Oxidative stress as a possible pathogenic cofactor of post-menopausal osteoporosis: Existing evidence in support of the axis oestrogen deficiency-redox imbalance-bone loss

Introduction

Post-menopausal osteoporosis (PO) is one of the major health problems associated with menopause-related oestrogen deprivation1. The disease is characterized by a reduced bone mass and an impairment of bone architecture, with both leading to an increased risk in fragility fractures (FFs). FFs, in turn, are significantly associated to a high rate of disability and mortality12.

According to an epidemiological study, 22 million women and 5.5 million men living in the European Union suffered from PO in 2010, with more than 3.5 million new FFs sustained3. The incidence of osteoporosis in Asia is also steadily increasing mostly because this region is witnessing a rapid rise in elderly population. In particular, India is expected to have the highest total increase in people aged over 50 yr (from 120 million in 2013 to 620 million in 2050)4. Moreover, the female population is estimated to go through menopause earlier than Caucasians (mean age 46 vs 51 yr). This fact, along with the expected increase in female life expectancy (from 68 to 73 yr in the period 2011-2021), will extend the time span of post-menopausal period and, hence, the likelihood of related disturbances14.

Since PO is a worldwide health issue, an effort should be made to better clarify the underlying pathogenic mechanism(s) so as to devise effective preventive strategies. One of the most intriguing hypotheses focuses on the pivotal role of oxidative stress (OxS) as a link between gonadal failure and clinical onset and progression of PO567. By definition, OxS is the result of disruption of a finely tuned balance between antioxidants and oxidants, with the latter group including reactive oxygen species (ROS), reactive nitrogen species (RNS) and other less abundant substances89. The main aim of this review is to illustrate and discuss the current evidence on the association between post-menopausal oestrogen withdrawal, uncontrolled ROS generation and PO. Furthermore, it is meant to provide a critical overview of the most significant epidemiological studies on the effects of dietary antioxidants on bone health and to devise a strategy to overcome the limitations emerged and controversial results.

Post-menopausal osteoporosis: From oestrogen decline to disease onset

To preserve structural integrity, the skeleton necessities to continuously remodel and repair the micro-cracks that take place in the two types of osseous tissue: the ‘spongy’ cancellous and the compact cortical bone. Bone remodelling cycle occurs through the balanced activities of its constituent cell types: bone-forming osteoblast, the bone-degrading osteoclast and the osteocyte, the primary trigger of the entire turnover process10.

Despite pivotal role of oestrogen on the bone, it is still difficult to identify the mechanism(s) by which these hormones modulate bone metabolism. The oestrogens are able to block bone resorption through two mechanisms: both by direct interaction with osteocytes and osteoclast and by regulation of T-cell and osteoblast formation and activity1112. As described in some reviews111314, the biological axis constituted by receptor activator of nuclear factor k-B (RANK), its natural ligand (RANKL) and decoy receptor for RANKL, osteoprotegerin (OPG), is the molecular link between oestrogen deficiency and bone loss15. Noteworthy, RANK/RANKL/OPG axis also appears to play a key role in mediating the widely hypothesized connection between immune system and bone, central concept of osteoimmunology field1314.

Osteoclast precursors differentiate under the influence of macrophage colony-stimulating factor and RANKL13. The latter cytokine, expressed in both membrane-bound (in osteoblasts) and soluble forms, promotes differentiation of osteoclast precursors and activates mature osteoclast to reabsorb bone, through the interaction with RANK16. The osteoclastogenic effect can be prevented by OPG, which is produced by osteoblasts and stromal cells and competes with RANKL for RANK binding14. In addition, oestrogens can decrease osteoclast precursor responsiveness to RANKL15 by binding to nuclear factor-κβ (NF-κβ), which prevents the production of RANKL and promotes the release of OPG. Finally, oestrogens interfere with osteoclast signalling pathways downstream to RANK, thereby inhibiting osteoclast differentiation14. As a logical consequence, oestrogen withdrawal affects the ‘healthy’ balance between RANKL and OPG, with the result of prolonging osteoclast lifespan and accelerating bone resorption14.

