Evidence that hydrogen peroxide, a component of oxidative stress, induces high-anxiety-related behaviour in mice

Accepted Manuscript Title: Evidence that hydrogen peroxide, a component of oxidative stress, induces high-anxiety-related behaviour in mice Authors: Jaouad Bouayed, Rachid Soulimani PII: DOI: Reference:

S0166-4328(18)31052-0 https://doi.org/10.1016/j.bbr.2018.11.009 BBR 11634

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

23 July 2018 24 September 2018 6 November 2018

Please cite this article as: Bouayed J, Soulimani R, Evidence that hydrogen peroxide, a component of oxidative stress, induces high-anxiety-related behaviour in mice, Behavioural Brain Research (2018), https://doi.org/10.1016/j.bbr.2018.11.009 This is a PDF file 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 final 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.

Evidence that hydrogen peroxide, a component of oxidative stress, induces high-anxietyrelated behaviour in mice

Jaouad Bouayed *, Rachid Soulimani Université de Lorraine, Neurotoxicologie Alimentaire et Bioactivité, Rue du Général

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Delestraint, Campus Bridoux, 57070 Metz, France

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* Corresponding author: E-mail address: [email protected] (J. Bouayed).

 -H2O2 induces anxiogenic behaviour in mice.

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H2O2, a ROS, plays a causal role in the genesis of anxiety.

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 -H2O2 induces hyperactivity in mice.

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Highlights

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ABSTRACT

The link between oxidative stress and high-anxiety-related behaviour is uncontested; but the

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cause-effect relationship has yet to be completely elucidated. Here, the behavioural effects of

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hydrogen peroxide (H2O2), given to mice (n=10 per group) in drinking water at 1%, were assessed in the light/dark choice test, the open field, the elevated-plus maze and the holeboard test. Compared to controls (drinking only water), subacute exposure (10-15 days) of

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mice to H2O2, the major component of reactive oxygen species (ROS) and the precursor of potent oxidants (hydroxyl radical and hypochlorous acid), affected emotional responses by inducing an anxious behaviour associated with hyperactivity. Our findings clearly showed that H2O2-treated mice exhibited anxiogenic behaviour in the light/dark choice test and in the hole-board test. Moreover, H2O2-treated mice displayed a hyperactive behaviour, revealed by

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a significant increase in the number of crossings made in the open field test relative to controls. Although H2O2-exposed mice made significantly less head-dippings in the open arms than controls, H2O2-induced hyperactivity may have blurred anxiogenic-like behaviour in H2O2-treated mice in the elevated-plus maze. Our findings provide the evidence that H2O2, an oxidizing component, caused high-anxiety-related behaviour associated with hyperactivity

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in mice. Antioxidants may play a role in preventing or attenuating oxidative stress-related

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anxiety.

Keywords: Anxiety; Hydrogen peroxide (H2O2); Hyperactivity; Oxidative stress; Reactive

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oxygen species (ROS).

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INTRODUCTION Anxiety is an aversive emotional state signaling an internal conflict or occurring when the danger is potential, not well defined, or even inexistent (Weinberger, 2001; Steimer, 2002; Belzung, 2007; Bouayed et al., 2009). Anxiety is essential for survival of individuals and conservation of species following its adaptive anticipatory response to threating stimuli;

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however this evolutionarily conserved response can also be maladaptive and thus debilitating (Weinberger, 2001; Steimer, 2002; Belzung, 2007; Sokolowska and Hovatta, 2013; Fedoce et

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al., 2018). For instance, anxiety disorders are affecting 18% of the population in the US

(Fedoce et al., 2018). Many studies have established a link between anxiety and oxidative stress, which corresponds to an imbalance between oxidant production and antioxidant

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protection, in favour of oxidation (Bouayed et al., 2009; Bouayed, 2011; Hovatta et al., 2010).

