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The female longevity phenomenon. Hypotheses on some molecular and cell biology aspects
m ~ t ~ s ~ ~nj Mechanisms of Ageing and Development 72 (1993) 89-95
ELSEVIER S('IENTIFI( P U B L I S H F R S IRI L A N I )
Brief review
The female longevity phenomenon. Hypotheses on some molecular and cell biology aspects Mauro Magnani*, Augusto Accorsi lstituto di Chimica Biologica 'G. Fornaini', Universith degli Studi di Urbino. Via Saffi 2, 61029 Urbino, Italy
(Received 26 December 1992; revision received 4 August 1993; accepted 26 August 1993)
Abstract
The most relevant theories on the sex-dependent longevity at population level are critically re-examined in the light of the knowledge available today on the aging process at cellular and molecular level. The aim of this perspective is to help in the understanding of the cellular and molecular bases of longevity and to indicate the most suitable biological models of investigation. Key words." Aging; Longevity; Gender gap
1. Introduction
In humans, the c o m m o n belief that females live longer than males has been supported by statistical analyses that have also demonstrated the existence o f such a difference in other animal species [1-6]. These studies have stimulated research on the biological basis o f the phenomenon and, at present, it seems possible to express reasonable hypotheses on some molecular and cell biology aspects. Before doing so it is important to state the following points: (1) The difference in the expectation o f life between males and females, also termed * Corresponding author. 0047-6374/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0047-6374(93)01398-R
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'gender gap', has changed over the years. It appears to be different in different developed countries and to depend on several parameters such as geography, pro-capita income, etc. [1-4]. (2) The higher survival of females in comparison with males, observed in animals [5,6], does not seem useful in explaining this finding in humans [1]. In fact, examination of the survival curves of laboratory rodents shows a spectrum of variable effects of sex on longevity up to species where the male has a longer lifespan [1]. (3) The 'gender gap' would not be substantially modified even though a decrease in the commoner causes of death (cardiovascular, diabetic and tumor pathologies) [7]. On the bases of the considerations reported above and taking into account the extensive reading of the literature on the subject, we have come to the conclusion that: (a) the current 'gender gap' may not be solely attributed to an advantage or a disadvantage linked to the XX- or XY-genotype, respectively (since it may vary in time and space) and (b) the 'gender gap' does exist even if all environmental, behavioral, and social influences are taken into account. In this review, we will try to critically re-examine some of the theories and hypothesis formulated to explain the 'gender gap'. During the sixties, it was proposed that sex-dependent longevity could be attributed to stronger life-stress for men than for women, as reflected in the higher incidence of cardiovascular diseases in males [8]. In the seventies smoking was claimed to be the reason for the 'gender gap' [9,10]. Female hormones were later indicated as protecting factors, though contrasting data have been reported [11]. The latter theory was founded on the observation that the lower incidence of cardiovascular disease mortality observed in women before menopause becomes similar to that of men after the climateric period. According to these previous considerations, we want to stress that all these theories are inadequate to clarify the phenomenon, although lessening it appropriately. The genetic theory is one of the oldest in trying to explain the gender gap. It should be underlined that the X-chromosome contains several genes not involved with female sex or phenotype determination. Moreover, besides the well-known random inactivation of the X-chromosome during embryogenesis and development, a non-random selection in cell proliferation seems to occur. Finally, the reported [13,14] reactivation of the X-chromosome could offer a selective advantage in female survival. Some outlines of the genetics of female longevity may also be useful in this discussion, since there is experimental evidence supporting the important role played by the genotype in the phenomenon. In fact, by assuming that the second Xchromosome is the major factor in determining the longer life of females, no 'gender gap' should be observed in the inbred strain of experimental animal homozygous for all loci. In such a case the difference in inactivation probability between the two Xchromosomes should not exist, as they are exactly alike. Inbred mouse strains actually have the same survival pattern for males and females [1]. Gene recombination frequency is another sex-dependent event. The genetic map assembled by recombina-
