) equal to that used to make the test solution. At the end of the exposure, roots were thoroughly washed and 1 mm of the root tip portion was fixed in 4% glutaraldehyde (prepared in sodium cacodylate buffer, pH 6.8) for 1 h at room temperature and post fixed with 1% osmium tetroxide (same buffer) for 1 h. Fixed root tips were washed with cacodylate buffer, dehydrated in an ethanol series (30–100%), and embedded in Aralditeepon medium (9). Thin sections were cut with glass knives on an LKB IV ultramicrotome and stained with uranyl acetate and lead citrate to examine under a Philips EM400 transmission electron microscope. To quantify the effect of deltamethrin on the cortical microtubules, 10 preprophase cells, selected at random, from five root tips were
FIG. 1. Concentration-wise root growth retardation in the deltamethrin-treated Allium roots and EC50 determination. EC50 5 0.87 ppm.
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137
FIG. 2. Electron micrograph of a normal cell wall with intact plasma membrane. Homogeneous electron density in the microfibrillar region of cell wall. 317,600.
examined to count microtubules. The statistical significance of results was calculated through Student’s t test. RESULTS
EC50 Value The treatment of deltamethrin (0.25–2.84 ppm) induced concentration-wise root growth retardation, without obvious morphological changes in the Allium roots. The EC50 value of deltamethrin was determined to be 0.87 ppm (Fig. 1). In the present experiment 1.0 ppm of deltamethrin has been used as EC50 with two other concentrations, 0.5 and 2.0 ppm, for comparison of effects. Ultrastructural Effects Cytoplasmic organelles. Cell wall, plasma membrane, and Golgi apparatus appeared to be highly sensitive to deltamethrin exposure. The
electron-dense microfibrillar appearance of the normal control cell wall was observed to be disturbed in 6-h treatment with deltamethrin (1 and 2 ppm; Figs. 2 and 3). Intact plasma membrane as seen in control cells appeared to be protruded into the cytoplasm and separated from the cell wall (Fig. 3). Vesicles extruded from cytoplasm were frequently noted in protruded portion of plasma membrane (Fig. 3). In comparison with control cells, vacuolization of cytoplasm was found to be increased in the treated cells. The cortical microtubules alongside the plasmalemma were seem to be undisturbed at 6-h treatment (Fig. 3). The treatment of similar concentrations of deltamethrin (0.5–2 ppm) exhibited drastic structural changes in the cell wall and plasma membrane at 24 h. The microfibrillar array of the cell wall appeared to be amorphous (Fig. 4). Extensive dehisence of the plasma membrane in these treated cells formed a wide gap from the
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FIG. 3. Electron micrograph of a deltamethrin-treated cell showing initial stage of plasma membrane dehiscence and sporadic protrusions (arrows). Loss of microfibrillar array of cell wall and accumulation of vesicles in the spaces formed between the cell wall and the plasma membrane. Vacoules (V) are also visible (2 ppm for 6 h). 328,800.
cell wall (Fig. 4). The cortical microtubules alongside the plasmalemma observed at 6-h treatment were not seen in the cells exposed for 24 h. The normal stacked cisternae of Golgi apparatus dictyosomes did not reveal any structural change in 6-h treatment of deltamethrin except conspicuous accumulation of vesicles (Figs. 5 and 6). However, root tip cells exposed for 24 h to deltamethrin (1 and 2 ppm) revealed various aberrations in dictyosome structure. Most of these cells were found to contain deformed or degenerated dictyosomes surrounded by numerous vesicles (Figs. 6 and 7). Frequently the middle or last cisternae (toward maturing phase) of the dictyosomes were found to be highly dilated like a sack (Fig. 8). The shape and structure of mitochondria and
proplastids were observed to be normal in deltamethrin-treated cells. Interphase and mitosis. Interphase nuclei, other than condensation of chromatin materials did not show any visible change at 6-h treatment. However, a number of cells were found to contain presumptive cytoplasmic inclusions in the nuclei after 24-h exposure to deltamethrin. The presence of numerous cortical microtubules marked the preprophase stage of mitosis in normal root meristem cells (Fig. 9). At 6h treatment, deltamethrin (0.5–2 ppm) did not induce any visible effect on preprophase microtubules, while at 24-h exposure, the number of cortical microtubules in the preprophase band was significantly reduced, particularly with 1 and 2 ppm of deltamethrin (Table 1, Fig. 10).
