DOI 10.1515/hmbci-2013-0039 Horm Mol Biol Clin Invest 2013; 16(2): 65–72
Jose Viña*, Juan Gambini, Francisco José García-García, Leocadio Rodriguez-Mañas and Consuelo Borrás
Role of oestrogens on oxidative stress and inflammation in ageing Abstract: Females live longer than males. We have shown that this could be in part due to the higher levels of oestrogens in females, which protect them against ageing, by upregulating the expression of antioxidant, longevity-related genes. However, the low concentration of oestrogens makes it unlikely that they exhibit significant antioxidant capacity in the organism. Our results show that physiological concentrations of oestrogens activate the expression of manganese superoxide dismutase and glutathione per oxidase by oestrogen receptors and the mitogen-activated protein kinase and nuclear factor kappa B pathway. More over, when considering oestrogen replacement therapy, it is of utmost importance to take into account when to start the therapy after menopause. We have shown that only early-onset administration of oestrogen replacement therapy is effective on oestrogen deprivation associated with oxidative and metabolic stress. This is due to a change in oestrogen receptor distribution after oestrogen deprivation. Oestrogens are also involved in inflammatory processes. Their role on inflammation is very complex, because their effects are different depending on the doses and also on the oestrogen receptor distribution. Keywords: anti-inflammatory effect; antioxidant; longevity; oestrogen receptors. *Corresponding author: Jose Viña, Professor, MD, Faculty of Medicine, Department of Physiology, University of Valencia, Av. Blasco Ibañez, 15, Valencia 46010, Spain, Phone: +34 96 386 46 50, Fax: +34 96 386 46 42, E-mail: [email protected] Juan Gambini and Consuelo Borrás: Faculty of Medicine, Department of Physiology, University of Valencia, Valencia, Spain Francisco José García-García: Division of Geriatric Medicine, Hospital Virgen del Valle, Complejo Hospitalario de Toledo, Toledo, Spain Leocadio Rodriguez-Mañas: S° de Geriatría Hospital Universitario de Getafe Ctra. de Toledo, Km. 12,5, Getafe, Spain
Introduction Menopause is associated with reduced functioning of the ovaries due to aging, resulting in lower levels of oestrogens.
As oestrogens are involved in many physiological functions, their decrease is involved in some menopause symptoms. Moreover, there is also a role of oestrogens on aging. Females live longer than males, and this difference in longevity can be attributed, at least in part, to oestrogens. Estrogens act as antioxidants by up-regulating the expression of antioxidant, longevity-related genes. Estrogens have been also related to inflammation. They can interact with its receptors to modulate different inflammatory factors such as cytokines, and even inducible nitric oxide synthase. These aspects are reviewed in the following paragraphs in depth.
Females live longer than males A major aim of gerontology is to find adequate models to understand ageing. The fact that in many species, including humans, females live longer than males [1, 2] offers a very interesting possibility to study differential ageing, i.e., why is it that one gender has approximately a 10% increase in average longevity over the other. This is by no means a universal phenomenon. In many species, males and females have the same longevity or males even live longer than females [3, 4] (see below). An important point is that the difference in longevity between women and men has occurred in all advanced as well as primitive societies. For instance, Table 1 indicates that the difference in longevity in Spain in 1900 and in 2000 always favoured women [1]. Moreover, the difference between women and men increased when average life expectancy approached the value of about 80 years as observed today. In fact, in 1900, i.e., when average life span was approximately 33 years, women lived only 3.8% longer than men. However, in 1992 when average life span was around 75–80 years, the increased longevity between women and men went up to almost 10%. This is by no means a characteristic of a given country, i.e., Spain, but rather it is a general phenomenon that there is a significant increase in the life expectancy of women vs. men in Sweden [5]. This has been calculated to around 8–10%
66 Viña et al.: Oestrogens and ageing Table 1 Life expectancy over time by gender in Spain. Year 1900 1960 1980 1992 2020
Men
Women
%
3.8 7.1 8.1 9.9 7.8
33.8 67.4 72.5 73.7 77.7
35.1 72.2 78.6 81.0 83.8
Data taken from reference [1].