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tion frequencies is about 90% longer in females than in males. This difference has been observed in all species having a sex determining system based on two chromosomes. An extreme example is represented by Drosophila in which XY males have no recombination. It has been suggested that these variations mirror gene (or gene region) transcription differences during gametogenesis, as a result of parental imprinting [15]. In other words, the selective activation of genes in masculine and feminine germinal cell lines could determine sites with crossing-over potency, thus explaining the discrepancies seen by comparing the gene maps of the two genders. If this hypothesis were confirmed, the possible advantages responsible for the 'gender gap' would also be pointed out at cellular and molecular levels. At present, it is known that several genes are under genomic imprinting, that is they may change theirexpression pattern depending on maternal or paternal transmission. In order to investigate this question more thorougly we shall now attempt to link the observations and the speculations reported above with the commonest theories of aging. It is clear that the longevity of a certain population, e.g. the Italians, depends on several parameters besides the genotype, and at the beginning of this century it was different from today. On the contrary, the longevity of a normal cell line, though influenced by 'in vitro' culture conditions, is able to divide a precise number of times. The exhaustion of this replicative potential has been considered to be representative of senescence at the cellular level [16]. The mean life span of a mono- or multicellular system, although under genetic control, can be influenced by the environment and it is accompanied by timedependent modifications that, after all, constitute the aging process itself. Therefore knowledge of the cellular and molecular aspects of these modifications may help in understanding the cellular and molecular basis of longevity, and thus some relevant theories of aging will be examined.
2. AGE, the theory of crosslinking Glucose and other reducing sugars may 'non-enzymatically' combine with proteins by a series of reactions that yield a family of 'advanced glycosylation endproducts' (AGE) with the ablity to cross-react. These crosslinks have been determined in several proteins and, in the case of collagen, their number increases in a linear manner as the subject becomes older [17]. Non-enzymatic glycosylation also occurs in intracellular proteins and increases during cell aging [18]. In diabetics the quantity of crosslinks is higher than in controls of comparable age [17]. Reducing sugars may also form adducts with DNA that have been reported to be mutagenic in bacteria [19]. However, this mechanism does not seem to correlate positively with the 'gender gap' because of the prevalence of diabetes among women [20]. On the other hand it has been shown [17] that the incubation of collagen and elastin with high glucose concentrations produces crosslinks in amounts that are directly proportional to the concentration of LDL in the experimental system. This may create potential sites of atheromatous plaque formation and, as men have higher plasma LDL than women (better to say LDL/HDL ratio), may contribute to the higher risk of cariovascular pathologies in males. The mechanism, in this case, raises the possibility of sharing in the 'gender gap' [21].
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3. The theory of mitochondrion participation The age-dependent decrease of respiration and ATP production have been known for a long time [22]; however, now the genome structure and function, and mitochondrial aspects are attracting a lot of interest. In humans the mitochondrial genome is a circular DNA made up of 16 569 bp, codifying for 13 protein subunits of the oxidative phosphorylation complex, for two mRNAs and for 22 tRNAs belonging to the protein synthesis system of the miochondrion. In many human pathologies deletions of mitochondrial DNA have been reported. Further, the accumulation of mutations in this genome, mainly deletions, has been suggested to be a relevant event in cellular aging [23,24] which reasonably accounts for the energy production decrease of senescent cells. The mitochondria and their genomes are trasmitted through generations together with the egg cell cytoplasm and their genes are solely inherited on the mother's side. In Parkinson's disease a deletion of 4977 bp in the mitochondrial DNA has been detected [24] and the same has been identified in normal elderly subjects. The latter pathology is more preponderantly displayed by men than by women (this may be due to a higher resistance of females to developing the illness), but when women are affected by Parkinson's disease their life expectation becomes similar to that of men [25]. Hence, deletions of mitochondrial DNA such as those occuring in aging may sometimes remove the 'gender gap'. This dos not necessarily mean that the cited mitochondrial DNA sequence is responsible for the 'gender gap'. In reality there are a number of factors, among them mitochondrial, which may potentially influence the frequency of mutations and deletions in mitochondrial DNA. Among these the formation of free radicals is one of the most probable.
