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EFFECTS OF HALOTHANE ON DNA SYNTHESIS AND MITOSIS IN ROOT TIP MERISTEMS OF VICIA FABA
Br. J. Anaesth. (1974), 46, 653
EFFECTS OF HALOTHANE ON DNA SYNTHESIS AND MITOSIS IN ROOT TIP MERISTEMS OF VICIA FABA C. J. GRANT, J. N. POWELL AND SHEILA G. RADFORD SUMMARY
It was proposed by Ostergren (1944) that many of the pharmacological actions of anaesthetic agents were associated with conformational changes in protein molecules. In his view these actions included not only narcosis in its strictest sense, but also "narcosis" of the cell cycle, this consisting of an over-all inhibitory or "poison" effect, and a more specific colchicine-like arrest of metaphase associated with disruption of the mitotic spindle and with contraction of chromosomes (c-mitosis). Ostergren's protein-change theory of narcosis has recently received strong support (Nunn, 1972; Eyring, Woodbury and di Arrigo, 1973), and the association between narcosis and c-mitosis (Andersen, 1966), and between narcosis and depression of various aspects of the metabolism of replicating cells (Geddes, 1971) have been amply confirmed. However, recent studies of the effect of halothane on the different stages of the cell cycle have led to some confusion. In 1969 Bruce and Traurig presented data from an autoradiographic study of the incorporation of tritiated thymidine into rat gut mucosa, from which they concluded that only the " S " phase of the cell cycle was affected. Delay at this point alone could account for the depression of mitotic index (MI) that had been found in the chick embryo neural tube after exposure to either 1.2 or 1.6% halothane (Snegireff, Cox and Eastwood, 1968), though this depression was not marked and was not seen after exposure to 2% halothane. It was then reported that in hepatoma and Hela cells depression of DNA synthesis by halothane was minimal over a short term (Ishii and Corbascio, C. J. GRANT, M.A., D.PHIL.; J. N. POWELL, M.B., B.CH., F.F.A.RX.S.; SHEILA G. RADFORD, A.LM.L.T.; Departments
of Botany and Anaesthetics, University of Bristol.
1971) and a c-mitotic effect of halothane was demonstrated in Vicia seedlings (Nunn, Lovis and Kimball, 1971), leading to the suggestion that the persistence of labelled mitotic figures in the original study by Bruce and Traurig might be attributed to such a metaphase arrest. However, the marked reductions in MI which accompany the c-mitosis in Vicia cannot be explained on the basis of metaphase arrest alone (Powell et al., 1973) and depression of DNA synthesis has now been demonstrated, after prolonged exposure to halothane, in PHA-stimulated lymphocytes (Bruce, 1972a; Cullen, Sample and Chretien, 1972), though the effect of a 2-hour exposure to 2% halothane appears to be reversible (Powell and Radford, 1971). We now report further studies of the action of halothane on the cell cycle in Vicia. These show that, in addition to both the " S " phase and mitosis, the "G 2 " phase is affected. METHODS AND MATERIALS
Treatments were carried out on primary roots of 11-day-old Vicia faba seedlings (Sutton's Prolific Longpod) which had been grown and standardized as described by Evans, Neary and Tonkinson (1957). 1% halothane in air was prepared as described by Powell et al. (1973), and was passed through all vessels and solutions for a minimum period of 1 hour before these were used. Experiment I. A. Control. Forty roots were immersed for 30 min in 500 ml of an aerated solution of 2 /tCi/ml tritiated tbymidine (Specific Activity 5.0Ci/mM; Radiochemical Centre, Amersham, U.K.) in tap
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on July 3, 2015
Further investigations on cell division and the cell cycle in Vicia faba show that halothane has a depressant effect on cells in all the phases of mitosis and also that it delays or inhibits the passage of cells through " S " and "G 2 ". An effect on cells in " G j " is not excluded. Comparison with colchicine emphasizes the widespread effects of halothane in contrast to the stage-specific effect of colchicine. The partial division synchrony induced by a short halothane exposure is discussed.
654
BRITISH JOURNAL OF ANAESTHESIA
Experiment II. Twenty roots were immersed in tap water aerated with 1 % halothane. Samples fixed after 0, 1, 2 and 3 hours of treatment. 1.5-mm tips were later made into permanent feulgen-stained squash preparations. The slides were then coded and randomized before being scored for the phases of mitosis. RESULTS
Experiment I. Mitotic index (fig. 1). In continuous halothane there was a rapid decline in MI over the first 5 hours to less than 20% of the control value. The MI then continued to decrease at a slower rate with a transient recovery at 10 hours. Re-examination of the decoded slides showed that many of the metaphases seen at 10 hours were labelled only in the late replicating hetero-chromatic regions of the chromosomes, indicating that they had been in late " S " at the time of labelling. After the 2-hour exposure to halothane, the mitotic index continued to decrease for a further 3 hours. This was followed by a series of waves of division during which the mitotic index increased above control values. Metaphase labelling index (fig. 2). Continuous halothane caused a marked delay in the appearance of labelled metaphases compared with controls, the peak being small and transient. After the 2-hour exposure to halothane, there was an initial delay,
4 S 6 7 I 9 Time since labelling (hours) •—•control;
• — • continuous halothane;
ID
II
12
>.—A halolhane loi first 2 hours only
FIG. 2. The effects of halothane (1%) on metaphase labelling index.