over the last 250 years, i.e., from 1750 to present times. In all cases, women always outlived men by a significant proportion. One could argue that this observed difference in longevity was due to sociological rather to biological reasons. For instance, difference in the rate of smoking or in the type of work could account for some of the difference in longevity observed [6, 7]. The following facts make this assumption unlikely: first, the differences are always observed in all kinds of societies where social differences are very marked. Second, this not only occurs in humans but also in animals (see Figure 1), albeit not in all species. Therefore, it is reasonable to assume that there must be biological differences that explain the increased longevity of women as opposed to men. We would like to point out here another critical fact reported in Table 1: the increase in longevity in the 20th century has gone from approximately 34 years to approximately 80 years. Thus, this increase in longevity, independently of gender, is very remarkable. In fact, the average life span in Spain almost tripled in less than a century. This outstanding change had never occurred previously in recorded history, and, to our knowledge, it is highly unlikely that it will happen again in the foreseeable future. And this leads us to consider another very important point, that of dependency. Since a vast portion of the population reaches ages well above 65, it is very important 120
Survival (%)
100 80 60
% males
40
% females
20 0
0
5
10
15
20
25
30
35
Age (months)
Figure 1 Gender differences in life expectancy in Wistar rats.
40
that we age successfully. In fact, the progress from independent healthy ageing to frailty and, eventually, to dependency is a very important one, which must be minimised by all means. Figure 2 shows that, in the European Community, the proportion of persons aged over 65 who are dependent on care by others in 2010 is approximately 25%; however, the predicted proportion of the population aged 65 that is dependent on others will be about 50% in 2050. This means that, of all the population of age 65, half will be dependent on others half of their lives. Any effort to understand ageing with an aim to improving the quality of life of the aged population should be vigorously tackled. Lowering the proportion of frail people and, eventually, of dependent people is a major task for gerontology and geriatrics [8]. As stated above, the differences in longevity between genders offer interesting possibilities to understand ageing. Animals with a very similar genetic background may differ in their average life span by as much as 10%. Paramount among these differences is that which happens in the human species. However, in order to find proof that this is not only due to sociological changes or to peculiarities between societies (e.g., whether women smoke more or less than men, etc.), one must understand the differences in longevity in animal species. One of the most widely used laboratory animals is the Wistar rat. In this rat, we and others have observed that females live longer than males, the maximal life span being approximately 10% longer (see Figure 1) [2]. In a similar fashion, using Fisher 344 rats, i.e., the same species but a different strain, in which longevity is also higher in females than in males, Jang et al. [9] observed that males produce more reactive oxygen species than females. However, the higher longevity of females as compared with males is not a universal phenomenon. In other species of rodents such as mice, some strains show a higher longevity of males when compared with females, for instance, the C57BL6 [4]. Moreover, a variation of this strain, i.e., C57B16J mice, which was used by Sanz et al. [3], shows no differences in longevity between sexes. In contrast, the Swiss albino mouse shows an increased longevity in females when compared with males [10]. So not only do we have different gender-specific longevity in different species, but also in different strains of the same species. This offers a unique opportunity to study comparative ageing, i.e., whether there are hormonal differences, different sensitivity or reactivity to hormones, or different fundamental molecular mechanisms of ageing. Since we are dealing with the same species, only with different genders, we are faced with optimal animal models to understand fundamental gerontology as well
Viña et al.: Oestrogens and ageing 67
83.8
Women
17 81.7
16 80.0 15
50
Men
14 1990 1992 1994 1996 1998 2000 2002 2004 Year
Proportion (%)
Number of years
55 84.6
19 18
Dependency
Mean lifespan
20
45 40 35 30 25 20 1990
2000
2010
2020
2030
2040
2050
Year
Figure 2 Proportion of persons aged over 65 who are dependent on others for care. Taken from the EUROSTAT and European Council Meeting in Lisbon in 2000.
