Adrenalectomy Causes Oxidative Damage and ... - Springer Link

International Journal of Stress Management, Vol. 5, No. 1, 1998

Adrenalectomy Causes Oxidative Damage and Monoamine Increase in the Brain of Rats and Enhances Immobilization Stress-Induced Oxidative Damage and Neurotransmitter Changes Jiankang Liu,1 Isao Yokoi,2 Stephanie J. Doniger,1 Hideaki Kabuto,2 Akitane Mori,2 and Bruce N. Ames1,3

The paradox that increased levels of glucocorticoids can either enhance or suppress the organism's defense against stress, has been an obstacle to formulating a unified picture of glucocorticoid function. To clarify the glucocorticoid paradox, we examined male Sprague-Dawley rats exposed to immobilization stress and/or bilateral adrenalectomy (ADX), and measured oxidative damage to lipid, protein, and DNA, as well as monoamine neurotransmitter turnover. ADX, which is similar to stress, induces an increase in lipid peroxidation and protein oxidation, accompanied by increased monoamine neurotransmitter turnover in several regions of the brain of rats. The effect of ADX is greater than that induced by short-term immobilization stress. In addition, ADX enhances stress-induced oxidative damage and increase of monoamine neurotransmitter turnover. These results, together with our previous finding that long-term stress causes oxidative damage to the brain, suggest that stress levels of glucocorticoids, or levels lower than basal, cause oxidative damage. However, basal levels of glucocorticoids appear to buffer against oxidative damage. These findings provide possible mechanisms to understand the glucocorticoid paradox, and support the stress-oxidative hypothesis of aging acceleration.

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Division of Biochemistry and Molecular Biology, 401 Barker Hall, University of California, Berkeley, California. 2 Department of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, Okayama, Japan. 3 Correspondence should be directed to Bruce N. Ames, Division of Biochemistry & Molecular Biology, 401 Barker Hall, University of California, Berkeley, California 94720-3202.

39 1072-5245/98/0100-0039$15.00/0 © 1998 Human Sciences Press, Inc.

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Liu, Yokoi, Doniger, Kabuto, Mori, and Ames

KEY WORDS: adrenalectomy; immobilization stress; oxidative damage; monoamine neurotransmitters; glucocorticoids; lipid peroxidation; protein oxidation; oxidative DNA damage; glutamine synthetase.

INTRODUCTION Various stresses produce, or are associated with, diseases such as gastric/ duodenal ulcers, hypertension, cancer, and cardiovascular/cerebrovascular disease (Glavin, 1985). A limbic-hypothalamic-pituitary-adrenocortical (HPA) system has been conceptualized as an integrating unit which controls the physiological and behavioral response to stress (Carpenter and Gruen, 1984). Increased levels of glucocorticoids (GCs) have been traditionally ascribed the physiological function of enhancing the organism's defenses against stress. However, it has become increasingly clear that GCs at moderate to high levels generally suppress the defense mechanism. This GC paradox remains a major obstacle to formulating a unified picture of GC function. Different hypotheses have been proposed. The glucocorticoid hypothesis of brain aging, proposed in the late 1970s, suggests that stress can damage the hippocampus, and that the damage plays a significant role in hippocampal degeneration over the course of aging (Landfield and Eldridge, 1994; Stein and Sapolsky, 1992). Munck et al. (1984) suggested that the stress-induced increase in GC levels does not protect against the source of stress itself, but rather protects against the body's normal reactions to stress, thereby preventing those reactions from overshooting and threatening homeostasis. In our previous studies, we found that immobilization stress induces oxidative damage to lipids and enzymes, changes membrane fluidity, and decreases antioxidant defenses in the rat brain (Liu and Mori, 1994). More recently, we have shown that immobilization stress causes oxidative damage to lipids, proteins, and DNA in the brain of rats (Liu et al., 1996a). Glutathione protects against this damage and can be supplemented or depleted, affecting damage accordingly (Liu and Mori, 1994; Liu et al., 1994). These results prompted us to suggest that stress may accelerate the aging process by causing oxidative damage (stress-oxidative hypothesis of aging acceleration) (Liu et al., 1996b). Thus far, there has been no report on the effect of adrenalectomy (ADX), with and without stress on oxidative damage in the brain. Based on the hypothesis of the involvement of oxidants in stress, it might be possible to understand the mechanisms underlying the GC paradox in stress by examining oxidative damage to the brain with stress and ADX. In the present study, rats were subjected to immobilization stress, to bilateral ADX, or to both. The levels of oxidative damage to lipid, protein, and DNA were assayed in the cerebral cortex, striatum, hippocampus, midbrain, hypothalamus, pons-medulla oblongata, and cerebellum.

