Brain Catalase Activity Inhibition as Well as Opioid

Receptor Antagonism Increases Ethanol-Induced HPA

Axis Activation
Ral Pastor, Carles Sanchis-Segura, and Carlos M.G. Aragon
Background: Growing evidence indicates that brain catalase activity is involved in the psychopharma-
cological actions of ethanol. Recent data suggest that participation of this enzymatic systemin some ethanol
effects could be mediated by the endogenous opioid system. The present study assessed whether brain
catalase has a role in ethanol-induced activation of the HPA axis, a neuroendocrine system modulated by
the endogenous opioid neurotransmission.
Methods: Swiss male mice received an intraperitoneal injection of the catalase inhibitor 3-amino-1,2,4-
triazole (AT; 01 g/kg), and 0 to 20 hr after this administration, animals received an ethanol (04 g/kg;
intraperitoneally) challenge. Thirty, 60, or 120 min after ethanol administration, plasma corticosterone
levels were determined immunoenzymatically. In addition, we tested the effects of 45 mg/kg of cyanamide
(another catalase inhibitor) and 0 to 2 mg/kg of naltrexone (nonselective opioid receptor antagonist) on
ethanol-induced enhancement in plasma corticosterone values.
Results: The present study revealed that AT boosts ethanol-induced increase in plasma corticosterone
levels in a dose- and time-dependent manner. However, it did not affect corticosterone values when
measured after administration of saline, cocaine (4 mg/kg, intraperitoneally), or morphine (30 mg/kg,
intraperitoneally). The catalase inhibitor cyanamide (45 mg/kg, intraperitoneally) also increased ethanol-
related plasma corticosterone levels. These effects of ATand cyanamide on ethanol-induced corticosterone
values were observed under treatment conditions that decreased significantly brain catalase activity. In-
deed, a significant correlation between effects of catalase manipulations on both variables was found.
Finally, we found that the administration of naltrexone enhanced the levels of plasma corticosterone after
the administration of saline or ethanol.
Conclusions: This study shows that the inhibition of brain catalase increases ethanol-induced plasma
corticosterone levels. Results are discussed together with previous findings suggesting a putative linkage
between brain ethanol metabolism and the endogenous opioid system to explain some of the neuroendo-
crine effects of ethanol.
Key Words: Brain Catalase, Ethanol, Corticosterone, Acetaldehyde, Naltrexone, Endogenous Opioid System.
E
THANOL HAS A variety of effects on the neuroendo-
crine function and, in particular, activates the HPA
axis, resulting in dose-related increases in plasma cortico-
sterone levels (Aragon et al., 1987; Ogilvie et al., 1997;
Rivier, 1999). In this regard, it has been suggested that
ethanol activates the HPA axis via central mechanisms
(reviewed by Rivier, 1996) that are dependent on the acti-
vation of the paraventricular nucleus (PVN) of the hypo-
thalamus (Ogilvie et al., 1998). This activation leads to
increased corticotrophin-releasing factor (CRF) and vaso-
pressin (VP) that act synergistically to release adrenocor-
ticotrophin hormone (ACTH) from the anterior lobe of the
pituitary. After release, ACTH induces the secretion of
glucocorticoids, such as corticosterone, from the adrenal
gland (Rivier, 1996; Ryabinin et al., 2002). However, the
precise mechanism by which ethanol activates the PVN,
thereby stimulating the HPA axis, remains unclear.
Several major neurotransmitter systems contribute to the
dynamic modulation of the PVN. Evidence has demon-
strated that the endogenous opioid system, in particular the
-endorphin projection from the hypothalamic arcuate nu-
cleus (ARC) to the PVN, exerts an inhibitory control to the
CRF-containing neurons in the PVN (Oswald and Wand,
2004; Yajima et al., 1986). According to this notion, the
administration of the nonselective opioid receptor antago-
nist naloxone induces a rise in ACTH and glucocorticoid
levels as a result of the blockade of this inhibitory compo-
nent (Oswald and Wand, 2004; Yajima et al., 1986).
From the Area de Psicobiologa, Universitat Jaume I, Castell, Spain.
Received for publication February 18, 2004; accepted September 13,2004.
This study was supported by grants from CICYT (BSO2002-00631) and
from the Red de Trastornos Adictivos, Ministerio de Sanidad y Consumo
(G03/005), Spain. R.P. was supported by a fellowship from the Agencia
Valenciana de Ciencia y Tecnologa, Generalitat Valenciana, Spain.
Reprint requests: Carlos M.G. Aragon, PhD, Area de Psicobiologa, Uni-
versitat Jaume I, Campus de Riu Sec, 12071 Castell, Spain; Fax: 34-964-
729267; E-mail: aragon@psb.uji.es.
Copyright 2004 by the Research Society on Alcoholism.