followed by a series of waves of labelled metaphases. Labelling of interphase nuclei (table I). The presence of halothane during or after labelling did not significantly alter the proportion of interphase nuclei that carried label 2 hours after labelling. Experiment II (fig. 3). Exposure to halothane produced a 40% increase in metaphase count at 1 hour; however, this was not
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water. The roots were then rinsed and returned to aerated tap water. B. Continuous halothane. Forty roots were immersed for 30 min in a similar 3 H TdR solution through which 1% halothane was being passed. They were then rinsed and placed in tap water aerated with 1 % halothane. C. Halothane for 2 hours only. Forty roots were labelled for 30 min as in A, rinsed and placed for 2 hours in tap water aerated with 1 % halothane. They were returned to aerated tap water. Roots from each of these regimes were fixed at hourly intervals after labelling. 1.5-mm tips were later made into feulgen-stained squash preparations, which were covered with Kodak AR10 stripping film and exposed for 6 weeks. The slides, after being 4 8 Time since start of treatment (hours) developed and made permanent, were coded and O—O colchicine; • — • continuous halothane 1 0 A—A halothane for first randomized before being scored for: (i) mitotic 2 hours only index (MI); (ii) metaphase labelling index; (iii) the FIG. 1. The effects of halothane (1%) and colchicine percentage of interphase cells carrying labelled (0.1%) on mitotic index. (Halothane data, Experiment I; colchicine data, Evans, Neary and Tonkinson, 1957.) nuclei at 2 hours after labelling.
EFFECTS OF HALOTHANE ON DNA SYNTHESIS AND MITOSIS TABLE I.
Proportion on interphase cells with labelled nuclei 2 hours post-labelling.
Mean per cent (SD)
Experiment IA IB IC
36.6 (8.1) 32.4 (4.5) 35.2 (6.6)
9 9 6
Analysis of variance shows no significant between treatments at the 5 % level.
Prophase...
Metaphase
Ana-telophase
Mitosis ...
n
-o
50-
-100
K H 0 1 2 3
•—•
0 1 2 3 0 1 2 3 0 1 2 3 D u r a t i o n of t r e a t m e n t (hours)
halothanc 1.0;', i
O—O c o l c h i c i n e 0.1'/,
FIG. 3. The effects of halothane (1%) and colchicine (0.1%) on the phases of mitosis. (Halothane data, Experiment II; colchicine data, Evans, Neary and Tonkinson, 1957.)
maintained subsequently and was followed by a progressive reduction. By 3 hours anaphase cells had nearly vanished, those which were left having a curious "slow motion" appearance with contracted, though scattered, chromosomes; telophase, however, had not decreased to a corresponding extent at this time. DISCUSSION
(a) Halothane and the stages of the cell cycle. In figure 1 the effects of halothane on mitotic index are compared with those of colchicine (data from Evans, Neary and Tonkinson, 1957). Colchicine has no direct effect on the entry of cells into prophase but, by disrupting the spindle fibres, holds chromosomes at c-meta/anaphase for up to 6 hours before a delayed restitution telophase nucleus is
formed. As a result there is an initial linear increase in MI followed after about 6 hours by a plateau when rates of entry to and exit from mitosis reach an approximate balance (Evans, Neary and Tonkinson, 1957). In contrast, halothane treatment causes a rapid fall in MI which indicates that cells already in mitosis when treatment begins are able to complete the process, although at a reduced rate; and also that the rate of entry of "G 2 " cells to prophase is decreased. Halothane-induced acceleration of mitosis as an alternative explanation for the decrease in MI is excluded by the autoradiographic evidence (fig. 2) which shows that cells in late " S " when treatment begins reach metaphase in 10 hours, twice as long as control cells. The transient nature of this wave of cells labelled in late " S " indicates that cells in earlier stages of " S " and in " G j " at the start of treatment are unable to complete the cell cycle in the presence of halothane. In the light of these results, and since both Nunn, Louis and Kimball (1971) and Powell et al. (1973) have referred to the induction of "colchicine-type mitosis" by halothane, the initial effect of halothane on the distribution of cells between the various phases of mitosis was investigated (Experiment II). This is shown in figure 3 together with that of colchicine (data from Evans, Neary and Tonkinson, 1957) over 3 hours. Two important distinctions can be made between the actions of colchicine and halothane. Firstly, the more rapid decrease of ana/telophase cells in colchicine shows that this drug has a minimal effect on cells which have passed metaphase, whereas halothane has a delaying effect on both anaphase and telophase. Secondly, the failure of metaphase cells to continue their build-up after the first hour in halothane confirms that entry of cells into diis phase is progressively inhibited. Since there is no corresponding build-up of prophase cells and the MI decreases before treated " S " cells are expected to arrive at mitosis ("G 2 " in untreated cells takes approximately 4 hours), inhibition must be occurring in "G 2 " to a greater extent than in mitosis. The actions of halothane thus differ from those of eolchicine and are more akin to those of less specific mitotic inhibitors such as para-dichlorobenzine and 8-hydroxy-quinoline (Evans, Neary and Tonkinson, 1957; Powell et al., 1973). With regard to the effects of halothane on interphase cells, although we have shown (table I) that
