Reactive Oxygen Species (ROS), Nanoparticles, and Endoplasmic Reticulum (ER) Stress-Induced Cell Death Mechanisms

date.

Summary

Keywords: Reactive oxygen and nitrogen species (RONS); Free radicals; Nanoparticles (NPs); Endoplasmic reticulum (ER) stress; Ecotoxicology; Nanotoxicology; Oxidant; Oxidative stress; Oxidative damage; Antioxidants; Redox

During recent years there has been much interest in the use of nanoparticles (NPs) for in vitro studies as well as for the delivery of drugs and contrast agents in living cells for animals and humans. The fact that many diseases have been found to be associated with oxidative stress, oxidative damage, and generation of reactive oxygen and nitrogen species (RONS) established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to the complex pathophysiology of oxidative stress in humans. The unifying factor in determining toxicity and carcinogenicity of heavy metals is the generation of reactive oxygen species (ROS). The formation of RONS is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The roles of RONS (

) produced during tissue pathogenesis and in response to viral or chemical toxicants induce a complex series of downstream adaptive and reparative events driven by associated oxidative and nitrative stress. ROS are produced by cellular metabolic activities and environmental factors, such as air pollutants, environmental pervasiveness, commercial use, or cigarette smoke. ROS are highly reactive molecules because of unpaired electrons in their structure, react with several biological macromolecules in cells, such as carbohydrates, nucleic acids, lipids, and proteins, and alter their functions. ROS also affects the expression of several genes by the upregulation of redox-sensitive transcription factors and chromatin remodeling via alteration in histone acetylation/deacetylation. Regulation of redox state is critical for cell viability, activation, proliferation, and organ function.

The toxic manifestations of heavy metals are caused primarily due to imbalance between pro-oxidant and antioxidant homeostasis, which is termed oxidative stress. Besides, heavy metals have high affinity for thiol groups containing enzymes and proteins, which are responsible for normal cellular defense mechanisms. Long-term exposure to these metals could lead to apoptosis. Studies show that supplementation of antioxidants along with a chelating agent proves to be a better treatment regimen than monotherapy with chelating agents.

We have summarized different methods that can be used to investigate cellular uptake of NPs and the pitfalls in such studies. Some NPs are known to induce endoplasmic reticulum (ER) stress and lead to cell death. It is clear that cells during development are sensitive to damage by ROS. Disruption of development may result from general macromolecular damage that can cause cell death, and also from the oxidation of specific key developmental molecules. Most studies report NPs to be found within cellular structures that are likely to be endosomes or lysosomes, and in some studies this has been addressed in more detail. NPs of varying size and composition may be taken up by various endocytic mechanisms ending up in different intracellular trafficking pathways. The structural alteration of metallic NPs leads to different biological functions, specifically resulting in different potentials for the generation of ROS. ROS and ER stress signaling play important roles in a number of physiological processes, including the intracellular killing of bacteria by neutrophil granulocytes, detoxification by the liver, prostaglandin production, and certain cell-signaling processes. To this end it is necessary to increase our understanding of how these particles are taken up and transported within the cells, and to what extent they are metabolized and secreted.

RONS may act as a regulator of cellular responses to extracellular environmental signaling. Furthermore, increasing evidence indicates that ROS may act as a mediator of lipid accumulation, which is associated with dramatic changes in the transcriptome, proteome, and metabolome. RONS play important roles in a number of physiological processes, including the intracellular killing of bacteria by neutrophil granulocytes, detoxification by the liver, prostaglandin production and certain cell-signaling processes. Marked metabolic changes occur in cells during development. ROS production levels in a cell are likely determined by the metabolic activity associated with a particular cellular process. On the other hand, antioxidant activity may be a consequence of the ROS production level. The complex mechanisms that cells have to determine the redox state make it difficult to assign a definitive developmental function to ROS based only upon altering ROS or antioxidant levels. Considering a passive ROS function may be the mitochondrial signal that contributes to the correct development of embryos from very early stages. Although this observation supports an active role of ROS in development, a convincing determination of a developmental function depends on evidence indicating a requirement of ROS in specific processes during embryogenesis. Genetic manipulations directed to determine the active functions of ROS may bring light to this difficult task.

, which are highly reactive molecules.

ER stress is a complex protein-folding and trafficking organelle and has been implicated in human pathologies. Alteration and discrepancy in the ER environment can affect the protein-folding process and hence can result in the production of misfolded proteins. The accumulation of misfolded proteins causes cellular damage and elicits ER stress. Apoptosis and autophagic and regulated necrosis are major forms of programmed mechanisms of cell death in living mammalian cells. The cell death and phagocytosis regulatory networks offer a multitude of targets for toxic chemicals. The roles of redox mediators cause multiple human disorders, including neurodegenerative diseases, diabetes mellitus, atherosclerosis, inflammation, ischemia, and kidney and liver diseases. Redox signaling and oxidative stress constitute fundamental regulatory mechanisms not only in highly prevalent pathologies but also in less explored areas of biomedicine such as those related to fibrosis, cell migration and proliferation, hearing loss, schizophrenic disorders, insulin resistance, drug metabolism, lung hypoxia, or renal hemodynamics. In some cases, the underlying molecular mechanisms of this redox sensitivity have been deciphered; in others we are just beginning to open the door. It is clear that the pervading nature of RONS reactivity will continue to offer surprises and explanations of complex pathologies such as those covered in this book.

Mechanisms of toxicity, cytotoxicity, genotoxicity, ecotoxicology, nanotoxicology, and nanopathology for free radicals and NPs inside the macromolecule’s cells have been discussed. A critical evaluation of counteractive antioxidant defense and future needs for safe environmental heavy metal ions and nanotechnology has been performed. The effective use of antioxidants as therapeutic agents against certain infections is a realistic possibility that is beginning to be applied against viruses.

In this book we discuss the possibilities, challenges, and pitfalls of studying endocytic pathways involved in the cellular uptake of NPs. Thus the use of pharmacological inhibitors, expression of mutated proteins, use of siRNAs, and colocalization experiments in such studies are critically evaluated. Although the main focus is on cellular uptake, aspects of intracellular transport, recycling of NPs to the cell exterior, disturbance of cellular functions, and metabolism of NPs are also discussed. Pharmacological inhibitors are often used to investigate which endocytic mechanism is responsible for the cellular uptake of NPs. This approach is far too often based on the assumption that these inhibitors have specific effects on a given endocytic mechanism, but as discussed throughout the book, this is normally not the case.

This book reviews and discusses induced cell death mechanisms caused by the roles of RONS, ER stress, free radicals, and NP mechanisms. Exposure to NPs has been shown to induce ER stress, showing as alterations in ER morphologies and activation of ER stress pathways both in vivo and in vitro. Furthermore, modulation of ER stress by chemicals has been shown to alter NP-induced toxicity, which confirmed ER stress as the mechanism for NP-induced toxicity. Meanwhile, exposure of cancer cells to ER stress-inducing NPs or NPs containing ER stress inducers could lead to ER stress-mediated apoptosis, whereas alleviation of ER stress by NPs with antioxidant properties has been shown as a strategy to cure metabolic diseases. Thus modulation of ER stress by NPs could be a potential way for disease therapy.

The book explains RONS, NPs, free radicals, and ER stress-induced cell death mechanisms. An update of the recent strategies for treatment with a possible beneficial role of antioxidant supplementation to achieve optimum effects is discussed. Also explained are the effects of interactions between antioxidant-exerted protective effects defense therapy and ROS.