4. The theory of free radicals The excited oxygen species such as hydrogen peroxide, superoxide and hydroxylic radicals are formed 'in vivo' as a consequence of the aerobic metabolism and following radiation exposure. In spite of the fact that cells have developed several enzymatic and non-enzymatic systems to counteract these highly reactive oxygen species, part of them escape cellular defences and may transitorily or permanently damage proteins, lipids and nucleic acids. It has been suggested that oxidative injury favours the aging process [26] as well as pathological conditions such as cancer, chronic inflammation, ischemia, autoimmune diseases, etc. High rates of DNA oxidative damage have been reported to be related to high metabolic activity and to low lifespan in organisms [27-29]. Mitochondria are particularly exposed to free radicals since they utilize more than 90% of the oxygen consumed by the organism. Furthermore, they are not protected by the usual antioxidant agents [30] and their DNA, besides not being shielded by histones, lacks an efficient DNA repairing system. The addition of antioxidants (e.g. 2-mercaptoethylamine) to the diet of experimental animals has been demonstrated to lessen the 'gender gap' in their offspring [26,31]. This finding raises the possibility of a better protection against oxidative harm at the embryonai stage, when one of the two female X-chromosomes has not yet been inactivated. This speculation is strengthened by the presence on the X-chromosome of
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the gene codifying for glucose 6-15hosphate dehydrogenase, an enzyme involved in NADPH production which, in turn, maintains high levels of reduced glutathione, the effective intracellular scavenger of hydrogen peroxide. The same advantages could be enjoyed by women when genes of the X-chromosome are reactivated by aging [13]. Anyhow, the latter mechanism is still under debate [32,38]. Finally, a short mention of human plasma antioxidants could be worth while. Among them we cite ascorbate, urate, bilirubin, a-tocopherol, protein thiols, etc. All are present in physiological conditions and act as scavengers of oxidants. The same action is displayed by carotenoids, a large family of molecules (more than 600), about 10% of which may serve as vitamin A precursors [34]. The capability of flcarotene and other carotenoids to face free radicals, thus preventing oxidative damage, must be linked to the higher serum/~-carotene concentration in women than in men [35]. Whether or not this difference is important in determining the 'gender gap' is unknown at present.
5. Recent evidence possibly relevant to the 'gender gap' In quiescent and senescent cells, cellular proliferation normally stops at the G1 phase of the cell cycle. Two proteins, namely p34 cdc2 and cyclin, have been found to be essential in cell cycle regulation. Both are expressed a little or not at all in senescent human fibroblasts [37]. In addition, during cell cycle progression the transition from metaphase to anaphase is induced by cyclin degradation, a process that takes place by an ubiquitin-dependent proteolytic system [38]. The gene coding one of the enzymes that binds ubiquitin to cyclin (and to other proteins), named El, has been mapped on the X-chromosome [39,40]. In the case of the real possibility that the genes on the X-chromosome may escape inactivation during senescence [13], the replicative advantage for double X- versus single X-containing cells is clearly evident.