Lymphocytes T and B may play an important role in the complicated network of biological interactions underlying oestrogen control of bone homeostasis1314. In particular, T-cells have been deemed to be the master regulators of both life cycle and metabolism of osteoclasts/osteoblasts. These immune cells are capable of inducing bone loss through the production of several bone-reabsorbing cytokines including RANKL, interleukin (IL)-1, IL-6 and tumour necrosis factor-α (TNF-α). Moreover, oestrogen decline has a direct effect on the synthesis of cytokines in T-cells, as shown in experiments on ovariectomized (OvX) rodents14 and in a few human studies1718. Our review does not aim at describing in detail the mechanisms implicated in the immune regulation of osteoporosis caused by oestrogen deficiency, which will not be dealt in-depth.

Pro-inflammatory cytokines are not the only factors that have been shown to be altered in a context of bone loss induced by oestrogen deficiency. Converging pre-clinical and epidemiological findings indicate that an excessive accumulation of oxidant substances, in particular, ROS, might contribute to the imbalance between bone resorption and formation that underlies PO development.

Reactive oxygen species (ROS) versus antioxidants: Perpetual struggle behind ageing processes and related diseases

ROS, the most abundant biological oxidants, can subtract electrons from all the biomolecules, including DNA, proteins, carbohydrates and lipids9. The modifications induced by these species are potentially toxic for cell biology because the induced damage easily spreads in the neighbouring cells if not adequately counteracted by the antioxidant system. A clear example of the contagious cytotoxicity of ROS is represented by the lipid peroxidation of cell membranes. Abstraction of one single electron from the polyunsaturated fatty acids incorporated in phospholipids triggers a chain reaction that diffuses throughout the lipid bilayer and finally leads to an irreversible structural and functional alteration of the plasmatic membranes9.

ROS are constantly produced in different cell sites: first, by mitochondria during the indispensable process of oxidative phosphorylation and, to a lesser extent, by cytosol, membrane, peroxisome and endoplasmic reticulum, and hence, their impact on cell health is not always deleterious. Accordingly, it is now well established that the dichotomy ROS-oxidation causes biological damage and, if perpetrated, disease, only upon exceeding a critical threshold91920. A number of endogens (e.g. inflammation and dysmetabolic syndrome) and environmental factors (e.g. pollution, smoking and nutrition) can accelerate the production of ROS, thereby increasing the risk of pathological conditions9. Moreover, the severity of the effects does not only depend on the quantity but also on the quality of the oxidant species involved. In fact, ROS family includes various members ranging from highly unstable/reactive-free radicals (i.e. molecules lacking one or more electrons) to poorly reactive non-radical molecules, such as hydrogen peroxide (H₂O₂)920. The production of this compound depends on the enzyme superoxide dismutase (SOD). It catalyzes the dismutation of peroxide radical (O₂⁻), which in turn is produced by different enzymes located in the mitochondria, cytosol or membrane9. In an hypothetical classification based on the antioxidant power, SOD and other enzymes are able to neutralize H₂O₂, i.e. catalase (CAT) and glutathione peroxidase (Gpx) most likely occupy the first place21. These three enzymes have been indicated as the first line of defence, thanks to their unique ability to directly scavenge ROS in vivo2021. There are other endogen enzymatic and non-enzymatic molecules which effectively contrast such reactive species as glutathione (natural substrate of Gpx), thioredoxin, coenzyme Q and uric acid (UA, the most abundant antioxidant in plasma). The core of the antioxidant protective system belongs to a complex network of enzymes, signalling molecules and transcription factors that result from the evolutionary adaptation of cells to the risk of living in an oxygen-rich environment9.

Dietary antioxidants, such as vitamins E (also named α-tocopherol) and C, β-carotene and lycopene, are also important in this context. Even though almost all these molecules show a detectable antioxidant capacity in experimental conditions, only α-tocopherol seems to be really effective in vivo21. This property depends on its relatively rapid kinetics of reaction, as well as on its high bodily concentration, especially in high ROS-vulnerable cell membranes21. Growing pre-clinical evidence has highlighted the beneficial effects of a highly variegate category of food antioxidants: the polyphenols21. Several members of this family, in particular, curcumin (turmeric) and resveratrol (grapes), have the peculiar capacity to stimulate a general xenobiotic response in target cells. In fact, these activate multiple defensive genes, such as those encoding SOD and CAT921.