The book comprises 24 chapters covering the following subjects in detail:

1.Pathophysiological, toxicological, and immunoregulatory roles of reactive oxygen and nitrogen species

2.Biological mechanisms of reactive oxygen species

3.Cellular signaling pathways with reactive oxygen species

4.Manganese as the essential element in oxidative stress and metabolic diseases

5.Affected energy metabolism is the primal cause of manganese toxicity

6.Heavy metals and free radical-induced cell death mechanisms

7.Cytotoxic mechanisms of xenobiotic heavy metals on oxidative stress

8.Oxidative stress and oxidative damage-induced cell death

9.Cell death mechanisms—apoptosis pathways and their implications in toxicology

10.Programmed cell death mechanisms and nanoparticle toxicity

11.Endoplasmic reticulum stress and associated reactive oxygen species in disease pathophysiology applications

12.Endoplasmic reticulum stress-induced cell death mechanisms

13.Modulation of endoplasmic reticulum stress of nanotoxicology for nanoparticles

14.Nanoparticle cellular uptake and intracellular targeting on reactive oxygen species in biological activities

15.Metal nanoparticles and particulate matter induce toxicity

16.Mechanisms for nanoparticle-mediated oxidative stress

17.Nanotechnological modifications of nanoparticles on reactive oxygen and nitrogen species

18.Medical imaging of the complexity of nanoparticles and reactive oxygen species dynamics in vivo for clinical diagnosis applications

19.Titanium dioxide nanoparticle-induced cytotoxicity and genotoxicity—generation of reactive oxygen species and cell damage

20.Toxicity of ZnO nanoparticle-induced reactive oxygen species and cancer cells

21.Silver nanoparticle-induced cellular cytotoxicity, genotoxicity, DNA damage, and cell death

22.Correlations between oxidative stress and aligning nanoparticle safety assessments

23.Effects of interactions between antioxidant defense therapy and reactive oxygen species

24.Food and Drug Administration breakthrough—drug therapy designations for clinical evidence

In general, more research is needed on the potential human and ecological effects of RONS, ER stress, free radicals, and NPs.

Chapter 1

Pathophysiological, toxicological, and immunoregulatory roles of reactive oxygen and nitrogen species (RONS)

Abstract

Plants and animals produce reactive oxygen species (ROS) in response to infection. ROS and reactive nitrogen species (RNS) produced during tissue pathogenesis and in response to viral or chemical toxicants induce a complex series of downstream adaptive and reparative events driven by the associated oxidative and nitrative stress. As highlighted by all the studies, ROS and RNS can promote diverse biological responses associated with a spectrum of disorders, including neurodegenerative/neuropsychiatric and cardiovascular diseases. Similar pathways are implicated during the process of liver and skin carcinogenesis. Mechanistically, reactive oxygen and nitrogen species drive sustained cell proliferation, cell death, including both apoptosis and necrosis, formation of nuclear and mitochondrial DNA mutations, and in some cases stimulation of a proangiogenic environment. In this chapter, evidence is presented in diverse tissues, including the liver, brain, heart, and skin, to highlight common themes.

Also in this chapter it has been demonstrated that the ROS level was critical both in the pathogenesis of psoriatic dermatitis (PD) and in the regulation of regulatory T cell (Treg) function. It has been also demonstrated that tissue hyperoxygenation through hyperbaric oxygen therapy (HBOT) attenuated a murine model of PD. In conclusion, it has been proposed that HBOT might attenuate psoriasis through enhancing Treg function.

Keywords

Reactive oxygen species; Reactive nitrogen species; Inflammation; Carcinogenesis; Neurotoxicology; Hepatotoxicity; Immunoregulatory; Regulatory T cells (Tregs); Psoriatic dermatitis (PD); Hyperbaric oxygen therapy (HBOT)

Apoptosis is characterized by chromatin condensation, phosphatidylserine translocation, and caspase activation. Neuronal apoptotic death involves the participation of reactive oxygen species (ROS), which have also been implicated in necrotic cell death. In the role of different ROS in neuronal death, a superoxide anion was produced by incubating cells with xanthine and xanthine oxidase plus catalase, a singlet oxygen was generated with rose Bengal and luminic stimuli, and hydrogen peroxide was induced with glucose and glucose oxidase. Cultured cerebellar granule neurons died with the characteristics of apoptotic death in the presence of a superoxide anion or singlet oxygen. These two conditions induced caspase activation, nuclear condensation, phosphatidylserine translocation, and a decrease in intracellular calcium levels. On the other hand, hydrogen peroxide led to a necrosis-like cell death that did not induce caspase activation, phosphatidylserine translocation, or changes in calcium levels. Cell death produced by both singlet oxygen and superoxide anion, but not hydrogen peroxide, was partially reduced by an increase in intracellular calcium levels. These results suggest that formation of specific ROS can lead to different molecular cell death mechanisms (necrosis and apoptosis) and that ROS formed under different conditions could act as initiators or executioners of neuronal death.

In this chapter, we illustrate the pivotal role played by oxidative and nitrative stress in cell death, inflammation, and pain and its consequences for toxicology and disease pathogenesis. Examples are presented from five different perspectives ranging from in vitro model systems through to in vivo animal model systems and clinical outcomes.

1.1 Oxidative and nitrative stress in toxicology and disease

Inflammation and the role of ROS and reactive nitrogen species (RNS) are discussed initially followed by illustrative examples from diverse tissue and model systems. The concept behind the session was to seek cross-fertilization between the different illustrative examples, revealing common themes. Inflammation begins when tissues react to a local irritation usually caused by a physical injury, by infection, or by exposure to a toxicant. Fluid and accompanying white blood cells traverse the vascular barrier leading to swelling, erythema, further inflammation, and attraction of further white blood cells (Fig. 1.1). The resolution phase of inflammation involves repair and cell proliferation ultimately leading to tissue regeneration. During the inflammatory phase, there is a burst of respiration leading to the creation and release of free radicals, including production of both ROS and RNS. ROS and RNS utilize three main pathways of signaling, which result in damage to DNA, proteins, and lipids. Lipid peroxidation triggers the arachidonic acid cascade, with the production of cell proliferation-stimulating eicosanoids. While free radicals induce DNA damage directly, by-products of the arachidonic acid pathway such as malondialdehyde (MDA) and 4-hydroxynonenol (4-HNE) also are DNA-damaging agents. Free radicals can also modify enzyme systems involved in DNA repair and alter cell death signaling by modification of caspases and modulation of cell survival pathways regulated by key molecules such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) [1, 2].

Fig. 1.1 Inflammation and the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in tissue damage. Inflammation begins with a reaction to an irritant or infection that is characterized by movement of fluid and white blood cells into extravascular tissue. This is followed by tissue repair and regeneration and involves cell proliferation. Associated with these processes are the release of free radicals, such as ROS and RNS. This can activate a process called lipid peroxidation and the arachidonic acid cascade, with the production of cell proliferation-stimulating eicosanoids. Also, DNA-damaging agents, such as malondialdehyde (MDA) and 4-hydroxynonenol (4-HNE), are by-products of the arachidonic acid cascade. The free radicals can also damage DNA and modify the structure and function of cancer-related proteins. OH , superoxide; NO , nitric oxide; ONOO − , peroxynitrite; N 2 O 3 , nitrous anhydride.