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on July 3, 2015
100-
differences
655
656
(b) Recovery from a short exposure. The data illustrated in figures 1 and 2 indicate that some cells at the end of " S " when treatment begins are delayed for the period of exposure to
halothane and then proceed normally through "G 2 " and mitosis. The wavelike nature of further recovery is a more complex phenomenon, which may result from either the differential blocking of a number of sequential metabolic events or the differential response of cells in different regions of the meristem (e.g. stele, cap initials, quiescent centre (Clowes, 1961a)). Evidence for the latter comes from numerous references in the literature to the heterogeneity of the primordial cell populations in Vicia {aba (Howard and Dewey, 1960; Rasch, Rasch and Woodard, 1967; Davidson and MacLeod, 1968), and other plants such as Zea (Clowes, 1961b) and Trillium (Grant, 1965; Boothroyd and Mark, 1970). There is also evidence that these populations may respond differentially to stimuli such as X-rays (Clowes, 1963) or colchicine and auxin (Webster and Davidson, 1969). This subject has been reviewed by Barlow (1973). Because of the anatomical complexity of the root-tip meristem, there is little direct evidence in Vicia for the former. However, in "simple" cultures of both animal and plant cells, there is an increasing body of evidence for the existence of numerous stage specific enzyme syntheses at which blocking might occur (Mitchison, 1973). Also the action of anaesthetic agents does appear to exhibit specificity at a molecular level (Nunn, 1972; Brammall, Beard and Hulands, 1973). It seems that both explanations may apply in this instance. Studies of the recovery from halothane of mammalian cells in culture are currently in progress. REFERENCES
Andersen, N. B. (1966). The effects of C.N.S. depressants on mitosis. Acta Anaesthesiol. Scand. (Suppl.), 22, 3. Barlow, P. W. (1973). Mitotic cycles in root meristems; in The Cell Cycle, in Development and Differentiation (eds. Balls, M., and Billett, F. S.), pp. 133-165. C.U.P. Boothroyd, E. R., and Mark, N. M. (1970). Mitotic cycles in root tip cells of two species of Trillium (Liliaceae). Can. J. Genet. Cytol, 12, 750. Brammall, A., Beard, D., and Hulands, G. H. (1973). The effects of inhalational anaesthetic agents on the enzyme glutamate dehydrogenase. Br. J. Anaesth., 45, 923. Bruce, D. L. (1972a). Halothane inhibition of phytohaemagglutinin-induced transformation of lymphocytes. Anesthesiology, 36, 201. (1972b). Normal thymidine entry into halothanetreated lymphocytes. Anesthesiology, 37, 588. Traurig, H. H. (1969). The effect of halothane on the cell cycle in rat small intestine. Anesthesiology, 30, 401. Clowes, F. A. L. (1961a). Apical Meristems. Oxford: Blackwell Scientific Publications. (1961b). Duration of the mitotic cycle in a meristem. J. Exp. Bot., 12, 283.
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up to 2 hours of halothane treatment has no effect on the number of nuclei capable of fixing 3 H TdR, it is not possible to say whether there was a reduction in the amount of thymidine fixed per nucleus. Our observations in this respect are compatible with those of Cullen, Sample and Chretien (1972) and Bruce (1972a,b), who showed that halothane does not interfere with the transport of thymidine into cytoplasm and nuclei but that it does partially block its incorporation into chromosomal DNA in PHAstimulated lymphocytes. Such a block may explain why the cells in early " S " or " G j " cannot complete the cycle in Vicia. In Vicia, therefore, all stages of the cell cycle are affected by halothane with the possible exception of "G^', which is technically more difficult to investigate. There is no reason, however, to suppose that " G L " is preferentially spared. Halothane is seen to be different from those agents which are known to block specific parts of the cycle, for example colchicine, 5-amino-uracil, which accumulates cells at the beginning of " S " (Prensky and Smith, 1965) and the radiomimetic nitroso-alkyl-urethanes which attack chromosomes in the process of replication but have no effect on them during "G 2 " (Grant and Heslot, 1966). The conclusions we have drawn from this study differ from those of Bruce and Traurig (1969) who suggested that, in rat gut epithelium, halothane had no effect on the passage of cells through "G 2 ". At 2 hours post-labelling in this system their results showed no significant difference between the labelling indices in the controls and in the rats treated with 0.5% halothane (P>0.1 by f-test). However, we note that in these rats at 1 hour post-labelling only 13.4% of mitoses were labelled in the presence of halothane, compared with 31.8% of mitoses in controls (P<0.001). In other words, the increase in labelling index has been significantly delayed, but because of the relatively short time spent in "G 2 " the delayed plateau of maximum labelling due to the extension of this stage by halothane cannot be demonstrated by hourly fixation. There is, therefore, no inconsistency between our results and those of Bruce'and Traurig, and "G 2 " is seen to be affected by halothane in both plants and animals.
