Accepted Manuscript

Title: Oxidative stress in anxiety and comorbid disorders Authors: Iiris Hovatta, Juuso Juhila, Jonas Donner PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: S0168-0102(10)02779-3 doi:10.1016/j.neures.2010.08.007 NSR 3191 Neuroscience Research 15-7-2010 20-8-2010 23-8-2010

Please cite this article as: Hovatta, I., Juhila, J., Donner, J., Oxidative stress in anxiety and comorbid disorders, Neuroscience Research (2010), doi:10.1016/j.neures.2010.08.007 This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Manuscript

Review article

Oxidative stress in anxiety and comorbid disorders

Iiris Hovatta a, b, c, Juuso Juhila a, b, and Jonas Donner a, b, c

Department of Mental Health and Substance Abuse Services, National Institute for Health and Welfare, PO box 30, FIN-00271 Helsinki, Finland

Corresponding author: Iiris Hovatta, PhD

Research Program of Molecular Neurology Biomedicum PO box 63

FIN-00014 University of Helsinki Finland

fax +358-9-471-71964

email iiris.hovatta@helsinki.fi

Number of pages Number of figures Number of tables

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Department of Medical Genetics, Haartman Institute, Biomedicum PO box 63, FIN-00014 University of Helsinki, Finland

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Research Program of Molecular Neurology, Faculty of Medicine, Biomedicum PO box 63, FIN-00014 University of Helsinki, Finland (emails: iiris.hovatta@helsinki.fi, juuso.juhila@helsinki.fi, jonas.donner@helsinki.fi)

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ABSTRACT Anxiety disorders, depression, and alcohol use disorder are common neuropsychiatric diseases that often occur together. Oxidative stress has been suggested to contribute to their etiology. Oxidative stress is a consequence of either increased generation of reactive oxygen species or impaired enzymatic or nonenzymatic defense against it. When excessive it leads to damage of all major classes of macromolecules,

detrimental to the proper functioning of the brain include mitochondrial dysfunction, altered neuronal signaling, and inhibition of neurogenesis. Each of these can further contribute to increased oxidative stress, leading to additional burden to the brain. In this review, we will provide an overview of recent work on oxidative stress markers in human patients with anxiety, depressive, or alcohol use disorders, and in relevant animal models. In addition, putative oxidative stress-related mechanisms important for neuropsychiatric diseases are discussed. Despite the considerable interest this field has obtained, the

largely unknown. Since this pathway may be amenable to pharmacological intervention, further studies are

KEYWORDS: anxiety-like behavior, depression, alcohol use disorder, brain, antioxidant

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warranted.

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detailed mechanisms that link oxidative stress to the pathogenesis of neuropsychiatric diseases remain

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and therefore affects several fundamentally important cellular functions. Consequences that are especially

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1. Introduction Oxidative phosphorylation, which takes place in mitochondria of the cell, is the major source of ATP in aerobic organisms. The downside of this important process is that as a byproduct, it may produce free radicals, such as some reactive oxygen species (ROS) and reactive nitrogen species (RNS). They have both beneficial and harmful roles in the cell. At low or moderate concentrations, they take part in normal physiological processes such as cellular response to injury or infection, signaling, and mitosis (Valko et al.,

ROS/RNS, cells exhibit harmful conditions of oxidative and nitrosative stress. On one hand, oxidative stress arises when generation of ROS/RNS is increased and exceeds the cellular detoxification and damage repair capacity. On the other hand, oxidative stress results from impaired oxidative defense mechanisms, such as depletion of enzymatic (e.g., superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase

1). Either way, the consequence of oxidative stress is increased damage to all major groups of cellular

necrosis (Aksenova et al., 2005, Berk et al., 2008, Filomeni and Ciriolo, 2006). A role for oxidative stress-related damage is well established in several pathological conditions, such as cardiovascular disease, ischemia/reperfusion, cancer, diabetes, neurological and psychiatric disorders, and in normal aging (Halliwell, 2006, Ng et al., 2008, Valko et al., 2007). Possible involvement of oxidative stress

sensitive to oxidative damage for a number of reasons (Halliwell, 2006). The brain consumes large amounts of oxygen and therefore produces a comparatively large amount of free radical by-products, and it has relatively modest antioxidant defenses. In addition, the brain is rich in lipid substrates for oxidation, and iron and copper ions that catalyze free radical reactions are abundant. Also, some neurotransmitters are reducing agents. The involvement of oxidative stress in neurodegenerative disorders (Halliwell, 2006,

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in psychiatric and neurological disorders has caught particular interest, as the brain is considered especially

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macromolecules (proteins, lipids, carbohydrates, and nucleic acids), which may result in apoptosis or

[GPX]) and non-enzymatic (e.g., glutathione [GSH], vitamins A, C, and E, and selenium) antioxidants (Figure

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2007). However, when the pro-oxidant/antioxidant balance is disturbed towards higher concentrations of

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Reynolds et al., 2007), in psychiatric disorders in general (Ng et al., 2008), and in anxiety (Bouayed et al., 2009) has been discussed in recent reviews. Anxiety, depressive, and alcohol use disorders are highly comorbid mental disorders, as shown by epidemiological studies (Kessler et al., 2008, Pirkola et al., 2005). In particular, anxiety and depression have co-occurring and related symptoms that may be due to an underlying shared genetic basis (Hettema, 2008). In the Finnish population-wide Health 2000 Survey, the annual prevalences of anxiety-, depressive-, and

35.9 % of the anxiety disorder patients had a comorbid depressive disorder (major depressive disorder and/or dysthymia), and 22.4 % a comorbid alcohol use disorder (alcohol abuse and/or dependence). These disorders are commonly diagnosed and classified for research purposes according to the Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, 2000). The core feature of anxiety

can further be divided into diagnostic subcategories based on specific features regarding the focus, course

generalized anxiety disorder (GAD), phobias, and posttraumatic stress disorder (PTSD). Major depressive

Alcohol use disorder is discerned by recurrent and continued use of alcohol despite social problems and failure to fulfill daily obligations, tolerance effects, and withdrawal symptoms. Oxidative stress-related molecular mechanisms may represent one unifying factor influencing the etiology of anxiety, depression and alcohol use disorders. In this review, we highlight key supportive observations from animal models and human studies for the involvement of oxidative stress in these disorders and further discuss the potential underlying mechanisms. 2. Oxidative stress markers and antioxidant levels in human studies

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disorder (MDD) is distinguished by feelings of sadness and emptiness, anhedonia, and loss of energy.

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and onset of the anxiety, and these include panic disorder (PD), obsessive-compulsive disorder (OCD),

disorders is exaggerated anxiety that causes distress, disability and loss of quality of life. Anxiety disorders

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alcohol use disorders were 4.1 %, 6.5 %, and 4.5 %, respectively (Pirkola et al., 2005). In the same study,

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The direct measurement of free radical concentrations is difficult due to their short half-lives and low concentrations, and therefore measurements of metabolites of reactive species, antioxidant levels, antioxidant enzyme activities, and markers of oxidative damage (lipid peroxidation, protein carbonylation, and DNA damage) are commonly used to quantify levels of oxidative stress (Berk et al., 2008). In humans, these parameters have been evaluated in a number of studies that establish a link between oxidative stress and anxiety, depressive, and alcohol use disorders (Table 1). Results are mainly based on studies of plasma, serum, and blood cells investigating oxidative stress on a systemic level, while fewer post mortem studies of specific brain regions exist.

One of the few specific free radicals measured in several studies is nitric oxide (NO), which interestingly is both a ROS and a neuronal second messenger involved in modulation of, among other physiological functions, noradrenaline and dopamine release, learning and memory, wakefulness, and food intake and

disorders have been reported (Table 1), suggesting that it might be involved in oxidative stress through

In absence of direct measurements of free radicals, the most studied marker of oxidative stress-related cellular damage in psychiatric diseases is lipid peroxidation. Increased levels of blood or urine lipid peroxidation have consistently been reported in studies examining patients with OCD, social phobia (SP), PD, anxiety symptoms, depressive disorders, and alcohol dependence (Table 1). Only a few studies

MDD patients (Forlenza and Miller, 2006), and in leukocytes of females with anxiety or depressive symptoms (Irie et al., 2001). Increased DNA damage has also been observed in patients with bipolar disorder (Andreazza et al., 2007). To our knowledge, no studies have examined protein carbonylation and this issue should be addressed in future experiments.