Although inflammation and the associated generation of free radicals can have a deleterious effect on the host, these processes also play a normal physiological role, especially in the elimination of invading pathogenic organisms. Since free radicals are ubiquitous in our body and are generated during normal physiological processes, multiple defense systems have evolved to protect our cells from these potentially damaging free radicals. Antioxidant enzymes and scavenger systems serve to remove free radicals, whereas DNA repair pathways are triggered to repair damage to DNA after it has occurred. It is clear that in certain situations of toxicant exposure or disease, the generation of ROS and RNS and the associated oxidative and nitrative stress are key mediators of inflammation, cell proliferation, and cell death.

1.2 Oxidative and nitrative stress: Role in the response to liver toxicants (Roberts)

The liver is the front line of defense and is therefore a target organ for many toxicants. Persistent inflammation plays a pivotal role in the response of liver to both chemical and viral damage, ranging from transient liver injury and repair through to hepatocarcinogenesis (Fig. 1.2).

Fig. 1.2 Liver damage, inflammation, and cancer: lessons from PPARα. This schematic depicts the pathway from toxicant exposure to inflammation and ultimately to tumor development. Inflammation can be triggered by toxicant exposure or by viral infection; if this inflammation persists it can progress to the development of tumors. Similarly, exposure of rodent liver to peroxisome proliferators causes activation of PPARα and hepatic growth changes ultimately leading to hepatocellular carcinoma. Cytokines such as TNFα and survival signaling molecules such as NFκB are implicated in both processes.

There is a major drive to understand the mechanisms of toxicant and viral-induced liver disease to facilitate prevention and treatment and also to understand the molecular basis of individual susceptibility. WHO mortality data for the Americas [3] show liver cirrhosis is responsible for 4% of deaths in the 45–64 age group, making it the third most common cause of death. As illustrated by these figures, cirrhosis itself is a major health issue that is exacerbated by the association of chronic cirrhosis with quite frequent progression to hepatocellular adenoma and carcinoma.

There are multiple causes of cirrhosis and associated carcinogenesis, with the majority of cases attributed to alcohol, viral infection, or a fatty diet [4]. The statistics surrounding hepatitis B infection are shocking; approximately one-third of the world’s population (∼ 2 billion people) have been infected with hepatitis B, with 600,000 deaths each year due to the acute or chronic consequences of viral infection, and 25% of adults who become chronically infected during childhood later die from liver cancer or cirrhosis [3].

Accumulating evidence implicates the dedicated hepatic macrophage, the Kupffer cell, as a key mediator of the response to chemical and viral insult, as well as other liver stresses such as transplantation [5]. Examination of the liver architecture [6] shows that the location of Kupffer cells, which are distributed around the key interface between the parenchyma, the sinusoidal space, and the portal space, allow these specialized macrophages to respond to incoming toxicants by stimulation of parenchyma.

The primary mechanism for the stimulation of parenchyma is via release of cytokines [5, 7] and ROS [8, 9] and RNS [10]. The release of ROS by Kupffer cells was well illustrated by Lin et al. [11] who showed an increase in hepatic ROS during d-galactosamine-induced acute liver injury and apoptosis. Interestingly, staining for 4-HNE, which identified oxidized protein, revealed colocalization with lipid accumulation in these livers [11]. Similarly, Powell et al. [10] reported that a subtoxic dose of acetaminophen significantly increased nitrotyrosine adducts in rat liver in vivo, especially in the centrilobular region, as a marker for peroxynitrite formed by the combination of ROS, superoxide anion, and nitric oxide (NO). Thus Kupffer cells orchestrate the hepatic response to chemical and viral insult via production and release of many mediators, including ROS and RNS. So, how do these mediators affect both normal liver biology and, more importantly, the adverse response to toxicants?

Toxicants cause tissue damage via diverse mechanisms, many of which involve activation of cell survival and apoptosis pathways. Two broad categories of apoptosis networks, referred to as the intrinsic and extrinsic pathways, have been identified [1]. The extrinsic pathway is driven by signaling from death receptors such as the tumor necrosis factor alpha (TNFα) receptor and Fas through the death-inducing signaling complex (DISC) and caspase-8. The intrinsic pathway is triggered by cellular stress and signals through survival kinases to the mitochondria, which then engage caspase-9 through cytochrome-c release. Both pathways converge on caspase-8 leading to cellular execution [1]. Oxidative and nitrative species can affect these pathways at several key points; RNS and ROS can modulate survival-signaling molecules such as JNK and p38 MAPK [1, 2]. Similarly, RNS such as NO can induce DNA damage, leading to p53 stabilization and engagement of apoptosis pathways. Conversely, NO downregulates nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), which protects from apoptosis often via perturbation of hepatocyte proliferation and death via apoptosis and necrosis [6].

1.2.1 Carcinogenesis and inflammation

As described, Kupffer cells detect and respond to viral and chemical stress by release of ROS, RNS, and cytokines that in turn regulate cell survival and proliferation. So how would this perturbation of cell growth lead to carcinogenesis? These concepts are well described in the early publications on multistage carcinogenesis models [12, 13], as summarized briefly here. First, to proliferate, a cell has to survive; thus any cell that should have died due to DNA damage but persists due to perturbation of signaling remains to be stimulated into proliferation possibly with an enhanced risk of mutation due to missense-mismatch repair [14]. These cells are then subjected to further mitotic stimulation and genetic instability progressing through to cancer. Overall, this explanation leads to the interesting question: Is cancer linked to unresolved inflammation?

One of the most interesting papers to address this point looked at the immune pathogenesis of hepatocellular carcinoma (HCC) in response to hepatitis B virus (HBV) infection [15]. As mentioned earlier, HCC is a common complication of HBV infection and it has long been assumed that the clonal integration of viral DNA into hepatocytes would play a key role in the process. However, these authors showed that HCC occurred in the absence of viral transactivation and insertional mutagenesis. In contrast, the immune response to HBV was shown to be critical for tumor development. Overall, these data suggest that the HCC accompanying chronic hepatitis may result from the immune response to viral infection possibly mediated by Kupffer cells. In this respect, nongenotoxic and viral carcinogenesis may share common features.

1.2.2 Crosstalk with PPARα?

In addition to the regulation of hepatocyte survival and proliferation by ROS and RNS released by Kupffer cells, there is the possibility of complex interplay between ligand-activated receptors such as the peroxisome proliferator-activated receptors (PPARs), which appear to play different roles in Kupffer cells, versus the hepatocytes. PPARα is the receptor for the peroxisome proliferators (PPs), a diverse group of rodent nongenotoxic hepatocarcinogens of broad environmental and therapeutic significance [16]. In rodents, PPs induce hepatocyte proliferation and suppress apoptosis mediated by PPARα, ultimately leading to clonal expansion and tumors. Both in vitro and in vivo experiments have shown that the hepatic response to PPs is absolutely dependent upon Kupffer cells [17] but interestingly, Kupffer cells do not have to express PPARα to support these changes in proliferation. Indeed, in a separate series of experiments it was demonstrated that PPARα is absent in Kupffer cells that instead express PPARγ [18].

Overall, it is clear that oxidative and nitrative stress play a pivotal role in the response to liver toxicants and viral injury. This is often orchestrated by the complex interplay between the nonparenchymal Kupffer cells and hepatocytes with crosstalk between survival mechanisms and transcriptional regulation. Further development of our understanding of these mechanisms provides increased opportunities in the future for vital work on prevention, intervention, and treatment.