6. Conclusions and perspectives The 'gender gap', that is the difference in the life expectancy between males and females, even if quantitatively different from that evaluated by recent statistical analysis, does have genetic, molecular and cellular bases that are little understood. The possibilities taken into consideration in this essay suggest some mechanisms that are worthy of further investigation. From the genetic point of view, parental imprinting and the difference in recombination frequency between males and females deserve attention. Observations at the molecular level indicate that the mitochondrial genome deletions and the effects of free radicals seem to play an important role in determining the 'gender gap'. Finally, from the cellular point of view evidence from mechanisms regulating the progression of the cell cycle and the location on the X-chromosome of genes involved in such a process deserves attention. All these considerations suggest the need to develop suitable biological models to ascertain the hypotheses described above. In our laboratory, as in others, the red blood cell has, for a log time, been chosen as a model for studying the aging process [41]. Erythrocytes have a life span that correlates with that of the considered animal species [42], have different survival
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time in the circulation of males and females [43], and are suitable for molecular and cellular studies during both the aging of the cell and of the individual [41]. For these reasons, the possibility of utilizing this model to investigate some aspects of the fascinating 'gender gap' phenomenon should be considered. This short review does not aim at covering all the literature and the theories in the field, but simply to underline considerations, experiments and speculations produced during recent years that can help in finding new directions in the search for facts useful in explaining one of the most intriguing biological phenomena, i.e. the gender gap. 7. Acknowledgement The authors thank Professor G. Novelli and Dr L. Chiarantini for their fruitful discussion and the important contribution to the draft of the manuscript. Research performed in the authors' laboratory was been partly granted by the Italian C.N.R., P.F. Invecchiamento (INV 93.1.360). 8. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
D.W.E. Smith, Is greater female longevity a general finding among animals? Biol. Rev. Cambridge Phil. Soc., 64 (1989) 1-12. G.H. Miller, Is the longevity gender gap decreasing? N. Y. State J. Med., 86 (1986) 59-60. S. Seely, The gender gap: why do women live longer than men? Int. J. Cardiol., 29 (1990) 113-119. A.J. Silman, Why do women live longer and is it worth it? Br. Meal J., 294 (1987) 1311-1312. A. Comfort, The Biology o f Senescence. Elsevier, New York, 1979. A.D Lopez, Sex differential in mortality. W.H.O. Chronicle, 38 (1984) 217-224. S.J. Olshansky, B.A. Carues and C. Cassel, In search of Methuselah: estimating the upper limits to human longevity. Science, 250 (1990) 634-640. R.H. Rosenman and M. Friedman, Association of specific overt behavior pattern with blood and cardiovascolar findings. Circulation, 24 (1959) 1174. S.H, Preston, An international comparison of excessive adult mortality. Pop. Study, 24 (1970) 5-20. R.D. Retherford, Tobacco smoking and the sex mortality differential. Demography. 9 (1972) 203-216. H. Sobel, Endocrine changes associated with maturation and their possible role in the natural history of ischemic heart disease. J. Am. Coll. Health Assoc., 22 (1973) 101-109. R.A. Fisher, The Cancer Controversy. Oliver Boyd, Edinburgh, 1959. K.A. Wareham, M.E. Lyon, P.H. Gleinister and E.D. Williams, Age related reactivation of an Xlinked gene. Nature, 327 (1987) 725-727. R. Holliday, Ageing. X-chromosome reactivation. Nature, 327 (1987) 661-662 B.J. Thomas and R. Rothstein, Sex, maps, and imprinting. Cell, 64 (1991) 1-3. A. Macieira-Coelho, Biology of normal proliferating cells in vitro. Relevance /or In Vivo Aging. Karger, Basel, 1988, pp. 1-218. A. Cerami, Aging of proteins and nucleic acids: what is the role of glucose? Trends Biochem. Sci., 11 (1986) 311-314. G. Fornaini, M. Magnani, A. Fazi, A. Accorsi, V. Stocchi and M. Dacha, Regulatory properties of human erythrocyte hexokinase during cell ageing. Arch. Biochem. Biophys., 239 (1985) 352-358. A.T. Lee and A. Cerami, Elevated glucose 6-phosphate levels are associated with plasmid mutation in vivo. Proc. Natl. Acad Sci. U.S.A., 84 (1987) 8311-8314. A.E. Renold, D.H. Mintz, W.A. Muller and G.F. Cahill Jr, Diabetes mellitus. In J.B. Standbury, J.B. Wingaarden and D.S. Fredrickson (eds), The Metabolic Basis of Inherited Disease, McGrawHill, New York, 1978, pp. 80-205.