BRITISH JOURNAL OF ANAESTHESIA
EFFECTS OF HALOTHANE ON DNA SYNTHESIS AND MITOSIS
Mitchison, J. M. (1973). Differentiation in the cell cycle; in The Cell Cycle, in Development and Differentiation (eds. Balls, M., and Billet, F. S.); pp. 1-11. C.U.P. Nunn, J. F. (1972). Anaesthesia and the living cell: specificity of action. Acta Anaesthesiol. Scand., 16, 169. Lovis, J. D., and Kimball, K. L. (1971). Arrest of mitosis by halothane. Br. J. Anaesth., 43, 524. Ostergren, G. (1944). Colchicine mitosis, chromosome contractions, narcosis and protein chain folding. Hereditas, 30, U29. Powell, J. N., Grant, C. J., Robinson, S. M., and Radford, S. G. (1973). A comparison with halothane of the hormonal and anaesthetic properties of ethylene in plants. Br. J. Anaesth., 45, 682. Radford, S. G. (1971). Immuno-suppressive effect • of surgery. Lancet, 2, 1091. Prensky, W., and Smith, H. H. (1965). The mechanism of 5-amino-uracil-induced synchrony of cell division in Vicia faba root meristems. J. Cell Biol., 2A, 401. Rasch, R. W., Rasch, E. M., and Woodard, J. W. (1967). Heterogeneity of nuclear populations in root meristems. Caryologia, 20, 87. Snegireff, S. L., Cox, J. R., and Eastwood, D. W. (1968). The effect of nitrous oxide, cyclopropane or halothane on neural tube mitotic index, weight, mortality and gross anomaly rate in the developing chick embryo; in Toxicity of Anaesthetics (ed. Fink, B. R.). Baltimore: Williams & Wilkins Co. Webster, P. L., and Davidson, D. (1969). Changes in the duration of the mitotic cycle induced by colchicine and indol-3yl-acetic acid in Vicia faba roots. J. Exp. Bot., 20, 671.
PAKISTAN ASSOCIATION OF ANAESTHETISTS General Secretary: (Pakistan branch)
Secretary: (U.K. branch)
Dr M. SALIM, MB, BS, MCPS (PAK.), DA (LOND.), FFARCS (ENG. AND IREL.),
Dr M. A. SANDHU, MB, BS(PB.), DA, FFARCS (ENG.),
Bungalow No. 5/287, Mandi-Bahaud-Din, District Gujrat, Pakistan. Tel.: 241.
North Staffordshire Royal Infirmary, Stoke-on-Trent, Staffs., U.K. Tel.: 44161 (Stoke-on-Trent).
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Clowes, F. A. L. (1963). X-irradiation of root meristems. Ann. Bot. N.S., 27, 243. Cullen, B. F., Sample, W. F., and Chretien, P. B. (1972). The effect of halothane on phytohaemagglutinininduced transformation of human lymphocytes in vitro. Anesthesiology, 36, 206. Davidson, D., and MacLeod, R. D. (1968). Heterogeneity in cell behaviour in primordia of Vicia faba. Chromosoma, 26, 470. Evans, H. J., Neary, G. J., and Tonkinson, S. M. (1957). The use of colchicine as an indicator of mitotic rate in broad bean root meristems. J. Genet., 55, 487. Eyring, H. J., Woodbury, W., and d'Arrigo, J. S. (1973). A molecular mechanism of general anesthesia. (Editorial.) Anesthesiology, 38, 415. Geddes, I. C. (1971). Cellular metabolism in relation to anaesthesia; in General Anaesthesia, 3rd edn. (eds. Gray, C. T., and Nunn, J. F.). London: Butterworths. Grant, C. J. (1965). Chromosome aberrations and the mitotic cycle in Trillium after X-irradiation. Mutat. Res.. 2, 247. Heslot, H. (1966). Chromosome aberrations and the chromosome cycle in Vicia faba after treatments with nitroso-methyl-urethane and nitroso-ethyl-urethane. Chromosomes Today, 1, 118. Howard, A., and Dewey, D. L. (1960). Variation in the period preceding deoxyribonucleic acid synthesis in beanroot cells; in The Cell Nucleus (ed. J. S. Mitchell), pp. 155-160. London: Butterworths. Ishii, D. N., and Corbascio, A. N. (1971). Some metabolic effects of halothane on mammalian tissue culture cells in vitro. Anesthesiology, 34, 427.