DOI: 10.1097/01.ALC.0000148107.64739.76
0145-6008/04/2812-1898$03.00/0
ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH
Vol. 28, No. 12
December 2004
1898 Alcohol Clin Exp Res, Vol 28, No 12, 2004: pp 18981906
It is interesting that it has been demonstrated that eth-
anol exposure produces a release of -endorphin from
hypothalamic neural cultures (Pastorcic et al., 1994; Reddy
et al., 1995) as well as from hypothalamic tissue explants
(de Waele et al., 1994; Gianoulakis, 1990). Moreover, re-
cent microdialysis studies have shown an increase in the
-endorphin content in ARC-projecting sites (i.e., nucleus
accumbens) (Marinelli et al., 2003; Olive et al., 2001) after
an acute ethanol administration. These studies have defin-
itively established that an acute ethanol exposure promotes
the hypothalamic release of -endorphin both in vitro and
in vivo. Therefore, the hypothalamic -endorphin secretion
derived from ethanol administration could be considered as
a candidate to participate in the mechanism by which eth-
anol modifies PVN activity.
The ethanol-induced release of -endorphin, however,
seems to be mediated by the activity of brain catalase, the
main enzymatic pathway oxidizing ethanol to acetaldehyde
in the brain (Aragon et al., 1992; Aragon and Amit, 1993;
Gill et al., 1992). Specifically, it has been shown that cata-
lase inhibition produces a significant reduction in
-endorphin release from hypothalamic cultures after eth-
anol exposure (Reddy et al., 1995). Moreover, in vivo evi-
dence of a brain catalase-mediated -endorphin release
induced by ethanol is provided because the ethanol-derived
decrease in plasma luteinizing hormone levels (an effect
directly mediated by the -endorphin release from the
ARC) is prevented by the inhibition of brain catalase ac-
tivity (Sanchis-Segura and Aragon, 2002).
Strong evidence supports the notion that brain catalase
mediates some of the psychopharmacological effects of
ethanol. Inhibition of brain catalase activity, by means of
pharmacological or genetic manipulations, results in a re-
duction of some ethanol-induced effects, including volun-
tary ethanol consumption (Aragon and Amit, 1992; Koech-
ling and Amit, 1994; Tampier et al., 1994), ethanol-
stimulated locomotion (Aragon et al., 1989; Aragon and
Amit, 1993; Correa et al., 2001; Escarabajal et al., 2000;
Pastor et al., 2002; Sanchis-Segura et al., 1999a), and
ethanol-induced conditioned taste aversion (Aragon et al.,
1985). Also, several studies have shown that the increase in
brain catalase activity boosts the locomotor-stimulating ef-
fect of ethanol (Correa et al., 1999; Sanchis-Segura et al.,
1999b). However, although participation of brain catalase
in the behavioral effects of ethanol is described extensively,
little research has assessed involvement of this enzymatic
system in other physiological effects of ethanol.
The present study was designed to elucidate the involve-
ment of brain catalase in ethanol-induced HPA axis acti-
vation. Given the precedents, two different hypothesis can
be postulated. First, because previous studies have found
that brain catalase has a role in mediating ethanol-induced
release of -endorphin from the ARC, increased ethanol-
induced HPA axis activation after brain catalase inhibition
can be expected. This effect could be understood as a result
of the decrease in the inhibitory modulation of
-endorphin at the level of PVN neurons. Second, it could
be possible that the activation of the HPA axis induced by
ethanol depends, as other ethanol effects, on its central
metabolism via brain catalase. If true, then the inhibition of
brain catalase would lead to a decrease in the ethanol-
derived HPA axis activation.
Several experiments using the catalase inhibitor 3-amino-
1,2,4-triazole (AT) were designed to assesses the role of
brain catalase in ethanol-induced HPA axis activation.
Also, we used cyanamide (CY), another brain catalase
inhibitor (Sanchis-Segura et al., 1999a), to corroborate AT
data. Finally, we explored whether the effects of the non-
selective opioid receptor antagonist naltrexone paralleled
those obtained with catalase manipulations on the same
parameters. HPA axis activation was evaluated by means of
determinations of plasma corticosterone levels, one of the
most solid indexes of the activation of this axis.
MATERIALS AND METHODS
Subjects
Four-week-old male Swiss-Albino mice, purchased from Janvier Spain,
S.A. (Barcelona, Spain), were used in this study. Animals were housed in
groups of four per cage with standard laboratory rodent chow (Panlab
S.L., Barcelona, Spain) and tap water ad libitum. Mice were acclimated for
2 weeks to the animal colony before experimentation. Because basal
corticosterone levels are age dependent (Silveri and Spear, 2004), all
animals were used at the same age (7 weeks). The colony lived in a
humidity-controlled (50%) and temperature-controlled (22 1C) envi-
ronment under a 12-hr light/dark cycle (on at 1:00 PM, off at 1:00 AM).
For CY experiments, however, animals were placed in a colony room
where the 12-hr light cycle started at 7:00 AM. Experiments were con-
ducted 2 hr after the light cycle started. Mice that were used for cortico-
sterone determination were fasted overnight. All experimental procedures
complied with the European Community Council Directive (86/609/ECC)
for the use of laboratory animals.