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Why do women outlive men? Hosp. Practice, 23 (1988) 50-64, D.R. Sanadi, Metabolic changes and their significance in aging, In C.E. Finch and L. Hayflick (eds), Handbook of the Biology o f Aging, Van Nostrand Reinhold Co., New York, 1977, pp. 73-98. A. Linnane, S. Marzuki, T. Osawa and M. Tanaka, Mitochondrial DNA mutations as an important contributor to aging and degenerative diseases. Lancet, (1989) 642-645. S. Ikebe, M, Tanaka, K. Ohono, W. Sato, K. Hattori, T. Kondo, Y. Mizuno and T. Ozawa, Increase of deleted mitochondrial DNA in the striatum in Parkinson's disease and senescence. Biochem. Biophys. Res. Commun., 170 (1990) 1044-1048. S.G. Diamond, C.H. Markham, M.M. Hoehn, F.H. McDowell and M.D. Muenter, An examination of male-female differences in progression and mortality of Parkinson's disease. Neurology, 40 {1990) 763-766. D. Harman, The aging process. Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 7124-7128. R. Adelman, R.L. Saul and B.N. Ames, Oxidative damage to DNA: relation to species metabolic rate and life span. Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 2706-2708. C. Richter, J. Park and B,N. Ames, Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc, Natl. Acad. ScL U.S.A., 85 (1988) 6465-6467. R.G. Cutler, Peroxide-producing potential of tissues: inverse correlation with longevity of mammalian species. Proc. Natl. Acad Sci. U.S.A., 82 (1985) 4798-4802. M.A. Horrum, D. Harman and R.B. Tobin, Free radical theory of aging: effects of antioxidants on mitochondrial function. Age, 10 (1987) 58-61. D. Harman and D.E. Eddy, Free radical theory of aging: beneficial effect of adding antioxidants to the maternal mouse diet on life span of offspring; possible explanation of the sex difference in longevity. Age, 2 (1979) 109-122. B.R. Migeon, J. Axelman and A.H. Beggs, Effect of ageing on reactivation of the human X-linked HPRT locus. Nature, 335 (1988) 93-96. R. HoUiday, X-chromosome reactivation and ageing. Nature, 337 (1989) 311. A. Bendich and J.A. Olson, Biological actions of carotenoids. FASEB ,L, 3 (1989) 1927-1932. R.S. Parker, Carotenoids in human blood and tissues. Am. ,L Clin. Nutr., 119 (1989) 101-104. M. Rockstein, M. (ed.) Theoretical Aspects of Aging. Academic Press, New York, 1974, pp. 1-192. G.H. Stein, L.F. Drullinger, R. S. Robertorye, O.M. Perreira-Smith and J.R. Smith, Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation. Proc. Natl. Acad Sci. U.S.A., 88 (1991) 11012-11016. M. Glotzer, A.W. Murray and M.W. Kirschner, Cyclin is degraded by the ubiquitin pathway. Nature, 349 (1991) 132-137. M, Ohtsubo and T. Nishimoto, The gene coding an ubiquitin-activating enzyme may locate on Xchromosome. Biochem. Biophys. Res. Cornmun., 153 (1988) 1173-1178. E. Zacksenhaus and R. Sheinin, Molecular cloning, primary structure and expression of the human X-linked A1S9 gene cDNA which complements the ts AIS9 mouse L cell defect in DNA replication. EMBO J., 9 (1990) 2923-2929. M. Magnani and A. De Flora, Red Blood Cell Aging. Plenum Press, New York, 1991, pp, 1-383. D. Rohme, Evidence for a relationship between longevity of mammalian species and life span of normal fibroblasts in vitro and erythrocytes in vivo. Proc. Natl. Acad Sci, U.S.A., 78 ( 1981 ) 5009-5013. D. Danon, Aging of erythrocyte, in V.J. Cristofalo (ed.), Handbook of Cell Biology of Aging, CRC Press, New York, 1985, pp. 317-340.