1.3 Characterization of oxidative stress using neuronal cell culture models (Smith)

The brain consumes more oxygen under physiological conditions than other organs, thereby increasing its susceptibility to oxidative stress since the generation of higher levels of ROS can lead to pathological changes when these are in excess of the buffering capacity of endogenous antioxidant systems [19]. Excessive stimulation of glutamate receptors, especially N-methyl-d-aspartate (NMDA) receptors, also contributes significantly to the production of ROS and/or NO. Glutamate toxicity is known to underlie many neurological disorders, including Alzheimer’s, Parkinson’s, and Huntington’s diseases and stroke [20]: with mitochondrial dysfunction triggering neuronal death pathways and subsequent neuronal loss in such conditions [21]. Fully elucidating the mechanisms involved remains pivotal in the development of strategies to ameliorate these neurodegenerative disorders [19].

Much data pertaining to oxidative and nitrative stress in the nervous system come from postmortem findings and experimental mammalian in vivo studies. In view of the complexities of the nervous system, however, mechanistic studies of oxidative stress have benefited also from using reductive in vitro culture models. Effects on neuronal and glial components can be investigated in both combined mixed cultures and using individual cell types in culture to monitor specific responses. The merits and limitations of different cell systems appropriate to neurotoxicological approaches have been reviewed [22].

Continuous cell lines are available for the elucidation of the death pathways activated by oxidative stress insults. The dopaminergic neuronal cell line, SH-SY5Y, derived from a human neuroblastoma, has been used in many studies since treatments can be applied both to proliferating undifferentiated cells and to cultures triggered to differentiate [23]. SH-SY5Y cells have also been used successfully in work demonstrating the protection afforded by the naturally occurring antioxidants, resveratrol and oxyresveratrol, against oxidative stress induced by the parkinsonian mimetic 6-hydroxydopamine [24].

Primary cultures, maintained either as neuronal and glial cell cocultures or as neuronal- or astrocyte-enriched cultures, have the advantage of expressing a phenotype more closely resembling the in vivo state. They can be routinely prepared from most brain areas in which oxidative stress is of interest, including cortical regions [25]. Postnatal cerebellar granule neurons (CGNs), maintained in culture for at least a week, undergo extensive regrowth of neurite processes and express functional glutamate receptors. As such, they remain one of the most frequently used model systems for in vitro investigation of excitotoxicity and oxidative stress. The cerebellum comprises over 95% of this neuronal type and therefore represents a source of highly purified and homogeneous neurons that are known to release glutamate as their principal neurotransmitter [26–28]. Extensive data on oxidative stress, signaling pathways, cell death, and neuroprotection have been generated in many studies using this culture model, including those illustrated in Fig. 1.3 and described next.

Fig. 1.3 A summary diagram of the oxidative stress insults applied to primary cultures of postnatal cerebellar granule neurons [26], the mechanistic and signaling pathways investigated, and the neuroprotective strategies employed to attenuate neuronal damage and loss (see text for details of the individual studies).

Fatokun et al. [29, 30] reported on the oxidative stress induced by hydrogen peroxide and glutamate. Means of attenuating the damage resulting from exposure of CGNs to the oxidative stressors were investigated by administrating the adenosine receptor A1 agonist N6-cyclopentyladenosine and the A2A antagonist ZM241385, 4-(2-[7-amino 2{furyl}{1,2,4}trichloro{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol, which proved effective in protecting CGNs. The death pathways triggered were a mixed profile of both apoptosis and necrosis [30]. The free radical-generating system of xanthine and xanthine oxidase, frequently used as a source of the superoxide anion to cause oxidative stress leading to cellular death, was investigated in CGN cultures [29]. Addition of superoxide dismutase failed to prevent damage by xanthine and xanthine oxidase, while catalase completely ameliorated it. When applied alone, xanthine oxidase significantly lowered cell viability, an effect that was blocked by allopurinol and catalase, but not by superoxide dismutase. These observations suggest that hydrogen peroxide, rather than the superoxide anion, mediates the main stress leading to cell death in this system [29].

The role of stimulation of NMDA receptors in the cell death induced by mitochondrial disruption following exposure to 3-nitroproprionic acid (3-NPA) and potassium cyanide (inhibitors of respiratory chain complexes II and IV, respectively) was demonstrated using primary cultured CGNs: the NMDA channel blocker MK-801 and the antagonist 2-amino-5-phosphopentanoic acid proving effective, although resistance to blockade by kynurenic acid suggested NMDA receptor activation independent of the glycine site [31]. Protection of cultured CGNs against subsequent lethal challenges of 3-NPA and glutamate toxicity was also achieved by preconditioning with sublethal doses of NMDA [27]. Less severe protocols, in which neurons were preconditioned by use of longer sublethal exposures with the potassium channel blocker 4-aminopyridine (4-AP) and the GABAA receptor antagonist bicuculline, have been shown to protect cortical neurons from oxidative stress by upregulating bcl-2 expression and by stimulating the phosphorylation of the cAMP response element binding protein (CREB) [25]. CREB phosphorylation increases in CGNs preconditioned with 4-AP, although activation of bcl-2 and caspase-3 levels did not differ significantly from untreated controls [32].

Investigation of the signaling pathways activated by oxidative stress has been the focus of studies of a number of neurotoxic intermediates generated by the metabolism of tryptophan to nicotinamide via the kynurenine pathway [33]. Neuronal loss, resulting from treatments with 3-hydroxykynurenine and 3- or 5-hydroxyanthranilic acids, appeared to be mediated by H2O2 since the effects were prevented by catalase, but not by superoxide dismutase. Activation of the p38 pathway was demonstrated, whereas caspase-3 levels were not increased, indicating the involvement of a caspase-3-independent mechanism [33]. Neuronal culture also permits the quantification of neurite outgrowth and its modification by oxidative stressors, which offers an additional endpoint for assessing damage in in vitro studies [22].

Despite the acknowledged limitations implicit in the simplicity of neuronal culture models, primary cells such as CGNs have proved most valuable in generating data that extends our understanding of the mechanisms underlying the response of the nervous system to oxidative stress. When taken together with in vivo studies, it is clear that in vitro culture systems can play a vital role in the development of novel drug therapies targeting pathways associated with oxidative stress, with potential relevance to the management of a number of neurodegenerative conditions.

1.4 Nitrative stress and glial-neuronal interactions in the pathogenesis of Parkinson’s disease (Tjalkens and Stephen Safe)

Parkinson’s disease (PD) is a severely debilitating movement disorder resulting from progressive degeneration of dopaminergic neurons within the substantia nigra pars compacta of the midbrain.

Unfortunately, pharmacologic treatment for PD has not progressed beyond the use of dopamine mimetics, such as l-dopa, that only transiently alleviate motor symptoms. Furthermore, chronic use of l-dopa is associated with its own array of resultant pathologies such as dyskinesia, cardiac arrhythmia, ischemic injury, and cerebral vascular dysfunction. Ultimately, individuals suffering from PD will progress to the end stage of the disease, which is characterized by significant gait abnormalities and frequent falls, as well as a deficit in nonmotor functions resulting in dementia, psychosis, and other autonomic disturbances [34].

Currently, a precise etiology explaining PD remains to be discovered but recent research has revealed features of the disease that represent realistic targets for neuroprotective chemotherapeutic intervention that could mitigate the progressive loss of dopaminergic neurons [35]. Among these observations are the presence of chronic inflammation and sustained expression of inducible nitric oxide (NOS2), accompanied by activation of the surrounding astrocytes and microglia [36].

