Considerable evidence implicates oxidative stress in the pathophysiology of many complications of human pregnancy, and this topic has now become a major focus of both clinical and basic science research. Oxidative stress arises when the production of reactive oxygen species overwhelms the intrinsic anti-oxidant defences. Reactive oxygen species play important roles as second messengers in many intracellular signalling cascades aimed at maintaining the cell in homeostasis with its immediate environment. At higher levels, they can cause indiscriminate damage to biological molecules, leading to loss of function and even cell death. In this chapter, we will review how reactive oxygen species are generated and detoxified in the human placenta, and what roles they may play at homeostatic concentrations. We will then consider their involvement in normal placental development, and in complications ranging from miscarriage to pre-eclampsia and premature rupture of the membranes.
1
Introduction
Oxygen is often referred to as the Janus gas, as it has both positive benefits and potentially damaging side-effects for biological systems. Reactivity allows oxygen to participate in high-energy electron transfers, and hence support the generation of large amounts of adenosine-5-triphosphate (ATP) through oxidative phosphorylation. This is necessary to permit the evolution of complex multicellular organisms, but also renders it liable to attack any biological molecule, be it a protein, lipid or DNA. Consequently, our body is under constant oxidative attack from reactive oxygen species (ROS). A complex system of antioxidant defences has evolved that generally holds this attack in balance. On occasions, however, this balance can be perturbed, leading to oxidative stress. Because of the multiple and diverse effects that oxygen toxicity can have on a cell, oxidative stress is best defined in broad terms as an alteration in the pro-oxidant–antioxidant balance in favour of the former that leads to potential damage. Oxidative stress is now recognised to play a central role in the pathophysiology of many different disorders, including complications of pregnancy.
The concept of a pro-oxidant–antioxidant balance is central to an understanding of oxidative stress for several reasons. Firstly, it emphasises that the disturbance may be caused through changes on either side of the equilibrium (e.g. abnormally high generation of ROS or deficiencies in the antioxidant defences). Secondly, it highlights the homeostatic concentrations of ROS. Although ROS first came to the attention of biologists as potentially harmful by-products of aerobic metabolism, it is now recognised that they play important roles as secondary messengers in many intracellular signalling pathways. Finally, the concept of a balance draws attention to the fact that there will be a graded response to oxidative stress. Hence, minor disturbances in the balance are likely to lead to homeostatic adaptations in response to changes in the immediate environment, whereas more major perturbations may lead to irreparable damage and cell death. The boundary between normal physiological changes and pathological insults is therefore inevitably indistinct.
The definition of oxidative stress provided above is necessarily broad because the outcome depends in part on the cellular compartment in which the ROS are generated. There are many potential sources of ROS, and the relative contributions of these will depend on the environmental circumstances prevailing. As the reactions of ROS are often diffusion-limited, the effects on cell function depend to a large extent on the biomolecules in the immediate vicinity. Different insults will therefore generate different outcomes.
A further feature of oxidative stress that affects its clinical presentation is that it rarely occurs in isolation. It is now appreciated that complex interactions take place between oxidative and other forms of cell stress, such as endoplasmic reticulum (ER) stress. The clinical manifestation will therefore depend on the balance of metabolic activities in a particular cell type or organ, and so may vary from system to system.
In this review, we will consider the main reactive oxygen species and their generation, the principal antioxidant defences, and then how oxidative stress may be manifested at the maternal–fetal interface during human pregnancy.
2
Reactive oxygen species
The term ‘reactive oxygen species’ is applied to both free radicals and their non-radical intermediates. Free radicals are defined as species containing one or more unpaired electrons, and it is this incomplete electron shell that confers their high reactivity. Free radicals can be generated from many elements, but in biological systems it is those involving oxygen and nitrogen that are the most important ( Fig. 1 ).