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FIG. 4. Large gaps between plasma membrane and cell wall. Microfibrillar region appears amorphous. Breaks are visible in plasma membrane (arrows; 2 ppm for 24 h). 322,000.
The characteristic parallel array of spindle microtubules of normal metaphase cells appeared to be disturbed within 6 h of exposure to deltamethrin (1 and 2 ppm). Chromosomes of these metaphase cells were found to be scattered all over the cytoplasm, instead of linearly arranged at the equitorial plate. Analysis of metaphase cells exposed 24 h displayed randomly dispersed short microtubules lacking pole-to-pole continuity. The normal telophase stage of mitosis revealed an enormous number of phragmoplast microtubules and associated dictyosomederived vesicles at the site of cell wall formation. The cross walls laid down were complete and linear (Fig. 11). The telophase cells exposed to deltamethrin (0.5 to 2 ppm) for 24 h revealed incomplete cell wall formation (Fig. 12). When magnified, these cells were found to contain short and misdirected microtubules and vesicles unevenly scattered at the site of wall formation
(Fig. 13). The cell walls, wherever laid down in treated cells, were either irregular or incomplete. DISCUSSION
In the present study, A. cepa root meristem cells have been used to elucidate the toxicity potential of a commercially formulated synthetic pyrethroid insecticide, deltamethrin. Present findings validate the use of root meristem cells of A. cepa as an appropriate model for studying the biological effects of environmental chemicals at subcellular levels. Further determination of EC50 values by measuring the root growth retardation is a simple yet reliable index of the cytotoxic potential of xenobiotics used in the present study (8, 10). The present electron microscopic findings are in agreement with our previous communication in which deltamethrin was shown to induce various spindle-related abnormalities in Allium root meristem cells (6). Most mitotic disruptors,
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FIG. 5. Photomicrograph of a normal dictyosome showing stacked cistrnae and no accumulation of secretory vesicles. 395,000.
including herbicides and microtubule polymerizing drugs, irrespective of their chemical structure and mode of action, induce structural alterations in cytoplasmic organelles, particularly in the cell wall and plasma membrane (11– 13). Present findings indicate that the action of deltamethrin on microtubules is partly similar to that of colchicine and vinblastine. These
TABLE 1 Effect of Deltamethrin on the Density of Cortical Microtubules in the Preprophase Band of the Root Meristem Cells of Allium cepa Concentration of deltamethrin in ppm
Period of exposure in h
No. of cortical microtubules/ preprophase band
0 1 2
24 24 24
80.42 6 2.72 19.57 6 3.05** 06.67 6 1.16**
Note. Values are mean 6SE from 10 preprophase cells. ** P , 0.001.
chemicals, in addition to affecting microtubules, disturb the fibrillar orientation of cell wall and cause extensive dehisence of plasma membrane (11). Further, structural alteration of cytoplasmic organelles with the treatment of deltamethrin is quite possible as its intracellular localization in the plant cells has been demonstrated in the cytoplasm (7). Our findings suggest that in the early phase of treatments, deltamethrin stops intracellular transport of dictyosome-derived vesicles through its action on the microfilamentous elements, which leads to the accumulation of vesicles in the cytoplasm. Similar accumulation of secretory vesicles, blebbed from dictyosomes, has been reported in plant cells exposed to a fungal product, cytochalasin B, and were interpreted to be the effect of the compound on the microfilamentous elements (actin filaments) which persist in plant cell cytoplasm in association with microtubules throughout cell division
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141
FIG. 6. Photomicrograph of a deltamethrin-treated cell showing degenerated cisternal membranes of dictyosomes (arrow) and accumulation of numerous secretory vesicles (1 ppm for 24 h). 346,000.