Sex differences in free radical production In many species in which females live longer than males, the former produce approximately half the amount of mitochondrial peroxide than the latter [2]. It is very important to note that not all species behave the same in terms of gender-related longevity and that there are some species in which males live longer than females (see below). As stated above, in the Wistar rat, our experimental rat model, as well as in humans, females do indeed live longer than males by approximately a 10% increase in the average life span. In the early 2000s, we measured peroxide production by mitochondria from female and from male Wistar rats and found that females produce approximately half the amount of peroxides than males. This was completely reversed when rats were ovariectomised, thus tracing the differential gender effect on radical production to ovarian hormones. We then measured the free radical production of mitochondria from ovariectomised females that had been treated with oestrogens. In this case, oestradiol was able to reverse the effect of ovariectomy and the rate of peroxide production was similar to that of females (see Figure 3). The increase in radical production resulted in a significantly lower damage to mitochondrial DNA in females than in males. In fact, the level of 8-oxo-deoxyguanosine was four-fold higher in males than in females [2].
However, Ali et al. [4] showed that, in those strains of mice such as the black C57BL6 that they were using in which males live longer than females, it is males that produce fewer oxidants than females [4]. The authors measured not only oxidant production by determining dihydroethidium oxidation in the brain but also EPR signals in the brain mitochondria of their mice. Thus, in this strain of mice in which males live longer than females it is males that produce fewer radicals. Far from contradicting results from a laboratory, these results nicely confirm our results. The claim by all of us is that the gender that lives longer produces fewer radicals independently of whether it is males or females who live longer. The critical test will be to see why oestrogens promote the expression of antioxidant genes in a given species and do not promote that expression in other species or strains. A third confirmation of this hypothesis came from the laboratory of Sanz et al. [3]. These researchers studied a particular strain derived from C57B6J mice in which males Males Females 0.18 nmol H2O2/mg prot.min
as the hormonal regulation of the ageing process [2–4]. We centred our attention to gender oxidative stress differences to explain gender life-span differences, but, of course, there may be many other factors involved in these gender life-span differences.
0.16 0.14
Ovariectomised Estrogen replaced
0.12 0.10 0.08 0.06 0.04 0.02 0.00
Figure 3 Oxidant production by hepatic mitochondria from males and females: effect of ovariectomy and oestrogen replacement therapy. Data are expressed as mean ± SD of n = 6 rats. The statistical difference is indicated as follows: **p
Jose Viña*, Juan Gambini, Francisco José García-García, Leocadio Rodriguez-Mañas and Consuelo Borrás
Role of oestrogens on oxidative stress and inflammation in ageing Abstract: Females live longer than males. We have shown that this could be in part due to the higher levels of oestrogens in females, which protect them against ageing, by upregulating the expression of antioxidant, longevity-related genes. However, the low concentration of oestrogens makes it unlikely that they exhibit significant antioxidant capacity in the organism. Our results show that physiological concentrations of oestrogens activate the expression of manganese superoxide dismutase and glutathione per oxidase by oestrogen receptors and the mitogen-activated protein kinase and nuclear factor kappa B pathway. More over, when considering oestrogen replacement therapy, it is of utmost importance to take into account when to start the therapy after menopause. We have shown that only early-onset administration of oestrogen replacement therapy is effective on oestrogen deprivation associated with oxidative and metabolic stress. This is due to a change in oestrogen receptor distribution after oestrogen deprivation. Oestrogens are also involved in inflammatory processes. Their role on inflammation is very complex, because their effects are different depending on the doses and also on the oestrogen receptor distribution. Keywords: anti-inflammatory effect; antioxidant; longevity; oestrogen receptors. *Corresponding author: Jose Viña, Professor, MD, Faculty of Medicine, Department of Physiology, University of Valencia, Av. Blasco Ibañez, 15, Valencia 46010, Spain, Phone: +34 96 386 46 50, Fax: +34 96 386 46 42, E-mail: [email protected] Juan Gambini and Consuelo Borrás: Faculty of Medicine, Department of Physiology, University of Valencia, Valencia, Spain Francisco José García-García: Division of Geriatric Medicine, Hospital Virgen del Valle, Complejo Hospitalario de Toledo, Toledo, Spain Leocadio Rodriguez-Mañas: S° de Geriatría Hospital Universitario de Getafe Ctra. de Toledo, Km. 12,5, Getafe, Spain
Introduction Menopause is associated with reduced functioning of the ovaries due to aging, resulting in lower levels of oestrogens.