Drugs
AT was purchased from Sigma-Aldrich Qumica (Madrid, Spain). AT
was dissolved at a concentration of 0.125, 0.25, 0.5, or 1 g/10 ml. Those
solutions of AT were used for preparing their respective doses to maintain
a constant volume of injections (10 ml/kg body weight). CY (Sigma-
Aldrich Qumica) was dissolved at a concentration of 45 mg/10 ml. Nal-
trexone hydrochloride (Sigma-Aldrich Qumica) was prepared at a con-
centration of 1 or 2 mg/10 ml. Ethanol (Panreac S.A., Barcelona, Spain)
was diluted at 20% (v/v) from 96% solutions. Cocaine hydrochloride and
morphine sulfate (Sigma-Aldrich Qumica) were dissolved at a concentra-
tion of 4 and 30 mg/10 ml, respectively. Saline solution was administered
to control groups. All solutions were freshly prepared in saline. Injections
were administered intraperitoneally.
Procedure
Effects of AT on ethanol-induced increase in plasma corticosterone. In the
first study, the time pattern of ethanol effects on plasma corticosterone
levels in AT-pretreated animals was assessed. Sixty mice were used in this
experiment. Animals (n 10 per group) received an AT (0 or 1 g/kg)
administration and 5 hr later received an injection of 2 g/kg of ethanol.
Blood samples were collected 30, 60, or 120 min after ethanol treatment.
To evaluate effects of AT on the increase in corticosterone induced by
several doses of ethanol, we conducted a second study. Animals (a total of
80, n 10 per group) received an injection of saline or AT (1 g/kg) and
BRAIN CATALASE ACTIVITY AND PLASMA CORTICOSTERONE LEVELS 1899
5 hr received an ethanol challenge (0, 1, 2, or 4 g/kg). Corticosterone levels
were determined 60 min after ethanol injection because this interval
showed the highest effect of AT in the first experiment.
In a third study, a wide range of AT doses were used to describe further
the effect of this catalase inhibitor on ethanol-induced corticosterone
changes. Fifty animals were randomly assigned to various pretreatment
groups (n 10 per group) corresponding to various doses of AT (0, 0.125,
0.25, 0.5, or 1 g/kg). Five hours after AT pretreatment, ethanol (2 g/kg)
was administered to the animals. Plasma corticosterone levels were mea-
sured 60 min after ethanol challenge. AT dose and time conditions were
selected accordingly with previous studies (Aragon et al., 1989; Pastor et
al., 2002).
Finally, we assessed the temporal pattern of AT effects on plasma
corticosterone levels in a subsequent challenge with ethanol. Animals (a
total of 50, n 10 per group) received an injection of AT (1 g/kg) and 0,
2.5, 5, 10, or 20 hr received ethanol (2 g/kg) administration. Blood samples
were collected 60 min after ethanol treatment, and corticosterone levels
were determined.
Effects of AT on cocaine- or morphine-induced increase in plasma corti-
costerone. This experiment evaluated whether effects of AT are shared by
other drugs that can increase plasma corticosterone levels or, conversely,
are specifically related to the mechanism by which ethanol increases this
endocrine outcome. Eighty mice were used in this experiment. Saline or
AT (1 g/kg) was administered to mice (n 10/group), and 5 hr later,
animals received an injection of saline, cocaine (4 mg/kg), or morphine (30
mg/kg). One hour later, trunk blood samples were collected and cortico-
sterone levels were determined. Doses of cocaine and morphine were used
according to previous studies (Mantsch et al., 2000; Sanchis-Segura and
Aragon, 2002).
Effects of CY on ethanol-induced increase in plasma corticosterone. This
experiment was designed to assess further the role of brain catalase in
regulating ethanol-derived effects on corticosterone levels. CY dose and
time conditions paralleled previous studies in which CY has revealed an
effect on some ethanol-induced behaviors directly related to brain catalase
levels (Sanchis-Segura et al., 1999a). Therefore, animals (a total of 40; n
10 per group) received saline or CY (45 mg/kg) and 10 hr later received
an injection of saline or ethanol (2 g/kg). Blood samples were collected 60
min after the ethanol challenge.
Effects of naltrexone on ethanol-induced increase in plasma corticoste-
rone. In this experiment, the nonselective opioid receptor antagonist
naltrexone was administered to mice to explore its effects on ethanol-
induced enhancement in plasma corticosterone levels. Animals (a total of
60; n 10 per group) received an injection of saline or naltrexone (1 or
2 mg/kg) and, 30 min later, were challenged with ethanol (0 or 2 g/kg).
Samples for corticosterone determinations were collected 60 min after
ethanol administration. Naltrexone treatment conditions were used ac-
cording to previous studies demonstrating an effect of this compound on
several ethanol-induced behaviors (Sanchis-Segura et al., 2004).