A central paradigm in the redox field is that ROS/RNS become pathological players only when they overwhelm the opposing forces of the innate (endogen) and acquired (dietary) antioxidants. The rupture of this redox homeostasis is the prelude to OxS, a condition that severely affects cell integrity and biology and predisposes to several diseases919.

Oestrogen deficiency: Starting point of oxidative stress-induced bone-degenerative processes heading to post-menopausal osteoporosis

OxS is considered the common pathogenic factor for the ‘menopausal syndrome’ that includes several disturbances (e.g. vasomotor complains, cognitive impairment and urogenital dystrophy) and diseases, such as PO. The oestrogen decline associated with menopausal transition is believed to be the ‘spark’ that triggers OxS.

Implication of oxidative stress in post-menopausal osteoporosis pathogenesis: Experimental and epidemiological evidence

Anti-osteoporotic therapies targeting oxidative stress: Current state of the art

The questions that inevitably arise from the reported evidence are: how can we translate the research findings and acquired knowledge into clinical practice? And, in line with the aim of this review, is there any evidence that targeting OxS could be beneficial in terms of prevention and treatment of PO?

At present, there are various effective drug treatments for the management of PO and for the prevention of subsequent FFs61. Pharmacologic intervention is appropriate in case of previous fractures or osteoporosis at increased risk of fracture based on a fracture risk assessment tool, i.e. WHO FRAX (http:www.shef.ac.uk/FRAX)62. Whereas bisphosphonates, denosumab and teriparatide are used for the treatment of patients with a full-blown osteoporosis, selective oestrogen receptor modulators may be considered as the first-line treatment in preventing bone loss in women within 10 years of the menopause, side by side with the menopausal hormonal therapy (MHT) and the most recent combination of bazedoxifene and oestrogen63.

Convergent data from research studies and clinical trials indicate that MHT normalizes turnover and preserves BMD at all skeletal sites, thereby decreasing vertebral and non-vertebral fractures significantly64. Despite the initial concern with the safety of such treatment, recent studies reported encouraging results in this field. In particular, a 10 yr randomized trial completed in 200865 showed a significantly lower risk of mortality, heart failure and myocardial infarction without any apparent increase in risk of cancer in carefully selected women younger than 60 yr old receiving MHT early after menopause6164. Epidemiologic evidence is consistent with the data from animal studies on OvX rats receiving oestrogen replacement therapy (ERT). López-Grueso et al66 showed that ERT could prevent redox imbalance and counteract the typical post-surgical dysmetabolic profile only when administered early after oophorectomy. Based on these results, the authors postulated that ERT could upregulate the antioxidant enzymes and hence promote longevity. Unfortunately, to the best of our knowledge, there is no longitudinal study supporting this hypothesis in humans.

Along with pharmacological interventions, a reduction in the risk of osteoporosis should be achieved through lifestyle interventions, including exercise, adequate intake of calcium and proteins and avoidance of risk factors, such as sedentary lifestyle, smoking and alcohol abuse67. According to wide-acknowledged guidelines, an adequate intake of calcium and vitamin D is mandatory to achieve peak bone mass and prevent post-menopausal bone loss68. Nutrition can also be the source of several antioxidants, with potential beneficial effects on bone health. However, the reliability of the studies on the possible benefit of dietary antioxidants on bone homeostasis, fracture risk and markers of bone turnover is hampered by several drawbacks. First, the long-term effects of phytochemicals have not been assessed in randomized controlled clinical trials on post-menopausal women. Second, there are only a few interventional studies, while observational research is highly heterogeneous in terms of study design and outcome measures. Finally, the studies published so far may produce biased results as these have mainly relied on dietary and food questionnaires and have not objectively assessed body stores through blood or urine analyses. Taking into account these multiple caveats, it is worth a brief overview of the reported findings.