1.4.1 Neuroinflammation and PD

Neuroinflammation is now understood to play a critical role in the progression of PD. Unfortunately, current therapies do not address this problem, being focused on ameliorating the symptoms of dopamine loss rather than on targeting the underlying causes of injury to dopaminergic neurons. Thus there remains no disease modifying therapeutic that has been approved for the treatment of PD. Although much attention has been devoted to understanding the role that activated microglia have in neuroinflammation, it has become clear that a more complex interplay between microglia and astrocytes underlies the neuroinflammatory phenotype observed in PD [37]. Both microglia and astrocytes produce neurotoxic levels of inflammatory mediators such as TNFα and NO that are increased in PD patients and in animal models of the disease [38]. However, recent clinical trials aimed at suppressing microglial-mediated inflammation have, to date, yielded poor results. Thus a better understanding of fundamental inflammatory pathways and cell-cell interactions between microglia and astrocytes is required to decipher the pathogenesis of neuroinflammation in degenerative central nervous system (CNS) disorders such as PD. Although the proinflammatory role played by microglia in PD has been extensively studied, the neuroinflammatory phenotype of activated astrocytes is less well understood.

Astrocytes have diverse and critical functions in the CNS that include providing metabolic substrates to neurons, antioxidant protection, and the synthesis of trophic factors essential for the survival and function of neurons [39, 40]. However, many neurological disease states, including PD, Alzheimer’s disease, and ischemic injury, are typically accompanied by varying degrees of astrocyte activation, or astrogliosis. While the exact cause of astrogliosis in PD is unknown, several reports have suggested that the activation of astrocytes is due to secretion of inflammatory cytokines, such as TNFα and interferon gamma, by the surrounding microglial cells [41]. While some degree of activation is likely, beneficial, reactive astrogliosis results in neuronal injury. Astrogliosis results in increased production of various neurotoxic inflammatory mediators, including NO, that contribute to the progressive loss of nigrostriatal neurons. Supporting a deleterious role for excessive NO production in PD are postmortem observations of increased NOS2 expression in patients diagnosed with PD [42], as well as reports that deletion of the Nos2 gene in mice confers protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated neurotoxicity [41].

1.4.2 Regulation of neuroinflammatory genes in astrocytes

Expression of NOS2 in response to diverse neurotoxicants is highly dependent upon the NF-κB signaling pathway. Recent studies demonstrated that NF-κB is required for expression of NOS2 in activated astrocytes following stimulation with various inflammatory cytokines and neurotoxicants [43, 44]. Multiple signaling pathways activate NF-κB through the IκB kinase complex, leading to phosphorylation and degradation of the inhibitory IκB subunit and nuclear translocation of the transcriptionally active p65 subunit. Ensuing induction of Nos2 mRNA then typically requires binding of p65 to enhancer sequences on the Nos2 promoter, acetylation of histones, and removal of constitutively bound nuclear corepressor proteins such as NCoR2 by the nuclear proteasome [45]. Increasing attention is being given to NF-κB as a therapeutic target for blocking neuroinflammation in PD because of its central role in inflammatory signaling in glia. Recent data from transgenic animals containing microglial- and astrocyte-specific gene deletion of NF-κB support the neuroprotective efficacy of blocking this signaling pathway in models of inflammatory neurodegeneration [46]. The importance of NF-κB-dependent transcriptional regulation of inflammatory genes in PD was highlighted in studies demonstrating that the orphan nuclear receptor, Nurr1, normally inhibits NF-κB-regulated neuroinflammatory genes in glia and its deficiency associates with loss of dopaminergic neurons [47]. Deficiency in the expression of Nurr1 is also associated with a late-onset form of PD [48], suggesting that nuclear regulators of NF-κB are linked to inflammatory activation of glia and loss of dopaminergic neurons.

1.4.3 Therapeutic strategies to interdict neuroinflammation

Suppressing neuroinflammation has emerged as a potential strategy for treating disorders such as PD. Specifically, modulation of nuclear orphan receptors has been examined as a possible approach for suppressing inflammatory gene expression in astrocytes using traditional thiazolidinedione (TZD) ligands of PPARγ [49]. The TZD ligand rosiglitazone appears to antagonize NF-κB by stabilizing NCoR2 at the proximal p65 enhancer element in RAW macrophages [50], and another drug in this series, pioglitazone, confers partial neuroprotection in MPTP-induced parkinsonism, preserving dopaminergic cell bodies in the substantia nigra but not dopaminergic fibers in the striatum [49]. The work presented here (Fig. 1.4) demonstrates that a novel para-substituted diindolylmethane compound, 1,1-bis(3′-indolyl)-1-(p-t-butyl)methane (DIM-C-pPhtBu), suppresses protein nitration in dopaminergic neurons in vivo caused by exposure to the neurotoxin MPTP. It was also recently reported that DIM-C-pPhtBu blocked NF-κB-dependent expression of NOS2 in astrocytes and protected cocultured neurons from astrocyte-mediated apoptosis following treatment with MPTP and inflammatory cytokines [43]. Furthermore, the mechanism underlying DIM-C-pPhtBu-mediated inflammatory suppression differs from the transrepressive mechanism described for rosiglitazone [50], presenting a potentially novel avenue for suppression of inflammatory genes and neuroprotection using this class of compounds. The antineuroinflammatory efficacy of DIM-C-pPhtBu was further examined in astrocytes derived from a unique transgenic mouse expressing a reporter construct consisting of high-affinity NF-κB consensus elements driving expression of enhanced green fluorescent protein. DIM-C-pPhtBu potently suppressed MPTP-dependent NF-κB activation in transgenic astrocytes isolated from these transgenic mice that directly correlated with inhibition of NOS2 expression [43]. Thus inhibition of key molecular signaling pathways responsible for the induction of NOS2, such as NF-κB, may offer a strategy for preventing neuronal injury resulting from excessive production of NO.

Fig. 1.4 Inhibition of NF-κB prevents protein nitration in dopaminergic neurons in vivo. C57Bl/6 mice were treated with MPTP (4 × 10 mg/kg, 2 h intervals) in the presence or absence of DIM-C-pPhtBu (DIM-C) (four daily doses by intragastric gavage, 50 mg/kg) and sacrificed after 7 days. Frozen sections were stained by immunofluorescence for tyrosine hydroxylase (green) , 3-nitrotyrosine ( red ; an indicator of peroxynitrite formation), and nuclear morphology (DAPI; blue ). (A–D) Control; (E–H) MPTP; (I–L) MPTP + DIM-C-pPhtBu. Yellow staining the merged images in D, H, and L indicates colocalization.

1.5 Oxidative and nitrative stress in multistage carcinogenesis (Robertson)

The murine model of multistage skin carcinogenesis has been used to define the sequential genetic κ and epigenetic mechanisms responsible for the development of human squamous cell carcinomas [51]. This classic model of skin inflammation and carcinogenesis has also been used to identify chemicals that have potential carcinogenic activity and is a primary screening tool to evaluate the toxicological profile of drugs under development. The process of murine multistage carcinogenesis is operationally defined as consisting of discreet steps of initiation, promotion, and malignant progression. Initiation is the first step in inducing growth of skin tumors and occurs following treatment of the dorsal skin of genetically susceptible mice with a chemical or physical carcinogen such as 7,12-dimethylbenz[a]anthracene (DMBA) or exposure to ultraviolet (UV) light in the 290–320 nm range, which is UVB light derived from solar irradiation. Exposure to DMBA induces stable, permanent, and heritable genetic alterations that consist of specific point mutations within the cellular v-Ha-ras gene, while exposure to UVB light induces signature mutations in the p53 tumor suppressor gene. These mutations occur in label retaining epidermal keratinocytes located within both the basal epidermis and in the bulge region of the hair follicles of the dermis, which are now recognized to represent the stem cell compartments of adult skin [52].

