Consuelo Borras

Mitochondria from females exhibit higher antioxidant gene expression and lower oxidative damage than males

We have investigated the differential mitochondrial oxidative stress between males and females to understand the molecular mechanisms enabling females to live longer than males. Mitochondria are a major source of free radicals in cells. Those from female rats generate half the amount of peroxides than those of males. This does not occur in ovariectomized animals. Estrogen replacement therapy prevents the effect of ovariectomy. Mitochondria from females have higher levels of reduced glutathione than those from males. Those from ovariectomized rats have similar levels to males, and estrogen therapy prevents the fall in glutathione levels that occurs in ovariectomized animals. Oxidative damage to mitochondrial DNA in males is 4-fold higher than that in females. This is due to higher expression and activities of Mn-superoxide dismutase and of glutathione peroxidase in females, which behave as double transgenics overexpressing superoxide dismutase and glutathione peroxidase, conferring protection against free-radical-mediated damage in aging. Moreover, 16S rRNA expression, which decreases significantly with aging, is four times higher in mitochondria from females than in those from males of the same chronological age. The facts reported here provide molecular evidence to explain the different life span in males and females.

Delayed ageing through damage protection by the Arf/p53 pathway.

The tumour-suppressor pathway formed by the alternative reading frame protein of the Cdkn2a locus (Arf) and by p53 (also called Trp53) plays a central part in the detection and elimination of cellular damage, and this constitutes the basis of its potent cancer protection activity. Similar to cancer, ageing also results from the accumulation of damage and, therefore, we have reasoned that Arf/p53 could have anti-ageing activity by alleviating the load of age-associated damage. Here we show that genetically manipulated mice with increased, but otherwise normally regulated, levels of Arf and p53 present strong cancer resistance and have decreased levels of ageing-associated damage. These observations extend the protective role of Arf/p53 to ageing, revealing a previously unknown anti-ageing mechanism and providing a rationale for the co-evolution of cancer resistance and longevity.

Decreasing xanthine oxidase‐mediated oxidative stress prevents useful cellular adaptations to exercise in rats

Reactive oxygen or nitrogen species (RONS) are produced during exercise due, at least in part, to the activation of xanthine oxidase. When exercise is exhaustive they cause tissue damage; however, they may also act as signals inducing specific cellular adaptations to exercise. We have tested this hypothesis by studying the effects of allopurinol‐induced inhibition of RONS production on cell signalling pathways in rats submitted to exhaustive exercise. Exercise caused an activation of mitogen‐activated protein kinases (MAPKs: p38, ERK 1 and ERK 2), which in turn activated nuclear factor κB (NF‐κB) in rat gastrocnemius muscle. This up‐regulated the expression of important enzymes associated with cell defence (superoxide dismutase) and adaptation to exercise (eNOS and iNOS). All these changes were abolished when RONS production was prevented by allopurinol. Thus we report, for the first time, evidence that decreasing RONS formation prevents activation of important signalling pathways, predominantly the MAPK–NF‐κB pathway; consequently the practice of taking antioxidants before exercise may have to be re‐evaluated.

Telomerase Reverse Transcriptase Delays Aging in Cancer-Resistant Mice

Telomerase confers limitless proliferative potential to most human cells through its ability to elongate telomeres, the natural ends of chromosomes, which otherwise would undergo progressive attrition and eventually compromise cell viability. However, the role of telomerase in organismal aging has remained unaddressed, in part because of the cancer-promoting activity of telomerase. To circumvent this problem, we have constitutively expressed telomerase reverse transcriptase (TERT), one of the components of telomerase, in mice engineered to be cancer resistant by means of enhanced expression of the tumor suppressors p53, p16, and p19ARF. In this context, TERT overexpression improves the fitness of epithelial barriers, particularly the skin and the intestine, and produces a systemic delay in aging accompanied by extension of the median life span. These results demonstrate that constitutive expression of Tert provides antiaging activity in the context of a mammalian organism.

17β-oestradiol up-regulates longevity-related, antioxidant enzyme expression via the ERK1 and ERK2[MAPK]/NFκB cascade

Females live longer than males. Oestrogens protect females against aging by up‐regulating the expression of antioxidant, longevity‐related genes such as glutathione peroxidase (GPx) and Mn‐superoxide dismutase (Mn‐SOD). The mechanism through which oestrogens up‐regulate those enzymes remains unidentified, but may have implications for gender differences in lifespan. We show that physiological concentrations of oestradiol act through oestrogen receptors to reduce peroxide levels in MCF‐7 cells (a mammary gland tumour cell line). Oestradiol increases MAP kinase (MAPK) activation as indicated by ERK1 and ERK2 phosphorylation in MCF‐7 cells, which in turn activates the nuclear factor kappa B (NFκB) signalling pathways as indicated by an increase in the p50 subunit of NFκB in nuclear extracts. Blockade of MAPK and NFκB signalling reduces the antioxidant effect of oestradiol. Finally, we show that activation of MAPK and NFκB by oestrogens drives the expression of the antioxidant enzymes Mn‐SOD and GPx. We conclude that oestradiol sequentially activates MAPK and NFκB following receptor activation to up‐regulate the expression of antioxidant enzymes, providing a cogent explanation for the antioxidant properties of oestrogen and its effects on longevity‐related genes.

