79
THE LANCET
Ageing
at the
Cellular Level
do human beings, and most other higher animals, age while many organisms, including higher plants, can live and propagate indefinitely ? Mammalian cells in culture can "transform" to become apparently immortal, whereas human diploid fibroblasts cannot. In subcultures normal growth continues for a long time but is followed by senescence and death. Unlike rodent cells, human cells do not undergo spontaneous transformation into permanent lines. Various explanations have been offered for the finite lifespan of human cells in culture. One is that human fibroblasts have a built-in genetic programme leading after a defined number of divisions to "terminal differentiation".1 On this hypothesis all the cells in any population would be of approximately the same age and would therefore be expected to have nearly the same life expectancy. In fact, individual cells taken from a population of fibroblasts are highly variable in their doubling potential.22 To account for these and other observations, HOLLIDAY and his colleagues3 have proposed a commitment theory of fibroblast ageing which states that, in a population of potentially immortal cells, there is a given probability that mitosis will give rise to fibroblasts that are irreversibly committed to senescence and death. During the period between commitment and senescence cells are assumed to maintain normal growth, so that uncommitted cells are diluted by committed ones and may ultimately be lost on subculturing. The model was tested by variation of the population size at different passage levels, and the results were compatible with the commitment theory. When cultures contain equal numbers of distinguishable kinds of cell (with enzyme or chromosome markers) they multiply at equivalent rates for many subcultures, but one or other cell type frequently becomes predominant at the end of the lifespan. The biochemical explanation of ageing that has attracted most attention is ORGEL IS4 suggestion that errors in protein synthesis can lead to an irreversible increase in errors and a final lethal catastrophe. Ageing of fibroblasts is accompanied by alterations or defects in enzymes, genes, chromosomes, D.N.A. replication, and repair,3 all of which might be expected if abnormal macromolecules are accumulating. ORGEL’s hypothesis has How and
1 2
why
Hayflick, L. Am. J. med Sci. 1973, 265, 433. Martin, G. M., Sprague, C. A., Norwood, T. H., Pendergrass, W. R. Am. J Path 1974, 74, 137
3. Holliday, R, Huschtcha, L. I., Farrant, G. M., Kirkwood, T. B. L. Science,
1977, 198, 366.
4
Orgel, L. E
Proc natn. Acad. Sci., U.S.A. 1970,
67, 1476.
lately been invoked by KIRKWOODs in an evolutionary interpretation of ageing. To provide the genetic diversity upon which evolution acts to ensure survival, the organism must make occasional errors in copying its genome. Too many mistakes would be a serious handicap, so what is needed is a tolerable level of error. Given enough time, selection ensures that organisms with optimal error levels will dominate. KIRKWOOD suggests that the stability of translation must be very high in germ-line cells, which contribute information to subsequent generations, but that error regulation may be much less stringent in somatic cells. Accuracy can be achieved only through the expenditure of energy, either by direct or kinetic "proof-reading" or by destruction of erroneous products or cells containing them. Ageing is envisaged as the result of an energy-saving switch-off of the mechanisms responsible for high accuracy in translation at or around the time of differentiation of somatic cells from the germ line. This may leave the somatic cells unstable so that a small random increase in errors commits them to an eventual error catastrophe. Thus ageing at the cellular level continues to provide material for hypothesis and experimentation. The commitment theory opens up the possibility of isolating populations enriched with committed or uncommitted cells. In this way the populations might be propagated indefinitely, which would be not only of theoretical interest to gerontologists but also of practical usefulness in studies of somatic cell genetics as well as propagation of viruses for vaccination. These are at present seriously impeded by the senescence of human diploid fibroblasts. The commitment theory is also relevant to the survival in vivo of clones of immunocompetent cells-see, for example, the apparent "terminal differentiation" in parasitic infec-
tions.6 WET OR DRY INHALERS?
WHAT is the truth about aerosol toxicity? As with chloroform, saccharin, cyclamates, and asbestos, as well as sugar and animal fats, facts and fiction have been so closely admixed by interested parties-political, environmental and commercial-that the disinterested observer has difficulty in discerning the data on which to make a judgment. Chlorofluorocarbons (c.F.c.s) are used not only as propellants in all sorts of aerosol cans but also in foam-plastic manufacture and quick freezing of foods. In medicine they have been used as propellants for drug inhalers, particularly asthma drugs such as isoprenaline and salbutamol, and also for making frozen sections in pathology laboratories. The direct toxic effects which have caused most concern are cardiac dysrhythmias and myocardial depression, possibly involving sensitisation of the myocardium to circulatory catechola5. Kirkwood, T. B. L. Nature, 1977, 270, 301. 6. Corsini, A. C., Clayton, C., Askonas, B. A , 1977, 29, 122.