The second stage of skin carcinogenesis is tumor promotion, which is induced by multiple applications of the tumor promoter 12-O-tetradecanolyphorbol-13-acetate or by multiple exposures to UVB light. While initiation is characterized by genetic alterations, there are multiple epigenetic changes that are critical to the process of tumor promotion. As an example, tumor promoters stimulate inflammation characterized by the production of reactive intermediates and proinflammatory mediators with contributions by multiple cell types, including epidermal keratinocytes, leukocytes, and vascular endothelial cells (Fig. 1.5). Multiple types of proinflammatory cytokines, including interleukin-1 alpha, TNF, and granulocyte-macrophage stimulating factor, are produced during tumor promotion [53–56]. To add to the proinflammatory microenvironment in the skin during growth of papillomas and conversion to malignant carcinomas, the process of tumor promotion is associated with the production of ROS, including the cell permeant mediator hydrogen peroxide [57–59]. ROS are produced in sufficient quantities during tumor promotion to result in the formation of signatures of oxidant-mediated DNA damage, detectable by the presence of the sentinel DNA adduct 8-oxo-deoxyguanosine.

Fig. 1.5 Schematic of the roles of reactive oxygen species (ROS), reactive nitrogen species (RNS) oxidative DNA damage, proinflammatory cytokines, and vascular endothelial growth factor (VEGF) in multistage skin carcinogenesis.

In addition to production of ROS, NO and peroxynitrite (ONOO−) play a role in the process of tumor promotion in the skin [54, 55], and are collectively defined as RNS. Peroxynitrite, formed by the interaction of ROS with NO and detectable by the presence of nitrotyrosine, was produced by infiltrating leukocytes within the dermis during tumor promotion [54, 55]. In addition, there was a notable absence of NOS2 in hyperplastic epidermis, consistent with the known antiproliferative effect of NO.

In contrast to the localization of NOS2 to infiltrating leukocytes in the dermis, NOS, also known as endothelial NOS, was localized specifically to the vascular endothelium during tumor promotion. Additionally, the NOS inhibitor L-NG-monomethyl-arginine was found to block vascular permeability, suggesting that targeting NOS3 effectively inhibits the early changes in vessel permeability that are characteristic of the angiogenic switch during skin carcinogenesis. Data generated showed that vascular endothelial growth factor (VEGF) regulates vascular permeability at early times during tumor promotion and at later times in this process mediates the robust tumor-associated angiogenesis that is a requirement for formation of skin papillomas and their malignant progression to form carcinomas [60].

Taken together, these studies suggest that the proinflammatory signatures of tumor promotion are regulated in part by proinflammatory cytokines, as well as by ROS and RNS. Furthermore, NO and VEGF are interlinked and are an integral part of the process of multistage skin carcinogenesis (Fig. 1.5).

1.6 Role of peroxynitrite in the pathogenesis of doxorubicin-induced cardiotoxicity (Szabo)

Doxorubicin is a broad-spectrum antitumor anthracycline antibiotic that is commonly used to treat a variety of cancers, including severe leukemias, lymphomas, and solid tumors. It continues to be an effective and widely used broad-spectrum chemotherapeutic agent. However, its clinical use is limited because of its serious dose-limiting cardiotoxicity [61–64]. Clinical and experimental investigations suggested that increased oxidative stress associated with an impaired antioxidant defense status plays a critical role in subcellular remodeling, calcium-handling abnormalities, and alteration of cardiac energetics and subsequent cardiomyopathy and heart failure associated with doxorubicin treatment [65–70].

Increased expression of NOS2 and formation of nitrotyrosine, a marker of RNS, have been shown to be present in cardiomyocytes after doxorubicin exposure [69–71]. The causative role of peroxynitrite in the cardiotoxic effects of doxorubicin is evidenced by the protective effects of peroxynitrite-neutralizing agents in experimental models of doxorubicin cardiotoxicity. For instance, studies with FP15, an N-PEGylated-2-pyridyl iron porphyrin, a potent peroxynitrite decomposition catalyst, demonstrated that FP15 exerts cardioprotective effects in various rodent models of doxorubicin cardiotoxicity [71]. FP15 attenuated the development of cardiac dysfunction, increased survival, and reduced the doxorubicin-induced increase in serum lactate dehydrogenase and creatine phosphokinase activities, consistent with protection against peroxynitrite-mediated necrosis of cardiac myocytes. FP15 also abolished tyrosine nitration in the hearts of doxorubicin-treated animals [71]. Although tyrosine nitration can be induced by alternative pathways, not just by the reaction of peroxynitrite with protein tyrosine residues [72], the increase in nitrotyrosine in myocytes of doxorubicin-treated mice, and its abolishment by FP15, suggests that a causative link exists between oxidative and nitrosative stress and cardiotoxicity of doxorubicin.

FP15 also prevented doxorubicin-induced cardiac lipid peroxidation and activation of matrix metalloproteases (MMPs). MMPs are significant contributors to the development of various pathophysiological conditions, including dilated cardiomyopathy and congestive heart failure. The activation of proMMPs is triggered by peroxynitrite generation, via an extensive S-glutathiolation reaction [73]. By inhibiting this reaction, peroxynitrite decomposition catalysts may reduce MMP activation. In addition to direct oxidation, peroxidation and nitration reactions, and MMP activation, likely additional downstream, cytotoxic mechanisms elicited by peroxynitrite during doxorubicin-induced cardiac injury include DNA damage and activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), as well as the inhibition of myofibrillar creatine kinase [69–71]. Consistent with these findings, direct, pharmacological inhibition of PARP activation, as well as genetic deletion of the PARP gene, has been shown to be effective in protecting against doxorubicin-induced cardiotoxicity in rodent models [71, 74]. Peroxynitrite, via activation of PARP, can also induce myocardial calcium overload and contractile dysfunction in doxorubicin-treated hearts [75]. Although peroxynitrite can also induce the activation of myocardial MMPs, this effect is independent of PARP activation [76, 77].

1.6.1 Molecular mechanisms of peroxynitrite formation

A recent series of in vitro and in vivo studies have evaluated in detail the molecular mechanisms of peroxynitrite formation and cardiotoxicity in response to doxorubicin exposure, and tested the effect of various peroxynitrite-neutralizing molecules [77]. Using a well-established mouse model of doxorubicin-induced heart failure, marked increases in myocardial apoptosis (caspase-3 and caspase-9 expression/activity, cytochrome-c release, transferase-mediated dUTP nick end labeling, etc.), NOS2 gene expression, generation of NO and mitochondrial superoxide generation, nitrotyrosine formation, MMP2 and MMP9 gene expression, and PARP activation were demonstrated, without major changes in expression of the multiple other genes linked to ROS and RNS production, including NOX1, NOX2, p22phox, p40phox, p47phox, p67phox, xanthine oxidase, NOS1 and NOS3 genes. These changes were associated with a decrease in myocardial contractility after doxorubicin treatment. Pretreatment of mice with two different peroxynitrite decomposition catalysts (FeTMPyP and MnTMPyP) markedly attenuated doxorubicin-induced myocardial apoptosis and dysfunction [77]. Doxorubicin also increased mitochondrial superoxide and nitrotyrosine generation in a dose-dependent manner, and apoptosis/necrosis in cardiac-derived H9c2 and/or human coronary artery endothelial cells as measured by flow cytometry and fluorescence microscopy. The doxorubicin- and/or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular nitrotyrosine formation, and was abolished by FeTMPyP/MnTMPyP. Likewise, the doxorubicin-induced cell death was also attenuated by selective NOS2 inhibitors. Importantly, coadministration of various NO donors and doxorubicin, but not NO donors alone, dramatically enhanced doxorubicin-induced cell death with concomitant increased nitrotyrosine formation and decreased mitochondrial superoxide generation [77]. These studies lend additional support to the hypothesis that peroxynitrite, and not NO or superoxide, is the major trigger of doxorubicin-induced cell death (Fig. 1.6). It is hoped that future experimental therapies will be designed around neutralization of peroxynitrite, or around modulation of the pathways leading to peroxynitrite generation.