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However, from available data, it cannot be inferred yet whether oxidative stress is the

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cause or rather the consequence of high anxiety levels and anxiety disorders (for more details

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see Bouayed et al., 2009 review, Bouayed, 2011 and Fedoce et al., 2018 review). Although

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oxidants at high levels are plausible disruptors of physiological mechanisms regulating anxiety (Bouayed et al., 2009; Hovatta et al., 2010; Bouayed, 2011), oxidative stress

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disturbances could also be a simple consequence of psychological distress of anxiety, which might activate pro-oxidant mechanisms that in turn stimulate enzymatic and non-enzymatic

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antioxidant defense mechanisms. We have previously reported a strong positive correlation between oxidative status of

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granulocytes and anxiety-like behaviour in the dark/light choice test in Swiss albino male mice (Bouayed et al., 2007). In other reports, we have also comparatively evaluated the oxidative status of mice in this strain with contrasting levels of anxiety, and we have found a strong link between intracellular oxidative stress metabolic pathways and the expression of anxiety-related behaviour (Rammal et al., 2008a and 2008b). These observations were in

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support of the initial findings of Hovatta et al. (2005) suggesting a close relationship between antioxidant mechanisms and anxious phenotypes in six inbred mouse strains. Hovatta et al. (2005) also hypothesized that some specific antioxidant-related enzymes regulate anxiety in mice, as they found that local overexpression in the murine brain of glutathione reductase 1 and glyoxalase 1 genes resulted in an increase of anxiety behaviour, while inhibition of

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glutathione reductase 1 expression produced low-anxiety in mice. The overexpression of

glyoxalase 1 and glutathione reductase 1 was induced in vivo by a lentiviral vector and not an

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excessive formation of reactive oxygen species (ROS), which raised questions on the exact mechanism by which these enzymes modulate anxiety levels (Bouayed et al., 2009). In contrast, Krömer et al. (2005) and Ditzen et al. (2006) found that glyoxalase 1 was

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upregulated in the strain with low- than rather high-trait anxiety. Moreover, Masood et al.

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(2008) found that oxidative stress leads to anxious behaviour in mice. In this last study,

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oxidative stress was induced by glutathione depletion in mice, inhibiting gamma-

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glutamylcysteine synthetase by buthionine-S,R-sulfoximine (BSO). Thus, glutathione

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depletion may be responsible for a myriad of cellular stresses including oxidative, carbonyl and nitrosative stresses, as glutathione is an important determinant of the oxygen, nitrogen

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and dicarbonyl metabolisms (Amicarelli et al., 2003; Delattre et al., 2005; Thornalley, 2006c; Valko et al., 2007). High levels of ROS causes oxidative damage to cellular structures

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(Halliwell, 2006a and 2006b), and reactive nitrogen species induce nitrosylation reactions that can alter the structure of proteins and thus inhibit their normal function (Lohinai et al., 1998;

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Valko et al., 2007). Excessive accumulation of reactive dicarbonyl compounds leads to protein and nucleotide damage by dicarbonyl glycation (Thornalley, 2006a, 2006b and 2006c). An important hallmark of oxidative stress is hydrogen peroxide (H2O2). It is widely accepted that when the level of H2O2, exceeds the antioxidative capacity of catalase and

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glutathione peroxidase in living organisms, a state termed oxidative stress can result (Ratnam et al., 2006; Valko et al., 2007). Besides its oxidizing property and its capacity to pass freely cellular membranes, H2O2 is a precursor of ROS, especially hydroxyl radical (OH•) and hypochlorous acid (HOCl), which are potent oxidants (Delattre et al., 2005; Valko et al., 2007; Bouayed and Bohn, 2010 and 2012).

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We hypothesized that ROS would impact anxiety levels in mice. To test our

hypothesis, the research objective in the current investigation was to assess anxiety-related

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behaviour of mice orally exposed to H2O2, the major component of ROS, in a battery of behavioural tests.

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MATERIALS AND METHODS

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Animals and treatment

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Twenty Swiss albino male mice (CD1) aged 9 weeks at the receipt from the breeder

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company (Charles River, France) and weighing 40-45 g were used. The animals were housed

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under a 12-h light: 12-h dark schedule (lights on starting at 8:00 p.m.) with water and food ad libitum (SD Dietex - France). Animal rooms were at a constant temperature of 21±2 °C and

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relative humidity of 55±10%. Experiments began after a 2-week period of acclimatization. During the dark phase (1 h after lights “off”) of the light/dark cycle, tests on animals were

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performed in a silent and isolated room under dim red lighting for a maximum of 3 h per day of experimentation (i.e., experiments were finished 4 h after lights “off” to avoid circadian