(14, 15). Deltamethrin affects the actin filaments which possibly direct the vesicle flow either independently or in association with microtubules. On the other hand, degeneration and deformation of dictyosomes appearing in the later phases of treatment, indicates the effect of deltamethrin on the intercisternal elements. In plant cells, intercisternal elements seem to be responsible for the structural integrity of dictyosomes as are microtubules in animal cells (14, 16). This is more evident when microtubule disruptors, such as antipolymerizing drugs, which are normally shown to induce structural aberrations in the Golgi apparatus of animal cells, do not show structural alterations in the dictyosomes of plant cells (14, 16, 17). However, further studies are required to confirm the effect of deltamethrin on the microfilaments using rhodamine-labeled phalloidin stain or antibodies against actin.
Significant and dose-dependant reduction of cortical microtubules in the preprophase band suggests the effect of deltamethrin on the polymerization of microtubules (11, 19). The disruption of metaphase microtubules as observed in the present study possibly leads to the formation of mitotic aberrations visible at the light microscopic level (6). Such functional impairment of spindle microtubules has been reported in plants with several pesticidal chemicals (6, 10, 12, 13). Similarly, induction of binucleate cells due to the loss of phragmoplast microtubules is a characteristic effect of mitotic disruptors (6, 11, 18). Cytokinesis in plant cells is a complex process in which dictyosome-derived vesicles are regularly deposited by the phragmoplast microtubules. Deltamethrin inhibits this process in the root meristem cells through its action on microtubules and dictysomes; consequently, irregular or incomplete cell walls are formed, which appear
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FIG. 7. Magnified view of a tangentially sectioned dictyosome showing accumulation of vesicles. (2 ppm for 6 h). 3100,000.
ANTIMITOTIC EFFECTS OF DELTAMETHRIN
FIG. 8. Photomicrograph of a deltamethrin-treated Golgi apparatus dictyosome showing fused cisternal membranes and dilation of last cisterna (arrow). 380,000.
FIG. 9. Normal preprophase stage showing numerous microtubules in the cortical band (arrows). 3110,000.
143
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FIG. 10. Deltamethrin-treated preprophase cell showing loss of microtubules (arrows) in the cortical band (1 ppm for 24 h). 380,000.
FIG. 11. A normal telophase cell. Cell wall formation is regular and complete. Dictyosomes are visible near the new cell wall (arrows). 319,200.
ANTIMITOTIC EFFECTS OF DELTAMETHRIN
FIG. 12. (a) A light microscope photomicrograph showing binucleate cell exposed to deltamethrin. (1 ppm for 24 h). 31000. (b) Telophase cell exposed to deltamethrin showing incomplete deposition of cell wall components (arrows, 1 ppm for 24 h). 38500.
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FIG. 13. A magnified EM photomicrograph of a deltamethrin-treated telophase cell showing incomplete and irregular deposition of vesicles. Vesicles seem to be misdirected (arrows). Dictyosomes are not visible in phragmoplast region. Very few microtubules are visible (arrows, 1 ppm for 24 h). 336,000.
as binucleate cells. Such effects indicate subcellular targets of toxicity of synthetic pyrethorids like deltamethrin in nontarget organisms, which shall help in understanding its mode of action in plants and animals. ACKNOWLEDGMENTS The authors thank Dr. P. K. Seth, Director, I. T. R. C., Lucknow, India for his keen interest in this study and Dr. Deepak K. Agarwal for critical review of the manuscript.
REFERENCES 1. J. P. Leahay, “The Pyrethroid Insecticides,” Taylor & Francis, London, 1985. 2. J. Miyamato, Degradation, metabolism and toxicity of synthetic pyrethroids, Environ. Health Pers. 14, 15 (1976). 3. R. Kavlok, N. Chernoff, R. Baron, R. Linder, E. Rogers, B. Casver, J. Dilley, and V. Simmon, Toxicity studies with decamethrin, a synthetic pyrethroid insecticide, J. Environ. Pathol. Toxicol. 2, 751 (1979).