Plasma Corticosterone Determination
The protocol for experiments involving corticosterone measurement
was determined from pilot studies (data not shown). According to these
data, we did not habituate the animals to handling and injections because
in our laboratory, no statistically significant differences were found in
basal levels. Moreover, our basal levels paralleled literature values of
plasma corticosterone in heterogeneous mice (Wenger et al., 2003).
Treatment injections were conducted in the colony room. Five minutes
before blood sample collection, mice were transferred from the colony
room, in their home cages, to a room where they were killed by decapi-
tation under ether anesthesia. Trunk blood samples were collected in
heparinized (15 units/ml of blood) Eppendorf tubes and centrifuged at
4000 rpm for 10 min. Supernatant was taken and stored at 20C until
corticosterone determination. Plasma corticosterone levels were mea-
sured using a commercially available enzymatic immunoassay (Rodents
Corticosterone Enzyme Immunoassay System, OCTEIA Corticosterone;
Immunodiagnostic Systems LTD, Boldon, England). The ng/ml of blood
corticosterone concentration was determined using a nonlinear (logarith-
mic) adjustment from the standard curve.
Brain Catalase Activity Determination
Encephalic activity of catalase was determined in three experiments. In
the first, several AT doses were assessed on brain catalase activity. Exper-
imentally naive mice (a total of 30; n 6 per group) were treated with AT
(0, 0.125, 0.25, 0.5, or 1 g/kg), and brain samples were collected for
determining brain catalase activity 5 hr later.
In the second study, we evaluated the effect of the time interval elapsed
after the AT administration on brain catalase activity. Thirty animals
received an injection of 1 g/kg of AT, and 0, 2.5, 5, 10, or 20 hr (n 6 per
group) after treatment, brain catalase levels were measured.
Finally, the effect of CY on brain catalase activity was also determined.
Two different groups of six animals were administered saline or CY (45
mg/kg) respectively. Brains were removed 10 hr after administration, and
catalase activity was measured.
Brain catalase activity was measured following Aebi (1974). Briefly,
animals were perfused through the heart with 50 ml of heparinized (876
units/liter) isotonic saline solution under ether anesthesia. Whole brains
were removed and homogenized in phosphate buffer [0.05 M (pH 7.0)]
with digitonin (0.01%). Supernatant aliquots from centrifuged brain ho-
mogenates (10,000 rpm for 10 min) were used to determine brain catalase
levels. Catalase activity was assayed spectrophotometrically by measuring
the decrease in absorbance of H
2
O
2
at 240 nm [()
240
0.00394 mmol
1
mm
1
].
ALDH Activity Determination
Determination of the effect of CY (0 or 45 mg/kg) on hepatic ALDH
activity was carried out to discard that the effect of CY on plasma
corticosterone levels could be a consequence of peripheral accumulation
of acetaldehyde derived from inhibited liver ALDH. Ten hours after
administration of saline or 45 mg/kg of CY, animals were killed as de-
scribed for catalase assay. After perfusion, livers were quickly excised,
rinsed in saline, blotted dry, and stored at 80C. On the day of the assay,
livers were homogenized in 0.25 M of sucrose that contained 0.1 mM of
EDTA to make 10% homogenates. The samples were mixed thoroughly
and then centrifuged at 800 g at 4C for 10 min. The clear supernatants
were used to obtain nuclei-free samples. Supernatants (1.4 ml) were then
centrifuged at 10,000 g at 4C for 10 min. The mitochondrial pellet was
resuspended in 0.4 ml of 0.25 M of sucrose1% Triton X-100. ALDH
activity was measured spectrophotometrically by following the production
of NADH at 340 nm [()
340
6.3 L mmol
1
mm
1
). Following
Gill et al. (1996), the assay mixture contained 50 mM of sodium pyro-
phosphate (pH 8.8), 1 mM of NAD, 2 M of rotenone, 0.2 mM of
4-methylpyrazole, and 1 mM of magnesium chloride. The assay mixture
was incubated with 0.1 ml of enzyme supernatant in a 25C water bath for
20 min. The reaction was started by the addition of 0.1 ml of the substrate
acetaldehyde (50 M for low K
m
). Each reaction was followed over a
10-min period. ALDH activity was expressed as nmol NADH produced/
min/mg protein.
Statistical Analysis
Data were analyzed by means of one- or two-way ANOVA. When a
significant interaction was found (p 0.05), post hoc comparisons were
made using Fishers least significant difference test. Pearsons correlation
index was used to assess the relation between plasma corticosterone levels
and brain catalase activity. CY effect on brain catalase activity and on
hepatic ALDH activity was analyzed using independent Students t test.
Statistica 6.1 (Statsoft Inc., Tulsa, OK) was used for statistical analysis.
1900 PASTOR ET AL.
RESULTS
Effects of AT on Ethanol-Induced Plasma Corticosterone
Levels
Figure 1 shows the effect of AT on ethanol-induced
plasma corticosterone values determined at different times
after ethanol (2 g/kg) administration. A two-way ANOVA
[AT treatment (0 or 1 g/kg) time after ethanol challenge
(30, 60, or 120 min)] revealed a significant effect of AT
[F
(1,51)
14.78, p 0.01] and a significant effect of the
time elapsed after the ethanol administration [F
(2,51)

10.27, p 0.01]. However, it did not show a significant
interaction (p 0.05). Thus, AT boosted the effect of
ethanol in increasing plasma corticosterone levels, and this
effect was evident 30, 60, and 120 min after ethanol
challenge.