Ogilvie,
B. M. Clin. exp. Immun.
80
mines; and lately there has also been alarm about their long-term effect on the atmosphere which might increase risks of skin cancer. Reviewing the toxicity of C.F.C.S, McLean2 concludes that the main risk is associated with misuse among "sniffers", who inhale them for kicks and may die with cardiac dysrhythmias. There seems to be no hazard in household use ofc.F.c. propellants in spray cans, nor does there seem to be an increased risk of sudden death among workers who make refrigerators, frozen food, or foam plastic. There has been a report of palpitations among pathology staff preparing frozen sections3 but the inhaled-air concentration of the particular c.F.c. was many times lower than that required to produce experimental dysrhythmias in animals: this matter needs prospective investigation. The possibility that industrial pollutants might influence ozone concentrations in the stratosphere cannot be dismissed but, as McLean points out, there is probably greater cause for concern about the oxides of nitrogen in the air, to which we are subjected and about which we do not hear so much. Furthermore, there has already been a great reduction in the use of c..c. propellants in aerosol cans. A good manufacturer will exclude any unnecessary ingredient of a preparation, especially if that ingredient is suspected of an undesirable effect. Most of us, no doubt, had come to accept that aerosol preparations of drugs for inhalation required some propellant or vehicle for satisfactory delivery. The introduction of the ’Spinhaler’ for delivery of disodium cromoglycate in the form of ’Intal’ showed that this was not true. Now a device which delivers salbutamol as a drug powder in a lactose base for inhalation has been described,4 and first clinical trials5.6 show that it is well tolerated with bronchodilator properties similar to those of the conventional pressurised aerosol. If further controlled studies confirm these results with salbutamol, then the onus will be on the manufacturers of other such drugs to produce preparations free of c.F.c. propellants. As McLean concludes : "Economic disruption that would be caused by a sudden ban on CFC spray cans would be unjustified, but the replacement of CFC spray cans by alternatives over the next three years would seem practical and desirable." INTRAVENOUS VERSUS INHALED SALBUTAMOL AN uncomfortable hiatus in the management of acute asthma often occurs shortly after admission, whilst the beneficial effects of corticosteroids are awaited. During this time, other agents such as aminophylline and beta agonists play a vital role. Selective j3 agonists such as salbutamol and terbutaline are now available for both parenteral and inhaled use, and the choice between these routes of administration may sometimes pose problems. Williams and Seatonhave compared the effects of salbutamol given intravenously and by inhalation during the first few days of hospital admission for acute severe asthma. Patients were first given salbutamol 5 mg by positive-pressure nebulisation and severe
1. Lancet, 1975, , 1073. 2. McLean, A. E M. Br. J. clin. Pharmac. 1977, 4, 663. 3. Speizer, F. E., Wegman, D. H., Ramirez, A. New Engl. 624. 4 Hallworth, G W. Br. J. clin. Pharmac. 1977, 4, 689. 5. Duncan, D , et al. ibid. p. 669. 6 Hartley, J. P. R. ibid. p. 673. 7. Williams, S., Seaton, A. Thorax, 1977, 32, 555.
J.
Med.