ELSEVIER S('IENTIFI( P U B L I S H F R S IRI L A N I )
Brief review
The female longevity phenomenon. Hypotheses on some molecular and cell biology aspects Mauro Magnani*, Augusto Accorsi lstituto di Chimica Biologica 'G. Fornaini', Universith degli Studi di Urbino. Via Saffi 2, 61029 Urbino, Italy
(Received 26 December 1992; revision received 4 August 1993; accepted 26 August 1993)
Abstract
The most relevant theories on the sex-dependent longevity at population level are critically re-examined in the light of the knowledge available today on the aging process at cellular and molecular level. The aim of this perspective is to help in the understanding of the cellular and molecular bases of longevity and to indicate the most suitable biological models of investigation. Key words." Aging; Longevity; Gender gap
1. Introduction
In humans, the c o m m o n belief that females live longer than males has been supported by statistical analyses that have also demonstrated the existence o f such a difference in other animal species [1-6]. These studies have stimulated research on the biological basis o f the phenomenon and, at present, it seems possible to express reasonable hypotheses on some molecular and cell biology aspects. Before doing so it is important to state the following points: (1) The difference in the expectation o f life between males and females, also termed * Corresponding author. 0047-6374/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0047-6374(93)01398-R
90
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'gender gap', has changed over the years. It appears to be different in different developed countries and to depend on several parameters such as geography, pro-capita income, etc. [1-4]. (2) The higher survival of females in comparison with males, observed in animals [5,6], does not seem useful in explaining this finding in humans [1]. In fact, examination of the survival curves of laboratory rodents shows a spectrum of variable effects of sex on longevity up to species where the male has a longer lifespan [1]. (3) The 'gender gap' would not be substantially modified even though a decrease in the commoner causes of death (cardiovascular, diabetic and tumor pathologies) [7]. On the bases of the considerations reported above and taking into account the extensive reading of the literature on the subject, we have come to the conclusion that: (a) the current 'gender gap' may not be solely attributed to an advantage or a disadvantage linked to the XX- or XY-genotype, respectively (since it may vary in time and space) and (b) the 'gender gap' does exist even if all environmental, behavioral, and social influences are taken into account. In this review, we will try to critically re-examine some of the theories and hypothesis formulated to explain the 'gender gap'. During the sixties, it was proposed that sex-dependent longevity could be attributed to stronger life-stress for men than for women, as reflected in the higher incidence of cardiovascular diseases in males [8]. In the seventies smoking was claimed to be the reason for the 'gender gap' [9,10]. Female hormones were later indicated as protecting factors, though contrasting data have been reported [11]. The latter theory was founded on the observation that the lower incidence of cardiovascular disease mortality observed in women before menopause becomes similar to that of men after the climateric period. According to these previous considerations, we want to stress that all these theories are inadequate to clarify the phenomenon, although lessening it appropriately. The genetic theory is one of the oldest in trying to explain the gender gap. It should be underlined that the X-chromosome contains several genes not involved with female sex or phenotype determination. Moreover, besides the well-known random inactivation of the X-chromosome during embryogenesis and development, a non-random selection in cell proliferation seems to occur. Finally, the reported [13,14] reactivation of the X-chromosome could offer a selective advantage in female survival. Some outlines of the genetics of female longevity may also be useful in this discussion, since there is experimental evidence supporting the important role played by the genotype in the phenomenon. In fact, by assuming that the second Xchromosome is the major factor in determining the longer life of females, no 'gender gap' should be observed in the inbred strain of experimental animal homozygous for all loci. In such a case the difference in inactivation probability between the two Xchromosomes should not exist, as they are exactly alike. Inbred mouse strains actually have the same survival pattern for males and females [1]. Gene recombination frequency is another sex-dependent event. The genetic map assembled by recombina-
M. Magnani, A. Aecorsi/Meeh. Ageing Dev. 72 (1993) 89-95
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tion frequencies is about 90% longer in females than in males. This difference has been observed in all species having a sex determining system based on two chromosomes. An extreme example is represented by Drosophila in which XY males have no recombination. It has been suggested that these variations mirror gene (or gene region) transcription differences during gametogenesis, as a result of parental imprinting [15]. In other words, the selective activation of genes in masculine and feminine germinal cell lines could determine sites with crossing-over potency, thus explaining the discrepancies seen by comparing the gene maps of the two genders. If this hypothesis were confirmed, the possible advantages responsible for the 'gender gap' would also be pointed out at cellular and molecular levels. At present, it is known that several genes are under genomic imprinting, that is they may change theirexpression pattern depending on maternal or paternal transmission. In order to investigate this question more thorougly we shall now attempt to link the observations and the speculations reported above with the commonest theories of aging. It is clear that the longevity of a certain population, e.g. the Italians, depends on several parameters besides the genotype, and at the beginning of this century it was different from today. On the contrary, the longevity of a normal cell line, though influenced by 'in vitro' culture conditions, is able to divide a precise number of times. The exhaustion of this replicative potential has been considered to be representative of senescence at the cellular level [16]. The mean life span of a mono- or multicellular system, although under genetic control, can be influenced by the environment and it is accompanied by timedependent modifications that, after all, constitute the aging process itself. Therefore knowledge of the cellular and molecular aspects of these modifications may help in understanding the cellular and molecular basis of longevity, and thus some relevant theories of aging will be examined.