Under physiological conditions, the most common oxygen free radical is the superoxide anion (O 2 •− ), and mitochondria are considered the principal source. The transfer of electrons along the enzymes of the respiratory chain is not totally efficient, and leakage of electrons on to molecular oxygen, in particular from complexes I and III, results in the formation of O 2 •− . The rate of formation is determined by the number of electrons present on the chain, and so is elevated under conditions of hyperoxia and of raised glucose, as in diabetes. Paradoxically, it is also increased under conditions of hypoxia, when the reduced availability of oxygen to act as the final electron acceptor for complex IV causes electrons to accumulate. Under normal conditions, 2% of oxygen consumed is converted to O 2 •− in the mitochondria rather than being reduced to water. Because of its charge, O 2 •− is membrane impermeable, and so remains within the mitochondrial matrix.
Similarly, superoxide can also be generated through leakage of electrons from the shorter electron transport chain within the ER. The formation of disulphide bonds during protein folding is an oxidative process, and about 25% of O 2 − within cells is generated within the ER. This can increase in cells with a high secretory output, and also under conditions of ER stress when repeated attempts to refold misfolded proteins may take place.
Other sources of superoxide under physiological conditions include the enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which generates substantial quantities throughout pregnancy but particularly in early pregnancy, cytochrome P450, and other oxido-reductases. Hence, various growth factors, drugs and toxins cause increased generation of ROS. Under pathological conditions, the enzyme xanthine dehydrogenase becomes an important contributor. This enzyme degrades purines, xanthine and hypoxanthine to uric acid and, under normal conditions, uses NAD + as the electron recipient. However, under hypoxic conditions it is proteolytically cleaved to the oxidase form, which donates electrons to molecular oxygen. This enzyme plays a key role in the reperfusion phase of ischaemia–reperfusion injury, when its action is augmented by the build up of hypoxanthine as a result of ATP breakdown during the hypoxic period.
Superoxide is detoxified by the superoxide dismutase enzymes, which convert it to hydrogen peroxide. Hydrogen peroxide is not a free radical, and so is less reactive than O 2 − . However, it comes under the term of ROS as it is intimately involved in the generation and detoxification of free radicals. As it is non-polar, it is able to diffuse through cell and organelle membranes, and hence acts widely as a second messenger in signal transduction pathways. Hydrogen peroxide is in turn detoxified to water by the enzymes catalase and glutathione peroxidase. It is important that the antioxidant enzymes act in concert, as an imbalance in the concentrations of O 2 − and hydrogen peroxide can result in the formation of the much more dangerous hydroxyl ion (OH ). This reaction is catalysed by free ferrous ions in the Fenton reaction. The hydroxyl ion has an estimated life of 10 −9 s, and reacts with any biological molecule in its immediate vicinity in a diffusion-limited manner. Because it is so highly reactive there is no known scavenger of OH .
Excessive generation of superoxide can also lead to interactions with nitric oxide (NO ) to form peroxynitrite (ONOO − ). Peroxynitrite is a powerful pro-oxidant. As it is capable of diffusing up to 5 μm, it may affect neighbouring cells.
2
Reactive oxygen species
The term ‘reactive oxygen species’ is applied to both free radicals and their non-radical intermediates. Free radicals are defined as species containing one or more unpaired electrons, and it is this incomplete electron shell that confers their high reactivity. Free radicals can be generated from many elements, but in biological systems it is those involving oxygen and nitrogen that are the most important ( Fig. 1 ).
Under physiological conditions, the most common oxygen free radical is the superoxide anion (O 2 •− ), and mitochondria are considered the principal source. The transfer of electrons along the enzymes of the respiratory chain is not totally efficient, and leakage of electrons on to molecular oxygen, in particular from complexes I and III, results in the formation of O 2 •− . The rate of formation is determined by the number of electrons present on the chain, and so is elevated under conditions of hyperoxia and of raised glucose, as in diabetes. Paradoxically, it is also increased under conditions of hypoxia, when the reduced availability of oxygen to act as the final electron acceptor for complex IV causes electrons to accumulate. Under normal conditions, 2% of oxygen consumed is converted to O 2 •− in the mitochondria rather than being reduced to water. Because of its charge, O 2 •− is membrane impermeable, and so remains within the mitochondrial matrix.