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cycle bias). All animal procedures were carried out in accordance with the relevant European Union regulations (Directive 2010/63/EU). The mice were randomly divided into two groups of 10 animals and treated for 15 days as follows: group 1 was given only food and water ad libitum (control mice); Group 2 was given food and H2O2 (1% in water) ad libitum (H2O2-treated mice). H2O2 (1%) was

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freshly prepared daily from the H2O2 stock solution (35%) purchased from Across Organics, France. During this study, external appearance of mice orally exposed to H2O2 were not different compared to controls. On day 10, the effects of H2O2 on the anxiety were evaluated by using the light/dark choice test. On day 11, the effects of H2O2 on the locomotor activity were evaluated by using the open field test. On day 14, the effects of H2O2 on the anxiety

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were evaluated by using the elevated-plus maze. On day 15, the effects of H2O2 on the anxiety

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were evaluated by using the hole-board test. On day 16, the mice were weighed.

Behavioural studies The light/dark choice test:

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The light/dark box apparatus consisted of two compartments (light/dark, surface ratio

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3:2), divided into 15 squares: 9 x 9 cm in total. The dark box (black PVC, 27 x 18 x 29 cm)

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was illuminated by a dim red light and was divided into six squares. The lit box (white PVC,

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27 x 27 x 29 cm) was illuminated by a white light located 1.50 m above the device and was

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divided into 9 squares. The two compartments communicated by means of a small door (7 x 7 cm). All the experiments were carried out in a dark room and after each test the light/dark

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box was cleaned with a 10% ethanol solution. Each test took 5 min. At the beginning of the test, each animal was placed in the lit box with its head facing the door of the dark box. The

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amount of time spent in the lit box, the number of entries into the lit box (all four feet) and the

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latency time were recorded after the first entry in the dark box (Bouayed et al., 2007).

The open field test We have employed the open field test to evaluate the locomotor activity of mice according to the method previously described by Grova et al. (2007). Locomotor activity was assessed on a circular open-field platform (diameter, 50 cm) with 27 cm high walls. The floor

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of apparatus was divided into 36 squares having the same surface (50 cm2). Mice were placed individually into the open-field, and after a 1-min for habituation, during 6 min period, the number of squares visited was recorded.

The elevated plus-maze

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All parts of the apparatus were made of dark polyvinyl plastic. The maze was elevated

to a height of 50 cm and had two open (30 x 5 cm) and two closed arms (30 x 5 x 15 cm),

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arranged in a manner that the arms of the same type were opposite to each other and connected by an open central square (5 x 5 cm). To prevent mice from falling of the open

arms, a rim (2.5 mm high and 8 mm deep) surrounded the perimeter of the open arms. At the

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beginning of the 5-min test session (Bouayed et al., 2007), mice naive to the apparatus were

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placed individually in the central square of the maze, facing one of the closed arms. An entry

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in the arm was counted when the animal placed all four paws into the arm. The total number

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of visits to the open arms, the total number of visits to the closed arms, the cumulative time

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spent in the open arms and the frequency of head-dipping (dipping the head below the level of the maze floor) were recorded. The results were expressed as number of entries into closed

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arms, percentage of time spent in the open arms, percentage of entries into open arms and

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number of head-dips. After each test, the apparatus was cleaned with a 10% ethanol solution.

The hole-board test

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The maze consisted of an elevated arena (one meter above the floor) with 16 equally

spaced holes (3.5 cm internal diameter, four lines of four holes each). Animals were placed individually in the centre of the hole-board and were allowed to explore the maze for 5 min. The number of head-dips was recorded in each of the three different parts of the maze (outside the maze, peripheral holes and central holes). A head-dip was scored if both eyes

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disappeared into the hole (do-Rego et al., 2006). This head-dipping behaviour is known as an indicator of the exploratory rate of mice in the maze and correlates with anxiety levels (Takeda et al., 1998).

Statistical analysis

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Results are presented as mean ± S.E.M. Data that did not conform to a Gaussian

distribution were analyzed using Mann–Whitney U-tests to compare one group to another.

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Data that did conform to a Gaussian distribution were analyzed using Student’s t-tests. We also performed repeated measures ANOVA, with treatment as between-subject fixed factor

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with p < 0.05 were considered statistically significant.