Fig. 1.6 Schematic diagram of doxorubicin-induced cardiotoxicity: role of superoxide, NO, and peroxynitrite. Doxorubicin initially increases mitochondrial superoxide and, consequently, the generation of other ROS (e.g., H 2 O 2 ) in cardiomyocytes and/or endothelial cells by redox cycling. Increased doxorubicin-induced ROS generation in cardiomyocytes triggers the activation of the transcription factor NF-κB, leading to enhanced NOS2 expression and NO generation. NO reacts with superoxide to form peroxynitrite both in the cytosol and mitochondria, which, in turn, induces cell damage via lipid peroxidation, inactivation of enzymes and other proteins by oxidation and nitration, and activation of stress-signaling pathways (e.g., MAPK), MMPs, and PARP-1, among others. In the mitochondria, peroxynitrite, in concert with other ROS/RNS, impairs various key mitochondrial enzymes, leading to more sustained intracellular ROS generation (persistent even after doxorubicin is already metabolized), triggering further activation of transcription factor(s) and NOS2 expression, resulting in the amplification of oxidative/nitrosative stress. In the mitochondria, peroxynitrite also triggers the release of proapoptotic factors (e.g., cytochrome- c and apoptosis-inducing factor) mediating caspase-dependent and -independent cell death pathways, which are also pivotal in doxorubicin-induced cardiotoxicity. Peroxynitrite, in concert with other oxidants, also causes strand breaks in DNA, activating the nuclear enzyme PARP-1. Once excessive oxidative and nitrosative stress-induced DNA damage occurs, overactivated PARP initiates an energy-consuming cycle by transferring ADP-ribose units from NAD + to nuclear proteins, resulting in the rapid depletion of intracellular NAD + and ATP pools, slowing the rate of glycolysis and mitochondrial respiration, eventually leading to cellular dysfunction and death, mostly by necrosis. Overactivated PARP may also facilitate the expression of a variety of inflammatory genes leading to increased inflammation (PARP-1 is a known coactivator of NF-κB) and associated oxidative stress, thus facilitating the progression of cardiovascular dysfunction and heart failure. PARG , poly(ADP-ribose) glycohydrolase. (Reproduced with permission from P. Mukhopadhyay, M. Rajesh, S. Bátkai, Y. Kashiwaya, G. Haskó, L. Liaudet, C. Szabó, P. Pacher, Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro, Am. J. Physiol. Heart Circ. Physiol. 296 (2009) H1466–H1483.)

1.7 Immunoregulatory role of ROS

It has been demonstrated that imiquimod-induced psoriatic dermatitis (PD) was attenuated in elevated levels of ROS, whereas it was aggravated in lowered levels of ROS [78]. This observation provides experimental evidence supporting the immunoregulatory role of ROS that is contradictory to the traditional concept [78]. Traditionally, ROS is implicated in the progression of inflammatory diseases by promoting cellular damage and tissue destruction as well as aging. At the moment, it is necessary to establish a new conceptual framework where the recent observations and traditional concept can be compromised.

Psoriasis is a chronic inflammatory skin disease resulting from immune dysregulation. Tregs are important in the prevention of psoriasis. Traditionally, ROS are known to be implicated in the progression of inflammatory diseases, including psoriasis, but many recent studies suggest the protective role of ROS in immune-mediated diseases. In particular, severe cases of psoriasis vulgaris have been reported to be successfully treated by HBOT, which raises the tissue level of ROS. Also, it was reported that Treg function was closely associated with ROS level. However, it has only been investigated in lowered levels of ROS so far. Thus, in this study, to clarify the relationship between ROS level and Treg function, as well as their role in the pathogenesis of psoriasis, imiquimod-induced PD in association with Treg function has been investigated both in elevated and lowered levels of ROS by using knockout mice, such as glutathione peroxidase-1−/− and neutrophil cytosolic factor-1−/− mice, as well as by using HBOT or chemicals, such as 2,3-dimethoxy-1,4-naphthoquinone and N-acetylcysteine. The studies consistently showed Tregs were hyperfunctional in elevated levels of ROS, whereas they were hypofunctional in lowered levels of ROS. In addition, imiquimod-induced PD was attenuated in elevated levels of ROS, whereas it was aggravated in lowered levels of ROS. For the molecular mechanism that may link ROS level and Treg function, the expression of an immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO), which is induced by ROS, in PD lesions has been investigated. Taken together, it was implied that appropriate elevated levels of ROS might prevent psoriasis through enhancing IDO expression and Treg function.

Psoriasis is a chronic, remitting, and relapsing inflammatory skin disease that is often associated with systemic manifestation, especially arthritis [79]. Currently, psoriasis is managed by topical treatment with steroids, immunosuppressants, and several other agents. Psoralen and ultraviolet A photochemotherapy and other systemic therapies, such as using methotrexate, cyclosporine, oral retinoids, and biological therapies, are also in practice. However, many psoriasis patients are still suffering from frequent relapse, adverse drug effects, and other untoward reactions such as the development of nonmelanoma skin cancer [80]. Thus it is still desirable to develop another therapeutic strategy that is more effective and does not induce side effects.

Psoriasis is known to develop as a result of immune dysregulation, in particular hyperfunction of T-helper 17 (Th17) cells [81, 82]. In the steady state, immune homeostasis is maintained by Tregs that suppress immune effectors, including Th17 cells. It was also reported that psoriasis is associated with impaired suppressive function of Tregs [83, 84]. Therefore to restore the dysregulated immune status in psoriasis, it is necessary to suppress immune effectors, including Th17 cells, and/or to enhance Tregs.

Traditionally, ROS are known to be implicated in the progression of many inflammatory diseases [85–87]. As ROS are highly reactive and interact with many biomolecules, they are likely to destroy biological structures, promoting cellular damage and tissue destruction. In contrast, recent evidence is accumulating on the protective role of ROS in immune-mediated diseases. Autoimmune arthritis was aggravated in rodents with lower levels of ROS than wild-type (WT) mice due to defects in the ROS-producing enzyme system, such as mutation in the neutrophil cytosolic factor (Ncf)-1 or NADPH oxidase-(NOX)2 [88–90]. In humans, too, many autoimmune diseases develop more frequently in chronic granulomatous disease patients with lower levels of ROS than normal persons due to a defect in ROS-producing NOX [91]. On the contrary, experimentally induced asthmatic inflammation was attenuated in mice with higher levels of ROS than WT mice due to the defect of a ROS-metabolizing enzyme, glutathione peroxidase-1 (GPx-1) [92]. Atherosclerotic lesions induced by a high-fat diet were also decreased in GPx-1−/− mice [93]. In addition, experimental colitis was attenuated in mice with a higher level of ROS due to a defect in a nonenzymatic antioxidant, peroxiredoxin II [94].