1975, 293,
flow response was measured over the next twenty minutes. Salbutamol 200 ;ag was then injected intravenously and any further response was recorded. The procedure was repeated each day. It transpired that inhaled salbutamol was often totally ineffective during the first phase of an acute severe asthmatic attack, whereas intravenous salbutamol almost always produced useful bronchodilatation. However, once patients began to cough and to raise their sputum, inhaled salbutamol proved just as effective as intravenous. One simple mechanical explanation for this failure of inhaled salbutamol is that the drug does not penetrate small airways, which in severe asthma are plugged with viscid mucus and narrowed by oedema and smoothmuscle spasm. Another possibility is that acute asthma is accompanied by partial p-receptor blockade-the hypothesis advanced by Cookson and Reed8 and elaborated by Szentivanyi.9 In fact, the p-blockade hypothesis receives no support from the latest work since intravenous salbutamol was useful even in the early stages of acute asthma. Moreover, further work from the Cardiff group showed that asthmatics during severe attacks were nevertheless able to mount a normal metabolic response to intravenous salbutamol, with appropriate rise in free fatty acids and insulin levels, again arguing
peak
against generalised p-receptor blockade. 10 These investigations support the use of intravenous salbutamol as an alternative to aminophylline in the early stages of acute severe asthma." The ideal dose has not been established, but 4 fLg/kg by slow intravenous infusion over 10 minutes achieved good bronchodilatation without harmful side-effects. CAROTID BODIES IT is about fifty years since Corneel Heymans stumbled on the chemical sensitivity of the carotid body, if stumbled is the right word to use when a vigorous and vigilant observer deliberately embarks on an eccentric experiment to see what will happen.12 He injected a tiny dose of cyanide into each carotid artery of an ancesthetised dog; one carotid body happened to have been previously denervated, and only the injection on the other side provoked panting. The carotid body behaves as an hypoxia sensor and so, under hypoxic conditions, instead of simply dying like normal aerobic tissues, it generates an increased impulse traffic which serves as an index of the partial pressure of oxygen in the arterial bloodthough also reflecting in part the concentration of hxmoglobin, the pH, and the PC02. Further, the impulse traffic does not change only as a final warning in extreme hypoxia; it changes all the way down from pressures as high as 200 mm Hg (28 kPa)-pressures presumably never naturally encountered by any mammal." How this unusual feat is performed is not yet agreed. Besides two main types of cell, the carotid body contains both efferent and afferent nerve-endings, but uncertainty persists about which are which, and the electron microscope does not reveal conclusively which way transmission occurs between a cell and a nerve-ending8. Cookson, D. U., Reed, C. E. Am. Rev. resp. Dis. 1963, 88, 636. 9. Szentivanyi, A. J. Allergy, 1968, 42, 203. 10. Nogrady, S. G., Hartley, J. P. R., Seaton, A. Thorax, 1977, 32, 559. 11 Williams, S. J., Parrish, R. W., Seaton, A. Br. med. J. 1975, iv, 685. 12. Neil, E. Archs int Pharmacodyn. Ther. 1973, 202, suppl. p. 283. 13. Biscoe, T. J. Am Rev. resp. Dis. 1977, 115, no. 6, part 2, p. 189.
THE LANCET
Ageing
at the
Cellular Level
do human beings, and most other higher animals, age while many organisms, including higher plants, can live and propagate indefinitely ? Mammalian cells in culture can "transform" to become apparently immortal, whereas human diploid fibroblasts cannot. In subcultures normal growth continues for a long time but is followed by senescence and death. Unlike rodent cells, human cells do not undergo spontaneous transformation into permanent lines. Various explanations have been offered for the finite lifespan of human cells in culture. One is that human fibroblasts have a built-in genetic programme leading after a defined number of divisions to "terminal differentiation".1 On this hypothesis all the cells in any population would be of approximately the same age and would therefore be expected to have nearly the same life expectancy. In fact, individual cells taken from a population of fibroblasts are highly variable in their doubling potential.22 To account for these and other observations, HOLLIDAY and his colleagues3 have proposed a commitment theory of fibroblast ageing which states that, in a population of potentially immortal cells, there is a given probability that mitosis will give rise to fibroblasts that are irreversibly committed to senescence and death. During the period between commitment and senescence cells are assumed to maintain normal growth, so that uncommitted cells are diluted by committed ones and may ultimately be lost on subculturing. The model was tested by variation of the population size at different passage levels, and the results were compatible with the commitment theory. When cultures contain equal numbers of distinguishable kinds of cell (with enzyme or chromosome markers) they multiply at equivalent rates for many subcultures, but one or other cell type frequently becomes predominant at the end of the lifespan. The biochemical explanation of ageing that has attracted most attention is ORGEL IS4 suggestion that errors in protein synthesis can lead to an irreversible increase in errors and a final lethal catastrophe. Ageing of fibroblasts is accompanied by alterations or defects in enzymes, genes, chromosomes, D.N.A. replication, and repair,3 all of which might be expected if abnormal macromolecules are accumulating. ORGEL’s hypothesis has How and
1 2
why
Hayflick, L. Am. J. med Sci. 1973, 265, 433. Martin, G. M., Sprague, C. A., Norwood, T. H., Pendergrass, W. R. Am. J Path 1974, 74, 137
3. Holliday, R, Huschtcha, L. I., Farrant, G. M., Kirkwood, T. B. L. Science,
1977, 198, 366.