Why females live longer than males? Importance of the upregulation of longevity-associated genes by oestrogenic compounds

Females live longer than males in many mammalian species, including humans. Mitochondria from females produce approximately half the amount of H2O2 than males. We have found that females behave as double transgenics overexpressing both superoxide dismutase and glutathione peroxidase. This is due to oestrogens that act by binding to the estrogen receptors and subsequently activating the mitogen activated protein (MAP) kinase and nuclear factor kappa B (NF‐κB) signalling pathways. Phytoestrogens mimic the protective effect of oestradiol using the same signalling pathway. The critical importance of upregulating antioxidant genes, by hormonal and dietary manipulations, in order to increase longevity is discussed.

Dietary soy isoflavone-induced increases in antioxidant and eNOS gene expression lead to improved endothelial function and reduced blood pressure in vivo

Epidemiological evidence suggests that populations consuming large amounts of soy protein have a reduced incidence of coronary heart disease (1–5). The cardiovascular risks associated with conventional hormone replacement therapy in postmenopausal women (5–7) have precipitated a search for alternative estrogen receptor modulators. Here we report that long‐term feeding of rats with a soy protein‐rich (SP) diet during gestation and adult life results in decreased oxidative stress, improved endothelial function, and reduced blood pressure in vivo measured by radiotelemetry in aged male offspring. Improved vascular reactivity in animals fed an SP diet was paralleled by increased mitochondrial glutathione and mRNA levels for endothelial nitric oxide synthase (eNOS) and the antioxidant enzymes manganese superoxide dismutase and cytochrome c oxidase. Reduced eNOS and antioxidant gene expression, impaired endothelial function, and elevated blood pressure in animals fed a soy‐deficient diet was reversed after refeeding them an SP diet for 6 months. Our findings suggest that an SP diet increases eNOS and antioxidant gene expression in the vasculature and other tissues, resulting in reduced oxidative stress and increased NO bioavailability. The improvement in endothelial function, increased gene expression, and reduced blood pressure by soy isoflavones have implications for alternative therapy for postmenopausal women and patients at risk of coronary heart disease.

Mitochondrial biogenesis in exercise and in ageing.

Mitochondrial biogenesis is critical for the normal function of cells. It is well known that mitochondria are produced and eventually after normal functioning they are degraded. Thus, the actual level of mitochondria in cells is dependent both on the synthesis and the degradation. Ever since the proposal of the mitochondrial theory of ageing by Jaime Miquel in the 70s, it was appreciated that mitochondria, which are both a target and a source of radicals in cells, are most important organelles to understand ageing. Thus, a common feature between cell physiology of ageing and exercise is that in both situations mitochondria are critical for normal cell functioning. Mitochondrial synthesis is stimulated by the PGC-1alpha-NRF1-TFAM pathway. PGC-1alpha is the first stimulator of mitochondrial biogenesis. NRF1 is an intermediate transcription factor which stimulates the synthesis of TFAM which is a final effector activating the duplication of mitochondrial DNA molecules. This pathway is impaired in ageing. On the contrary, exercise, particularly aerobic exercise, activates mitochondriogenesis in the young animal but its effects on mitochondrial biogenesis in the old animal are doubtful. In this chapter we consider the interrelationship between mitochondrial biogenesis stimulated by exercise and the possible impairment of this pathway in ageing leading to mitochondrial deficiency and eventually muscle sarcopenia.

Theories of ageing

Ageing is a universal, intrinsic, progressive and deleterious process. Understanding it is of major interest to scientist, physicians as well as to the general population. Critical to this understanding is to formulate comprehensive theories of aging with high predictive and explanatory power. More than 300 theories have been postulated and are reviewed here. The free radical theory of ageing is one of the most prominent and well studied. It was further developed by one of us (JM) in what has become known as the mitochondrial theory of ageing. These theories provide new experimental approaches to further develop our understanding of the phenomenon of ageing. IUBMB Life, 59: 249‐254, 2007

Genistein, a soy isoflavone, up-regulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NFκB

We have previously reported that estrogens up‐regulate longevity‐associated genes. As recent evidence has shown that estrogen replacement therapy is associated with an increased risk of cardiovascular disease, we have studied the effects of genistein, a soy isoflavone with a similar structure to estradiol, on the expression of antioxidant, longevity‐related genes. MCF‐7 cells (human mammary gland tumor cell line) were incubated for 48 h with 0.5 μM genistein, a concentration found in the plasma of populations consuming diets rich in soy protein. Peroxide levels were determined by fluorimetry, activation of extracellular‐signal regulated kinase (ERK1/2), and nuclear factor κB (NFκB)‐signaling pathways by Western blot analysis and ELISA, respectively, and mRNA expression of antioxidant genes by real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR). Inhibition of basal peroxide levels in MCF‐7 cells by genistein was prevented by pretreatment of cells with the estrogen receptor antagonist tamoxifen. Phosphorylation of extracellular regulated kinase (ERK)1/2 led to an activation of NFκB, as indicated by increased p50 subunit expression in nuclear extracts, and increased mRNA levels of the antioxidant enzyme manganese‐superoxide dismutase (MnSOD). Inhibition of ERK1/2 abrogated genistein‐mediated NFB activation and elevated expression of MnSOD. Our molecular studies may provide a basis to determine the effects of genistein and other soy protein‐derived products on longevity in both animals and the human population.—Borrás, C., Gambini, J., Gómez‐Cabrera, M. C., Sastre, J., Pallardó, F. V., Mann, G. E., Viña, J. Genistein, a soy isoflavone, upregulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NFB. FASEB J. 20, E1476 –E1481 (2006)