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examining oxidative DNA damage exist, and they show increased damage to circulating DNA in serum of

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multiple mechanisms.

drinking (Herken et al., 2006). However, increased, decreased, and unaltered levels of NO in the studied

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In general, activities of antioxidant enzymes appear increased in anxiety, depression, and alcohol use disorder (Table 1). Some discrepant and negative results exist both across and within phenotypes, possibly due to many studies carried out with limited sample sizes or due to heterogeneity of study samples and conditions. The strongest and most consistent support exists for increased activity of SOD, possibly reflecting its crucial role as the first enzyme of the superoxide radical detoxification pathway, although SOD is also the most intensively studied antioxidant enzyme. Several studies have detected increased activities of the other enzymes involved in superoxide radical detoxification, including CAT, glutathione reductase (GSR), and GPX, but decreased activity of GPX has also been reported. Notably, most of the studies

occurs more readily by the GPX system at low concentrations, and by CAT at higher concentrations (Halliwell, 2007). In a complementary approach, peripheral blood gene expression profiling of PTSD patients revealed differential expression of enzymes related to ROS-metabolism, including downregulation of thioredoxin reductase and SOD (Zieker et al., 2007). Interestingly, the same study also reported downregulation of the xc(-) cystine-glutamate exchanger, which is responsible for the cellular uptake of cysteine, the rate-limiting precursor of glutathione (GSH) synthesis. Reports of increased antioxidant enzyme activities may in general reflect activated oxidative defenses due to increased ROS generation, and adaptation to continuous increased oxidative challenge. In contrast, decreased activities may represent situations where excessive oxidative challenge and high ROS levels have lead to consumption of the antioxidant enzymes. For instance, decreased GPX activity may reflect depletion of its essential component selenium and antioxidant cofactor GSH. Blood levels of specific antioxidants appear in general to be decreased in anxiety, depression, and alcohol use disorder, suggesting depletion of non-enzymatic antioxidant defenses (Table 1). In particular, decreased levels of vitamin E and GSH have been observed. Several studies have reported decreased blood total antioxidant capacity, a measure of the additive effects of specific antioxidant components, such as free sulfhydryl (-SH) groups, vitamins C and E, uric acid, and bilirubin (Erel, 2004). In contrast, increases in total 6

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measuring CAT found no change in its activity. This observation may reflect the fact that disposition of H2O2

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antioxidant status were observed in OCD and PD (Ersoy et al., 2008, Selek et al., 2008). Similarly, discrepant results from studies evaluating general total oxidant status or oxidative stress index (total oxidant status/total antioxidant status) in blood exist. It was hypothesized that increased total antioxidant capacity could reflect reactive increase in defense mechanisms, as rebound decreases in total oxidant levels were also observed (Selek et al., 2008). Such rebound phenomena only in certain studied patient groups could explain some of the discrepancies between phenotypes.

mechanisms in anxiety disorders, major depression, and alcohol use disorder, but to what extent and how these represent state or trait markers has not yet been conclusively resolved. Some studies have addressed the effect of antidepressant treatment on oxidative stress markers in patients with anxiety or depressive disorders (Atmaca et al., 2004, Bilici et al., 2001, Ersoy et al., 2008, Herken et al., 2006, Herken et al., 2007).

the absence of healthy controls receiving treatment or patient groups receiving placebo interpretation of

3. Oxidative stress markers and antioxidant levels in animal models Oxidative stress markers and antioxidant concentrations have been evaluated in numerous animal studies to investigate the role of oxidative stress in anxiety-like and depressive-like behavior (Table 2). Oxidative stress is also strongly implicated as one mediator of ethanol-induced brain damage in studies of acute and chronic ethanol intake. Animal models offer the advantages of access to samples of specific brain regions, minimization of genetic heterogeneity when inbred strains are used, and possibilities to control environmental effects and administer specific compounds. Several paradigms to induce anxiety-like and depression-like behaviors, such as immobilization stress, swim stress, chronic mild stress, maternal separation, olfactory bulbectomy, and social defeat paradigms are widely used (Bourin and Hascoet, 2003, Lister, 1987, Prut and Belzung, 2003). Pharmacologically validated 7

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these results is difficult.

In several of these studies, oxidative stress was diminished in patients after drug treatment. However, in

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Taken together, findings from human studies clearly support involvement of altered oxidative stress-related

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rodent models to measure anxiety-like and depression-like behavior, such as the elevated plus maze, light/dark box, open field, forced swim, and the tail suspension tests exist. Both psychological distress (e.g., communication box paradigm (Matsumoto et al., 1999)) and physical stress (e.g., immobilization stress (Zafir and Banu, 2009)) modulate antioxidant defenses and increase oxidative damage in the brain (Table 2). Studies of markers of oxidative stress-related cellular damage in animals strongly suggest that not only is there increased damage to lipids, proteins, and DNA in the brain after stress but also after ethanol

treatment (Table 2). Notably, these effects seem to be brain region-specific. The effect of stress on specific free radicals has not been evaluated as extensively as oxidative stress damage. Increased generation of O2-

mild stress treatment (Lucca et al., 2009), whereas increased NO levels were found in mouse whole brain (Matsumoto et al., 1999), rat hippocampus (Harvey et al., 2004), and rat serum after different stress paradigms (Kamper et al., 2009). Outbred Swiss albino mice show positive correlation between trait anxiety and intracellular ROS levels in cerebellum and hippocampus (Rammal et al., 2008a), and in peripheral blood cells (Rammal et al., 2008b). Increased ROS levels were also observed in isolated synaptosomes of rats fed with ethanol diet for eight weeks (Montoliu et al., 1994). Activity of several antioxidant enzymes are modulated by stress or ethanol drinking (Table 2). As in human studies, both increased and decreased activities have been reported. Comparison of the studies is challenging due to differences in experimental setup (stress model, brain regions studied, ethanol dosage and mode of administration). In general, there is a relative abundance of reports of decreased antioxidant enzyme activities after stress in rodents (Table 2), compared to the findings from studies of human patients with anxiety or depressive disorders (Table 1). This observation suggests that the animal stress paradigms may model conditions of antioxidant enzyme depletion due to extreme oxidative challenge to a greater extent than their compensatory upregulation to boost oxidative defenses. As in human studies, there is also evidence for depletion of antioxidants in animal models of stress and excessive ethanol drinking (Table 2). GSH is the only antioxidant assessed in multiple studies, and its levels were consistently decreased after 8

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was observed in submitochondrial particles of rat hippocampus, prefrontal cortex and cortex after chronic

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restraint stress, chronic mild stress, olfactory bulbectomy, and ethanol treatment. Depleted GSH levels may explain the decreased enzyme activities of GPX and GSR observed in some studies (Atif et al., 2008, Zafir and Banu, 2009). In addition to investigations focusing on specific markers of oxidative stress, more global brain proteomic and gene expression studies also support a connection between oxidative stress and anxiety. Glyoxalase 1 (GLO1), a detoxification enzyme, is downregulated in the brain of two separate mouse strains selectively

et al., 2010). In addition, several other proteins related to oxidative stress metabolism are either upregulated (glutathione S-transferase M1, and sirtuin 2) or downregulated (glutaredoxin 3, peroxiredoxin 6, and quinoid dihydropteridine reductase) in one of the anxious mouse strains (Szego et al., 2010). Contrary to these findings, brain gene expression levels and enzyme activities of GLO1 and GSR correlate

local overexpression of Glo1 and Gsr in the cingulate cortex of inbred mice increases anxiety-related

anxiety (Hovatta et al., 2005). The difference in Glo1 expression between inbred mouse strains is due to a

2009). The importance of GLO1 in mental disorders has recently been supported by two studies in human patients. GLO1 expression is reduced in patients with major depression or bipolar disorder when in depressive state, but not during remission (Fujimoto et al., 2008). In schizophrenia, rare genetic forms of GLO1 have been associated with decreased enzyme activity and increased carbonyl stress (Arai et al., 2010). These studies provide an excellent starting point to more detailed studies aiming to reveal the molecular mechanisms involved. Findings from knockout mouse models of oxidative stress pathway genes may help shed light on the mechanisms related to the regulation of behavior. Mice deficient in the life span determinant Shc1 gene, a regulator of mitochondrial ROS metabolism and apoptosis pathway, exhibit decreased intracellular ROS 9