4
Orgel, L. E
Proc natn. Acad. Sci., U.S.A. 1970,
67, 1476.
lately been invoked by KIRKWOODs in an evolutionary interpretation of ageing. To provide the genetic diversity upon which evolution acts to ensure survival, the organism must make occasional errors in copying its genome. Too many mistakes would be a serious handicap, so what is needed is a tolerable level of error. Given enough time, selection ensures that organisms with optimal error levels will dominate. KIRKWOOD suggests that the stability of translation must be very high in germ-line cells, which contribute information to subsequent generations, but that error regulation may be much less stringent in somatic cells. Accuracy can be achieved only through the expenditure of energy, either by direct or kinetic "proof-reading" or by destruction of erroneous products or cells containing them. Ageing is envisaged as the result of an energy-saving switch-off of the mechanisms responsible for high accuracy in translation at or around the time of differentiation of somatic cells from the germ line. This may leave the somatic cells unstable so that a small random increase in errors commits them to an eventual error catastrophe. Thus ageing at the cellular level continues to provide material for hypothesis and experimentation. The commitment theory opens up the possibility of isolating populations enriched with committed or uncommitted cells. In this way the populations might be propagated indefinitely, which would be not only of theoretical interest to gerontologists but also of practical usefulness in studies of somatic cell genetics as well as propagation of viruses for vaccination. These are at present seriously impeded by the senescence of human diploid fibroblasts. The commitment theory is also relevant to the survival in vivo of clones of immunocompetent cells-see, for example, the apparent "terminal differentiation" in parasitic infec-
tions.6 WET OR DRY INHALERS?
WHAT is the truth about aerosol toxicity? As with chloroform, saccharin, cyclamates, and asbestos, as well as sugar and animal fats, facts and fiction have been so closely admixed by interested parties-political, environmental and commercial-that the disinterested observer has difficulty in discerning the data on which to make a judgment. Chlorofluorocarbons (c.F.c.s) are used not only as propellants in all sorts of aerosol cans but also in foam-plastic manufacture and quick freezing of foods. In medicine they have been used as propellants for drug inhalers, particularly asthma drugs such as isoprenaline and salbutamol, and also for making frozen sections in pathology laboratories. The direct toxic effects which have caused most concern are cardiac dysrhythmias and myocardial depression, possibly involving sensitisation of the myocardium to circulatory catechola5. Kirkwood, T. B. L. Nature, 1977, 270, 301. 6. Corsini, A. C., Clayton, C., Askonas, B. A , 1977, 29, 122.
Ogilvie,
B. M. Clin. exp. Immun.
80
mines; and lately there has also been alarm about their long-term effect on the atmosphere which might increase risks of skin cancer. Reviewing the toxicity of C.F.C.S, McLean2 concludes that the main risk is associated with misuse among "sniffers", who inhale them for kicks and may die with cardiac dysrhythmias. There seems to be no hazard in household use ofc.F.c. propellants in spray cans, nor does there seem to be an increased risk of sudden death among workers who make refrigerators, frozen food, or foam plastic. There has been a report of palpitations among pathology staff preparing frozen sections3 but the inhaled-air concentration of the particular c.F.c. was many times lower than that required to produce experimental dysrhythmias in animals: this matter needs prospective investigation. The possibility that industrial pollutants might influence ozone concentrations in the stratosphere cannot be dismissed but, as McLean points out, there is probably greater cause for concern about the oxides of nitrogen in the air, to which we are subjected and about which we do not hear so much. Furthermore, there has already been a great reduction in the use of c..c. propellants in aerosol cans. A good manufacturer will exclude any unnecessary ingredient of a preparation, especially if that ingredient is suspected of an undesirable effect. Most of us, no doubt, had come to accept that aerosol preparations of drugs for inhalation required some propellant or vehicle for satisfactory delivery. The introduction of the ’Spinhaler’ for delivery of disodium cromoglycate in the form of ’Intal’ showed that this was not true. Now a device which delivers salbutamol as a drug powder in a lactose base for inhalation has been described,4 and first clinical trials5.6 show that it is well tolerated with bronchodilator properties similar to those of the conventional pressurised aerosol. If further controlled studies confirm these results with salbutamol, then the onus will be on the manufacturers of other such drugs to produce preparations free of c.F.c. propellants. As McLean concludes : "Economic disruption that would be caused by a sudden ban on CFC spray cans would be unjustified, but the replacement of CFC spray cans by alternatives over the next three years would seem practical and desirable." INTRAVENOUS VERSUS INHALED SALBUTAMOL AN uncomfortable hiatus in the management of acute asthma often occurs shortly after admission, whilst the beneficial effects of corticosteroids are awaited. During this time, other agents such as aminophylline and beta agonists play a vital role. Selective j3 agonists such as salbutamol and terbutaline are now available for both parenteral and inhaled use, and the choice between these routes of administration may sometimes pose problems. Williams and Seatonhave compared the effects of salbutamol given intravenously and by inhalation during the first few days of hospital admission for acute severe asthma. Patients were first given salbutamol 5 mg by positive-pressure nebulisation and severe
1. Lancet, 1975, , 1073. 2. McLean, A. E M. Br. J. clin. Pharmac. 1977, 4, 663. 3. Speizer, F. E., Wegman, D. H., Ramirez, A. New Engl. 624. 4 Hallworth, G W. Br. J. clin. Pharmac. 1977, 4, 689. 5. Duncan, D , et al. ibid. p. 669. 6 Hartley, J. P. R. ibid. p. 673. 7. Williams, S., Seaton, A. Thorax, 1977, 32, 555.