657
EFFECTS OF HALOTHANE ON DNA SYNTHESIS AND MITOSIS IN ROOT TIP MERISTEMS OF VICIA FABA C. J. GRANT, J. N. POWELL AND SHEILA G. RADFORD SUMMARY
It was proposed by Ostergren (1944) that many of the pharmacological actions of anaesthetic agents were associated with conformational changes in protein molecules. In his view these actions included not only narcosis in its strictest sense, but also "narcosis" of the cell cycle, this consisting of an over-all inhibitory or "poison" effect, and a more specific colchicine-like arrest of metaphase associated with disruption of the mitotic spindle and with contraction of chromosomes (c-mitosis). Ostergren's protein-change theory of narcosis has recently received strong support (Nunn, 1972; Eyring, Woodbury and di Arrigo, 1973), and the association between narcosis and c-mitosis (Andersen, 1966), and between narcosis and depression of various aspects of the metabolism of replicating cells (Geddes, 1971) have been amply confirmed. However, recent studies of the effect of halothane on the different stages of the cell cycle have led to some confusion. In 1969 Bruce and Traurig presented data from an autoradiographic study of the incorporation of tritiated thymidine into rat gut mucosa, from which they concluded that only the " S " phase of the cell cycle was affected. Delay at this point alone could account for the depression of mitotic index (MI) that had been found in the chick embryo neural tube after exposure to either 1.2 or 1.6% halothane (Snegireff, Cox and Eastwood, 1968), though this depression was not marked and was not seen after exposure to 2% halothane. It was then reported that in hepatoma and Hela cells depression of DNA synthesis by halothane was minimal over a short term (Ishii and Corbascio, C. J. GRANT, M.A., D.PHIL.; J. N. POWELL, M.B., B.CH., F.F.A.RX.S.; SHEILA G. RADFORD, A.LM.L.T.; Departments
of Botany and Anaesthetics, University of Bristol.
1971) and a c-mitotic effect of halothane was demonstrated in Vicia seedlings (Nunn, Lovis and Kimball, 1971), leading to the suggestion that the persistence of labelled mitotic figures in the original study by Bruce and Traurig might be attributed to such a metaphase arrest. However, the marked reductions in MI which accompany the c-mitosis in Vicia cannot be explained on the basis of metaphase arrest alone (Powell et al., 1973) and depression of DNA synthesis has now been demonstrated, after prolonged exposure to halothane, in PHA-stimulated lymphocytes (Bruce, 1972a; Cullen, Sample and Chretien, 1972), though the effect of a 2-hour exposure to 2% halothane appears to be reversible (Powell and Radford, 1971). We now report further studies of the action of halothane on the cell cycle in Vicia. These show that, in addition to both the " S " phase and mitosis, the "G 2 " phase is affected. METHODS AND MATERIALS
Treatments were carried out on primary roots of 11-day-old Vicia faba seedlings (Sutton's Prolific Longpod) which had been grown and standardized as described by Evans, Neary and Tonkinson (1957). 1% halothane in air was prepared as described by Powell et al. (1973), and was passed through all vessels and solutions for a minimum period of 1 hour before these were used. Experiment I. A. Control. Forty roots were immersed for 30 min in 500 ml of an aerated solution of 2 /tCi/ml tritiated tbymidine (Specific Activity 5.0Ci/mM; Radiochemical Centre, Amersham, U.K.) in tap
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on July 3, 2015
Further investigations on cell division and the cell cycle in Vicia faba show that halothane has a depressant effect on cells in all the phases of mitosis and also that it delays or inhibits the passage of cells through " S " and "G 2 ". An effect on cells in " G j " is not excluded. Comparison with colchicine emphasizes the widespread effects of halothane in contrast to the stage-specific effect of colchicine. The partial division synchrony induced by a short halothane exposure is discussed.
654
BRITISH JOURNAL OF ANAESTHESIA
Experiment II. Twenty roots were immersed in tap water aerated with 1 % halothane. Samples fixed after 0, 1, 2 and 3 hours of treatment. 1.5-mm tips were later made into permanent feulgen-stained squash preparations. The slides were then coded and randomized before being scored for the phases of mitosis. RESULTS
Experiment I. Mitotic index (fig. 1). In continuous halothane there was a rapid decline in MI over the first 5 hours to less than 20% of the control value. The MI then continued to decrease at a slower rate with a transient recovery at 10 hours. Re-examination of the decoded slides showed that many of the metaphases seen at 10 hours were labelled only in the late replicating hetero-chromatic regions of the chromosomes, indicating that they had been in late " S " at the time of labelling. After the 2-hour exposure to halothane, the mitotic index continued to decrease for a further 3 hours. This was followed by a series of waves of division during which the mitotic index increased above control values. Metaphase labelling index (fig. 2). Continuous halothane caused a marked delay in the appearance of labelled metaphases compared with controls, the peak being small and transient. After the 2-hour exposure to halothane, there was an initial delay,
4 S 6 7 I 9 Time since labelling (hours) •—•control;
• — • continuous halothane;
ID
II
12
>.—A halolhane loi first 2 hours only
FIG. 2. The effects of halothane (1%) on metaphase labelling index.