As oestrogens are involved in many physiological functions, their decrease is involved in some menopause symptoms. Moreover, there is also a role of oestrogens on aging. Females live longer than males, and this difference in longevity can be attributed, at least in part, to oestrogens. Estrogens act as antioxidants by up-regulating the expression of antioxidant, longevity-related genes. Estrogens have been also related to inflammation. They can interact with its receptors to modulate different inflammatory factors such as cytokines, and even inducible nitric oxide synthase. These aspects are reviewed in the following paragraphs in depth.
Females live longer than males A major aim of gerontology is to find adequate models to understand ageing. The fact that in many species, including humans, females live longer than males [1, 2] offers a very interesting possibility to study differential ageing, i.e., why is it that one gender has approximately a 10% increase in average longevity over the other. This is by no means a universal phenomenon. In many species, males and females have the same longevity or males even live longer than females [3, 4] (see below). An important point is that the difference in longevity between women and men has occurred in all advanced as well as primitive societies. For instance, Table 1 indicates that the difference in longevity in Spain in 1900 and in 2000 always favoured women [1]. Moreover, the difference between women and men increased when average life expectancy approached the value of about 80 years as observed today. In fact, in 1900, i.e., when average life span was approximately 33 years, women lived only 3.8% longer than men. However, in 1992 when average life span was around 75–80 years, the increased longevity between women and men went up to almost 10%. This is by no means a characteristic of a given country, i.e., Spain, but rather it is a general phenomenon that there is a significant increase in the life expectancy of women vs. men in Sweden [5]. This has been calculated to around 8–10%
66 Viña et al.: Oestrogens and ageing Table 1 Life expectancy over time by gender in Spain. Year 1900 1960 1980 1992 2020
Men
Women
%
3.8 7.1 8.1 9.9 7.8
33.8 67.4 72.5 73.7 77.7
35.1 72.2 78.6 81.0 83.8
Data taken from reference [1].
over the last 250 years, i.e., from 1750 to present times. In all cases, women always outlived men by a significant proportion. One could argue that this observed difference in longevity was due to sociological rather to biological reasons. For instance, difference in the rate of smoking or in the type of work could account for some of the difference in longevity observed [6, 7]. The following facts make this assumption unlikely: first, the differences are always observed in all kinds of societies where social differences are very marked. Second, this not only occurs in humans but also in animals (see Figure 1), albeit not in all species. Therefore, it is reasonable to assume that there must be biological differences that explain the increased longevity of women as opposed to men. We would like to point out here another critical fact reported in Table 1: the increase in longevity in the 20th century has gone from approximately 34 years to approximately 80 years. Thus, this increase in longevity, independently of gender, is very remarkable. In fact, the average life span in Spain almost tripled in less than a century. This outstanding change had never occurred previously in recorded history, and, to our knowledge, it is highly unlikely that it will happen again in the foreseeable future. And this leads us to consider another very important point, that of dependency. Since a vast portion of the population reaches ages well above 65, it is very important 120
Survival (%)
100 80 60
% males
40
% females
20 0
0
5
10
15
20
25
30
35
Age (months)
Figure 1 Gender differences in life expectancy in Wistar rats.