4. D. Ruzo, J. L. Engel, and J. E. Casida, Decamethrin metabolites from oxidative, hydrolytic and conjugative reactions in mice, J. Agric. Food Chem. 27, 725 (1979). 5. J. Miyamoto and N. Mikami, Degradation of pyrethroids in the fields, in “Pesticide Chemistry” (J. Miyamoto and P. C. Kearney, Eds.), pp. 173–189, Pergamon, Oxford, 1983. 6. L. K. S. Chauhan, T. S. S. Dikshith, and V. Sundararaman, Effect of deltamethrin on plant cell. I. Cytological effects on the root meristem cells of Allium cepa, Mutation Res. 171, 25 (1986). 7. A. Baeza-Squiban, F. Marano, X. Ronot, M. Alolphe, and S. Puisenx-Dao, Effects of deltamethrin and its commercial formulation decis on different cell types in vitro: Cytoxicity, cellular binding and intracellular localization, Pestic. Biochem. Physiol. 28, 103 (1987). 8. G. Fiskesjo, The Allium test: An alternative in environmental studies: The relative toxicity of metal ions, Mutation Res. 197, 243 (1988). 9. M. A. Hayat, “The Handbook of Electron Microscopic Techniques,” Van Nostrand–Reinhold, New York, 1972. 10. L. K. S. Chauhan and V. Sundararaman, Effects of substituted ureas on plant cell. I. Cytological effects of
ANTIMITOTIC EFFECTS OF DELTAMETHRIN
11.
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isoproturon on the root meristem cells of Allium cepa, Cytologia 55, 91 (1990). G. Hillman and A. Ruthman, Effect of mitotic inhibitors on the ultrastructures of root meristem cells, Planta 155, 124 (1982). L. K. S. Chauhan and V. Sundararaman, Effects of substituted ureas on plants cells. II. Ultrastructural effects of isoproturon on the root meristem cells of Allium cepa, Cytologia 55, 99 (1990). B. L. Armbruster, T. W. Molin, and M. W. Bugg, Effects of herbicide dithiopyr in cell division of wheat root tips, Pestic. Biochem. Physiol. 39, 110 (1991). H. H. Mollenhauer and D. J. Morre, The Golgi apparatus, in “Biochemistry of Plants: A Comprehensive Treatise” (P. K. Stumpf and E. E. Conn, Eds.), Vol. 1, pp. 437–488. Academic Press, New York, 1980. J. A. Traas, J. H. Doonam, D. J. Rawlins, P. J. Shaw, J. Watts, and C. W. Lloyd, An actin network is present
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in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus, J. Cell Biol. 105, 387 (1987). A. A. Rogalski and S. J. Singer, Association of Golgi apparatus with microtubules, J. Cell Biol. 99, 1092 (1984). J. Wehland, M. Henkart, R. Klausner, and I. V. Sandoval, Role of microtubules in the distribution of the Golgi apparatus: Effect of taxol and microinjected anti-alphatublin antibodies, Proc. Natl. Acad. Sci. USA 80, 4286 (1989). P. G. Bartels and J. I. Hilton, Comparison of trifluralin, oryzalin, pronamide, propham and colchicine, treatments on microtubules, Pestic. Biochem. Physiol. 3, 46 (1973). M. K. Upadhyaya and L. D. Nooden, Mode of dinitroaniline hebricide action. I. Analysis of the colchicinelike effects of dinitroaniline hebricides, Plant Cell Physiol. 18, 1319 (1977).