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and weighing as the repeated measure, to monitor body weight of mice. Mean differences

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RESULTS

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The light/dark choice test

Mann–Whitney U-tests revealed that H2O2-treated mice took significantly more time

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to leave the dark box (p = 0.04) and spent significantly less time in the lit box (p = 0.009) than

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control mice. There were no significant differences between groups with respect to number of

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transitions between two boxes (p > 0.05) (Fig. 1).

The open field test

Mann–Whitney U-test revealed that H2O2-treated mice made significantly more visits

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to squares than control mice (p=0.019) (Fig. 2).

The elevated plus-maze There were no significant differences between groups with respect to time spent in the open arms, the entries in the open arms and the number of closed arms entries (p > 0.05).

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Mann–Whitney U-test revealed that H2O2-treated mice made significantly less head-dips in the open arms than control mice (Fig. 3).

The hole-board test

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There were no significant differences between groups with respect to number of headdips made in centre board (p > 0.05). Student’s t-tests revealed that H2O2-treated mice showed

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significant decreases in the number of head-dips measured in both peripheral (p=0.005) and exterior board (p=0.027), and also in the total number of head-dips (p=0.0006) than control

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mice (Fig. 4).

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Effect of subacute exposure to H2O2 on body weight of mice

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Repeated measures ANOVA showed that treatment, time and interaction between

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these factors did not have significant effects on the body weight of mice (F (1,15)=0.033, p >

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0.05; F (1,15)=0.627, p > 0.05; F (1,15)=0.053, p > 0.05, respectively) (Fig. 5).

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DISCUSSION

In the present study, H2O2-treated mice exhibited an anxiogenic behaviour in the light/

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dark choice test as revealed by both a significant increase in the latency time and a significant decrease in the amount of time spent in the lit box versus control mice (Fig. 1). Furthermore,

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H2O2-treated mice displayed an anxious behaviour in the hole-board test as highlighted by a significant decrease in the number of head-dips made by these mice in the exterior, peripheral and total holes versus controls (Fig. 4). Moreover, H2O2-treated mice displayed hyperactivity in the open field test emphasized by a significant increase in the number of crossings versus control mice (Fig. 2). This hyperactivity may explain the fact that the number of transitions made by H2O2-treated mice between the two boxes of the light/dark test was elevated despite 9

the apparent increased anxiety in these mice (Fig. 1). In addition, hyperactivity induced by H2O2 may have blurred the anxiogenic-like behaviour in H2O2-treated mice in the elevatedplus maze (Fig. 3). This assumption was reinforced by the fact that H2O2-treated mice made significantly less head-dips in the open arms than control mice (Fig. 3). It must be emphasized here that our results ruled out any unspecific effect of weight loss on anxiogenic behaviour

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because subacute exposure to H2O2 had no significant effects on body weight of mice (Fig 5). Therefore, subacute exposure to prooxidant H2O2, a component of oxidative stress

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metabolism, induced high-anxiety-related behaviour associated with hyperactivity in mice.

do-Rego et al. (2006) have proposed that the hole-board test might replace the elevated plus maze because they found a high correlation between the scores performed by Swiss mice

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in these two tests. Additionally, they found that the light/ dark test correlated weakly with

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both elevated plus maze and the hole-board test, probably due to the fact that these two

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categories of tests do not mobilize the same phobias. It is worth mentioning that exploratory

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behaviour of rodents in the light/dark box test and in the elevated plus maze confounds

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locomotor activity and emotion of anxiety (Belzung and Le Pape, 1994; Belzung et al., 1999; Bourin and Hascoet, 2003). As prolonged exposure to H2O2 induced an increase in locomotor

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activity, it would be expected that H2O2-treated mice would exhibit a non-anxious phenotype rather than an anxious phenotype in the light/dark choice test. However, the exploratory

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behaviour corresponding to dipping the head into a hole appears not confounded by general locomotor activity (do-Rego et al., 2006). It is worth noting that the hole-board test was

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initially developed to assess the curiosity level in rodents (do-Rego et al., 2006). Nevertheless, head-dipping behaviour may be restrained by neophobia that may be also worsened by the fear of void, which permits to assess the anxiety level of rodents and to detect anxiolytic properties of drugs (do-Rego et al., 2006; Grova et al., 2007).