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copy number variant, the presence of which correlates positively with anxiety-like behavior (Williams et al.,

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behavior, while silencing of Glo1 decreases it, suggesting a causal link between Glo1 and Gsr expression and

positively with anxiety-related behavior across six inbred mouse strains (Hovatta et al., 2005). Furthermore,

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bred for high anxiety behavior compared to their respective low-anxiety strains (Kromer et al., 2005, Szego

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levels and oxidative stress markers, and also show decreased anxiety-like behavior (Berry et al., 2007). Mice lacking the phospholipid transfer protein (PLTP), a transfer factor for the antioxidant vitamin E, have depleted brain vitamin E levels, and increased levels of oxidative stress markers and show increased anxiety-like behavior (Desrumaux et al., 2005). Oxidative stress can also be induced by administration of compounds, such as L-buthionine-(S,R)-sulfoximine (BSO), which depletes cellular GSH levels by inhibiting its synthesis. Interestingly, treatment of mice or rats with BSO induces oxidative stress and increases anxiety-like behavior (Masood et al., 2009, Salim et al., 2010). The oxidative stress-induced anxiety is reduced by inhibition of NADPH oxidase pathway via PDE2 inhibition (Masood et al., 2009) or with

oxidative damage in the frontal cortex and induces anxiety-like behavior (Souza et al., 2007). To summarize, rodent models suggest that psychological and physical stress are associated with increased

lead to the observed oxidative damage to the brain. Also gene expression and proteomic studies in various

behavior. Genetic and pharmacological perturbations of oxidative stress have revealed some specific

4. Oxidative stress and accelerated telomere shortening Oxidative stress might also be involved in the etiology of neuropsychiatric diseases by causing accelerated telomere shortening. Telomeres are DNA-protein complexes at the ends of all chromosomes. They shorten gradually in each cell division, and therefore, telomere length might be considered as a biomarker of aging. Telomere length is maintained in germ and stem cells in which the activity of the telomerase enzyme is high. Oxidative damage is repaired less well in telomeric DNA than elsewhere in the chromosome, suggesting that oxidative stress contributes to telomere loss and telomere-driven replicative senescence (von Zglinicki, 2002). Accelerated telomere shortening in leukocytes has been observed in many complex 10

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pathways involved, and future work in this area should help elucidate additional mechanisms.

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mouse models of innate anxiety have implicated oxidative stress pathway in the regulation of anxiety-like

levels of free radicals, depleted antioxidant levels, and altered antioxidant enzyme activities, which may

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moderate treadmill exercise (Salim et al., 2010). In rats, intake of a highly palatable diet causes increased

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diseases, including cardiovascular diseases, diabetes, inflammatory diseases, schizophrenia, mood disorders, and anxiety disorders. Mechanisms that contribute to accelerated telomere shortening in vivo remain largely unknown. Oxidative stress shortens telomeres in vitro, as shown by mild stress induced in various ways, including chronic hyperoxia, treatment with homocysteine, low doses of tertbutylhydroperoxide or hydrogen peroxide (Dumont et al., 2000, Dumont et al., 2001, Lorenz et al., 2001, Vaziri et al., 1997, von Zglinicki et al., 1995, von Zglinicki et al., 2000, von Zglinicki, 2002, Xu et al., 2000). Importantly, antioxidant vitamin C and the free radical scavenger -phenyl-t-butylnitrone reverses the oxidative stress-induced telomere shortening in vitro (Furumoto et al., 1998, von Zglinicki et al., 2000). It has been hypothesized that DNA damage caused by mild oxidative stress might lead to the presence of unrepaired nucleotide or base damage, which interferes with the replication fork at telomeres and therefore leads to shortened telomeres (von Zglinicki, 2002).

stress in 62 healthy women (Epel et al., 2004). Also other studies have associated recent psychological distress with shorter telomere length (Damjanovic et al., 2007, Parks et al., 2009), although negative reports exist as well (Kananen et al., 2010, Tyrka et al., 2010). Interestingly, childhood maltreatment and stress, important risk factors for mental disorders later in life, have been associated to shorter telomere length at the adult age (Kananen et al., 2010, Tyrka et al., 2010). Regarding psychiatric disorders, shortened telomere length has been observed in schizophrenia (Kao et al., 2008, Yu et al., 2008), mood disorders

The next challenge is to identify molecular mechanisms that link oxidative stress to accelerated telomere shortening in neuropsychiatric disorders. This area warrants further studies, as telomere length might turn out to be one of the phenomena that links neuropsychiatric diseases to increased incidence of somatic illness in patients. 5. Mitochondrial dysfunction, oxidative stress and behavior 11

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(Simon et al., 2006), and anxiety disorders (Kananen et al., 2010).

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Epel et al. associated self-perceived stress to shorter leukocyte telomere length and increased oxidative

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Mitochondria are the main intracellular sites of ROS generation and are also targets for oxidative damage. Several genetic studies both in humans and in rodents have provided evidence for the involvement of mitochondrial dysfunction in neuropsychiatric diseases. Patients with some mitochondrial diseases, such as progressive external ophtalmoplegia (PEO) and mitochondrial recessive ataxia syndrome (MIRAS), have psychiatric symptoms, including anxiety and depression(Hakonen et al., 2005, Suomalainen et al., 1992). Both PEO and MIRAS can be caused by mutations in the nuclear encoded mitochondrial polymerase gamma (POLG) gene. Mutations in POLG, which is responsible for mitochondrial DNA replication, result in randomly distributed mtDNA point mutations. Interestingly, transgenic mice expressing mutant POLG specifically in

phenotype (Kasahara et al., 2006). Studies of another transgenic model with the Y955C POLG mutation causing PEO have suggested that one of the pathological mechanisms may be oxidative damage to mtDNA (Graziewicz et al., 2007). The effect of mtDNA damage to the forebrain neurons of mice and its consequences on behavioral phenotypes has also been studied in a transgenic mouse model that has been designed specifically to result in the induction of a high number of apyrimidinic sites to the mitochondrial genome (Lauritzen et al., 2010). Transgenic mice had progressive atrophy of the hippocampus accompanied with reduction of the number of neurons and increased apoptosis. Also, reduction in the postsynaptic density size on dendritic spines of CA1 pyramidal cells was observed. On a behavioral level, the transgenic mice had increased locomotor activity, but decreased cognitive ability and anxiety-like behavior. More generally, evidence for the involvement of mitochondrial variations on anxiety-like behavior in mice have been obtained from a model in which C57BL/6J mice with substituted mitochondria from FVN/NJ strain had increased anxiety-like behavior compared to C57BL/6J mice with either AKR/J mtDNA or their own mtDNA (Gimsa et al., 2009). In humans, decreased mitochondrial ATP production rate and deletions of mtDNA have been observed in patients with major depressive disorder comorbid with somatic illness (Gardner et al., 2003). A postmortem gene expression study of the dorsolateral prefrontal cortex of PTSD patients and controls using 12

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forebrain neurons have accumulation of mitochondrial DNA mutations and show a mood disorder-like

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mitochondria-focused microarrays found that the majority of the differentially expressed transcripts were related to mitochondrial dysfunction and oxidative phosphorylation (Su et al., 2008), conditions which may be associated with excessive ROS production. Another approach to study the specific mechanisms relating mitochondrial function to anxiety has been behavioral studies of knockout mice of mitochondria-located proteins. BCL2 is a mitochondrial membrane protein involved in apoptosis and Ca2+ homeostasis. Mice over-expressing Bcl2 in neurons have decreased

anxiety-like behavior (Einat et al., 2005). In a human genetic association analysis one SNP in BCL2 was associated with generalized anxiety disorder (Sipila et al., 2010). Interestingly, glucocorticoid receptors form a complex with BCL2 followed by translocation to mitochondria in response to corticosterone which leads to modulation of mitochondrial oxidation, membrane potential, and mitochondrial calcium holding

Mitochondrial dysfunction may also be one of the mechanisms mediating the pathogenetic effects of ethanol-induced oxidative stress. Chronic ethanol exposure decreases activities of mitochondrial enzymes involved in oxidative phosphorylation (e.g., complex III and ATP synthase) in adult (Marin-Garcia et al., 1995) and neonatal rat brain (Marin-Garcia et al., 1996). In addition, decreased activity of cytochrome c oxidase in the rat cerebellum has been reported after chronic ethanol treatment (Jaatinen et al., 2003) and in ethanol withdrawal (Jung et al., 2007). Gene expression profiling in the human nucleus accumbens and

individuals compared to controls (Flatscher-Bader et al., 2005), suggesting disrupted mitochondrial function. Ethanol-induced mitochondrial dysfunction may accelerate generation of ROS or affect Ca2+ homeostasis and rate of apoptosis, all of which are important mitochondrial functions needed for the normal maintenance of neuronal cells.