The effect of AT on ethanol-induced corticosterone lev-
els, using several doses of ethanol, is shown in Fig. 2. The
results of a two-way ANOVA [AT treatment (0 or 1 g/kg)
ethanol dose (0, 1, 2, or 4 g/kg)] showed a significant
effect for AT factor [F
(1,74)
25.56, p 0.01] and ethanol
dose factor [F
(3,74)
9.73, p 0.01] and for their interac-
tion [F
(3,74)
2.83, p 0.05]. Pairwise comparisons dem-
onstrated that, in the saline pretreated groups, ethanol (2
and 4 g/kg) produced a significant increase in the levels of
plasma corticosterone (p 0.01). Regarding the groups
that were pretreated with AT, ethanol (1, 2, and 4 g/kg) also
produced a significant increase in corticosterone levels (p
0.05 for 1 g/kg and p 0.01 for 2 and 4 g/kg) with respect
to control groups. It is interesting that AT significantly
boosted ethanol-induced increase in plasma corticosterone
levels at the ethanol doses of 2 g/kg (p 0.01) and 4 g/kg
(p 0.05) with respect to the corresponding saline pre-
treated groups. Furthermore, pretreatment with AT did
not modify the levels of corticosterone when animals were
administered saline (p 0.05).
Figure 3 displays the effect of several AT doses on
ethanol-induced (2 g/kg) plasma corticosterone values. A
one-way ANOVA revealed a significant effect of AT [F
(4,45)
3.58, p 0.05]. Post hoc analyses showed a significant
increase in ethanol-derived plasma corticosterone when
AT was administered at the doses of 0.5 and 1 g/kg (p
0.01). Moreover, the effect of AT was dose dependent
because statistical differences were found when the highest
(1 g/kg) and the lowest (0.125 g/kg) doses of AT were
compared (p 0.01).
Figure 4 depicts the effect of the time interval between
AT and ethanol (2 g/kg) administrations on corticosterone
values. The results of a one-way ANOVA yielded a signif-
icant effect of the period of time elapsed between AT and
Fig. 1. Effect of AT on ethanol-induced plasma corticosterone levels deter-
mined at different times after ethanol administration. Bars depict mean SEM
plasmatic corticosterone values (ng/ml) for all treatment groups (n 10). Animals
received an AT administration (0 or 1 g/kg) and received an injection of ethanol (2
g/kg) 5 hr later. Blood samples were collected 30, 60, or 120 min after ethanol
administration.
Fig. 2. Effect of several doses of ethanol on ethanol-induced plasma cortico-
sterone levels in AT-treated animals. Bars depict mean SEM plasma cortico-
sterone levels (ng/ml) for all treatment groups (n 10). Animals were administered
AT (0 or 1 g/kg) and received an injection of ethanol (0, 1, 2, or 4 g/kg) 5 hr later.
Blood samples were collected 60 min after ethanol administration. *p 0.05, **p
0.01, different from respective saline group.
Fig. 3. Effect of several doses of AT on ethanol-induced plasma corticosterone
levels. For all treatment groups (n 10), bars depict mean SEM plasma
corticosterone values (ng/ml). Animals were administered AT (0, 0.125, 0.25, 0.5,
or 1 g/kg) and received a challenge with ethanol (2 g/kg) 5 hr later. Blood samples
were collected 60 min after ethanol administration. **p 0.01, significantly
different from saline-treated group.
BRAIN CATALASE ACTIVITY AND PLASMA CORTICOSTERONE LEVELS 1901
ethanol treatments [F
(4,47)
6.58, p 0.01]. Post hoc
comparisons showed that AT, when administered 5 or 10 hr
before ethanol, significantly increased (p 0.01) the
ethanol-derived increase in plasma corticosterone levels.
Effects of AT on Cocaine- or Morphine-Induced Plasma
Corticosterone Levels
Consequences of administration of AT on cocaine- or
morphine-enhanced plasma corticosterone levels are pre-
sented in Fig. 5. Regarding the cocaine experiment, a two-
way ANOVA [AT pretreatment (0 or 1 g/kg) cocaine
treatment (0 or 4 mg/kg)] yielded only a significant effect of
the cocaine factor [F
(1,40)
17.46, p 0.01]. Thus, the
ability of cocaine to increase plasma corticosterone levels
appeared regardless of pretreatment conditions (AT or
saline). The same result was found with morphine admin-
istration. A two-way ANOVA was performed taking as
main factors the AT pretreatment (0 or 1 g/kg) and the
treatment with morphine (0 or 30 mg/kg). The results of
this ANOVA showed a highly significant effect of mor-
phine treatment [F
(1,40)
69.42, p 0.01], but it revealed
neither an effect of the AT pretreatment nor their
interaction.