followed by a series of waves of labelled metaphases. Labelling of interphase nuclei (table I). The presence of halothane during or after labelling did not significantly alter the proportion of interphase nuclei that carried label 2 hours after labelling. Experiment II (fig. 3). Exposure to halothane produced a 40% increase in metaphase count at 1 hour; however, this was not
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on July 3, 2015
water. The roots were then rinsed and returned to aerated tap water. B. Continuous halothane. Forty roots were immersed for 30 min in a similar 3 H TdR solution through which 1% halothane was being passed. They were then rinsed and placed in tap water aerated with 1 % halothane. C. Halothane for 2 hours only. Forty roots were labelled for 30 min as in A, rinsed and placed for 2 hours in tap water aerated with 1 % halothane. They were returned to aerated tap water. Roots from each of these regimes were fixed at hourly intervals after labelling. 1.5-mm tips were later made into feulgen-stained squash preparations, which were covered with Kodak AR10 stripping film and exposed for 6 weeks. The slides, after being 4 8 Time since start of treatment (hours) developed and made permanent, were coded and O—O colchicine; • — • continuous halothane 1 0 A—A halothane for first randomized before being scored for: (i) mitotic 2 hours only index (MI); (ii) metaphase labelling index; (iii) the FIG. 1. The effects of halothane (1%) and colchicine percentage of interphase cells carrying labelled (0.1%) on mitotic index. (Halothane data, Experiment I; colchicine data, Evans, Neary and Tonkinson, 1957.) nuclei at 2 hours after labelling.
EFFECTS OF HALOTHANE ON DNA SYNTHESIS AND MITOSIS TABLE I.
Proportion on interphase cells with labelled nuclei 2 hours post-labelling.
Mean per cent (SD)
Experiment IA IB IC
36.6 (8.1) 32.4 (4.5) 35.2 (6.6)
9 9 6
Analysis of variance shows no significant between treatments at the 5 % level.
Prophase...
Metaphase
Ana-telophase
Mitosis ...
n
-o
50-
-100
K H 0 1 2 3
•—•
0 1 2 3 0 1 2 3 0 1 2 3 D u r a t i o n of t r e a t m e n t (hours)
halothanc 1.0;', i
O—O c o l c h i c i n e 0.1'/,
FIG. 3. The effects of halothane (1%) and colchicine (0.1%) on the phases of mitosis. (Halothane data, Experiment II; colchicine data, Evans, Neary and Tonkinson, 1957.)
maintained subsequently and was followed by a progressive reduction. By 3 hours anaphase cells had nearly vanished, those which were left having a curious "slow motion" appearance with contracted, though scattered, chromosomes; telophase, however, had not decreased to a corresponding extent at this time. DISCUSSION
(a) Halothane and the stages of the cell cycle. In figure 1 the effects of halothane on mitotic index are compared with those of colchicine (data from Evans, Neary and Tonkinson, 1957). Colchicine has no direct effect on the entry of cells into prophase but, by disrupting the spindle fibres, holds chromosomes at c-meta/anaphase for up to 6 hours before a delayed restitution telophase nucleus is
formed. As a result there is an initial linear increase in MI followed after about 6 hours by a plateau when rates of entry to and exit from mitosis reach an approximate balance (Evans, Neary and Tonkinson, 1957). In contrast, halothane treatment causes a rapid fall in MI which indicates that cells already in mitosis when treatment begins are able to complete the process, although at a reduced rate; and also that the rate of entry of "G 2 " cells to prophase is decreased. Halothane-induced acceleration of mitosis as an alternative explanation for the decrease in MI is excluded by the autoradiographic evidence (fig. 2) which shows that cells in late " S " when treatment begins reach metaphase in 10 hours, twice as long as control cells. The transient nature of this wave of cells labelled in late " S " indicates that cells in earlier stages of " S " and in " G j " at the start of treatment are unable to complete the cell cycle in the presence of halothane. In the light of these results, and since both Nunn, Louis and Kimball (1971) and Powell et al. (1973) have referred to the induction of "colchicine-type mitosis" by halothane, the initial effect of halothane on the distribution of cells between the various phases of mitosis was investigated (Experiment II). This is shown in figure 3 together with that of colchicine (data from Evans, Neary and Tonkinson, 1957) over 3 hours. Two important distinctions can be made between the actions of colchicine and halothane. Firstly, the more rapid decrease of ana/telophase cells in colchicine shows that this drug has a minimal effect on cells which have passed metaphase, whereas halothane has a delaying effect on both anaphase and telophase. Secondly, the failure of metaphase cells to continue their build-up after the first hour in halothane confirms that entry of cells into diis phase is progressively inhibited. Since there is no corresponding build-up of prophase cells and the MI decreases before treated " S " cells are expected to arrive at mitosis ("G 2 " in untreated cells takes approximately 4 hours), inhibition must be occurring in "G 2 " to a greater extent than in mitosis. The actions of halothane thus differ from those of eolchicine and are more akin to those of less specific mitotic inhibitors such as para-dichlorobenzine and 8-hydroxy-quinoline (Evans, Neary and Tonkinson, 1957; Powell et al., 1973). With regard to the effects of halothane on interphase cells, although we have shown (table I) that
