Induction of Mitotic Abnormalities in Onion Root-Tips by Tobacco Smoke Condensate'

Transcription

1 Cytologia 38: , 1973 Induction of Mitotic Abnormalities in Onion Root-Tips by Tobacco Smoke Condensate' P. R. Bhalla, T. S. Kochhar and P. S. Sabharwal Thomas Hunt Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506, U.S.A. Received June 6, 1972 The association of chromosomal abnormalities with malignant disease in vivo has been demonstrated by many investigators (see Porter et al. 1969). In vitro studies with lung tissues obtained from human fetus and young kittens (Na kanishi et al. 1959, Awa et al. 1961), mouse lung fragments (Bouchard and May 1960) and the KB line of mammalian cells in culture (Thayer and Kensler 1964) have indicated that cigarette smoke fractions have definite cytologic influences. Quantitative evaluation of such effects should provide information which may be important to health related research on tobacco. It seems obvious that experimentation with plants is faster and easier than the standard mouse skin bioassay which is commonly employed in assessing the biolo gical effects of tobacco smoke fractions and their components. The present investi gation was, therefore, conducted to ascertain the influence of water-soluble extract of tobacco smoke condensate (TSC) on the mitotic process in young roots obtained from onion bulbs. An attempt was made to investigate the feasibility of utilizing plant tissues for a rapid bioassay to detect the cytological effects caused by TSC. Material and methods Commercially available bulbs of onion (Allium cepa) were utilized in the pre sent study. To obtain the roots, several bulbs were placed with their basal side facing downwards over small jars containing 60ml of glass-distilled water. The water in the jars was replaced at 24-hour intervals. After about 72hours, when the roots had emerged from the bulbs and were about one inch in length, the water was replaced by known concentrations of water-soluble extract of tobacco smoke con densate (TSC). The water-soluble extract of tobacco smoke condensate was prepared under the supervision of Dr. J. F. Benner (Department of Agronomy, University of Kentucky, Lexington, Kentucky) from the University of Kentucky Reference Cigarettes, 1R1. These cigarettes, which provide smoke of known composition, are 85mm long, non-filter, and are manufactured from a "typical" blend of tobacco 1 This study was carried out under contract No (73) with 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.

2 708 P. R. Bhalla, T. S. Kochhar and P. S. Sabharwal Cytologia 38 such as is used in commercially available cigarettes in the United States (Atkinson 1970, Benner 1970). The cigarettes were first equilibrated at 20 Ž and 60% re lative humidity and then smoked on a Borgwaldt Smoking Machine using a 35ml volume of 2 second duration at 1 minute intervals. The smoke was collected in a 3 1 flask containing 100ml of water cooled to 0 Ž. Maximum contact of the smoke with water was achieved by a specially designed pump, Chemap Vibro-mixer. When 840 cigarettes had been smoked, the aqueous solution was transferred to a pre-cooled graduated cylinder. The non-volatile residue was calculated by evapora tion of a 5ml portion of the solution on a rotary evaporator at a pressure of 30mm at 35 Ž with 50ml/minute stream of nitrogen. The flask was then rinsed with sufficient water several times to give a final concentration of 50mg/ml of non volatile residue. This water-soluble extract of tobacco smoke condensate (TSC) was subsequently diluted with distilled water to make the required concentrations to be tested. The concentrations employed for TSC treatment in the present investigation were 0.001%, 0.01%, 0.025%, 0.04%, 0.05%, 0.1%, and 0.25%. The roots were treated with TSC for 24hours. Root-tips, 1cm long, were fixed in 95% ethanol: acetic acid (3:1) at room temperature for 24hours. The root-tips were then washed with distilled water and treated with 1 N HCl at 60 Ž for 2minutes. They were then rinsed with distilled water several times, and stored in 45% acetic acid at room temperature. For each treatment 25 squash preparations were made from the apical 2mm of the root-tips. The cells were stained with aectocarmine. The data were recorded from three different microscope views of each preparation. These views were selected where the greatest number of dividing cells were observed. The mitotic index was calculated on the basis of number of dividing cells per 1000 cells. Ab normally dividing cells were recorded and the percentages of such abnormal divi sions were calculated. The qualitative nature of these abnormalities was also noted. Results During the course of the 24hour treatment, roots in control medium and 0.001% TSC showed some increase in length, but in higher TSC concentrations no root growth occurred; microscopically, the cells were normal with well defined nuclei. Cytological observations revealed that controls, 0.001%, 0.01% and TSC showed mitotic abnormalities in about 5-7% cells. At higher TSC concentrations of 0.04%, 0.05% and 0.1% changes were found in both the cytoplasm and the nucleus. Some cells displayed vacuolization; these cells were in the interphase stage, and had disorganized cytoplasm and irregularly shaped nuclei (Fig. 1). In addition, some preparations revealed that nuclei were pycnotic and nucleoli were hard to distinguish. The percent abnormally dividing cells varied in different TSC concentrations. In 0.04% TSC 12% dividing cells showed irregular mitotic divisions while in 0.05%, 0.1% and 0.25% of TSC 21, 37, and 6% cells displayed abnormalities respectively. The abnormalities were most