Adrenalectomy, Stress, and Oxidative Damage

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The general hypothesis that stress is involved in the genesis of behavioral impairment via its disruptive effects on brain monoamine neurotransmitters, has received a great deal of research attention. However, the effect of stress and/or ADX upon the regional brain metabolism of these monoamines and their major metabolites, and how they are related to oxidative damage are not well understood. In attempting to achieve a greater understanding of the complex relationship among stress, neurotransmitters, and oxidative damage, we also studied the brain regional alterations and turnover of the monoamine neurotransmitters, such as norepinephrine (NE), dopamine (DA), and serotonin (5-HT).

MATERIALS AND METHODS Animals and Immobilization Stress Male Sprague-Dawley rats (7-week-old) were obtained from Charles River Japan Inc. (Kumamoto, Japan). The animals were used for experiments after adaptation to the new environment for 1 week. The rats were randomly divided into four groups, each group consisting of eight rats. Group 1: sham-operated without immobilization stress; group 2: ADX without immobilization stress; group 3: sham-operated with 1 hour of immobilization stress; and group 4: ADX with 1 hour of immobilization stress. A bilateral ADX was performed on rats anesthetized with Nembutal. The body weight was monitored every week before and after the operation. Two weeks after the operation, the rats in the two stress groups were immobilized for 1 hour. The 1 hour immobilization stress was carried out at room temperature (22 ± 2°C) as described (Liu and Mori, 1994; Weininger, 1956). Upon completion of the immobilization, the animals were sacrificed and the brain was removed and dissected. The dissection was performed as described (Glowinski and Iverson, 1966). Seven regions were separated on an ice-cooled plate and immersed in liquid nitrogen immediately, and then kept under — 80°C until analysis. The simplified names for the seven regions are: cerebral cortex, striatum, hippocampus, midbrain, hypothalamus, pons-medulla oblongata, and cerebellum. The sodium balance of ADX animals is normally maintained by providing saline drinking water so that the rats can maintain sodium balance. ADX animals can die if not provided with saline. A simple index of the completeness of ADX is to remove the saline (replacing it with tap water) and observe the body weight. Completely ADX rats will lose a substantial amount of weight within days (Roy et al., 1990). Another set of rats subjected to the same treatment was used for the brain monoamine study. The rats were terminated by transcranial microwave irradiation. This procedure inactivates metabolic enzymes of monoamines such as

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monoamine oxidase and catechol-O-methyltransferase, thereby stabilizing the brain levels of monoamines and their metabolites.

Measurement of Malondialdehyde (MDA) Lipid peroxidation was assayed by measuring MDA production. Samples were homogenized in a buffer consisting of 100 mM NaCl, 30 mM Tris, 10 mM EDTA, 10 mM (3-mercaptoethanol, and 5% triton X-100, pH 8.0. The MDA adduct derived from pentafluorophenylhydrazine was assayed by gas chromatography-mass spectrometry (GC-MS) (Yeo et al., 1994).

Protein Carbonyl Determination Protein oxidation was assayed by measuring the protein carbonyl levels in the samples (Levine et al., 1994). The protein carbonyl content in the sample was determined after reaction with 2,4-dinitrophenyl hydrazine with a Shimadzu UV160 spectrometer recording at 360 nm, and calculated using a molar absorption coefficient of 22,000 M - 1 c m - 1 . Measurement of 8-hydroxy-2'-deoxyguanosine (oxo8dG) in the Nuclear DNA The nuclear DNA of the brain was isolated as described (Shigenaga et al., 1994). The level of oxo8dG, a measure of oxidative DNA damage, was determined by high performance liquid chromatography with electrochemical detection (HPLC-EC) (Shigenaga et al., 1994).

Glutamine Synthetase Activity Glutamine synthetase activity was determined by a spectrophotometric assay as described (Rowe et al., 1970; Smith et al., 1991).