2. AGE, the theory of crosslinking Glucose and other reducing sugars may 'non-enzymatically' combine with proteins by a series of reactions that yield a family of 'advanced glycosylation endproducts' (AGE) with the ablity to cross-react. These crosslinks have been determined in several proteins and, in the case of collagen, their number increases in a linear manner as the subject becomes older [17]. Non-enzymatic glycosylation also occurs in intracellular proteins and increases during cell aging [18]. In diabetics the quantity of crosslinks is higher than in controls of comparable age [17]. Reducing sugars may also form adducts with DNA that have been reported to be mutagenic in bacteria [19]. However, this mechanism does not seem to correlate positively with the 'gender gap' because of the prevalence of diabetes among women [20]. On the other hand it has been shown [17] that the incubation of collagen and elastin with high glucose concentrations produces crosslinks in amounts that are directly proportional to the concentration of LDL in the experimental system. This may create potential sites of atheromatous plaque formation and, as men have higher plasma LDL than women (better to say LDL/HDL ratio), may contribute to the higher risk of cariovascular pathologies in males. The mechanism, in this case, raises the possibility of sharing in the 'gender gap' [21].
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3. The theory of mitochondrion participation The age-dependent decrease of respiration and ATP production have been known for a long time [22]; however, now the genome structure and function, and mitochondrial aspects are attracting a lot of interest. In humans the mitochondrial genome is a circular DNA made up of 16 569 bp, codifying for 13 protein subunits of the oxidative phosphorylation complex, for two mRNAs and for 22 tRNAs belonging to the protein synthesis system of the miochondrion. In many human pathologies deletions of mitochondrial DNA have been reported. Further, the accumulation of mutations in this genome, mainly deletions, has been suggested to be a relevant event in cellular aging [23,24] which reasonably accounts for the energy production decrease of senescent cells. The mitochondria and their genomes are trasmitted through generations together with the egg cell cytoplasm and their genes are solely inherited on the mother's side. In Parkinson's disease a deletion of 4977 bp in the mitochondrial DNA has been detected [24] and the same has been identified in normal elderly subjects. The latter pathology is more preponderantly displayed by men than by women (this may be due to a higher resistance of females to developing the illness), but when women are affected by Parkinson's disease their life expectation becomes similar to that of men [25]. Hence, deletions of mitochondrial DNA such as those occuring in aging may sometimes remove the 'gender gap'. This dos not necessarily mean that the cited mitochondrial DNA sequence is responsible for the 'gender gap'. In reality there are a number of factors, among them mitochondrial, which may potentially influence the frequency of mutations and deletions in mitochondrial DNA. Among these the formation of free radicals is one of the most probable.