Similarly, superoxide can also be generated through leakage of electrons from the shorter electron transport chain within the ER. The formation of disulphide bonds during protein folding is an oxidative process, and about 25% of O 2 − within cells is generated within the ER. This can increase in cells with a high secretory output, and also under conditions of ER stress when repeated attempts to refold misfolded proteins may take place.
Other sources of superoxide under physiological conditions include the enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which generates substantial quantities throughout pregnancy but particularly in early pregnancy, cytochrome P450, and other oxido-reductases. Hence, various growth factors, drugs and toxins cause increased generation of ROS. Under pathological conditions, the enzyme xanthine dehydrogenase becomes an important contributor. This enzyme degrades purines, xanthine and hypoxanthine to uric acid and, under normal conditions, uses NAD + as the electron recipient. However, under hypoxic conditions it is proteolytically cleaved to the oxidase form, which donates electrons to molecular oxygen. This enzyme plays a key role in the reperfusion phase of ischaemia–reperfusion injury, when its action is augmented by the build up of hypoxanthine as a result of ATP breakdown during the hypoxic period.
Superoxide is detoxified by the superoxide dismutase enzymes, which convert it to hydrogen peroxide. Hydrogen peroxide is not a free radical, and so is less reactive than O 2 − . However, it comes under the term of ROS as it is intimately involved in the generation and detoxification of free radicals. As it is non-polar, it is able to diffuse through cell and organelle membranes, and hence acts widely as a second messenger in signal transduction pathways. Hydrogen peroxide is in turn detoxified to water by the enzymes catalase and glutathione peroxidase. It is important that the antioxidant enzymes act in concert, as an imbalance in the concentrations of O 2 − and hydrogen peroxide can result in the formation of the much more dangerous hydroxyl ion (OH ). This reaction is catalysed by free ferrous ions in the Fenton reaction. The hydroxyl ion has an estimated life of 10 −9 s, and reacts with any biological molecule in its immediate vicinity in a diffusion-limited manner. Because it is so highly reactive there is no known scavenger of OH .
Excessive generation of superoxide can also lead to interactions with nitric oxide (NO ) to form peroxynitrite (ONOO − ). Peroxynitrite is a powerful pro-oxidant. As it is capable of diffusing up to 5 μm, it may affect neighbouring cells.
3
Antioxidant defences
Enzymatic and non-enzymatic defences inhibit oxidant attack. The enzymatic defences all have a transition metal at their core, capable of taking on different valences as they transfer electrons during the detoxification process. Two isoforms of superoxide dismutase convert O 2 − to hydrogen peroxide, the manganese form that is restricted to the mitochondria, and the copper and zinc form that is located in the cytosol. The hydrogen peroxide is then broken down to water by the actions of catalase or glutathione peroxidase, a tetrameric selenoprotein.
The activity of glutathione peroxidase depends on the presence of reduced glutathione (GSH) as a hydrogen donor. Glutathione is the major cellular thiol redox buffer in cells, and is synthesised in the cytosol from l -glutamate, l -cysteine and glycine. GSH participates in a large number of detoxifying reactions forming glutathione disulfide, which is converted back to GSH by the action of glutathione reductase at the expense of NADPH. The latter is generated through the pentose phosphate pathway, of which glucose-6-phosphate dehydrogenase is the first enzyme. This enzyme is subject to common polymorphisms, and decreased activity may compromise GSH concentrations and lead to embryopathy.
The non-enzymatic defences include ascorbate (vitamin C) and α-tocopherol (vitamin E). These again act in concert, with ascorbate being necessary to regenerate reduced α-tocopherol. In addition, thiol compounds, such a thioredoxin, are capable of detoxifying hydrogen peroxide, but in turn require converting back to the reduced form by thioredoxin reductase. Ceruloplasmin and transferrin also play important roles by sequestering free iron ions and so inhibiting the Fenton reaction and production of OH .