Measurement of Monoamines and Their Metabolites The monoamines and their metabolites in the brain, namely, NE, DA, 5-HT, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) were analyzed by HPLC-EC (Kabuto et


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a/., 1992). Brain tissues were homogenized with 0.2 M HC1O4 and centrifuged at 1600 g for 15 min. The supernatant was further centrifuged at 11,000 g for 30 min, passed through a 0.45 um Millipore filter, and injected on to an analytical column using a mobile phase consisting of 100 mM potassium phosphate buffer (pH 3.1), 13% acetonitrile, 770 mg/ml sodium octanesulfonate and EDTA, at a flow rate of 0.8 ml/min. The monoamines and their metabolites were quantified against external standards and indexed to protein.

Protein Measurement Protein concentrations were measured with the bicinchoninic acid (BCA) method (Wiechelman et al., 1988) using the Pierce BCA Protein Assay Reagent kit (Rockford, IL) with a 96-well microtiter plate.

Statistical Analysis All the results, except those of hypothalamus, are from 7-8 rats in each group. Because the weight of hypothalamus is less than 100 mg, the results were obtained from pooled samples from two rat brains. Mean and SEM were calculated and multiple comparisons were performed using one-way analysis of variance (ANOVA).

RESULTS Lipid Peroxidation (MDA), Protein Oxidation (Protein Carbonyls), and Oxidative DNA Damage (oxo8dG) MDA. The results are shown in Fig 1. One-hour stress induced a significant increase in MDA in the cerebellum. ADX without stress caused significant increases in the hypothalamus; the ADX plus stress group caused a significant increase in the cerebral cortex and hippocampus over the ADX without stress group. Protein Carbonyls. Stress alone did not cause any significant change in the brain protein carbonyls. However, ADX caused an increase in the hippocampus in the unstressed group, and also a significant increase in the cerebral cortex and hippocampus in the stressed group (Fig. 2). oxo8dG. Small increases were seen in the cerebral cortex, hippocampus, midbrain, pons-medulla oblongata, and cerebellum by stress and/or ADX, but none of these values was statistically significant (Fig. 3).

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Fig. 1. Immobilization stress and/or ADX-induced changes of MDA in the regions of the rat brain (CX: cerebral cortex; CL: cerebellum; PM: pons-medulla oblongata; ST: striatum; MB: midbrain; HY: hypothalamus, and HP: hippocampus). Values are mean + SEM from 7-8 animals. The significance of differences was examined with one-way ANOVA and when P was < 0.05 are shown as connected groups.

Fig. 2. Immobilization stress and/or ADX-induced changes of protein carbonyl in the regions of the rat brain (abbrevations are the same as in Fig. 1). Values are mean + SEM from 7-8 animals. The significance of differences was examined with one-way ANOVA and when P was < 0.05 are shown as connected groups.


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Fig. 3. Immobilization stress and/or ADX-induced changes of oxo8dG in the regions of the rat brain (Abbrevations are the same as in Fig. 1). Values are mean + SEM from 7-8 animals. There were no differences among the four groups evaluated by one-way ANOVA.

Glutamine Synthetase Activity. The changes in all of the regions of the brain were quite similar. Stress alone did not cause any significant changes. However, ADX caused a significant decrease in both the control and stressed groups in the cerebral cortex, midbrain, and cerebellum, as well as a decrease in the stressed group in the hippocampus (Fig. 4). To get a clear picture of the effects of stress and/or ADX on oxidative damage, we summarized these parameters in Table 1. Monoamines and Their Turnover Monoaminergic and cholinergic mechanisms both contribute to stress-induced behavior deficits. Neurochemical adaptation occurs over the course of repeated exposure to stress with increased amine synthesis, rather than decreased utilization (Glavin, 1985). Chronic vs. acute stress and psychological vs. physical stress produce markedly dissimilar central neurochemical effects besides the known differences in their peripheral effects. Therefore, the effects of stress were examined on central monoamine activity, rather than on the usual peripheral measures of stress such as plasma corticosterone and gastric ulcers. The effects of stress and/or ADX are shown for the following monoamines and their metabolites: NE; DA, DOPAC, HVA, 5-HT, and 5-HIAA. NE. Stress did not induce many changes in NE. However, ADX alone