4. The theory of free radicals The excited oxygen species such as hydrogen peroxide, superoxide and hydroxylic radicals are formed 'in vivo' as a consequence of the aerobic metabolism and following radiation exposure. In spite of the fact that cells have developed several enzymatic and non-enzymatic systems to counteract these highly reactive oxygen species, part of them escape cellular defences and may transitorily or permanently damage proteins, lipids and nucleic acids. It has been suggested that oxidative injury favours the aging process [26] as well as pathological conditions such as cancer, chronic inflammation, ischemia, autoimmune diseases, etc. High rates of DNA oxidative damage have been reported to be related to high metabolic activity and to low lifespan in organisms [27-29]. Mitochondria are particularly exposed to free radicals since they utilize more than 90% of the oxygen consumed by the organism. Furthermore, they are not protected by the usual antioxidant agents [30] and their DNA, besides not being shielded by histones, lacks an efficient DNA repairing system. The addition of antioxidants (e.g. 2-mercaptoethylamine) to the diet of experimental animals has been demonstrated to lessen the 'gender gap' in their offspring [26,31]. This finding raises the possibility of a better protection against oxidative harm at the embryonai stage, when one of the two female X-chromosomes has not yet been inactivated. This speculation is strengthened by the presence on the X-chromosome of
M. Magnani, A. Accorsi/Mech. Ageing Dev. 72 (1993) 89-95
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the gene codifying for glucose 6-15hosphate dehydrogenase, an enzyme involved in NADPH production which, in turn, maintains high levels of reduced glutathione, the effective intracellular scavenger of hydrogen peroxide. The same advantages could be enjoyed by women when genes of the X-chromosome are reactivated by aging [13]. Anyhow, the latter mechanism is still under debate [32,38]. Finally, a short mention of human plasma antioxidants could be worth while. Among them we cite ascorbate, urate, bilirubin, a-tocopherol, protein thiols, etc. All are present in physiological conditions and act as scavengers of oxidants. The same action is displayed by carotenoids, a large family of molecules (more than 600), about 10% of which may serve as vitamin A precursors [34]. The capability of flcarotene and other carotenoids to face free radicals, thus preventing oxidative damage, must be linked to the higher serum/~-carotene concentration in women than in men [35]. Whether or not this difference is important in determining the 'gender gap' is unknown at present.
5. Recent evidence possibly relevant to the 'gender gap' In quiescent and senescent cells, cellular proliferation normally stops at the G1 phase of the cell cycle. Two proteins, namely p34 cdc2 and cyclin, have been found to be essential in cell cycle regulation. Both are expressed a little or not at all in senescent human fibroblasts [37]. In addition, during cell cycle progression the transition from metaphase to anaphase is induced by cyclin degradation, a process that takes place by an ubiquitin-dependent proteolytic system [38]. The gene coding one of the enzymes that binds ubiquitin to cyclin (and to other proteins), named El, has been mapped on the X-chromosome [39,40]. In the case of the real possibility that the genes on the X-chromosome may escape inactivation during senescence [13], the replicative advantage for double X- versus single X-containing cells is clearly evident.
6. Conclusions and perspectives The 'gender gap', that is the difference in the life expectancy between males and females, even if quantitatively different from that evaluated by recent statistical analysis, does have genetic, molecular and cellular bases that are little understood. The possibilities taken into consideration in this essay suggest some mechanisms that are worthy of further investigation. From the genetic point of view, parental imprinting and the difference in recombination frequency between males and females deserve attention. Observations at the molecular level indicate that the mitochondrial genome deletions and the effects of free radicals seem to play an important role in determining the 'gender gap'. Finally, from the cellular point of view evidence from mechanisms regulating the progression of the cell cycle and the location on the X-chromosome of genes involved in such a process deserves attention. All these considerations suggest the need to develop suitable biological models to ascertain the hypotheses described above. In our laboratory, as in others, the red blood cell has, for a log time, been chosen as a model for studying the aging process [41]. Erythrocytes have a life span that correlates with that of the considered animal species [42], have different survival
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time in the circulation of males and females [43], and are suitable for molecular and cellular studies during both the aging of the cell and of the individual [41]. For these reasons, the possibility of utilizing this model to investigate some aspects of the fascinating 'gender gap' phenomenon should be considered. This short review does not aim at covering all the literature and the theories in the field, but simply to underline considerations, experiments and speculations produced during recent years that can help in finding new directions in the search for facts useful in explaining one of the most intriguing biological phenomena, i.e. the gender gap. 7. Acknowledgement The authors thank Professor G. Novelli and Dr L. Chiarantini for their fruitful discussion and the important contribution to the draft of the manuscript. Research performed in the authors' laboratory was been partly granted by the Italian C.N.R., P.F. Invecchiamento (INV 93.1.360). 8. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
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