40
that we age successfully. In fact, the progress from independent healthy ageing to frailty and, eventually, to dependency is a very important one, which must be minimised by all means. Figure 2 shows that, in the European Community, the proportion of persons aged over 65 who are dependent on care by others in 2010 is approximately 25%; however, the predicted proportion of the population aged 65 that is dependent on others will be about 50% in 2050. This means that, of all the population of age 65, half will be dependent on others half of their lives. Any effort to understand ageing with an aim to improving the quality of life of the aged population should be vigorously tackled. Lowering the proportion of frail people and, eventually, of dependent people is a major task for gerontology and geriatrics [8]. As stated above, the differences in longevity between genders offer interesting possibilities to understand ageing. Animals with a very similar genetic background may differ in their average life span by as much as 10%. Paramount among these differences is that which happens in the human species. However, in order to find proof that this is not only due to sociological changes or to peculiarities between societies (e.g., whether women smoke more or less than men, etc.), one must understand the differences in longevity in animal species. One of the most widely used laboratory animals is the Wistar rat. In this rat, we and others have observed that females live longer than males, the maximal life span being approximately 10% longer (see Figure 1) [2]. In a similar fashion, using Fisher 344 rats, i.e., the same species but a different strain, in which longevity is also higher in females than in males, Jang et al. [9] observed that males produce more reactive oxygen species than females. However, the higher longevity of females as compared with males is not a universal phenomenon. In other species of rodents such as mice, some strains show a higher longevity of males when compared with females, for instance, the C57BL6 [4]. Moreover, a variation of this strain, i.e., C57B16J mice, which was used by Sanz et al. [3], shows no differences in longevity between sexes. In contrast, the Swiss albino mouse shows an increased longevity in females when compared with males [10]. So not only do we have different gender-specific longevity in different species, but also in different strains of the same species. This offers a unique opportunity to study comparative ageing, i.e., whether there are hormonal differences, different sensitivity or reactivity to hormones, or different fundamental molecular mechanisms of ageing. Since we are dealing with the same species, only with different genders, we are faced with optimal animal models to understand fundamental gerontology as well
Viña et al.: Oestrogens and ageing 67
83.8
Women
17 81.7
16 80.0 15
50
Men
14 1990 1992 1994 1996 1998 2000 2002 2004 Year
Proportion (%)
Number of years
55 84.6
19 18
Dependency
Mean lifespan
20
45 40 35 30 25 20 1990
2000
2010
2020
2030
2040
2050
Year
Figure 2 Proportion of persons aged over 65 who are dependent on others for care. Taken from the EUROSTAT and European Council Meeting in Lisbon in 2000.
Sex differences in free radical production In many species in which females live longer than males, the former produce approximately half the amount of mitochondrial peroxide than the latter [2]. It is very important to note that not all species behave the same in terms of gender-related longevity and that there are some species in which males live longer than females (see below). As stated above, in the Wistar rat, our experimental rat model, as well as in humans, females do indeed live longer than males by approximately a 10% increase in the average life span. In the early 2000s, we measured peroxide production by mitochondria from female and from male Wistar rats and found that females produce approximately half the amount of peroxides than males. This was completely reversed when rats were ovariectomised, thus tracing the differential gender effect on radical production to ovarian hormones. We then measured the free radical production of mitochondria from ovariectomised females that had been treated with oestrogens. In this case, oestradiol was able to reverse the effect of ovariectomy and the rate of peroxide production was similar to that of females (see Figure 3). The increase in radical production resulted in a significantly lower damage to mitochondrial DNA in females than in males. In fact, the level of 8-oxo-deoxyguanosine was four-fold higher in males than in females [2].
However, Ali et al. [4] showed that, in those strains of mice such as the black C57BL6 that they were using in which males live longer than females, it is males that produce fewer oxidants than females [4]. The authors measured not only oxidant production by determining dihydroethidium oxidation in the brain but also EPR signals in the brain mitochondria of their mice. Thus, in this strain of mice in which males live longer than females it is males that produce fewer radicals. Far from contradicting results from a laboratory, these results nicely confirm our results. The claim by all of us is that the gender that lives longer produces fewer radicals independently of whether it is males or females who live longer. The critical test will be to see why oestrogens promote the expression of antioxidant genes in a given species and do not promote that expression in other species or strains. A third confirmation of this hypothesis came from the laboratory of Sanz et al. [3]. These researchers studied a particular strain derived from C57B6J mice in which males Males Females 0.18 nmol H2O2/mg prot.min
as the hormonal regulation of the ageing process [2–4]. We centred our attention to gender oxidative stress differences to explain gender life-span differences, but, of course, there may be many other factors involved in these gender life-span differences.
0.16 0.14
Ovariectomised Estrogen replaced
0.12 0.10 0.08 0.06 0.04 0.02 0.00
Figure 3 Oxidant production by hepatic mitochondria from males and females: effect of ovariectomy and oestrogen replacement therapy. Data are expressed as mean ± SD of n = 6 rats. The statistical difference is indicated as follows: **p