Effects of AT on Brain Catalase Activity
The effect of several doses of AT on brain catalase
activity is shown in Table 1. The results of a one-way
ANOVA showed that AT reduces significantly brain cata-
lase activity [F
(4,24)
12.7, p 0.01]. Thus, post hoc
analysis showed that, when compared with the control
group, AT reduced the activity of brain catalase at the
doses of 0.125 (p 0.05), 0.25, 0.5, and 1 g/kg (p 0.01).
This effect of AT was dose dependent because pairwise
comparisons revealed differences between the AT doses of
0.5 or 1 g/kg and the dose of 0.125 g/kg (p 0.01).
Table 2 represents the effect of AT on the activity of
brain catalase determined at several times after its admin-
istration. A one-way ANOVA showed an effect of the
interval of time between the AT administration and the
brain catalase determination [F
(4,26)
5.05, p 0.01]. Post
hoc analysis revealed that the administration of 1 g/kg of
AT leads to a reduction of brain catalase activity when
measured 2.5 (p 0.05), 5, and 10 hr (p 0.01) after its
Fig. 4. Effect of time interval between AT and ethanol administrations on the
levels of plasma corticosterone. Bars depict mean SEM plasma corticosterone
levels (ng/ml) for all treatment groups (n 10). Mice received an injection of AT
(1 g/kg) and 0, 2.5, 5, 10, or 20 hr later received ethanol (2 g/kg) administration.
Sixty minutes after ethanol administration, blood samples were collected. **p
0.01, different from the group that received ethanol immediately after AT injection.
Fig. 5. Effect of AT on cocaine- or morphine-induced plasma corticosterone
values. Bars depict mean SEM plasma corticosterone levels (ng/ml) for all
treatment groups (n 10). Mice received an injection of AT (0 or 1 g/kg) and 5 hr
later received cocaine (0 or 4 mg/kg; A) or morphine (0 or 30 mg/kg; B) admin-
istration. Blood samples were collected 60 min after cocaine or morphine
injections.
Table 1. Effect of Several Doses of AT on Brain Catalase Activity
AT dose
(g/kg)
Brain catalase activity (mmol H
2
O
2
degraded/min/mg protein) p
0 1.14 0.04
0.125 0.88 0.03 0.05
0.25 0.77 0.05 0.01
0.5 0.65 0.06 0.01
1 0.62 0.07 0.01
Data are mean SEM of brain catalase activity (mmol H
2
O
2
degraded/min/mg
protein; n 6 per group). Brains were obtained 5 hr after AT administration.
1902 PASTOR ET AL.
injection. Brain catalase activity returned to control levels
20 hr after the AT administration.
Effects of CY on Ethanol-Induced Plasma Corticosterone
Levels, on Brain Catalase Activity, and on Hepatic ALDH
Activity
The administration of 45 mg/kg of CY enhanced the
effect of ethanol in increasing plasma corticosterone levels.
The results of a two-way ANOVA [CY pretreatment (0 or
45 mg/kg) ethanol treatment (0 or 2 g/kg)] confirmed this
conclusion, revealing a pretreatment [F
(1,22)
20.95, p
0.01], treatment [F
(1,22)
4.12, p 0.05], and interaction
effect [F
(1,22)
4.34, p 0.05]. Post hoc comparisons
revealed that CY increased corticosterone secretion only
when ethanol was administrated (p 0.01), showing no
effect on saline-treated animals (p 0.05). Specifically,
saline-saline levels were 18.7 2.9, saline-ethanol levels
were 40.5 5.12, CY-saline levels were 21.9 6.2, and
CY-ethanol levels were 76.3 12.2 ng/ml of corticosterone
in plasma. With regard to the effects of CY on brain
catalase activity (Table 3), an independent Students t test
revealed that 45 mg/kg of CY significantly reduced the
activity of this enzyme [t
(10)
6.18, p 0.01]. Finally,
consequences of CY administration on liver low Km ALDH
activity were also shown in Table 3. Independent Students
t test [t
(9)
1.63, p 0.05] did not reveal statistical differ-
ences when saline- and CY-treated animals were
compared.
Effects of Naltrexone Pretreatment on Ethanol-Induced
Increase in Plasma Corticosterone Levels
Figure 6 shows the effect of naltrexone on plasma corti-
costerone levels determined after saline or ethanol admin-
istration. A two-way ANOVA [naltrexone pretreatment (0,
1, or 2 mg/kg) ethanol treatment (0 or 2 g/kg)] yielded a
significant effect of naltrexone [F
(2,50)
3.37, p 0.05] as
well as of ethanol [F
(1,50)
16.35, p 0.01]; however, no
interaction between main factors was found. Thus, naltrex-
one administration increased plasma corticosterone levels
in both saline- and ethanol-treated mice; however, this
effect was more potent in saline-treated animals. Indeed, in
that group, the administration of 1 or 2 mg/kg of naltrexone
increased plasma corticosterone levels up to 69 and 119%
over baseline values, respectively, whereas the same doses
of this opioid receptor antagonist produced only a 38 and
40% increase in ethanol-treated mice.