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prefrontal cortex showed downregulation of genes encoding mitochondrial proteins in alcohol dependent

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capacity (Du et al., 2009).

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anxiety-like behavior (Rondi-Reig et al., 1997) while mice with a targeted mutation of Bcl2 show increased

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In summary, genetic defects or environmental factors, such as stress or diet, can cause mitochondrial dysfunction, which leads to increased oxidative stress or altered Ca2+ homeostasis (Figure 2). This in turn might alter neuronal signaling and further increase oxidative stress through accelerated ROS production. Genetic studies in human patients with mitochondrial disorders and mouse genetic studies using transgenic models have been especially useful to show that mechanisms related to mitochondrial dysfunction are involved in the pathogenesis of neuropsychiatric diseases. 6. The effect of oxidative stress on neuronal signaling and excitotoxicity

endogenous substances, and therefore it is one of the mechanisms contributing to neuronal degeneration.

transmitters or peptides) outweighs the inhibitory (mainly aminobutyric acid, GABA) signaling. Excessive

turn modulates mitochondrial Ca2+ homeostasis, and activates hydrolytic enzymes and apoptotic signaling pathways. Glutamate neurotoxicity is attributable in part to the activation of nitric oxide synthase (NOS) evoked by the increased intracellular Ca2+ concentration. Activation of NOS may lead to excessive formation of NO and consequently to oxidative stress (Dawson et al., 1994). This pathway has been shown to contribute to alcohol-induced neurotoxicity. In preclinical models, administration of ethanol increases glutamate release and decreases extracellular levels of GABA in the brain (Crews et al., 2004, Manto et al., 2005). In vitro, chronic ethanol treatment of cortical neurons leads to supersensitive N-methyl-D-aspartic acid (NMDA) receptors and increased intracellular Ca2+ concentration (Chandler et al., 1993, Hu and Ticku, 1995). Subsequent abstinence of ethanol also increases Ca2+ influx and stimulates NO production leading to neurotoxicity (Chandler et al., 1997) as described above. These findings from in vitro experiments suggest that ethanol-induced neurotoxicity is related to glutamate excitotoxicity and is at least partially mediated by oxidative stress-related events. However, multiple in vivo 14

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excitatory signaling leads to modulation of glutamate receptors and increased cellular Ca2+ influx. This in

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Excitotoxic neuronal damage may occur when the excitatory signaling (glutamate or other excitatory

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Excitotoxicity is the pathological process by which nerve cells are damaged and eventually killed by

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studies with NMDA or -amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor antagonists or calcium channel blockers have failed to prevent NMDA receptor-mediated toxicity especially in brain damage caused by chronic ethanol. These studies indicate that NMDA glutamate receptor-related excitotoxicity by itself cannot fully explain the neurodegeneration induced by ethanol exposure (Crews et al., 2004). NO-producing pathways and NO-mediated signaling are also linked to modulation of anxiety-like behavior, although results from different models have been contradictory as inhibition of NOS increased anxiety-like behavior in one study (Masood et al., 2009) and decreased it in another (Zhang et al., 2010). To conclude, while it is evident that glutamatergic and GABAergic systems modulate neuronal Ca2+ influx putatively stimulating NO production and leading to oxidative stress, additional work on specific mechanisms concerning the effect of oxidative stress on excitotoxic neuronal degeneration is needed. 7. Oxidative stress and inflammation

Increased levels of pro-inflammatory cytokines have been detected in patients with major depression, anxiety disorders and alcohol use disorder (Bob et al., 2010 , He et al., 2006, O'Donovan et al., 2010). Elevated interleukin-6 (IL-6) concentrations in serum or blood of patients with depression (Bob et al., 2010) and clinically anxious individuals (O'Donovan et al., 2010) have been observed. Chronic alcohol consumption in humans is associated with increases in serum tumor necrosis factor alpha (TNF) and IL-1 levels (McClain and Cohen, 1989, McClain et al., 1999). In addition, the expression level of another proinflammatory cytokine, monocyte chemoattractant protein 1, is higher in the brains of patients with alcoholism compared to controls (He and Crews, 2008). Neuronal degeneration can be caused, at least in part, by inflammatory processes activated by cytokines. Cytokines further activate pro-inflammatory signaling molecules, such as phospholipase A2 (PLA2) and arachidonic acid (AA) (Crews et al., 2004). The role of these signaling pathways has been studied in the cell culture models of neurological diseases involving inflammation. Mimicking of edema or ischaemia activates PLA2, which leads to increased release of AA (Lambert et al., 2006). Elevated AA can in turn generate ROS 15

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and additional pro-inflammatory signaling molecules, such as eicosanoids, that further promote inflammation and degeneration in the brain (Sun et al., 2004). AA may also have direct apoptotic effects (Caro and Cederbaum, 2006, Fang et al., 2008, Sun et al., 2004). Conversely, anti-inflammatory agent docosahexaenoic acid (DHA), a major component of brain membrane phospholipids, prevents neuronal apoptosis and plays an important role as an anti-oxidant agent (Bazan, 2007, Suganuma et al., 2010). Furthermore, brain concentrations of DHA are reduced after chronic ethanol exposure in cats and monkeys (Pawlosky and Salem, 1995, Pawlosky et al., 2001). Subchronic ethanol induces damage to neurons in rat brain slice culture. This effect can be prevented by the PLA2 pan-inhibitor mepacrine and is ameliorated by

inflammatory cytokine signaling may promote ROS generation and lead to oxidative damage, and this might be one mechanism that links inflammation to neuropsychiatric diseases.

cytokines. It is activated by ROS, cytokines and glutamate, and thought to be a mediator of oxidative stress

expression level of NFB has been reported in neurodegeneration caused by trauma, ischemia, and

Terai et al., 1996a, Terai et al., 1996b). NFB is a heterodimer of two subunits, usually p65 and p50 (Liou and Baltimore, 1993). Deletion of the p65 subunit in mice is embryonic lethal (Beg et al., 1995). Mice lacking p50 are vital and fertile, but have deficits in specific cognitive tasks and decreased anxiety-like behavior compared to control mice (Kassed and Herkenham, 2004). Interestingly, chronic NFB blocking by systemic administration of pyrrolilidine dithiocarbamate (PDTC) attenuates the higher levels of cytosolic and mitochondrial ROS generation in kidneys of spontaneously hypertensive rats when compared to similarly treated control rats (Elks et al., 2009). These findings suggest that NFB activation may induce oxidative stress and inflammation that leads to neuronal damage.