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(b) Recovery from a short exposure. The data illustrated in figures 1 and 2 indicate that some cells at the end of " S " when treatment begins are delayed for the period of exposure to
halothane and then proceed normally through "G 2 " and mitosis. The wavelike nature of further recovery is a more complex phenomenon, which may result from either the differential blocking of a number of sequential metabolic events or the differential response of cells in different regions of the meristem (e.g. stele, cap initials, quiescent centre (Clowes, 1961a)). Evidence for the latter comes from numerous references in the literature to the heterogeneity of the primordial cell populations in Vicia {aba (Howard and Dewey, 1960; Rasch, Rasch and Woodard, 1967; Davidson and MacLeod, 1968), and other plants such as Zea (Clowes, 1961b) and Trillium (Grant, 1965; Boothroyd and Mark, 1970). There is also evidence that these populations may respond differentially to stimuli such as X-rays (Clowes, 1963) or colchicine and auxin (Webster and Davidson, 1969). This subject has been reviewed by Barlow (1973). Because of the anatomical complexity of the root-tip meristem, there is little direct evidence in Vicia for the former. However, in "simple" cultures of both animal and plant cells, there is an increasing body of evidence for the existence of numerous stage specific enzyme syntheses at which blocking might occur (Mitchison, 1973). Also the action of anaesthetic agents does appear to exhibit specificity at a molecular level (Nunn, 1972; Brammall, Beard and Hulands, 1973). It seems that both explanations may apply in this instance. Studies of the recovery from halothane of mammalian cells in culture are currently in progress. REFERENCES
Andersen, N. B. (1966). The effects of C.N.S. depressants on mitosis. Acta Anaesthesiol. Scand. (Suppl.), 22, 3. Barlow, P. W. (1973). Mitotic cycles in root meristems; in The Cell Cycle, in Development and Differentiation (eds. Balls, M., and Billett, F. S.), pp. 133-165. C.U.P. Boothroyd, E. R., and Mark, N. M. (1970). Mitotic cycles in root tip cells of two species of Trillium (Liliaceae). Can. J. Genet. Cytol, 12, 750. Brammall, A., Beard, D., and Hulands, G. H. (1973). The effects of inhalational anaesthetic agents on the enzyme glutamate dehydrogenase. Br. J. Anaesth., 45, 923. Bruce, D. L. (1972a). Halothane inhibition of phytohaemagglutinin-induced transformation of lymphocytes. Anesthesiology, 36, 201. (1972b). Normal thymidine entry into halothanetreated lymphocytes. Anesthesiology, 37, 588. Traurig, H. H. (1969). The effect of halothane on the cell cycle in rat small intestine. Anesthesiology, 30, 401. Clowes, F. A. L. (1961a). Apical Meristems. Oxford: Blackwell Scientific Publications. (1961b). Duration of the mitotic cycle in a meristem. J. Exp. Bot., 12, 283.
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up to 2 hours of halothane treatment has no effect on the number of nuclei capable of fixing 3 H TdR, it is not possible to say whether there was a reduction in the amount of thymidine fixed per nucleus. Our observations in this respect are compatible with those of Cullen, Sample and Chretien (1972) and Bruce (1972a,b), who showed that halothane does not interfere with the transport of thymidine into cytoplasm and nuclei but that it does partially block its incorporation into chromosomal DNA in PHAstimulated lymphocytes. Such a block may explain why the cells in early " S " or " G j " cannot complete the cycle in Vicia. In Vicia, therefore, all stages of the cell cycle are affected by halothane with the possible exception of "G^', which is technically more difficult to investigate. There is no reason, however, to suppose that " G L " is preferentially spared. Halothane is seen to be different from those agents which are known to block specific parts of the cycle, for example colchicine, 5-amino-uracil, which accumulates cells at the beginning of " S " (Prensky and Smith, 1965) and the radiomimetic nitroso-alkyl-urethanes which attack chromosomes in the process of replication but have no effect on them during "G 2 " (Grant and Heslot, 1966). The conclusions we have drawn from this study differ from those of Bruce and Traurig (1969) who suggested that, in rat gut epithelium, halothane had no effect on the passage of cells through "G 2 ". At 2 hours post-labelling in this system their results showed no significant difference between the labelling indices in the controls and in the rats treated with 0.5% halothane (P>0.1 by f-test). However, we note that in these rats at 1 hour post-labelling only 13.4% of mitoses were labelled in the presence of halothane, compared with 31.8% of mitoses in controls (P<0.001). In other words, the increase in labelling index has been significantly delayed, but because of the relatively short time spent in "G 2 " the delayed plateau of maximum labelling due to the extension of this stage by halothane cannot be demonstrated by hourly fixation. There is, therefore, no inconsistency between our results and those of Bruce'and Traurig, and "G 2 " is seen to be affected by halothane in both plants and animals.