The preventive effect of vitamin E on bone erosion observed in animal models prompted a number of large epidemiological surveys on the possible effect of its intake from diet and/or supplements4669. In a study on 951 current-smoking post-menopausal women, high intake of vitamin E was associated with a significantly lower risk of hip fracture70. Accordingly, the supplementation with vitamin E was related to a reduced rate of fracture of any type in a large-scale epidemiological study (n=61,433 peri-menopausal women)71. In contrast with the aforementioned results, a longitudinal study conducted by MacDonald et al72 highlighted a direct association between increased vitamin E intake and greater femoral neck BMD loss. Finally, Wolf et al73 evaluated the effect of daily vitamin E intake (and serum level of the vitamin) in 11,068 elderly women and failed to show any significant association between vitamin E and BMD at any site in multivariate analysis. Besides vitamin E, many other studies evaluated the effect of other antioxidants on bone health. A large population-based case-control study showed that the intake of β-carotene and selenium, but not vitamin C, was related to a decreased rate of hip fracture48. Promising results also arose from the handful of small epidemiological studies on lycopene, one of the isomers of β-carotene, that is, especially present in tomato fruit. In particular, two studies indicated that daily consumption of this phytochemical may suppress bone resorption as suggested by the apparent inverse relation between lycopene intake and serum levels of resorption markers7475. Unfortunately, the encouraging pre-clinical findings obtained with other bioactive compounds, especially present in fruits, such as flavonoids, resveratrol and pectin, have not been adequately replicated in humans yet69. On the contrary, robust epidemiological evidence gathered in the last two decades yields a wide consensus around the positive impact of dietary intake of vegetables and, in particular, fruits on bone health6976.

Summary and novel roads ahead

The body of evidence supporting the pathogenic role of OxS in PO is mostly from in vitro or animal studies. Ageing and oestrogen deficiency represent the noxious combination that ‘breaches’ the first line of defence (i.e. antioxidant enzymes) against the potentially cytotoxic reactive species. The erosive activity of ROS on the bone has been well characterized. These chemically unstable molecules can affect bone health at various levels, influencing both indirectly (by stimulating the release of pro-absorptive cytokines) and directly the life cycle of osteoblasts/osteoclasts (Figure).

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Fig. 1

Cause-effect relationship between oestrogen decline, oxidative stress and post-menopausal osteoporosis. The menopause-associated decline in oestrogens (E2) (1) results in a decrease of systemic and local (bone) protection against reactive oxygen species attack (2). This effect is due to the ability of 17β-oestradiol to act as a direct antioxidant and, most likely, to simultaneously upregulate the expression of the antioxidant enzymes and to contrast the accumulation of proinflammatory (and thus pro-oxidant) visceral fat. The uncontrolled increase of reactive oxygen species (ROS) leads to oxidative stress (3) which, in turn, alters the balance between bone formation and resorption (4), thereby increasing the latter activity and contributing to the onset of post-menopausal osteoporosis (5).

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This putative connection between OxS and the clinical manifestations of the menopausal syndrome has sparked a constellation of observational studies exploring the beneficial effects of dietary antioxidants on the post-menopausal women. However, it is impossible to reach a firm conclusion as to the usefulness of phytochemicals in this context, due to the numerous methodological and design limitations as well as to the lack of randomized clinical trials. Nonetheless, taking into account also the safety of these natural compounds, we firmly believe, along with many others, that this could be the right road ahead.

The future interventional studies with antioxidants should address some criteria that have not been adequately considered so far. First, the modification of the usual diet (with increased intake of fruits and vegetables) or the use of antioxidant supplements might be suggested to accompany MHT to preserve correct redox homeostasis. It is now well accepted that no antioxidant is able to contrast oxidative challenge by itself. On the contrary, this task can be achieved only in concert with other antioxidants. A typical example in this field is α-tocopherol; after reacting with ROS, it becomes itself a free radical, which can be neutralized by the finely orchestrated synergic action of a network of endogen antioxidants, including glutathione and reduced glutathione77, and vitamin C. This consideration directly leads to the second benchmark to be addressed in the future trials: the interventions based on the administration of a single antioxidant are doomed to fail in the absence of an exhaustive picture of the individual redox profile. Such a tailored approach can be designed by measuring the peripheral levels of oxidative damage both overall and of the single antioxidants. Third and last points to take into account are that all types of antioxidant act as preventive, but not repairing agents. Translating these assumptions into practice: if the aim of a therapy is to effectively reduce the ‘weight’ of OxS contribution to PO development, then it must be prescribed to patients with normal or moderately low BMD, but not to patients with full-blown osteoporosis. The interventions aiming to reduce OxS may be accompanied with classical prevention strategies such as lifestyle modifications, avoidance of recognized risk factors and adequate intake of vitamin D, calcium and proteins.