Effects of Deltamethrin on the Ultrastructures of the Root Meristem Cells of Allium cepa L. K. S. Chauhan, P. N. Saxena, and S. K. Gupta1 Cell Biology Section, Industrial Toxicology Research Centre, P.O. Box 80, Mahatma Gandhi Road, Lucknow 226001, India Received October 29, 1998; accepted April 28, 1999 The EC50 value of commercially formulated deltamethrin (Decis 2.8% EC) was determined to be 0.87 ppm against Allium root growth. Root meristem cells were exposed to deltamethrin (0.5–2 ppm) for 6 to 24 h and examined by transmission electron microscopy. Deltamethrin induced various morphological abnormalities in the cell wall, plasma membrane, and Golgi apparatus dictyosomes. The microfibrillar arrangement of cell wall appeared to be amorphous in treated cells. The plasma membrane showed extensive dehiscence from the cell wall. The stacked cisternal membrane of dictyosomes were seem to be degenerated and the vesicles emanating from the dictyosomes were found to be accumulated around the degenerated dictyosome remnants. Sporadic dilation of dictyosome cisternae was also observed in the treated cells. Most nuclei in treated cells were observed to contain condensed chromatin material. The number of microtubles in the preprophase band was significantly reduced in 24 h treatment. Deltamethrin frequently disturbed the parallel array of spindle microtubules in metaphase cells. Telophase cells exposed to deltamethrin revealed incomplete, if not irregular, cell wall formation. Present findings suggest that the action of deltamethrin on microtubules is to an extent similar to that of microtubule depolymerizing drugs, which is the primary cause of toxicity leading to the induction of various mitotic aberrations. However, induction of structural and functional aberrations in the Golgi apparatus dictyosomes indicates the effect of deltamethrin on intercisternal elements and microfilaments, which possibly keeps the cisternal stack intact and directs the intracellular transport of vesicles. q1999 Academic Press
INTRODUCTION
Synthetic pyrethroids are photostable, environmentally compatible, and potent insecticides for widespread use in agriculture and veterinary and public health (1). Their usage is perferred over organochlorine and organophosphate insecticides in view of their safety to nontarget organisms (plants and animals) due to rapid hydrolytic disposition (2). The toxicity of deltamethrin (CAS No. 5291863-5; proper name S-alpha-cyano-3-phenoxybenzyl-1R-cis-3-2, 2-dibromoviny 1-2, 2-dimethylethylcyclopropane carboxylate) has been elucidated in animals (3, 4) and plants (5–7), indicating similarities in its toxicokinetic profile in higher eukaryotes. Considering Allium root metristem cells as a suitable model and also 1 To whom correspondence should be addressed. Fax: 0522-228227.
a recepient of these agricultural chemicals, we extended our previous cytological observations (6) to the ultrastructural level to elucidate the subcellular targets of deltamethrin toxicity. The observations are expected to improve our understanding of the mode of action of deltamethrin and establish Allium cepa as an appropriate model for biomonitoring of environmental chemicals at cellular and subcellular levels. MATERIALS AND METHODS
Chemicals Commercial formulation of deltamethrin (Decis 2.8% EC) was obtained from Hoechst India Ltd. Fixatives and other embedding mediums used in electron microscopy were procured from LADD Research Industries, England, and all other chemicals/reagents used in this study were purchased from E. Merck/Glaxo, India.
135 0048-3575/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
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EC50 Determination Following the standard protocol of Fiskesjo (8), healthy and clean onion bulbs were mounted on glass jars filled with aqueous concentrations (containing less than 0.6% DMSO) of deltamethrin (0.25–2.84 ppm) for rooting at 22 6 18C in B.O.D. incubator. A minimum of five bulbs was rooted in each concentration. Control bulbs were maintained in concentrations of DMSO equal to those used for test concentration. Every 24 h after each concentration was renewed and 5th day, root lengths from treated and control bulbs were measured. The average root growths were plotted against the test concentrations and the point showing 50% growth compared to controls (considered as 100%) was marked as the EC50 value. Electron Microscopy Fresh and healthy onion bulbs were thoroughly washed and rooted in tap water. Roots
1–2 cm long were exposed to 0.5, 1.0, and 2.0 ppm of deltamethrin for 6 or 24 h at 22 6 18C. Control bulbs were maintained in distilled water mixed with a quantity of DMSO (.0.1%) equal to that used to make the test solution. At the end of the exposure, roots were thoroughly washed and 1 mm of the root tip portion was fixed in 4% glutaraldehyde (prepared in sodium cacodylate buffer, pH 6.8) for 1 h at room temperature and post fixed with 1% osmium tetroxide (same buffer) for 1 h. Fixed root tips were washed with cacodylate buffer, dehydrated in an ethanol series (30–100%), and embedded in Aralditeepon medium (9). Thin sections were cut with glass knives on an LKB IV ultramicrotome and stained with uranyl acetate and lead citrate to examine under a Philips EM400 transmission electron microscope. To quantify the effect of deltamethrin on the cortical microtubules, 10 preprophase cells, selected at random, from five root tips were
FIG. 1. Concentration-wise root growth retardation in the deltamethrin-treated Allium roots and EC50 determination. EC50 5 0.87 ppm.