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Fig. 4. Immobilization stress and/or ADX-induced changes in glutamine synthetase activity in the regions of the rat brain (abbrevations are the same as in Fig. 1). Values are mean + SEM from 7—8 animals. Significances were determined with one-way ANOVA and when P was < 0.05 are shown as connected groups.

induced a significant increase in NE in the pons-medulla oblongata. Stress plus ADX also did not cause significant changes (Fig. 5). DA. There were no significant changes in DA in any of the brain regions (Data not shown). DOPAC. Stress alone induced a significant increase in the midbrain; ADX alone induced significant increases in the midbrain and hippocampus. ADX enhanced the stress-induced increase to more significant levels in the midbrain, hypothalamus, and pons-medulla oblongata (Fig. 6). HVA. Both stress and ADX alone induced some HVA increases, but only in the cerebellum, the ADX without stress induced a significant increase. ADX plus stress caused significant increases in hippocampus, midbrain, hypothalamus, pons-medulla oblongata, and cerebellum (Fig. 7). 5-HT. No significant changes were found (Data not shown). 5-HIAA. There were no significant changes (Data not shown). DA Turnover [(DOPAC+ HVA)/DA]. The evaluation of amine turnover could be achieved by calculating the ratio of the metabolites and the amine. The turnover reflects the synthesis, release, utilization, re-uptake, and metabolism. All the regions, except the hippocampus, showed a similar changing pattern. Stress alone induced significant increase in cerebral cortex, striatum, midbrain, hypothalamus, and pons-medulla oblongata. ADX alone caused a significant increase in the midbrain. Stress plus ADX caused significant increase in the

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Fig. 5. Immobilization stress and/or ADX-induced changes in NE in the regions of the rat brain (abbrevations are the same as in Fig. 1). Values are mean + SEM from 7-8 animals. Significance were calculated with one-way ANOVA and when P was < 0.05 are shown as connected groups.

Fig. 6. Immobilization stress and/or ADX-induced changes in DOPAC in the regions of the rat brain (abbrevations are the same as in Fig. 1). Values are mean + SEM from 7-8 animals. Significance were determined with one-way ANOVA and when P was < 0.05 are shown as connected groups.


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Fig. 7. Immobilization stress and/or ADX-induced changes in HVA in the regions of the rat brain (Abbrevations are the same as in Fig. 1). Values are mean + SEM from 7-8 animals. Significance were determined with one-way ANOVA and when P was < 0.05 are shown as connected groups.

cerebral cortex, striatum, midbrain, hypothalamus, pons-medulla oblongata, and cerebellum, suggesting that ADX enhanced the stress-induced increases in DA turnover in the brain (Fig. 8). 5-HT Turnover [5-HIAA/5-HT]. Stress alone induced significant increase in the cerebral cortex and striatum, and stress plus ADX showed that ADX enhanced these increases. There were no significant changes in the other regions (Fig. 9). DISCUSSION Consistent with long-term immobilization stress (8 hr) (Liu et al., 1996a), short-term immobilization stress (1 hr) also induces an increase in lipid peroxidation and protein oxidation in the brain, though there were differences in extent. ADX exhibits a stress-like activity in causing oxidative damage, and its effect is even greater in some parameters than those induced by 1 hr immobilization stress. ADX enhanced stress-induced oxidative damage such as in the hippocampus. Short-term immobilization stress and ADX cause similar increases in monoamine levels and turnover. Both effects are synergistic in some brain regions, suggesting that ADX does not protect the brain from stress-induced abnormal change of neurotransmitters: rather, ADX enhances the effect.

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Fig. 8. Immobilization stress and/or ADX-induced changes in DA turnover [(DOPAC + HVA)/DA] in the regions of the rat brain (abbrevations are the same as in Fig. 1). Values are mean + SEM from 7-8 animals. Significance were determined with one-way ANOVA and when P was < 0.05 are shown as connected groups.

Fig. 9. Immobilization stress and/or ADX-induced changes in 5-HT turnover (5-HIAA/5-HT) in the regions of the rat brain (abbrevations are the same as in Fig. 1). Values are mean + SEM from 7-8 animals. Significance were determined with one-way ANOVA and when P was < 0.05 are shown as connected groups.