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Alzheimers and Parkinsons diseases (Bethea et al., 1998, Kaltschmidt et al., 1997, Schneider et al., 1999,

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in neurodegeneration (Mattson and Camandola, 2001, O'Neill and Kaltschmidt, 1997). Increased

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Nuclear factor B (NFB) is a transcription factor associated with the induction of pro-inflammatory

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DHA supplementation (Brown et al., 2009). Taken together, there is evidence that enhanced pro-

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The role of NFB activation in relation to oxidative stress has also been extensively studied in alcoholic neuropathy (Crews et al., 2006, Crews and Nixon, 2009). Ethanol exposure increases NFBDNA binding in rat brain (Crews et al., 2006) and in brain slice cultures in vitro (Zou and Crews, 2006, Zou and Crews, 2010). In human astroglial cells, which normally regulate extracellular glutamate concentrations, ethanol enhances NFB-DNA binding and activation of inducible nitric oxide synthase (iNOS) (Davis and Syapin, 2004, Davis et al., 2005). Induction of NOS may enhance NO production and oxidative stress, and modulate anxietyrelated behavior as discussed in the previous section.

cAMP response element-binding (CREB) family is another class of transcription factors linked to inflammation. CREB promotes neuronal survival, protecting neurons from excitotoxicity and apoptosis through transcriptional activation of pro-survival factors (Lonze and Ginty, 2002, Mantamadiotis et al., 2002). In vivo, subchronic ethanol treatment decreases the expression of the phosphorylated form of CREB

anxiolytic effects of 5-HT1A serotonin receptor agonists and selective serotonin reuptake inhibitors (SSRIs).

decreasing NO levels (Zhang et al., 2010). This may have behavioral consequences as NO levels exert

A key event in alcohol-associated brain damage seems to be increased NFB-driven induction of oxidative stress enzymes, such as cyclooxygenase-2 (COX2) and NADPH oxidase (Crews and Nixon, 2009). They are

behavioral phenotypes in rodents. Chronic ethanol treatment induces Cox2 immunoreactivity in the rat brain (Knapp and Crews, 1999), and the expression of NADPH oxidase subunits gp91phox and p67phox in the mouse brain (Qin et al., 2008). The NADPH oxidase pathway has also been implicated in the modulation of oxidative stress-induced anxiety (Masood et al., 2009), as discussed in section 3 above.

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activated during systemic inflammation and are likely involved in inflammatory responses related to

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negative control on CREB phosphorylation and thus activation.

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Stimulation of 5-HT1A receptors downregulates nNOS expression via an unknown mechanism, thereby

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in the brain (Bison and Crews, 2003). Interestingly hippocampal nNOS and CREB mediate some of the

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In summary, activation of inflammatory pathways has been observed in patients with anxiety disorders, major depression, and alcoholism, and in experimental animal models for these disorders. On one hand, increased levels of pro-inflammatory cytokines seem to be involved, and on the other hand activation of inflammation-related transcription factors, such as NFB and CREB. These transcription factors in turn regulate the expression level of several inflammation-related enzymes including NOS, COX2, and NADPH oxidase that in turn enhance production of ROS. Of these, especially the NADPH oxidase pathway has been associated with the regulation of anxiety-like behavior. 8. Inhibition of neurogenesis by oxidative stress

A growing body of evidence shows that impaired neurogenesis is involved in the pathogenesis of

and reduce anxiety- and depression-like behaviors (Kempermann et al., 1997, Salam et al., 2009, van Praag

SSRIs that are used as the main pharmacotherapy for both anxiety disorders and depression seem to increase neurogenesis in animal models (Malberg et al., 2000). The detailed mechanisms concerning the effect of oxidative stress on neurogenesis remain unknown. Interestingly, chronic ozone exposure, which causes an increase of ROS, leads to reduced neurogenesis in the adult rat hippocampus (Rivas-Arancibia et al., 2010).

Treadmill exercise training prevents increased anxiety-like behavior induced by oxidative stress in rats (Salim et al., 2010), but it remains to be seen whether this behavioral effect is mediated by increased neurogenesis. Several studies have shown that subchronic ethanol administration decreases hippocampal neurogenesis in rats (Crews et al., 2006, Nixon and Crews, 2002), and that antioxidants can reverse this effect (Crews et al., 2006, Herrera et al., 2003). One of these antioxidants is BHT (butylated hydroxytoluene) and the beneficial effects of it has been associated with reduction of alcohol induced NFB-DNA binding (Crews et al., 2006), further implicating the NFB pathway as a mediator of the effects of 18

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et al., 1999, van Praag et al., 2005) and alcohol-induced brain damage (Leasure and Nixon, 2010). Also,

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neuropsychiatric illness. In preclinical studies enriched environment and exercise increase neurogenesis

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oxidative events. Also chronic alcohol exposure decreases neurogenesis and increases cell death in the dentate gyrus of hippocampus (Herrera et al., 2003), and similarly, the inhibition of neurogenesis was prevented by an antioxidant, ebselen. Ebselen is an organoselenium GPX mimetic, and a poor oxidative radical scavenger, but it inhibits lipid peroxidation and blocks the function of inflammatory enzymes, such as COX2 (Nakamura et al., 2002). In addition, in cultured hepatocytes and in mouse skin ebselen potentiates activities of phase II enzymes, including NAD(P)H:(quinone-acceptor) oxidoreductase 1 and glutathione S-transferase (Nakamura et al., 2002).

Taken together, oxidative stress seems to impair neurogenesis, as exemplified by studies carried out on the effects of ethanol in rodent models. However, it remains to be investigated how significantly oxidative stress reduces neurogenesis in humans and what is the significance of this mechanism in neuropsychiatric diseases. 9. Antioxidant-related clinical therapies

Oxidative stress pathways provide a tempting target for pharmacological intervention. Few human clinical trials of antioxidants have been published in anxiety disorders and depression so far, even though there is evidence from animal models suggesting that treatment with antioxidants can both reduce oxidative stress and decrease anxiety-like behavior (Salim et al., 2010). The implemented therapies are based on supplementation with antioxidants, such as vitamins E or C, or on reinforcement of endogenous antioxidant pathways with compounds such as N-acetylcysteine (NAC) (Berk et al., 2008). NAC is converted to cystine, a substrate for the glutamate/cystine antiporter of glial cells (Lafleur et al., 2006). Thus, glial cystine uptake causes export of glutamate and modulation of glutamatergic neurotransmission. Moreover, increased cellular cystine reinforces GSH-related antioxidant defenses, as it is the rate-limiting component of GSH synthesis (Berk et al., 2008). Also valproate and lithium that are used in the treatment of bipolar disorder, have antioxidant properties through increased GSH levels (Cui et al., 2007). A double-blind, placebo controlled trial of NAC in trichotillomania, a condition related to OCD, reported significant improvement of 19

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symptoms observable after 9 weeks of active use (Grant et al., 2009). Case studies have further reported symptom reduction by NAC in OCD (Lafleur et al., 2006), trichotillomania, and pathological nail biting and skin picking (Berk et al., 2009, Odlaug and Grant, 2007). Depressive symptoms in bipolar disorder were significantly reduced by 24 week adjunction of NAC to usual medication in another double-blind, placebo controlled trial (Berk et al., 2008). The efficacy of natural remedies, most of which have antioxidant properties, in treatment of anxiety disorders was recently reviewed, and the findings mainly suggested beneficial effects of passionflower in GAD and inositol in PD and OCD (Kinrys et al., 2009). Taken together, these findings suggest that targeting oxidative stress-related mechanisms may be beneficial in treatment of

understanding the detailed neurobiological mechanisms related to antioxidant supplementation and perturbation of oxidative stress pathways is a key to the development of new and safe treatment practices. 10. Summary and future directions

It has become evident in recent years that oxidative stress is involved in the pathogenesis of various neuropsychiatric diseases, including anxiety disorders, major depression, and alcohol use disorder. In human patients, depletion of specific antioxidants and decreased activity of antioxidant enzymes, as observed in the blood, leads to impaired defense against oxidative stress. However, animal models of these disorders mainly suggest decreased antioxidant enzyme activities in the brain. Consequences of oxidative stress include damage to macromolecules, of which especially lipid peroxidation has been extensively

shortening of leukocytes in anxiety disorders and depression, which may be due to increased oxidative stress.

Several mechanisms are likely involved in increased generation of reactive species and impairment of oxidative defenses, both of which contribute to increased oxidative stress. Both genetic and environmental factors, such as stress, diet, substance abuse, and physical exercise, have an impact on our susceptibility to 20

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studied. On a more general level, recent human genetic work has implicated accelerated telomere

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anxiety, and an additional augmentation to conventional antidepressant and behavioral therapy. However,

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oxidative stress. Various events including mitochondrial dysfunction, inflammation, alterations in glutamate or GABA signaling, and inhibition of neurogenesis may each contribute individually to increased oxidative stress which in turn impacts these very same factors leading to excessive oxidative stress, and resulting in damage to cellular macromolecules. Eventually the consequences will be increased apoptosis, neuronal degeneration, and brain damage, which contribute to the manifestation of neuropsychiatric illness in susceptible individuals. The detailed mechanisms, however, remain largely unknown.

combined. Functional genomics offer powerful tools to assess gene expression differences in tissue-specific and temporal manner. At the same time, a large-scale biochemical approach should be taken to monitor the oxidative stress status and resulting damage to various macromolecules. The recently developed metabolomic technologies should be of great advantage allowing simultaneous investigation of a large

develop accurate animal models for neuropsychiatric disorders, and they will be instrumental to the

then be directly tested for their relevance in human patients. The mechanisms that lead to and regulate

oxidative stress pathways are well amenable to pharmacological manipulation, which makes this pathway a putative target for pharmacological therapies for anxiety disorders and depression.