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137
FIG. 2. Electron micrograph of a normal cell wall with intact plasma membrane. Homogeneous electron density in the microfibrillar region of cell wall. 317,600.
examined to count microtubules. The statistical significance of results was calculated through Student’s t test. RESULTS
EC50 Value The treatment of deltamethrin (0.25–2.84 ppm) induced concentration-wise root growth retardation, without obvious morphological changes in the Allium roots. The EC50 value of deltamethrin was determined to be 0.87 ppm (Fig. 1). In the present experiment 1.0 ppm of deltamethrin has been used as EC50 with two other concentrations, 0.5 and 2.0 ppm, for comparison of effects. Ultrastructural Effects Cytoplasmic organelles. Cell wall, plasma membrane, and Golgi apparatus appeared to be highly sensitive to deltamethrin exposure. The
electron-dense microfibrillar appearance of the normal control cell wall was observed to be disturbed in 6-h treatment with deltamethrin (1 and 2 ppm; Figs. 2 and 3). Intact plasma membrane as seen in control cells appeared to be protruded into the cytoplasm and separated from the cell wall (Fig. 3). Vesicles extruded from cytoplasm were frequently noted in protruded portion of plasma membrane (Fig. 3). In comparison with control cells, vacuolization of cytoplasm was found to be increased in the treated cells. The cortical microtubules alongside the plasmalemma were seem to be undisturbed at 6-h treatment (Fig. 3). The treatment of similar concentrations of deltamethrin (0.5–2 ppm) exhibited drastic structural changes in the cell wall and plasma membrane at 24 h. The microfibrillar array of the cell wall appeared to be amorphous (Fig. 4). Extensive dehisence of the plasma membrane in these treated cells formed a wide gap from the
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CHAUHAN, SAXENA, AND GUPTA
FIG. 3. Electron micrograph of a deltamethrin-treated cell showing initial stage of plasma membrane dehiscence and sporadic protrusions (arrows). Loss of microfibrillar array of cell wall and accumulation of vesicles in the spaces formed between the cell wall and the plasma membrane. Vacoules (V) are also visible (2 ppm for 6 h). 328,800.
cell wall (Fig. 4). The cortical microtubules alongside the plasmalemma observed at 6-h treatment were not seen in the cells exposed for 24 h. The normal stacked cisternae of Golgi apparatus dictyosomes did not reveal any structural change in 6-h treatment of deltamethrin except conspicuous accumulation of vesicles (Figs. 5 and 6). However, root tip cells exposed for 24 h to deltamethrin (1 and 2 ppm) revealed various aberrations in dictyosome structure. Most of these cells were found to contain deformed or degenerated dictyosomes surrounded by numerous vesicles (Figs. 6 and 7). Frequently the middle or last cisternae (toward maturing phase) of the dictyosomes were found to be highly dilated like a sack (Fig. 8). The shape and structure of mitochondria and
proplastids were observed to be normal in deltamethrin-treated cells. Interphase and mitosis. Interphase nuclei, other than condensation of chromatin materials did not show any visible change at 6-h treatment. However, a number of cells were found to contain presumptive cytoplasmic inclusions in the nuclei after 24-h exposure to deltamethrin. The presence of numerous cortical microtubules marked the preprophase stage of mitosis in normal root meristem cells (Fig. 9). At 6h treatment, deltamethrin (0.5–2 ppm) did not induce any visible effect on preprophase microtubules, while at 24-h exposure, the number of cortical microtubules in the preprophase band was significantly reduced, particularly with 1 and 2 ppm of deltamethrin (Table 1, Fig. 10).