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H2O2 is mainly formed by dismutation of the superoxyde radical (O2•−), by the superoxide dismutase, or during mitochondrial respiration, by univalent reduction of O2•− (O22− + 2 H+) (Delattre et al., 2005; Halliwell, 2006; Valko et al., 2007). Albeit being of nonradical nature, H2O2 is more reactive than O2•− (Delattre et al., 2005; Halliwell, 2006). H2O2 is employed by cells in intra- and/or inter-cellular signalling following its ability to freely pass

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across cell membranes (Delattre et al., 2005; Halliwell, 2006). In vivo, catalase and

glutathione peroxidase permit to remove H2O2 present at physiologic micromolar

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concentrations (Dringen et al., 2005). However, H2O2 accumulation may lead to a state of oxidative stress (Ratnam et al., 2006; Delattre et al., 2005; Valko et al., 2007, Bouayed et al.,

2009, Bouayed and Bohn 2010). Mice lacking the enzyme glutathione peroxidase-1 (GPx-1)

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have developed central cataracts (Wang et al., 2009). Homozygous catalase (CAT) knockout

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mice, although apparently developing normally, have shown differential sensitivity to oxidant

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tissue injury in comparison to wild-type mice (Ho et al., 2004). At high level, H2O2 becomes a

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cytotoxic compound resulting in e.g. the damage of neurons, following its own oxidizing

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property and its ability to generate more highly reactive compounds including OH• and HOCl (Delattre et al., 2005; Halliwell, 2006a and 2006b; Valko et al., 2007, Bouayed and Bohn

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2010, Bouayed and Bohn 2012). Fenton reaction, catalysed by transition metals like Fe2+ and Cu+, or Haber-Weiss reaction, which uses superoxyde radical (O2•−) as the donor electron,

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convert H2O2 into the fearsomely reactive and biologically damaging OH•, while myeloperoxidase produces HOCl from H2O2 (Delattre et al., 2005; Valko et al., 2007,

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Bouayed and Bohn 2010, Bouayed and Bohn, 2012). The very short half-life of OH• radicals making them the very dangerous radicals, thus when produced in vivo they react close to their site of formation, with almost everything around it contrary to H2O2 which reacts with few molecules (Delattre et al., 2005; Halliwell, 2006a; Valko et al., 2007 Bouayed and Bohn 2010a, Bouayed and Bohn, 2012). OH• radicals are able to initiate lipid peroxidation, to

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oxidize amino acid side chains in proteins, and to cause DNA strand breaks and base modifications (Halliwell and Gutteridge, 1999; Delattre et al., 2005; Valko et al., 2007, Bouayed and Bohn 2010a, Bouayed and Bohn, 2012). Although oxidative stress markers were not assessed in this study, our results highlight the direct effects of H2O2, a component of oxidative metabolism, on anxiety-like behaviour. In vivo, excessive H2O2 accumulation

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provokes an overexpression of the glutathione redox system including glutathione reductase

and also a general overexpression of endogenous antioxidants including catalase

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(Splettstoesser and Schuff-Werner, 2002; Ratnam et al., 2006, Bouayed and Bohn 2010a,

Bouayed and Bohn, 2012). Thus, the results from our study provide evidence that H2O2, at non physiologic concentrations, which is a precursor of toxic oxygen metabolites (Delattre et

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al., 2005, Bouayed and Bohn 2010a, Bouayed and Bohn, 2012), induces high-anxiety-like

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behaviour associated with hyperactivity in mice. Our findings were in support of the findings

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of Hovatta et al. (2005) about the genesis of anxiety-like behaviour in mice from an

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overexpression of specific brain antioxidant enzymes including glutathione reductase.