The work by the authors is supported by the Academy of Finland, Sigrid Juselius Foundation, Yrj Jahnsson Foundation, Yrj and Tuulikki Ilvonen Foundation, Gyllenberg Foundation, Jalmari and Rauha Ahokas Foundation, Rosalind Frankling Young Investigator Award, and Biocenter Finland. We wish to thank the members of the Hovatta lab for discussion, and Petri Hyyti, Kaisa Manninen, and Tessa Sipil for critical reading of the manuscript. 21

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Acknowledgements

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oxidative stress and contribute to neuropsychiatric diseases are likely to be complex. However, some

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success of this system-wide approach. The specific models implicated by the experimental systems can

number of metabolites and signaling molecules. Considerable effort has been made in recent years to

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To distinguish pathogenetic mechanisms from adaptation and compensation, several approaches should be

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Figure captions: Figure 1. Major biochemical pathways of free radical production, and enzymatic and non-enzymatic antioxidative defenses. Abbreviations: CAT = catalase; GPX = glutathione peroxidase; GSSG/GSH = oxidized/reduced glutathione; GSR = glutathione reductase; NADP+/NADPH = oxidized/reduced nicotinamide adenine dinucleotide phosphate; NOS = nitric oxide synthase; RNS = reactive nitrogen species; SOD = superoxide dismutase.

Figure 2. Hypothetical mechanisms of oxidative stress-induced neuronal damage. Abbreviations: COX-2 = cyclooxygenase 2 ; CREB = cAMP response element-binding; GABA = aminobutyric acid; NADPH = nicotinamide adenine dinucleotide phosphate; NFB = Nuclear factor B; NO = nitric oxide; NOS = nitric oxide synthase; RNS = reactive nitrogen species; ROS = reactive oxygen species.

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Table 1
Table 1. Oxidative stress markers and antioxidant levels measured in anxiety, depressive, and alcohol use disorders. Finding Number of Marker Assessed in compared to Phenotype Reference cases/controls controls Oxidative stress markers Lipid peroxidation / MDA and TBARS Plasma Erythrocytes Plasma Serum Plasma Plasma Plasma Urine Erythrocytes Erythrocytes and plasma Plasma Serum Plasma Plasma Plasma in F, - in M in MD, - in anx. dis. OCD with or without MD OCD OCD OCD SP SP PD HADS MD MD MD Alc. dep. PTSD MD 27 OCD-MDD, 15 Kuloglu et al., 2002a OCD+MDD / 32 30 / 30 28 / 28 39 / 33 39 / 39 18 / 18 20 / 20 31 / 31 50 / 30 30 / 32 96 / 54 28 / 19 14 / 14 25 / 25 Ersan et al., 2006 Ozdemir et al., 2009 Chakraborty et al., 2009 Atmaca et al., 2004 Atmaca et al., 2008 Kuloglu et al., 2002b Ratnakar et al., 2008 Galecki et al., 2009 Bilici et al., 2001

Lipid peroxidation / HNE Lipid peroxidation / 8- iso -PGF2a

Geriatric 66 Depression Scale MD MD POMS Alc. dep. OCD STAI

DNA oxidation / 8-OHdG

NO generation / Total nitrite or nitrate

Plasma Serum Serum CSF PML Plasma Neutrophils PML Erythrocytes Erythrocytes Erythrocytes Erythrocytes Erythrocytes Seminal plasma Erythrocytes Serum Erythrocytes Erythrocytes Erythrocytes pfcx, and hp

MD or anx. dis. PD MD Alc. dep. MD MD BAI MD

an

Serum Leukocytes Pcx and cb Plasma Seminal plasma

us
28 / 28 39 / 39 18 / 18 20 / 20 27 50 / 30 29 / 30 30 / 32 96 / 54 35 / 35 7/7 32 / 20 14 / 14 15 / 27 6-22 / 3-21 36 / 20 28 / 19 39 / 39 18 / 18 50 / 30 29 / 30 6-22 / 3-21 20 / 20 14 / 14 27 30 / 32 26 / 43 35 / 35 28 / 19

Lipid peroxidation / Conjugated dienes Serum

35 / 35

Superoxide anion production

84 / 85 362 6-22 / 3-21 23 / 23 29 17 MD, 6 anx. dis. / 12 32 / 20 36 / 20 12 / 16 30 / 114 25 / 25 33 / 33 29 / 30

SOD activity

d
-

OCD with MD

27 OCD-MDD, 15 Kuloglu et al., 2002a OCD+MDD / 32 Ozdemir et al., 2009 Atmaca et al., 2004 Atmaca et al., 2008 Kuloglu et al., 2002b Eskiocak et al., 2005a Galecki et al., 2009 Szuster-Ciesielska et al., 2008 Bilici et al., 2001 Sarandol et al., 2007 Kodydkova et al., 2009 Michel et al., 2007 Herken et al., 2006 Tezcan et al., 2003 Srivastava et al., 2002 Gtz et al., 2001 Herken et al., 2007 Peng et al., 2005 Atmaca et al., 2004 Atmaca et al., 2008 Galecki et al., 2009 Szuster-Ciesielska et al., 2008 Gtz et al., 2001

Ac ce pt e

CAT activity

Serum Erythrocytes PML Fcx, tcx, cc, cm, and cb Serum Serum Erythrocytes Erythrocytes Erythrocytes Serum Fcx, tcx, cc, cm, and in fcx, in cc cb Erythrocytes Erythrocytes Erythrocytes Seminal plasma Erythrocytes PML Erythrocytes Serum

OCD SP SP PD STAI MD MD MD MD MD SOD1 in pfcx, MD SOD2 PD PTSD MD Alc. dep. MD Alc. dep. SP SP MD MD Alc. dep. OCD with or without MD PD PTSD STAI MD MD MD Alc. dep.

27 OCD-MDD, 15 Kuloglu et al., 2002a OCD+MDD / 32 Kuloglu et al., 2002b Tezcan et al., 2003 Eskiocak et al., 2005a Bilici et al., 2001 Srivastava et al., 2002 Kodydkova et al., 2009 Peng et al., 2005

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Suzuki et al., 2001

Sarandol et al., 2007 Peng et al., 2005 Tezcan et al., 2003 Selley et al., 2004 Dimopoulos et al., 2008 Kodydkova et al., 2009

Forlenza et al., 2006 Irie et al., 2001 Gtz et al., 2001 Atmaca et al., 2005 Eskiocak et al., 2006

Herken et al., 2006 Herken et al., 2007 Neiman et al., 1997 Srivastava et al., 2002 Selley et al., 2004 Arranz et al., 2007 Szuster-Ciesielska et al., 2008

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Erythrocytes GPX activity Erythrocytes Erythrocytes Erythrocytes Erythrocytes Erythrocytes and plasma Erythrocytes Erythrocytes Whole blood PML Erythrocytes Erythrocytes Serum Serum Erythrocytes Erythrocytes and plasma XDH activity PON1 activity Total oxidant status Serum Serum Serum Serum Serum Plasma Plasma Plasma Serum Plasma Plasma Plasma Serum Plasma Plasma Plasma Serum Serum Serum Plasma Seminal plasma Serum Fcx, tcx, na, pu, am, cc, cm, and cb Serum Seminal plasma Plasma Plasma Plasma Plasma Serum Serum

in erythrocytes, - in plasma

OCD OCD with or without MD SP SP PD MD PTSD MD MD MD OCD MD Alc. dep. MD MD

28 / 28

Ozdemir et al., 2009

27 OCD-MDD, 15 Kuloglu et al., 2002a OCD+MDD / 32 39 / 39 18 / 18 20 / 20 30 / 32 14 / 14 50 / 30 96 / 54 12 / 18 28 / 28 35 / 35 28 / 19 29 / 30 35 / 35 30 / 32 28 / 19 32 / 20 36 / 20 35 / 35 57 / 40 19 / 40 37 / 40 19 / 40 57 / 40 37 / 40 Atmaca et al., 2004 Atmaca et al., 2008 Kuloglu et al., 2002b Bilici et al., 2001 Tezcan et al., 2003 Galecki et al., 2009 Sarandol et al., 2007 Srivastava et al., 2002 Ozdemir et al., 2009 Kodydkova et al., 2009 Peng et al., 2005 Szuster-Ciesielska et al., 2008 Kodydkova et al., 2009 Bilici et al., 2001

in plasma, MD - in erythrocytes Alc. dep. PD MD MD MD PD OCD PD MD OCD MD OCD MD MD OCD MD MD OCD PD OCD STAI MD