ANTIMITOTIC EFFECTS OF DELTAMETHRIN
139
FIG. 4. Large gaps between plasma membrane and cell wall. Microfibrillar region appears amorphous. Breaks are visible in plasma membrane (arrows; 2 ppm for 24 h). 322,000.
The characteristic parallel array of spindle microtubules of normal metaphase cells appeared to be disturbed within 6 h of exposure to deltamethrin (1 and 2 ppm). Chromosomes of these metaphase cells were found to be scattered all over the cytoplasm, instead of linearly arranged at the equitorial plate. Analysis of metaphase cells exposed 24 h displayed randomly dispersed short microtubules lacking pole-to-pole continuity. The normal telophase stage of mitosis revealed an enormous number of phragmoplast microtubules and associated dictyosomederived vesicles at the site of cell wall formation. The cross walls laid down were complete and linear (Fig. 11). The telophase cells exposed to deltamethrin (0.5 to 2 ppm) for 24 h revealed incomplete cell wall formation (Fig. 12). When magnified, these cells were found to contain short and misdirected microtubules and vesicles unevenly scattered at the site of wall formation
(Fig. 13). The cell walls, wherever laid down in treated cells, were either irregular or incomplete. DISCUSSION
In the present study, A. cepa root meristem cells have been used to elucidate the toxicity potential of a commercially formulated synthetic pyrethroid insecticide, deltamethrin. Present findings validate the use of root meristem cells of A. cepa as an appropriate model for studying the biological effects of environmental chemicals at subcellular levels. Further determination of EC50 values by measuring the root growth retardation is a simple yet reliable index of the cytotoxic potential of xenobiotics used in the present study (8, 10). The present electron microscopic findings are in agreement with our previous communication in which deltamethrin was shown to induce various spindle-related abnormalities in Allium root meristem cells (6). Most mitotic disruptors,
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CHAUHAN, SAXENA, AND GUPTA
FIG. 5. Photomicrograph of a normal dictyosome showing stacked cistrnae and no accumulation of secretory vesicles. 395,000.
including herbicides and microtubule polymerizing drugs, irrespective of their chemical structure and mode of action, induce structural alterations in cytoplasmic organelles, particularly in the cell wall and plasma membrane (11– 13). Present findings indicate that the action of deltamethrin on microtubules is partly similar to that of colchicine and vinblastine. These
TABLE 1 Effect of Deltamethrin on the Density of Cortical Microtubules in the Preprophase Band of the Root Meristem Cells of Allium cepa Concentration of deltamethrin in ppm
Period of exposure in h
No. of cortical microtubules/ preprophase band
0 1 2
24 24 24
80.42 6 2.72 19.57 6 3.05** 06.67 6 1.16**
Note. Values are mean 6SE from 10 preprophase cells. ** P , 0.001.
chemicals, in addition to affecting microtubules, disturb the fibrillar orientation of cell wall and cause extensive dehisence of plasma membrane (11). Further, structural alteration of cytoplasmic organelles with the treatment of deltamethrin is quite possible as its intracellular localization in the plant cells has been demonstrated in the cytoplasm (7). Our findings suggest that in the early phase of treatments, deltamethrin stops intracellular transport of dictyosome-derived vesicles through its action on the microfilamentous elements, which leads to the accumulation of vesicles in the cytoplasm. Similar accumulation of secretory vesicles, blebbed from dictyosomes, has been reported in plant cells exposed to a fungal product, cytochalasin B, and were interpreted to be the effect of the compound on the microfilamentous elements (actin filaments) which persist in plant cell cytoplasm in association with microtubules throughout cell division
ANTIMITOTIC EFFECTS OF DELTAMETHRIN
141
FIG. 6. Photomicrograph of a deltamethrin-treated cell showing degenerated cisternal membranes of dictyosomes (arrow) and accumulation of numerous secretory vesicles (1 ppm for 24 h). 346,000.