Polymorphisms in the antioxidant enzymes or dietary restriction of micronutrients, such as selenium, can thus play an important role in predisposing to oxidative stress and complications of pregnancy.
4
Biological actions of reactive oxygen species
At homeostatic levels, ROS have diverse actions on cell function, including activation of redox-sensitive transcription factors and activation of protein kinases. These are described below.
4.1
Activation of redox-sensitive transcription factors
Activation of redox-sensitive transcription factors, such as AP-1, p53 and NF-κB regulate the expression of pro-inflammatory and other cytokines, cell differentiation and apoptosis. Under normal conditions NF-κB is held inactive by the binding of its inhibitory sub-unit IκB. However, under conditions of stress, IκB becomes phosphorylated and dissociates from NF-κB, which then translocates to the nucleus and activates expression of pro-inflammatory and other cytokines. Increased phosphorylation of IκB is observed in term placental explants subjected to hypoxia-reoxygenation in vitro, which provides a model for malperfusion of the placenta in vivo . Activation of the pathway is associated with increased tissue levels of the proinflammatory enzyme COX-2, interleukin 1ß, increased secretion of TNF-α, and activation of the apoptotic cascade as evinced by cleavage of caspase 3. All these effects can be blocked by the addition of vitamins C and E or sulfasalazine, an inhibitor of NF-κB activation.
4.2
Activation of protein kinases
With activation of protein kinases, cells respond to a variety of extracellular signals and stress through a family of mitogen-activated protein kinases (MAPK). Of this family, ROS-induced activation of extracellular regulated kinases (ERK1/2) generally promotes cell survival and proliferation, whereas stimulation of p38MAPK (p38) and stress-activated protein kinase–c-Jun amino terminal kinases (SAPK–JNK) mostly induces apoptosis. p38 and SAPK–JNK are activated by phosphorylation through an upstream kinase, apoptosis-regulating signal kinase 1 (ASK1). Under normal conditions ASK1 is held inactive by binding to thioredoxin, but O 2 − is capable of oxidising the thiol groups in the latter, leading to a conformational change and its release. Increased phosphorylation of p38, but not SAPK, is observed in the term placenta after labour compared with control participants delivered by caesarean section. ASK1 is also activated in explants exposed to either hypoxia-reoxygenation or hydrogen peroxide, and is inhibited by addition of vitamins C and E. Activation is associated with increased levels of the soluble receptor for vascular endothelial growth factor (sFlt-1), which has been implicated in the pathogenesis of pre-eclampsia. Levels of sFlt-1 can be reduced by the addition of vitamins C and E, or inhibitors of the p38 pathway. They can also be reduced by the addition of sulfasalazine, which indicates considerable interactions and mutual reinforcement between the NF-κB and MAPK signalling pathways in the placenta.
The above responses may be considered as physiological adaptive changes to alterations in the environment aimed at restoring homeostasis. More severe attack by ROS may lead to more extensive and irreparable cell damage, resulting ultimately in cell death through necrosis or apoptosis. These more pathological effects are mediated by opening of ion channels, lipid peroxidation, protein modifications and DNA oxidation. These are discussed below.
4.3
Opening of ion channels
Imbalances of ROS lead to loss of intracellular Ca 2+ homeostasis, with release of Ca 2+ ions from the endoplasmic reticulum and other stores. The calcium concentration within the ER lumen is much higher than in the cytosol, reaching millimolar levels. This concentration is maintained by pumps belonging to the sarco and endoplasmic reticulum calcium ATPase family, and is necessary for the correct functioning of the protein-folding machinery. ROS are able to activate calcium release channels in the ER membrane, which include the inositol-1,4,5,triphosphate receptor (IP 3 R) and the ryanodine receptor.