DISCUSSION
The present study demonstrates that mice that are pre-
treated with the catalase inhibitors AT and CY exhibit
higher plasma corticosterone levels after ethanol but not
saline administration. These effects were observed under
the same treatment conditions that produced a significant
decrease in brain catalase activity. Also, this study shows
that the administration of the general opioid receptor an-
tagonist naltrexone increases plasma corticosterone in both
saline- and ethanol-treated mice. On the basis of these
results and other data discussed below, we propose that the
effects of ethanol on plasma corticosterone levels are mod-
ulated by the activity of brain catalase putatively via mech-
anisms that could involve some components of the endog-
enous opioid system (e.g., -endorphins).
According to previous reports, an acute ethanol admin-
istration produced a significant increase in plasma cortico-
sterone levels (Aragon et al., 1987; Rivier, 1996, 1999). In
our study, this effect of ethanol was significantly increased
when animals were pretreated with AT. It is interesting that
this effect of AT on ethanol-induced plasma corticosterone
was found at the same dose and time conditions that pro-
duced its higher inhibition in brain catalase in the present
Table 2. Time Course for Brain Catalase Activity After AT (1 g/kg)
Administration
Hours after
AT
Brain catalase activity (mmol H
2
O
2
degraded/min/mg protein) p
0 1.07 0.03
2.5 0.71 0.03 0.05
5 0.61 0.04 0.01
10 0.63 0.04 0.01
20 0.97 0.16 NS
Data are mean SEM of brain catalase activity (mmol H
2
O
2
degraded/min/mg
protein; n 6 per group).
Table 3. Effect of CY on Brain Catalase and Liver ALDH Activity
Dose of
CY (mg/kg)
Brain catalase
activity
Liver ALDH
activity (low Km)
0 1.18 0.13 3.46 0.56
45 0.71 0.15
a
2.62 0.27
Data are mean SEM of brain catalase activity or liver ALDH (mmol H
2
O
2
degraded/min/mg protein or nmol/NADH produced/min/mg protein respectively;
n 6 per group). Brains were obtained 10 hr after CY challenge.
a
p 0.01 significantly different from saline group.
Fig. 6. Effect of naltrexone pretreatment on ethanol-induced corticosterone
levels. Bars depict mean SEM plasmatic corticosterone levels (ng/ml) for all
treatment groups (n 10). Mice received an injection of naltrexone (0, 1, or 2
mg/kg) and 30 min later received an injection of ethanol (0 or 2 g/kg). Blood
samples were collected 60 min after ethanol challenge.
BRAIN CATALASE ACTIVITY AND PLASMA CORTICOSTERONE LEVELS 1903
study. Also, under the same pretreatment conditions, AT
did not modify plasma corticosterone levels after saline,
cocaine, or morphine administration. Here, it is interesting
to emphasize that our results cannot be explained as result-
ant of AT-induced differences in blood ethanol concentra-
tions. Indeed, previous studies have shown that AT does
not alter blood ethanol levels in rodents when treated at the
doses of ethanol used in this study (Aragon et al., 1989;
Correa et al., 2001; Tampier and Quintanilla, 1991). More-
over, to date, there are no data suggesting that AT can
modify the pharmacokinetic or pharmacodynamic effects
of cocaine or morphine. Therefore, we propose that the
effects of AT on ethanol-mediated corticosterone changes
could be related to its ability to modify a central interaction
between catalase and ethanol.
This proposal was further supported by other data of the
present study. Specifically, the administration of 45 mg/kg
of CY reduced significantly brain catalase activity as well as
increased ethanol-induced plasma corticosterone levels,
again without modifying saline-mediated values. Similarly,
Kinoshita et al. (2001) demonstrated that 10 mg/kg of CY,
given 1 hr before ethanol challenge, increased HPA axis
activity induced by 1 g/kg of ethanol in rats. Nevertheless,
those authors suggested that the ability of CY to inhibit the
activity of ALDH, thus increasing peripheral acetaldehyde
concentrations, may explain the increase in ethanol-
induced HPA activation. In our study, however, ethanol
administration was given 10 hr after CY, and here we
demonstrate that activity of hepatic ALDH is not inhibited
at this time point. Therefore, converse to other studies
(Kinoshita et al., 2001), the ability of CY to enhance the
effects of ethanol on plasma corticosterone showed in the
present report seems to be related to its inhibitory effect on
brain catalase. Supporting this notion, we found an inverse
significant Pearsons correlation between the effects of AT
and CY on brain catalase activity and ethanol-induced
plasma corticosterone values (r
2
0.887, p 0.001).
Therefore, the conclusion so far is that catalase, through a
cerebral and ethanol-specific mechanism, affects at least
some of the neural actions of ethanol that determine
plasma corticosterone levels.