(14, 15). Deltamethrin affects the actin filaments which possibly direct the vesicle flow either independently or in association with microtubules. On the other hand, degeneration and deformation of dictyosomes appearing in the later phases of treatment, indicates the effect of deltamethrin on the intercisternal elements. In plant cells, intercisternal elements seem to be responsible for the structural integrity of dictyosomes as are microtubules in animal cells (14, 16). This is more evident when microtubule disruptors, such as antipolymerizing drugs, which are normally shown to induce structural aberrations in the Golgi apparatus of animal cells, do not show structural alterations in the dictyosomes of plant cells (14, 16, 17). However, further studies are required to confirm the effect of deltamethrin on the microfilaments using rhodamine-labeled phalloidin stain or antibodies against actin.
Significant and dose-dependant reduction of cortical microtubules in the preprophase band suggests the effect of deltamethrin on the polymerization of microtubules (11, 19). The disruption of metaphase microtubules as observed in the present study possibly leads to the formation of mitotic aberrations visible at the light microscopic level (6). Such functional impairment of spindle microtubules has been reported in plants with several pesticidal chemicals (6, 10, 12, 13). Similarly, induction of binucleate cells due to the loss of phragmoplast microtubules is a characteristic effect of mitotic disruptors (6, 11, 18). Cytokinesis in plant cells is a complex process in which dictyosome-derived vesicles are regularly deposited by the phragmoplast microtubules. Deltamethrin inhibits this process in the root meristem cells through its action on microtubules and dictysomes; consequently, irregular or incomplete cell walls are formed, which appear
142
CHAUHAN, SAXENA, AND GUPTA
FIG. 7. Magnified view of a tangentially sectioned dictyosome showing accumulation of vesicles. (2 ppm for 6 h). 3100,000.
ANTIMITOTIC EFFECTS OF DELTAMETHRIN
FIG. 8. Photomicrograph of a deltamethrin-treated Golgi apparatus dictyosome showing fused cisternal membranes and dilation of last cisterna (arrow). 380,000.
FIG. 9. Normal preprophase stage showing numerous microtubules in the cortical band (arrows). 3110,000.
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CHAUHAN, SAXENA, AND GUPTA
FIG. 10. Deltamethrin-treated preprophase cell showing loss of microtubules (arrows) in the cortical band (1 ppm for 24 h). 380,000.
FIG. 11. A normal telophase cell. Cell wall formation is regular and complete. Dictyosomes are visible near the new cell wall (arrows). 319,200.
ANTIMITOTIC EFFECTS OF DELTAMETHRIN
FIG. 12. (a) A light microscope photomicrograph showing binucleate cell exposed to deltamethrin. (1 ppm for 24 h). 31000. (b) Telophase cell exposed to deltamethrin showing incomplete deposition of cell wall components (arrows, 1 ppm for 24 h). 38500.
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FIG. 13. A magnified EM photomicrograph of a deltamethrin-treated telophase cell showing incomplete and irregular deposition of vesicles. Vesicles seem to be misdirected (arrows). Dictyosomes are not visible in phragmoplast region. Very few microtubules are visible (arrows, 1 ppm for 24 h). 336,000.
as binucleate cells. Such effects indicate subcellular targets of toxicity of synthetic pyrethorids like deltamethrin in nontarget organisms, which shall help in understanding its mode of action in plants and animals. ACKNOWLEDGMENTS The authors thank Dr. P. K. Seth, Director, I. T. R. C., Lucknow, India for his keen interest in this study and Dr. Deepak K. Agarwal for critical review of the manuscript.
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