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The changes found in MDA, protein carbonyl, oxo8dG, and glutamine synthetase activity suggest that stress and ADX causes oxidative damage in the brain of rats. An increased level of lipid peroxidation is the evidence most frequently quoted in support of involvement of oxidants in tissue damage (Halliwell, 1992). Protein carbonyl levels indicate oxidative damage to protein (Stadtman, 1992). Emotional stress also causes oxidative DNA damage (Adachi et al., 1993). Glutamine synthetase is among the enzymes which are rapidly induced by GCs. Decreased uptake of glutamate due to decreased glutamine synthetase activity, could result in neurotoxic effects of abnormally prolonged N-methyl-D-aspartate (NMDA) receptor activation (Smith et al., 1991). The hippocampus is damaged by GCs, and ADX attenuates the deleterious effects of GCs (Landfield and Eldridge, 1994; Lowy et al., 1993, 1994). ADX also attenuates hippocampal damage after neurological insults such as seizures, or hypoxia-ischemia (Stein and Sapolsky, 1988, 1992). ADX of rats chronically exposed to social stress completely blocked the effects of the stress on spatial learning (Issa et al., 1990). These results provide direct evidence for the importance of elevated GCs levels in mediating the effects of chronic stress on hippocampal aging (Bodnoff et al., 1995). ADX, instead of protecting, enhances stress-induced oxidative damage and the abnormality of neurotransmitter turnover in the brain, suggesting that basal level of GCs may buffer against stress-induced oxidative damage. ADX of adult male rats results in a nearly complete loss of hippocampal granule cells, and corticosterone replacement prevents both the ADX-induced granule cell loss and the attenuated physiological response (McNeill et al., 1991; Sloviter et al., 1989). This finding indicates a protective role for GCs in addition to the welldocumented deleterious effects of elevated levels of corticoids. There is both dentate neuron loss by ADX, and a significant loss of CA4 pyramidal neurons (Sapolsky et al., 1991). It is possible that GCs may increase antioxidant activity during stress. GCs could decrease oxygen radical production by inhibiting the synthesis of prostaglandins, which are involved in the inflammatory response, an important source of free radicals (Feng et al., 1995; Hruza and Pentland, 1993; Weidenfeld et al., 1987). They might also fluidize membranes due to their steroidal nature, thereby, stabilizing them against oxidative attack (Brackan et al., 1990). Beside the loss of granule cells, ADX also causes other abnormal changes which may be related to the increased oxidative damage and increased turnover of monoamine neurotransmitters. ADX sensitizes mice to the lethal effects of IL-1 and tumor necrosis factor (Bertini et al., 1988), as well as lowering the activity of mitochondrial enzymes and Na, K-ATPase, suggesting that GCs are essential for the long-term regulation of these enzyme activities (Djouadi et al., 1993). The mechanisms of the oxidative damage induced by stress and ADX may

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be quite different. Stress releases extracellular amino acids such as aspartate and glutamate (Andine et al., 1991; Itoh et al., 1993; Moghaddam, 1993), which could be secondary to a GC-dependent starvation of the neuron. Glutamate and aspartate have been shown to exhibit the most potent cytotoxic effects (Bondy and LeBel, 1993). The cytotoxic or "excitotoxic" effect of glutamate and aspartate may be mediated through the production of oxidants (Choi, 1992; Dugan and Choi, 1994). It has been argued that prolonged NMDA receptor activation by these excitatory amino acids (EAAs) will lead to the mobilization of mitochondrial calcium, which in turn can stimulate calcium dependent phospholipase A2 (PLA2), thus leading to activation of the arachidonic acid cascade, as well as other pathways leading to oxidant production, including nitric oxide and its reaction product, the oxidant peroxynitrite (Coyle and Puttfarcken, 1993; Dugan and Choi, 1994). Oxidant production, in addition to being a consequence of NMDA receptor activation, may, independently stimulate the release of EAAs leading to the sequence of events described above (Choi, 1988, 1992a,b; Lowy et al., 1993). The reported protective effect of ADX against the deleterious effect of GCs may be achieved by attenuating stress-induced elevation in the extracellular glutamate concentration in the brain. The mechanism for ADX-induced oxidative damage may be related to the loss of basal level of GCs which protect against normal, though potentially toxic substances, such as hormones, prostaglandins and other arachidonic acid metabolites, neutral proteinases, and lymphokines. For example, ADX-induced oxidative damage may be related to the inflammatory response and the interference in the feedback system of the HPA axis. Such an interference may lead to an increase in hypothalamic and pituitary activities that result in an increased release of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH), resulting in the subsequent generation of oxidants, such as by induction of cytochrome P450, which can lead to generation of oxygen radicals and consequent oxidative damage (Park et al., 1996). The ADX induces an increased release of IL-1 both in vitro and in vivo, which is consistent with a feedback mechanism between IL-1 and GC hormones (Platner, 1961). IL-1 is a potent HPA axis activator. Enhancement of the inflammatory response in ADX animals is at least partially due to the activation of PLA2, and not counteracted by an increase in circulating corticosteroid hormones. IL-1 stimulates PLA2 (Burch et al., 1988; Mugridge et al., 1991; Solito and Parente, 1989), leading to the release of free fatty acids and to generate oxidants such as superoxide and hydrogen peroxide (Liu et al., 1996b). Stress is associated with an increased release of monoamine neurotransmitters. A general increase in the levels, or in the turnover rate, of NE, DA, and 5-HT in different brain regions is seen in several stress models (Roth et al., 1988; Tanaka et al., 1982a,b, 1983; Weiss et al., 1981). Following their release, these neurotransmitters are taken up into the presynaptic nerve terminal or are degraded, either through spontaneous autoxidation or by monoamine oxidase


