Cytological Studies on the Responses of Onion Root - Tip Cells to Water-Soluble Tobacco Smoke Extracts from Various Experimental Cigarettes1

Transcription

1 Cytologia 40: , 1975 Cytological Studies on the Responses of Onion Root - Tip Cells to Water-Soluble Tobacco Smoke Extracts from Various Experimental Cigarettes1 D. K. Gulati, P. S. Sabharwal and P. R. Bhalla Thomas Hunt Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506, U. S. A. Received October 30, 1973 Recent work in our laboratory (Bhalla et al. 1973) has shown that water-soluble extract of tobacco smoke condensate (TSC) from the lr1 Reference cigarette (Uni versity of Kentucky) induced mitotic abnormalities in the root-tip cells of onion (Allium cepa). The present investigation details results on water-soluble extracts from experimental cigarettes (IA1, IA2, IA3, and IA4) of varying alkaloid contents. The alkaloid series was comprised of cigarettes with varying levels of nicotine. Nicotine variability is achieved through the use of special strains of genetically controlled low nicotine tobaccos (Benner 1970). Material and methods Locally available green onions (Allium cepa, family Liliaceae) were utilized in the present investigation. Green bulbs with their basal end downwards were placed into test tubes containing distilled water. The bulbs were in immediate contact with distilled water. After 72 hours most of the roots were about one inch in length. They were then treated with various concentrations of water-soluble tobacco smoke condensate (TSC) from IA1, IA2, IA3, and IA4 cigarettes. The TSC was prepared under the supervision of Dr. J. F. Benner (Tobacco and Health Research Institute, University of Kentucky, Lexington, Kentucky). The detailed method of TSC preparation was previously described (Kochhar et al. 1971, Bhalla and Sabharwal 1973). The concentrations of TSC utilized in the present investigation were 0.01%, 0.025%, 0.05%, 0.10%, and 0.25%. The onion roots were treated by placing the onion bulbs with roots dipping in small vials (Fig. 1) containing 15 ml of TSC solution for 24 hours. The root-tips from the controls and bulbs treated with various test solutions were fixed in 95% ethanol; acetic acid (3:1) at room temperature, for 24 hours. They were hydrolyzed with 1 N HC1 at 60 Ž for 3 minutes. The roots were then washed several times in distilled water and stored in 45% acetic acid. Squashes were made in acetocarmine for cytological observations. Three slides with a large 1 This study was carried out under contract no (73) with the Agricultural Research Service, U. S. Department of Agriculture, administered by the Athens, Georgia Area, Richard B. Russell Agricultural Research Center, Athens, Georgia, 30604, U.S.A.

2 384 D. K. Gulati, P. S. Sabharwal and P. R. Bhalla Cytologia 40 number of divisions were selected for recording data. The percentage of cells in mitosis in each treatment was determined on the basis of a minimum of 1000 cells. Observations on the percentage of abnormally dividing cells was recorded. The qualitative nature of these abnormalities was also observed. Each experiment was conducted twice and the values reported are the averages of these experiments. Results Fig. 1. Diagrammatic sketch showing TSC treatment of ger minating green onion bulb. TSC from all the four cigarettes induced mitotic abnormalities in the young roots of onion. With increasing concentrations, there was a progressive rise of mitotic abnormalities (Fig. 2). The varia tions within each treatment are also shown. As apparent from Fig. 2, TSC from IA2 proved most effective at 0.025% while TSC from IA4 was most potent at 0.01% and 0.05% in inducing mitotic abnormalities. However, at 0.25%, TSC from both IA2 and IA4 were equally effective and induced ab normalities in 100% of the dividing cells. TSC from IA1 and IM proved almost equally effective at Fig. 2. Effects of various concentrations of TSC from different experimental cigarettes in inducing mitotic abnormalities.

3 1975 Cytological Studies on the Responses of Onion Root-Tip Cells %, while at 0.025% and 0.05% TSC from IA 1 induced slightly more mitotic irregularities, as compared to TSC from IA3 (Fig. 2). The mitotic abnormalities induced by TSC of these experimental cigarettes included: the scattering of chro Figs , induction of scattering of chromosomes by 0.1% TSC from IM cigarettes. 4, in duction of lagging of chromosome in a meristematic cell of onion root by 0.05% TSC from IM cigarettes. 5, induction of binucleate cell by 0.25% TSC from IA2 cigarettes. 6, cell treated with 0.25% TSC from IA1 cigarettes showing one nucleus at interphase while other at metaphase.