BRITISH JOURNAL OF ANAESTHESIA
EFFECTS OF HALOTHANE ON DNA SYNTHESIS AND MITOSIS
Mitchison, J. M. (1973). Differentiation in the cell cycle; in The Cell Cycle, in Development and Differentiation (eds. Balls, M., and Billet, F. S.); pp. 1-11. C.U.P. Nunn, J. F. (1972). Anaesthesia and the living cell: specificity of action. Acta Anaesthesiol. Scand., 16, 169. Lovis, J. D., and Kimball, K. L. (1971). Arrest of mitosis by halothane. Br. J. Anaesth., 43, 524. Ostergren, G. (1944). Colchicine mitosis, chromosome contractions, narcosis and protein chain folding. Hereditas, 30, U29. Powell, J. N., Grant, C. J., Robinson, S. M., and Radford, S. G. (1973). A comparison with halothane of the hormonal and anaesthetic properties of ethylene in plants. Br. J. Anaesth., 45, 682. Radford, S. G. (1971). Immuno-suppressive effect • of surgery. Lancet, 2, 1091. Prensky, W., and Smith, H. H. (1965). The mechanism of 5-amino-uracil-induced synchrony of cell division in Vicia faba root meristems. J. Cell Biol., 2A, 401. Rasch, R. W., Rasch, E. M., and Woodard, J. W. (1967). Heterogeneity of nuclear populations in root meristems. Caryologia, 20, 87. Snegireff, S. L., Cox, J. R., and Eastwood, D. W. (1968). The effect of nitrous oxide, cyclopropane or halothane on neural tube mitotic index, weight, mortality and gross anomaly rate in the developing chick embryo; in Toxicity of Anaesthetics (ed. Fink, B. R.). Baltimore: Williams & Wilkins Co. Webster, P. L., and Davidson, D. (1969). Changes in the duration of the mitotic cycle induced by colchicine and indol-3yl-acetic acid in Vicia faba roots. J. Exp. Bot., 20, 671.
PAKISTAN ASSOCIATION OF ANAESTHETISTS General Secretary: (Pakistan branch)
Secretary: (U.K. branch)
Dr M. SALIM, MB, BS, MCPS (PAK.), DA (LOND.), FFARCS (ENG. AND IREL.),
Dr M. A. SANDHU, MB, BS(PB.), DA, FFARCS (ENG.),
Bungalow No. 5/287, Mandi-Bahaud-Din, District Gujrat, Pakistan. Tel.: 241.
North Staffordshire Royal Infirmary, Stoke-on-Trent, Staffs., U.K. Tel.: 44161 (Stoke-on-Trent).
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Clowes, F. A. L. (1963). X-irradiation of root meristems. Ann. Bot. N.S., 27, 243. Cullen, B. F., Sample, W. F., and Chretien, P. B. (1972). The effect of halothane on phytohaemagglutinininduced transformation of human lymphocytes in vitro. Anesthesiology, 36, 206. Davidson, D., and MacLeod, R. D. (1968). Heterogeneity in cell behaviour in primordia of Vicia faba. Chromosoma, 26, 470. Evans, H. J., Neary, G. J., and Tonkinson, S. M. (1957). The use of colchicine as an indicator of mitotic rate in broad bean root meristems. J. Genet., 55, 487. Eyring, H. J., Woodbury, W., and d'Arrigo, J. S. (1973). A molecular mechanism of general anesthesia. (Editorial.) Anesthesiology, 38, 415. Geddes, I. C. (1971). Cellular metabolism in relation to anaesthesia; in General Anaesthesia, 3rd edn. (eds. Gray, C. T., and Nunn, J. F.). London: Butterworths. Grant, C. J. (1965). Chromosome aberrations and the mitotic cycle in Trillium after X-irradiation. Mutat. Res.. 2, 247. Heslot, H. (1966). Chromosome aberrations and the chromosome cycle in Vicia faba after treatments with nitroso-methyl-urethane and nitroso-ethyl-urethane. Chromosomes Today, 1, 118. Howard, A., and Dewey, D. L. (1960). Variation in the period preceding deoxyribonucleic acid synthesis in beanroot cells; in The Cell Nucleus (ed. J. S. Mitchell), pp. 155-160. London: Butterworths. Ishii, D. N., and Corbascio, A. N. (1971). Some metabolic effects of halothane on mammalian tissue culture cells in vitro. Anesthesiology, 34, 427.
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