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catalyzed degradation. In either case, hydrogen peroxide and superoxide would appear to be by-products of catecholamine oxidation. It has been suggested that the pathways leading to catecholamine oxidation may be an important source of oxidants that lead to damage in catecholaminergic neuronal systems. Our results show that the turnover of monoamines are augmented by stress and ADX, and ADX also augments the immobilization-induced turnover of monoamine neurotransmitters, consistent with previous reports (Faulk et al., 1995; Pacak et al., 1993; Vetrugno et al., 1993). These results indicate that ADX stimulates several aspects of sympatho-neural function, including stressinduced increments in monoamine turnover. Since cortisol administration reverses the augmentation of catecholaminergic responses in ADX rats, thus, GCs appear to feedback inhibit stress-induced increments in the release and biosynthesis of monoamine neurotransmitters in the brain. Thus endogenous GCs restrain responses of monoamine turnover in sympathetic nerves in immobilization stress model (Kvetnansky and Mikulaj, 1970). In addition, the removal of the adrenal medulla and thus circulating catecholamines has major system effects which undoubtedly can also influence cerebral metabolism. On the other hand, monoamines and their metabolites are scavengers of free radicals, they may be able to affect the redox mediated balance at the glutamate receptors between synapse formation and synapse removal that may be a key factor in neurocomputational plasticity. Therefore, it has been proposed that the increase in monoamine turnover may also serve to defend against oxidants (Cohen, 1983; Liu and Mori, 1993; Liu et al., 1996b). The effect of deprenyl on Parkinson's disease appears to be to scavenge superoxide radicals by raising cellular levels of dopamine (Cohen, 1983). Thus, ADX causes, and also enhances, stress-induced oxidative damage and monoamine turnover in the brain. These results suggest that both stress levels and lower than basal levels of GCs probably cause oxidative damage, and that basal levels of GCs buffer against oxidative damage. While these findings are of considerable interest, further work is necessary to advance this study. For example, it is quite important to further understand the dynamic aspects of the parameters by examining several points following ADX and stress, to further establish the relationship between glucocorticoids and oxidative damage (and the neurotransmitter system) by administering corticosterone to the adrenalectomized animals, and to further study oxidative damage by analyzing oxidative biomarkers at cellular and subcellular levels. The authors are currently engaged in intensive studies of these activities.

ACKNOWLEDGMENTS We thank P. S. Timiras, R. Sapolsky, and H. C. Yeo for valuable comments and critical reading of the manuscript. This work was supported by the National

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Cancer Institute Outstanding Investigator Grant CA 39910, and National Institute of Environmental Health Sciences Center Grant ESO1896 to B. N. A., and by the Budgets for Comprehensive Research on Aging and Health of the Ministry of Health and Welfare of Japan to A. M. The animal stress experiments were performed at Okayama University in accordance with institutional rules.

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