4 386 D. K. Gulati, P. S. Sabharwal and P. R. Bhalla Cytologia 40 mosomes during metaphase (Fig. 3); lagging of chromosomes during anaphase (Fig. 4); breakage of chromosomes; stickiness at metaphase and bridging at an aphase. Such mitotic irregularities were observed in 5 to 7% dividing cells in the root-tip cells (controls) treated with distilled water. Table 1. Observations on the binucleate cells in onion root-tips treated with water-soluble extracts (TSC) from various experimental cigarettes. Percentages are based on 1000 cells Besides inducing mitotic irregularities, TSC from these cigarettes prevented cell wall formation in some cells. Binucleate cells (Fig. 5) were observed in all the treatments. In some instances one of the nuclei of binucleate cell undergoes further division (Fig. 6). Detailed observations are summarized in Table 1. As evident, TSC from IA2 induced binucleate cells in all concentrations utilized, while in TSC from IA1 and IA3 only higher concentrations (0.10% and 0.25%) were effective in inducing polynucleate cells. TSC from IA4 was not effective at 0.01% in inducing binucleate cells. Discussion Cigarette smoke is a mixture of a large number of diverse chemical compounds (Stedman 1968). The relative amounts of many smoke constituents depends upon the type of tobacco, the way it is smoked and the temperature of combustion (Diehl 1969). This leads to a question of whether or not tobacco smoke, with different tar and nicotine contents has differential biological activity? Studies by Kensler and Battista (1963), Thayer and Kensler (1964), and Wynder and Hoffman (1967) re vealed that cigarettes with filters reduce the cilia-toxic activity as measured by in vitro tests. The present investigation, however, utilizes water-soluble extract of tobacco smoke condensate (TSC) from 4 experimental cigarettes, differing in nicotine, phenols and tar yields (Table 2). The TSC from these cigarettes was tested on the Table 2. Tar*, nicotine* and total phenols* contents of 4 experimental cigarettes utilized in the present investigation * The values were determined and supplied by Dr. J. F. Benner, Assistant Director, To bacco and Health Research Institute, University of Kentucky, Lexington, Kentucky. ** Smoke values are for cigarettes smoked to a 30mm butt length.

5 1975 Cytological Studies on the Responses of Onion Root-Tip Cells 387 young roots of onion bulbs. After 24 hours, the root-tip cells were examined for mitotic abnormalities. In general, the percentage of mitotic irregularities increased with increasing concentrations of TSC. Although slight differences in the percentage of mitotic abnormalities were observed between the extracts of various cigarette condensates significant differential responses on the basis of nicotine, tar and phenol contents could not be ascertained. Among the mitotic abnormalities, breakage of chromosomes during metaphase, anaphase, stickiness at metaphase, and bridging at anaphase were frequently observed. Besides inducing mitotic abnormalities in the onion root-tip cells, TSC from various cigarettes utilized in the present investigation also somehow inhibited cell wall formation after the nucleus has divided and formed daughter nuclei (Table 1). Binucleate cells were observed when roots were treated with TSC from these cigaret tes. The present study provides a relatively simple and sensitive biological measure of the effect of water-soluble cigarette smoke condensate at the cellular level. The response is dose related but specificity of this system remains to be ascertained. Summary Young roots from onion bulbs were treated with different concentrations of water-soluble extracts of tobacco smoke condensate from 4 experimental cigarettes (IA1, IA2, IA3, and IA4). The cigarettes have varied in amounts of tar, nicotine and total phenols produced (Table 2). From the treated roots, meristematic cells were examined for various cytological parameters. TSC from all cigarettes induced mitotic irregularities. These abnormalities consisted of breakage of chromosomes during metaphase or anaphase, stickiness at metaphase and bridging at anaphase. The mitotic irregularities increased with increasing concentrations of TSC. There were no significant differences in potency of TSC from four experimental cigarettes in inducing mitotic abnormalities. In addition to the mitotic irregularities, TSC from different cigarettes inhibited cell wall formation which resulted in binucleate cells. Thus, the present studies clearly show that TSC from various cigarettes, yielding different amounts of tar and nicotine, have no differential cytological effects on onion root-tip cells. References Benner, J. F Tentative summary of leaf and smoke analysis of University of Kentucky reference and alkaloid series cigarettes. Proc. Tobacco and Health Conf., University of Kentucky, Lexington, Kentucky, p Bhalla, P. R., Kochhar, T. S. and Sabharwal, P. S Induction of mitotic abnormalities in onion root-tips by tobacco smoke condensate. Cytologia 39: and Sabharwal, P. S Investigations on the effect of tobacco smoke condensate on seed germination of higher plants. Environ. Pollut. 4: Diehl, H. S Tobacco and Your Health: The Smoking Controversy. McGraw-Hill Book Commpany, New York. Kensler, C. J. and Battista, S. P Factors affecting mammalian ciliary activity. Proc. Am. Assoc. Cancer Res. 4: 33.

6 388 D. K. Gulati, P. S. Sabharwal and P. R. Bhalla Cytologia 40 Kochhar, T. S., Bhalla, P. R. and Sabharwal, P. S In-vitro induction of vegetative buds by tobacco smoke condensate. Experientia 27: 591. Stedman, R. L The chemical composition of tobacco smoke. Chem. Rev. 68: Thayer, P. S. and Kensler, C. J Cigarette smoke: charcoal filters reduce components that inhibit growth of cultured human cells. Science 146: Wynder, E. L. and Hoffmann, D Tobacco and Tobacco Smoke. Academic Press, New York.