Relative to the effects of ethanol, the role of catalase has
been linked to its demonstrated ability to produce acetal-
dehyde in the brain (for a review, see Zimatkin and Dei-
trich, 1997). In this regard, whereas most of the neuro-
chemical actions of acetaldehyde remain unexplored, it has
been demonstrated that acetaldehyde formation via cata-
lase is necessary to observe a -endorphin release from the
hypothalamic ARC after ethanol exposure (Pastorcic et al.,
1994; Reddy and Sarkar, 1993; Reddy et al., 1995). Here,
we propose that this observation could probably explain the
results of the present study.
Plasma corticosterone levels initially depend on
corticotrophin-releasing hormone (CRH) neurons in the
PVN. These neurons receive both excitatory (serotonergic/
noradrenergic pathways) and inhibitory (mediated by
GABA/-endorphins) inputs from several brain areas, in-
cluding the ARC (Calogero, 1995; Oswald and Wand, 2004;
Tsagarakis et al., 1990), and the final status of those neu-
rons determine the release of ACTH and, consequently,
plasma corticosterone levels. Ethanol promotes an increase
in the activity of this axis through a neural mechanism that
remains unidentified and that seems to be independent of
brain catalase activity. However, ethanol administration
also leads to functional changes in some neurotransmitter
systems (-endorphins but perhaps also GABA) that simul-
taneously promote a negative modulation of its own exci-
tatory effects on the HPA. In agreement with this proposal,
we found that the administration of a general opioid re-
ceptor antagonist such as naltrexone enhanced HPA axis
activation in ethanol- but also in saline-treated mice. Ac-
cording to previous reports (Oswald and Wand, 2004; Ya-
jima et al., 1986), the effect of naltrexone in saline-treated
animals can be understood as resultant of the blockade of
the inhibitory effects of -endorphins tonically released
from the ARC over the CRH-synthesizing neurons located
at the PVN. In the case of ethanol and similar to the effects
of naltrexone on ethanol-induced changes in blood lutein-
izing hormone levels (Cicero et al., 1982), the increase in
-endorphins release from the ARC promoted by ethanol
(Gianoulakis, 1990) partially counteracted the effect of this
opioid receptor antagonist and, as expected, produced a
smaller increase in plasma corticosterone levels than in
saline-treated mice. This rationale can be initially surpris-
ing given the ability of morphine, an agonist at opioid
receptors, to increase corticosterone levels. However, it
should be noted that morphine produces this effect acting
at the but not receptor (Roy et al., 2001) and that low
doses of naltrexone as those used in the present study (1
and 2 mg/kg) affect the activity only of and opioid
receptor subtypes (Froehlich et al., 1991), those where
-endorphins preferentially bind (Herz, 1997). Thus, nal-
trexone administration, by blocking the / opioid recep-
tors, could prevent the inhibitory effect of -endorphins
released from the ARC over CRH-containing neurons of
the PVN and enhance the rise of corticosterone observed
after ethanol administration.
The effects of naltrexone on ethanol-induced increases in
plasma corticosterone levels showed a noteworthy parallel-
ism with those observed in the present study after the
administration of catalase inhibitors such as CY or AT.
Indeed, on the basis of evidence provided by previous
studies (Pastorcic et al., 1994; Reddy and Sarkar, 1993;
Reddy et al., 1995), a functional relationship between brain
catalase and -endorphins could be proposed. Thus, the
findings of the present study might be understood as a
result of a brain catalasedependent mechanism involving
acetaldehyde formation and, in a second step, -endorphin
release from the ARC, which finally interact with opioid
receptors at the CRH-releasing neurons at the PVN. In
other words, either catalase inhibition (which prevents the
release of -endorphins from the ARC after ethanol ad-
1904 PASTOR ET AL.
ministration) or the blockade of / opioid receptors would
lead to a decrease in the inhibitory control of these pep-
tides over CRH neurons at the PVN and, in consequence,
an increase in blood corticosterone levels. In this respect, it
is noteworthy that we have previously proposed a similar
neurofunctional system as responsible for the effects of
catalase manipulations on the changes in blood luteinizing
hormone levels induced by ethanol (Sanchis-Segura and
Aragon, 2002).
In summary, ethanol promotes an increase in plasma
corticosterone levels by a presently unknown excitatory
central mechanism that does not seem to be mediated by
brain catalase activity. However, brain catalase seems to
modulate negatively the HPA activation produced by eth-
anol. Furthermore, as proposed in previous studies (Miquel
et al., 2003; Sanchis-Segura and Aragon, 2002), the
catalase-dependent mechanisms may be related to ethanol-
induced activation of the endogenous opioid system, spe-
cially -endorphin neurons in the ARC. Future studies will
be devoted to explore further this putative relationship
between the results of ethanol oxidation via catalase (i.e.,
acetaldehyde formation) and the endogenous opioid
system.
ACKNOWLEDGEMENTS
We gratefully acknowledge the technical assistance provided by
A. Dosda and G. Caballer and the linguistic revision by Carrie M.
Leslie.
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