3 1973 Figs exposed ing Induction of Mitotic Abnormalities in Onion Root -Tips by TSC cell root-tip 1, to irregular 0.25% of a root-tip treated configuration tobacco smoke exposed with 0.25% marked) to of nuclei (see arrows) condensate. ~ % tobacco tobacco forming smoke 2, smoke non-dividing of cells at Note anaphase. ~ , a of chromosomes condensate. ~1405. condensate. a bridge in scattering 709 lagging a dividing chromosome a in root-tip a divid cell of a (arrow

4 710 P. R. Bhalla, T. S. Kochhar and P. S. Sabharwal Cytologia 38 numerous in metaphase, in which chromosomes were scattered in the cells in a disorganized manner (Fig. 2). Bridging or lagging of the chromosomes was also common in anaphase (Fig. 3). Table 1. Effect of different concentrations of TSC (%) on mitotic index in root-tips of Allium cepa (onion) after 24 hours of treatment Observations made on mitotic indices revealed that roots grown in controls, 0.001% and 0.01% TSC showed the same mitotic index. Table 1 summarizes the mitotic indices in the various concentrations of tobacco smoke condensate. It shows that from 0.001% TSC to 0.04% of TSC mitotic index declined from 0.11 to 0.02 while at higher concentrations of 0.05%,0.1% and 0.25% no further decrease was observed. Discussion Nakanishi et al. (1959) emphasized that little attention has been directed to studies based on cytological effects of cigarette smoke and its fractions. Several investigations have been undertaken since then. Bouchard and May (1960) ob served increased mitotic abnormalities in lung fragments that had been kept for 24hours in smoke condensate and subsequently grafted under the renal capsule of mice. Awa et al. (1961), using a human cell strain, observed more cell damage when cells were exposed to paper smoke than when exposed to smoke from toabcco, or from entire cigarettes. No definite conclusion could be drawn as to the effect of cigarette paper, however, since the paper was burnt in a manner quite different from the way in which it is burnt as part of a cigarette. Later, Mizutani (1962) using Nakanishi's cell strain could not confirm the findings of Nakanishi et al. about a change in chromosome number when the cells were exposed to cigarette smoke. A major problem of these investigations is that in vitro cultured cells are particularly sensitive to toxic effects of certain tobacco smoke components. A more durable living system would aid in evaluation of effects of tobacco smoke and its components upon living organisms. Studies with higher plants have demonstrated that tobacco smoke fractions induce mitotic abnormalities (Izard et al. 1970, Izard and Moree-Testa 1971, Izard 1972). We have earlier reported that tobacco smoke alters mitotic index and induces mitotic abnormalities in the root-tip cells of onion seedlings (Bhalla et al. 1972). In the present investigation onion root-tips were utilized to study the effect of water-soluble extract of tobacco smoke condensate upon the mitotic process. Since the plants are inexpensive, immobile, and can easily be grown in controlled conditions, they should prove excellent material as compared to animals. Water soluble extract of tobacco smoke condensate caused chromosomal abnormalities