These clinical and experimental observations implicated the immunoregulatory role of ROS [95]. In particular, Treg function seems to be closely linked to ROS level. Tregs isolated from mice with a lower level of ROS (Ncf1−/−) were hypofunctional, compared to WT Tregs [96]. Tregs were also hypofunctional in lowered levels of ROS prepared in vitro by the addition of antioxidants or NOX inhibitors [96]. Consequently, Treg function seems to be closely associated with ROS level, but so far it has only been investigated in lowered levels of ROS. Therefore the suppressive function of Tregs isolated from mice with a higher level (GPx-1−/−), as well as from mice with a lower level (Ncf1−/−), of ROS than WT mice has been investigated to clarify the relationship between ROS level and Treg function. Treg function in elevated and lowered levels of ROS prepared in vitro by using chemicals that increase or decrease ROS has also been investigated. In addition, the susceptibility of GPx-1−/− and Ncf1−/− mice to an experimentally induced PD has also been investigated. Next, for the purpose of immunomodulation, the susceptibility of WT mice with elevated and lowered levels of ROS prepared by tissue hyperoxygenation or administration of an antioxidant to the murine models of PD has been investigated as well.

HBOT is defined as breathing pure (100%) oxygen at increased atmospheric pressure. HBOT is a standard therapy for decompression sickness, gas embolism, and CO poisoning [97, 98]. HBOT is also effective for gas gangrene, anaerobic infection, diabetic foot, Buerger’s disease, and other oxygen-deficient conditions [99, 100]. In addition, HBOT has been proved effective in the healing of chronic wounds, such as radiation-induced soft tissue necrosis [101–104]. Meanwhile, many studies have reported the therapeutic or preventive effect of HBOT in various kinds of inflammatory or immune-mediated diseases, such as systemic lupus erythematosus [105, 106], atherosclerosis [107], collagen-induced arthritis [108], Crohn’s disease [109], ulcerative colitis [110, 111], and atopic dermatitis [112], although these diseases are not included in the current indication of HBOT. In particular, Butler et al. [113] reported successful treatment of two severe cases of psoriasis vulgaris by HBOT [113]. Inflammatory and antiinflammatory cytokines have been investigated in several studies [107], but the therapeutic mechanism of HBOT in immune-mediated diseases still remains enigmatic. Faleo et al. [114] reported that HBOT prevented autoimmune diabetes in nonobese diabetic (NOD) mice [114]. They showed for the first time that the frequency of FoxP3+ Tregs was increased in the spleen and pancreatic islets of the mice treated with HBOT. In consequence, it was suggested that Tregs may play an important role in the immunoregulatory mechanism of HBOT. It is well known that HBOT increases cellular levels of ROS according to tissue hyperoxia [115, 116]. Therefore it can be hypothesized that HBOT may modulate immune-mediated diseases by enhancing Treg function.

For the molecular mechanism of immunomodulation by ROS, tissue expression of IDO has been investigated. IDO is an immunoregulatory enzyme that can be induced by ROS [117–119]. Moreover, the enzyme activity of IDO can be enhanced in high levels of ROS, as the superoxide radical acts as a cofactor of IDO [120]. IDO catabolizes the essential amino acid tryptophan into the stable metabolite kynurenine [110]. Consequently, IDO depletes tryptophan from the environment, thus starving effector cells, and results in immune suppression. It was also found that tryptophan depletion resulted in inhibition of Th17 cell differentiation and expansion of Foxp3+ Tregs [118, 121, 122]. Therefore IDO might be a molecular cue that links ROS and Tregs, participating in the immunoregulatory mechanism of ROS. The results of the studies [78] consistently showed that Tregs were hyperfunctional and PD was attenuated in elevated levels of ROS, whereas Tregs were hypofunctional and PD was aggravated in lowered levels of ROS. In addition, it was implied that induction of IDO expression by ROS might contribute to the preparation of an immunosuppressive environment that prevents inflammatory reaction in the lesions of PD.

Another molecular mechanism investigated in the studies for the defense against ROS-induced tissue damage is IDO expression. IDO is primarily expressed in antigen-presenting cells, such as dendritic cells and macrophages. As an oxygenase, IDO can be induced by ROS [117–119]. In the study, IDO expression was only rarely expressed in the control skins, but substantially enhanced by HBOT, suggesting that ROS level was increased (Fig. 1.7). In elevated levels of ROS, the enzyme activity of IDO should be enhanced as the superoxide radical acts as a cofactor of IDO [120]. Therefore enhanced expression and activity of IDO from the beginning through hyperoxygenation by HBOT might contribute to the preparation of an immunosuppressive environment preventing inflammatory damage in PD [123, 124]. On the other hand, the IDO pathway is induced in many tissues during inflammation because IDO gene expression is induced by interferons [118, 119, 125]. In the present study, IDO expression was remarkably enhanced in the lesions of PD, compared with the control skins (Fig. 1.7). Accordingly, IDO expression should be enhanced during or after the inflammatory reactions in the lesions of PD, and might contribute to feedback regulation. In contrast, in the groups treated with HBOT, IDO expression was not significantly different before and after the development of PD, suggesting that IDO expression was not increased as a consequence of inflammatory reactions. Instead, early expression of IDO from the beginning might contribute to the prevention of PD. On the other hand, in the groups treated with N-acetyl cysteine (NAC), IDO expression was enhanced after the development of PD, but at a far lower level than that not treated. Thus it was suggested that treatment with NAC inhibited the expression of IDO in the lesions of PD although inflammatory reaction was developed. Consequently, NAC might inhibit feedback regulation of the inflammatory reaction by IDO, thus aggravating the inflammatory tissue damage.

Fig. 1.7 Treatment with HBOT enhanced, whereas administration with NAC reduced, IDO expression. (A) In the skins of the control mice treated with vehicle, IDO was very rarely expressed in the dermal layers, but treatment with HBOT increased IDO expression. On the other hand, no noticeable change was observed in the skins of the mice administered with NAC. In the lesions of imiquimod-induced PD, IDO expression was remarkably increased mainly in the dermal layer, but was also observed in the epidermal layer. Meanwhile, in the lesions of the mice treated with HBOT, IDO expression was not enhanced, compared with the control skins, and was substantially weaker compared with the PD lesions in the mice not treated. Administration with NAC also reduced IDO expression. Scale bar is 100 mm. (B) Image analysis showed a significant increase in the IDO-expressing area in the skins of the control mice treated with HBOT. Meanwhile, in the lesions of PD, IDO-expressing area was decreased by treatment with HBOT or NAC. Data are mean ± SD ( n  = 12). * P  = .05. (Based on Nonaudio: https://doi.org/10.1371/journal.pone.0091146.g006.)

Based on the observations made in the present study, we propose that tissue hyperoxygenation by HBOT might be an alternative therapeutic strategy for psoriasis. The current protocol of HBOT within 2 h at a maximum of 3 atm induces only reversible biochemical changes [126–128]. HBOT is quite safe, as major complications are very rare without sequelae, and there is no noticeable complication such as telangiectasia or skin cancer [129–131]. However, for practical application, several important points must be considered. First, at the moment we