J.
Med.
1975, 293,
flow response was measured over the next twenty minutes. Salbutamol 200 ;ag was then injected intravenously and any further response was recorded. The procedure was repeated each day. It transpired that inhaled salbutamol was often totally ineffective during the first phase of an acute severe asthmatic attack, whereas intravenous salbutamol almost always produced useful bronchodilatation. However, once patients began to cough and to raise their sputum, inhaled salbutamol proved just as effective as intravenous. One simple mechanical explanation for this failure of inhaled salbutamol is that the drug does not penetrate small airways, which in severe asthma are plugged with viscid mucus and narrowed by oedema and smoothmuscle spasm. Another possibility is that acute asthma is accompanied by partial p-receptor blockade-the hypothesis advanced by Cookson and Reed8 and elaborated by Szentivanyi.9 In fact, the p-blockade hypothesis receives no support from the latest work since intravenous salbutamol was useful even in the early stages of acute asthma. Moreover, further work from the Cardiff group showed that asthmatics during severe attacks were nevertheless able to mount a normal metabolic response to intravenous salbutamol, with appropriate rise in free fatty acids and insulin levels, again arguing
peak
against generalised p-receptor blockade. 10 These investigations support the use of intravenous salbutamol as an alternative to aminophylline in the early stages of acute severe asthma." The ideal dose has not been established, but 4 fLg/kg by slow intravenous infusion over 10 minutes achieved good bronchodilatation without harmful side-effects. CAROTID BODIES IT is about fifty years since Corneel Heymans stumbled on the chemical sensitivity of the carotid body, if stumbled is the right word to use when a vigorous and vigilant observer deliberately embarks on an eccentric experiment to see what will happen.12 He injected a tiny dose of cyanide into each carotid artery of an ancesthetised dog; one carotid body happened to have been previously denervated, and only the injection on the other side provoked panting. The carotid body behaves as an hypoxia sensor and so, under hypoxic conditions, instead of simply dying like normal aerobic tissues, it generates an increased impulse traffic which serves as an index of the partial pressure of oxygen in the arterial bloodthough also reflecting in part the concentration of hxmoglobin, the pH, and the PC02. Further, the impulse traffic does not change only as a final warning in extreme hypoxia; it changes all the way down from pressures as high as 200 mm Hg (28 kPa)-pressures presumably never naturally encountered by any mammal." How this unusual feat is performed is not yet agreed. Besides two main types of cell, the carotid body contains both efferent and afferent nerve-endings, but uncertainty persists about which are which, and the electron microscope does not reveal conclusively which way transmission occurs between a cell and a nerve-ending8. Cookson, D. U., Reed, C. E. Am. Rev. resp. Dis. 1963, 88, 636. 9. Szentivanyi, A. J. Allergy, 1968, 42, 203. 10. Nogrady, S. G., Hartley, J. P. R., Seaton, A. Thorax, 1977, 32, 559. 11 Williams, S. J., Parrish, R. W., Seaton, A. Br. med. J. 1975, iv, 685. 12. Neil, E. Archs int Pharmacodyn. Ther. 1973, 202, suppl. p. 283. 13. Biscoe, T. J. Am Rev. resp. Dis. 1977, 115, no. 6, part 2, p. 189.