The resultant release of Ca 2+ from the ER will activate diverse Ca 2+ -sensitive processes within the cell, including many of the signalling pathways above. It also has a profound effect on function. The loss of chaperone activity results in the accumulation of misfolded proteins within the lumen, leading to further generation of ROS as attempts are made to refold them. The accumulation will also stimulate the unfolded protein response (UPR), a highly conserved set of signalling pathways that aim to restore homeostasis, but, if this fails, will stimulate apoptosis. The relationship between oxidative and ER stress will be considered in greater detail later.
The rise in cytosolic Ca 2+ ion concentration will also adversely affect mitochondrial function, including an increase in their own production of ROS and opening of the permeability transition pore. Opening of the membrane permeability transition is promoted synergistically by increased Ca 2+ ions and oxidation of the thiol groups on proteins in the inner mitochondrial membrane. As a result, the mitochondrial membrane potential and ATP synthesis collapse. If mitochondria throughout the cell are affected, ATP concentrations fall precipitously, ionic homeostasis is lost and the cell undergoes primary necrosis. Involvement of a more limited number of organelles, or transient opening of the pore, may allow ATP to be maintained at levels sufficient to permit apoptosis to occur instead.
4.4
Lipid peroxidation
Hydroxyl radicals are capable of causing lipid peroxidation in the plasma membrane or that of any organelle that contains large quantities of polyunsaturated fatty acid side chains. By abstracting hydrogen from the hydrocarbon side-chain of a fatty acid, they create a carbon-centred radical, C . If oxygen is present, this may react to form a peroxyl radical (–C–O–O ), which in turn is capable of abstracting hydrogen from an adjacent fatty acid, so propagating the reaction.
Because vitamin E is lipid-soluble and possesses a hydrophobic tail, it tends to accumulate within the interior of lipid membranes. Here, it acts as the most important chain-breaker, as it reacts with lipid peroxyl radicals about four times faster than they can react with adjacent fatty acid side chains.
Evidence of lipid peroxidation can be detected using antibodies directed against one of the principal products, 4-hydroxynonenal. It can be efficiently detoxified in cells by the glutathione S-transferase group of enzymes, but high levels are associated with loss of membrane fluidity and function, and activation of the apoptotic cascade.
4.5
Protein modifications
Amino acids, both free and in proteins, are a target for oxidative damage. Direct oxidation of the side chains leads to the formation of carbonyl groups (aldehydes and ketones), and proline, argenine, lysine and threonine are particularly vulnerable to attack. The carbonyl products are stable, and their detection using enzyme-linked immunosorbent assay or western blotting is the most commonly used method to assay protein oxidation.
Abstraction of hydrogen ions from the thiol group of cysteine can lead to the formation of disulfide bonds and abnormal protein folding, in a manner analagous to the activation of ASK1. Abnormal folding can lead to loss of function, but also protein aggregation and cell death.
Finally, peroxynitrite will react with tyrosine residues to form 3-nitrotyrosine, which can again be detected immunohistochemically. At physiological levels, protein nitration is thought to be a selective and reversible process that leads to activation in a manner analogous to phosphorylation, but at higher levels can be detrimental. Protein nitration in the placenta can therefore have diverse effects, with both gain and loss of function.
4.6
DNA oxidation
DNA is attacked principally by OH radicals, and a variety of products can be generated through reactions with either the DNA bases or the deoxyribose sugars. For example, OH can add on to guanine to produce 8-hydroxy-2′-deoxyguanosine, which may be measured biochemically and detected immunohistochemically. Attacks on the sugar moieties may cause strand breakages, whereas those on histone proteins may lead to cross-linkages that interfere with chromatin folding, DNA repair and transcription. Mutation or aberrant gene expression may therefore result.
Mitochondrial DNA is particularly vulnerable to ROS attack owing to its proximity to the site of O 2 − generation from the electron transport chain, the lack of histone protection, and the minimal repair mechanisms that exist. Consequently, damage to mitochondrial DNA is extensive even under normal conditions, and mutations occur at five to 10 times the rate seen in nuclear DNA. As mitochondrial DNA encodes several proteins, including enzymes of the electron transport chain, mutations may lead to impaired energy production and the risk of further electron leakage, compounding the original stress.
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