Oxidative stress index

Antioxidants Vitamin E

an

us
96 / 54 30 / 30 42 / 26 49 30 / 30 96 / 54 96 / 54 28 / 28 19 / 40 35 / 40 34 35 / 35 6-22 / 3-21 19 / 40 34 37 / 40 19 / 40 33 / 33 50 / 30 57 / 40 96 / 54

Vitamin C Total carotenoids Selenium Ceruloplasmin GSH or GSH/GSSG ratio

Ac ce pt e

d
cb, and cm

M
Alc. dep. PD STAI OCD PD BAI MD MD MD

Total free -SH groups

Total antioxidant status/capacity

Abbreviations: 8-OHdG = 8-hydroxy-2 -deoxyguanosine; Alc. dep. = alcohol dependence; Am = amygdala; Anx. dis. = anxiety disorder; BAI = Beck Anxiety Inventory; CAT = catalase; Cb = cerebellum; Cc = corpus callosum; Cm = corpus mamillare; CSF = cerebrospinal fluid; F = females; Fcx = frontal cortex; GPX = glutahione reductase; GSH/GSSG = reduced/oxidized glutahione; GSR = glutahione reductase; HADS = Hospital Anxiety and Depression Scale; HNE = (E)-4-hydroxy-2-nonenal; Hp = hippocampus; M = males; MD = major depression; MDA = malondialdehyde; Na = nucleus accumbens; NO = nitric oxide; OCD = obsessive-compulsive disorder; Pcx = parietal cortex; PD = panic disorder; Pfcx = prefrontal cortex; PML = polymorphonuclear leukocytes; POMS = Profile of Mood States, Tension-Anxiety and Depression-Rejection subscales; PON1 = paraoxonase; PTSD = post-traumatic stress disorder; Pu = putamen; SOD = superoxide dismutase; SH = sulfhydryl; SP = social phobia; STAI = State Trait Anxiety Inventory; TBARS = thiobarbituric acid reactive substances; Tcx = temporal cortex; XDH = xanthine oxidase

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Gtz et al., 2001

Total peroxidase activity GSR activity

Peng et al., 2005 Herken et al., 2006 Herken et al., 2007 Kodydkova et al., 2009 Cumurcu et al., 2009 Ersoy et al., 2008 Selek et al., 2008 Ersoy et al., 2008 Cumurcu et al., 2009 Selek et al., 2008

Sarandol et al., 2007 Ersan et al., 2006 Maes et al., 2000 Owen et al., 2005 Ersan et al., 2006 Sarandol et al., 2007 Sarandol et al., 2007 Ozdemir et al., 2009 Ersoy et al., 2008 Virit et al., 2008 Eskiocak et al., 2005b Kodydkova et al., 2009

Ersoy et al., 2008 Eskiocak et al., 2005b Selek et al., 2008 Ersoy et al., 2008 Arranz et al., 2007 Galecki et al., 2009 Cumurcu et al., 2009 Sarandol et al., 2007

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Table 2

Table 2. Oxidative stress markers and antioxidant levels in animal models of anxiety, depressive, and alcohol use disorders Markers Oxidative stress markers Lipid peroxidation / MDA and TBARS Assessed in (findings compared to controls) Paradigm Reference

Pfcx (-), cx (), hp (-) Pfcx (-), cx (-), hp (-), st ( ), cb () Fcx (), hp (), st () Ccx (), cb (), pmo (-), st (-), mb (), hp (), ht (), plasma (), liver (), kidney (-) Brain () Brain () Brain () Brain (), liver (), heart (), serum () Brain (), liver (-), serum (-) Cx (), plasma () Brain (), isolated synaptosomes () Cx (-), st (-), hp (-), cb ( ), plasma (), liver (-) Serum () Brain () Cb (-)

CMS in rats CMS in rats Restraint stress in rats Immobilization stress in rats Chronic immobilization stress in rats Cold stress in rats Chronic immobilization stress combined with cold stress in rats Chronic restraint stress in rats Psychological distress in mice CMS in rats Chronic EtOH diet in rats Chronic EtOH treatment in rats CMS in rats Olfactory bulbectomy in rats Chronic EtOH diet in rats

Lucca et al., 2009a Lucca et al., 2009b Atif et al., 2008 Liu et al., 1996 Sahin et al., 2004 Sahin et al., 2004 Sahin et al., 2004 Zafir et al., 2009 Matsumoto et al., 1999 Eren et al., 2007 Montoliu et al., 1994 Calabrese et al., 1998

Lipid peroxidation / LSFP Lipid peroxidation / Conjugated dienes

cx (), st (-), hp (-), cb (), plasma (), liver () Brain () Brain () Brain ()

Chronic EtOH treatment in rats

Protein carbonylation

an

Pfcx (), cx (), hp (), st (), cb (-) Ccx (), cb (-), pmo (), st (-), mb (-), hp (-), ht (), plasma (), liver (), kidney (-) Brain (), liver (), heart () Brain () Brain () Brain ()

Cold stress in rats Sahin et al., 2004 Chronic immobilization stress combined Sahin et al., 2004 with cold stress in rats CMS in rats Lucca et al., 2009b Immobilization stress in rats Liu et al., 1996 Zafir et al., 2009 Sahin et al., 2004 Sahin et al., 2004 Sahin et al., 2004 Rouach et al., 1997 Bondy et al., 1995 Bondy et al., 1995 Liu et al., 1996 Rammal et al., 2008a Rammal et al., 2008b Montoliu et al., 1994 Lucca et al., 2009a Matsumoto et al., 1999 Harvey et al., 2004 Kamper et al., 2009 Rouach et al., 1997 Djordjevic et al., 2009 Djordjevic et al., 2009 Djordjevic et al., 2009 Sahin et al., 2004 Sahin et al., 2004 Sahin et al., 2004 Montoliu et al., 1994 Kamper et al., 2009 Lucca et al., 2009b Zafir et al., 2009 Grundmann et al., 2010 Tunez et al., 2010 Calabrese et al., 1998 Lucca et al., 2009b Sahin et al., 2004 Sahin et al., 2004 Sahin et al., 2004 Djordjevic et al., 2009 Djordjevic et al., 2009 Djordjevic et al., 2009

Proteolytic activity DNA oxidation / 8-OHdG ROS levels / DCF formation

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Superoxide anion production NO generation / Total nitrite or nitrate

Cb (-) Cx (-), cb (-), st ( ) Cx (), cb (-), st (-) Ccx (), cb (-), pmo (-), st (-), mb (-), hp (-), ht (), liver (-), kidney (-) Lymphocytes (), granulocytes (), monocytes () Neurons of ccx (), cb (), hp (); glia of ccx (-), cb (), hp (); lymphocytes (), monocytes (), granulocytes () Synaptosomes () Pfcx (), cx (), hp ()

Brain () Hp ()

Serum ( in F, - in M)

Nonheme and LMW-chelated iron Cb () SOD activity

Hp (SOD2 ; SOD1 -) Hp (SOD2 -; SOD1 -) Hp (SOD2 ; SOD1 -) Brain () Brain () Brain ()

CAT activity

Brain () Serum (-) Pfcx (), cx (), hp (), st (), cb (-) Brain (), liver (), heart (), serum () Hp (), ht () Brain () Cx (SOD1 ), st (-), hp (-), cb (SOD1 and SOD2 ), liver (-), plasma (SOD1 ) Pfcx (-), cx (), hp (), st (), cb () Brain () Brain () Brain () Hp () Hp (-) Hp (-)

Chronic restraint stress in rats Chronic immobilization stress in rats Cold stress in rats Chronic immobilization stress combined with cold stress in rats Chronic EtOH diet in rats Acute EtOH treatment in rats Subchronic EtOH treatment in rats Immobilization stress in rats

10 most vs. 10 least anxious mice among 100 tested animals 10 most vs. 10 least anxious mice among 100 tested animals Chronic EtOH diet in rats CMS in rats Psychological distress in mice Time-dependent sensitization stress in rats CMS in rats Chronic EtOH diet in rats Immobilization stress in rats Chronic psychosocial isolation in rats Immobilization stress combined with chronic psychosocial isolation in rats Chronic immobilization stress in rats Cold stress in rats Chronic immobilization stress combined with cold stress in rats Chronic EtOH diet in rats CMS in rats CMS in rats Chronic restraint stress in rats Chronic restraint stress in rats Olfactory bulbectomy in rats Chronic EtOH treatment in rats CMS in rats Chronic immobilization stress in rats Cold stress in rats Chronic immobilization stress combined with cold stress in rats Immobilization stress in rats Chronic psychosocial isolation in rats Immobilization stress combined with chronic psychosocial isolation in rats

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Chronic immobilization stress in rats

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Sahin et al., 2004

Kamper et al., 2009 Tunez et al., 2010 Rouach et al., 1997 Calabrese et al., 1998

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GPX activity

Brain () Fcx (), hp (), st () Brain (), liver (), heart (), serum () Hp (), ht () Cx (-), st (-), hp (-), cb ( ), liver (-) Brain () Brain () Brain () Brain (-) Cb (-) Cx (-), st (-), hp (-), cb (-), liver (-), plasma (-) Hp (protein levels ) Hp () Hp () Fcx (), hp (), st () Brain () Hp (), ht (-) Serum ( in M, in F) Cx () Serum ( in M - in F) Hp (protein levels ) Hp (protein levels ) Hp (-) Brain (-) Fcx (), hp (), st () Brain (), liver (), heart () Cx (-), st (-), hp (-), cb (-), liver (-), plasma ( )

GSR activity

Chronic EtOH treatment in rats

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Chronic EtOH diet in rats Restraint stress in rats Chronic restraint stress in rats Chronic restraint stress in rats Chronic EtOH administration in rats Chronic immobilization stress in rats Cold stress in rats Chronic immobilization stress combined with cold stress in rats Chronic EtOH diet in rats Chronic EtOH diet in rats Chronic EtOH treatment in rats Immobilization stress in rats Chronic psychosocial isolation in rats Immobilization stress combined with chronic psychosocial isolation in rats Restraint stress in rats Olfactory bulbectomy in rats Chronic restraint stress in rats CMS in rats CMS in rats CMS in rats Immobilization stress in rats Chronic psychosocial isolation in rats Immobilization stress combined with chronic psychosocial isolation in rats Chronic EtOH diet in rats Restraint stress in rats Chronic restraint stress in rats

Montoliu et al., 1994 Atif et al., 2008 Zafir et al., 2009 Grundmann et al., 2010 Calabrese et al., 1998 Sahin et al., 2004 Sahin et al., 2004 Sahin et al., 2004 Montoliu et al., 1994 Rouach et al., 1997 Calabrese et al., 1998 Djordjevic et al., 2009 Djordjevic et al., 2009 Djordjevic et al., 2009 Atif et al., 2008 Tunez et al., 2010 Grundmann et al., 2010 Kamper et al., 2009 Eren et al., 2007 Kamper et al., 2009 Djordjevic et al., 2009 Djordjevic et al., 2009 Djordjevic et al., 2009

GLUL activity

Antioxidants GSH or GSH/GSSG ratio

Fcx () , hp () , st () Brain (), liver (), heart () Brain () Brain () Brain () Cx () Brain () Brain () Cx (-), cb (-), st (-) Cx (), cb, st () Serum () Cx (-) Cx () Cb () Cx (-) Cb () Cb () Cb ()

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GST activity

Cb () Brain (-) Fcx (), hp (), st () Brain (), liver (), heart (), serum () Cb () Cx (-), cb (-), st (-) Cx (-), cb (-), st ( )

Chronic EtOH diet in rats Chronic EtOH diet in rats Restraint stress in rats Chronic restraint stress in rats Chronic EtOH diet in rats Acute EtOH treatment in rats Subchronic EtOH treatment in rats

Urate Vitamin A Vitamin C Vitamin E Beta-carotene Copper Selenium Zinc

Protein bound -SH groups

Ac ce pt e

Restraint stress in rats Chronic restraint stress in rats Chronic immobilization stress in rats Cold stress in rats Chronic immobilization stress combined with cold stress in rats CMS in rats Olfactory bulbectomy in rats Chronic EtOH diet in rats Acute EtOH treatment in rats Subchronic EtOH treatment in rats Chronic restraint stress in rats CMS in rats CMS in rats Chronic EtOH diet in rats CMS in rats Chronic EtOH diet in rats Chronic EtOH diet in rats Chronic EtOH diet in rats

Cx (), st (), hp (), cb (), liver (), plasma () Chronic EtOH treatment in rats Chronic EtOH treatment in rats

Non-protein bound -SH groups

Cx (-), st (-), hp (), cb, liver (), plasma (-)

Abbreviations: 8-OHdG = 8-hydroxy-2 -deoxyguanosine; CAT = catalase; Cb = cerebellum; Ccx = cerebral cortex; CMS = chronic mild stress; Cx = cortex; DCF = dichlorofluorescein; F = females; Fcx = frontal cortex; GLUL = glutamine synthetase; GPX = glutahione reductase; GSH/GSSG = reduced/oxidized glutahione; GSR = glutahione reductase; GST = glutathione S-transferase; Hp = hippocampus; Ht = hypothalamus; LMW = lowmolecular-weight; LSFP = Lipid soluble fluorescent products; M = males; Mb = midbrain; MDA = malondialdehyde; NO = nitric oxide; Pfcx = prefrontal cortex; PMO = pons-medulla oblongata; ROS = reactive oxygen species; SOD = superoxide dismutase; SH = sulfhydryl; St = striatum; TBARS = thiobarbituric acid reactive substances

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Atif et al., 2008 Zafir et al., 2009 Sahin et al., 2004 Sahin et al., 2004 Sahin et al., 2004

Montoliu et al., 1994 Atif et al., 2008 Zafir et al., 2009 Calabrese et al., 1998 Rouach et al., 1997 Montoliu et al., 1994 Atif et al., 2008 Zafir et al., 2009 Rouach et al., 1997 Bondy et al., 1995 Bondy et al., 1995

Eren et al., 2007 Tunez et al., 2010 Montoliu et al., 1994 Bondy et al., 1995 Bondy et al., 1995 Zafir et al., 2009 Eren et al., 2007 Eren et al., 2007 Rouach et al., 1997 Eren et al., 2007 Rouach et al., 1997 Rouach et al., 1997 Rouach et al., 1997 Calabrese et al., 1998 Calabrese et al., 1998

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Figure 1

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Figure 2

Mitochondrial dysfunction:
ROS mtDNA damage Ca2+ Membrane potential ATP production

Altered neuronal signaling:

Glutamate GABA Receptor modulation Ca2+ NOS and NO

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Genetic susceptibility Pathological states (e.g. anxiety disorders or depression) Physical and psychological stress

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Genetic and environmental factors:

Substance abuse Diet and exercise Aging

Inflammation:
Pro-inflammatory cytokines Pro-inflammatory signaling NFB activity CREB activity NOS, COX-2 and NADPH oxidase

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Oxidative stress:

ed

Increased generation of reactive species ROS, RNS and NO Impaired oxidative defences Antioxidant enzyme activities Antioxidants

Inhibition of neurogenesis:
Antioxidant protection

Ac

Accelerated telomere shortening

Antioxidant protection

Damage to macromolecules:
Protein carbonylation Protein oxidation DNA damage Lipid peroxidation

Apoptosis

Changes in plasticity Neurodegeneration Page 49 of 49 Brain damage