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Masood et al., (2008) found that treatment of mice with BSO, an inducer of oxidative stress, caused anxious behaviour through NADPH oxidase pathway. In our study, it has been

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indicated that H2O2, an oxidizing agent, may be a factor in the genesis of anxiety. Our results also are in good concordance with other reports showing indirect link between oxidative

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stress and anxious behaviour (Souza et al., 2007; Berry et al., 2007). Souza et al., (2007) demonstrated that the consumption of highly palatable diet leads to an obese phenotype,

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increased protein oxidation in the frontal cortex and appeared to induce anxiety-like behaviour in rats. Berry et al. (2007) showed that the deletion of the p66Shc gene in mice, which resulted in reduced of oxidative stress and extended life span, decreased anxiety-related behaviour in p66Shc– / – mice. It was noted previously that cellular oxidative damage disturbs many functions, including alterations of neurotransmitter levels (Lebel, 1991, Valko et al.,

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2007, Bouayed et al., 2009). Therefore, it is possible that H2O2-mediated oxidative damage in H2O2-treated mice affected the anxiety-related behaviour via alteration of neurotransmission, by acting for example on the level of neurotransmitters and/or by impairing receptor–ligand interaction. It is also possible that oxidative stress induces emotional stress via the alteration of the hypothalamic-pituitary-adrenal (HPA) axis function (Masood et al., 2008), which is

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implicated in the response to stress (Spencer, 2013a, 2003b, Soualeh et al., 2017), and also

that its dysfunction is associated with anxiety disorders (Mathew et al., 2008, Masood et al.,

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2008, Spencer et al., 2014). Oxidative stress may also affect, among others, corticolimbic regions involved in mediating and regulating anxiety (Mozhui et al., 2010, Hovatta et al., 2005, Souza et al., 2007).

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Our results emphasize the possible role of antioxidants in attenuating anxiety

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symptoms. In this respect, Salim et al. (2010) demonstrated that anxiety generated by BSO

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treatment of rats was reversed either by preventive treatment with the antioxidant tempol, or

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by moderate treadmill exercise. Masood et al. (2008) showed that the well-known anxiolytic

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diazepam does not fully reverse the anxiety generated by BSO treatment. Nevertheless, diazepam can abolish e.g. restraint stress-induced anxiety, although immobilization stress

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being a prooxidant (Bouayed, 2011). Interestingly, Masood et al. (2008) showed that oxidative stress-related anxiety could be reversed in mice after inhibition of NADPH oxidase

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or phosphodiesterase-2, an enzyme that is indirectly implicated in oxidative stress mechanisms. These findings emphasise that anxiety can result from physiological

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disturbances other than those produced by oxidative stress. Thus, antioxidants might be interesting in the case of oxidative stress-related anxiety. In summary, this investigation provides evidence that H2O2, a component of oxidative metabolism, may be one of the physiological factors causing emotional stress. As high anxiety levels are multifactorial conditions, our results raise the question of whether

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antioxidants might have a preventive and/or therapeutic action on high anxiety levels resulting from oxidative stress disturbances. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgments

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We gratefully acknowledge Dr. Torsten Bohn for corrections in the English language. References

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Figure legends Fig 1. Effects of subacute exposure to H2O2 compared to drinking water on the anxietyrelated behaviour of Swiss mice in the light/dark choice test. The latency of the first entry into the lit box (A), the number of entries into the lit box (B) and the time spent in the lit box (C)

0.01: statistically significant differences compared to control animals.

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are presented. Data are reported as mean ± SEM of 10 mice per group. *p < 0.05 and **p <

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Fig 2. Effects of subacute exposure to H2O2 compared to drinking water on the locomotor

activity of Swiss mice in the open field test. Data are reported as mean ± SEM of 10 mice per

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group. *p< 0.05: statistically significant difference compared to control animals.

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Fig 3. Effects of subacute exposure to H2O2 compared to drinking water on the anxiety-

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related behaviour of Swiss mice in elevated plus maze. The time spent in open arms (%) (A),

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the number of entries into open arms (%) (B), the number of closed arms entries (C), and the

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number of head-dips (D) are presented. Data are reported as mean ± SEM of 10 mice per

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group. *p < 0.05: statistically significant differences compared to control animals.

Fig 4. Effects of subacute exposure to H2O2 compared to drinking water on the anxiety-

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related behaviour of Swiss mice in the hole-board test. The number of head-dips in the exterior board (A), the number of head-dips in the peripheral board (B), the number of head-

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dips in the centre board (C), and the number of total head-dips (D) are presented. Data are reported as mean ± SEM of 10 mice per group. *p < 0.05, **p < 0.01 and ***p < 0.001 statistically significant differences compared to control animals.

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Fig 5. Effect of subacute exposure to H2O2 compared to drinking water on body weight of

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