5 1973 Induction of Mitotic Abnormalities in Onion Root-Tips by TSC 711 in these onion bulb root-tips. Vacuolization of the cytoplasm and nuclear pycnosis were seen in many cells following the treatment. At higher concentrations bridging of the chromosomes was quite common at anaphase stage. Also with increasing concentration of tobacco smoke condensate, the mitotic index declined. The present investigation, thus, demonstrated that water-soluble extract of tobacco smoke condensate has a marked effect on the mitotic index and the frequency of chromo somal irregularities in onion root-tips. We are currently working on the various subfractions of tobacco smoke condensate, attempting to find out the specific component or components responsible for such chromosomal irregularities, and the results will be reported subsequently. Summary Onion root-tips were used to study the effects of water-soluble extract of tobacco smoke condensate (TSC) on various cytological parameters. When roots were treated with different concentrations of TSC (0.04%, 0.1%, 0.05% and 0.25%) a number of abnormalities resulted. These included vacuolization of the cytoplasm as well as abnormal mitotic stages. The abnormalities consisted of scattering of chromosomes during metaphase, and bridging during anaphase. Also with in creasing concentrations of tobacco smoke condensate, the mitotic index declined. This investigation should be of importance in assessing the biological effects of tobacco smoke and its constituents. Acknowledgement The authors are thankful to Dr. R. B. Griffith, Director, Tobacco and Health Research Institute, University of Kentucky, for critical review of this paper. References Atkinson, W. O Production of sample cigarettes for tobacco and health research. Proc. Tobacco and Health Conference, University of Kentucky, Lexington, Kentucky. p Awa, A., Ohnuki, Y. and Pomerat, C. M Some comparative effects of smoked paper, tobac co and cigarettes on chromosomes in vitro. Texas Rept. Biol. Med. 19: Benner, J. F Tentative summary of leaf and smoke analysis of University of Kentucky Reference and alkaloid series cigarettes. Proc. Tobacco and Health Conference, Univer sity of Kentucky, Lexington, Kentucky. p Bhalla, P. R., Kochhar, T. S. and Sabharwal, P. S Induction of mitotic abnormalities in the root-tips of Allium cepa by tobacco smoke. Experientia 28: Bouchard, J. and May, R. M L'effect des goudrons de tabac sur les griffes sous-capsualires de poumon chez la souris. Arch. Anat. Microscop. Morphol. Exptl. 49: Izard, C. M., Valadud, D. and Moree-Testa, P Sur L'activite chromatoclasique et mito clasique de certines fractions de goudrons de fume'e de cigarette. C. R. Acad. Sc. Paris. 270: and Moree-Testa, P Activite mitoclasique et aberrations chromosomiques induites 1'a le p-aberrations indentifies dans une fraction de goudron de fume'e de cigarette. C. R.

6 712 P. R. Bhalla, T. S. Kochhar and P. S. Sabharwal Cytologia 38 Acad. Sc. Paris 272: Action de la N-methyl-N'-nitrosoguanidine et de la Ĉ-propiolactone sur la division cellulaire de Vicia faba en presence d'hydroquinone. C. R. Acad. Sc. Paris 274: Mizutani, M A preliminary study of the effect of cigarette smoke and air pollution upon cells in culture. Jour. Fac. Sci. Hokkiado Univ. Ser. VI. Zool. 15: Nakanishi, Y. H., Mizutani, M. and Pomerat, C. M Smoke condensates on lung cells in tissue culture with special reference to chromosomal changes. Texas Rept. Biol. Med. 17: Porter, I. H., Benedict, W. F., Brown, C. D. and Paul, B Recent advances in molecular pathology: A review, some aspects of chromosome changes in cancer. Exper. Mole. Path. 11: Thayer, P. S. and Kensler, C. J Cigarette smoke: charcoal filters reduce components that inhibit growth of cultured human cells. Science 146: