Reduction in the anxiolytic effects of ethanol by centrally formed acetaldehyde: the role of catalase inhibitors and acetaldehyde-sequestering agents.

RATIONALE: Considerable evidence indicates that brain ethanol metabolism mediated by catalase is involved in modulating some of the behavioral and physiological effects of this drug, which suggests that the first metabolite of ethanol, acetaldehyde, may have central actions. Previous results have shown that acetaldehyde administered into the lateral ventricles produced anxiolysis in a novel open arena in rats. OBJECTIVES: The present studies investigate the effects of centrally formed acetaldehyde on ethanol-induced anxiolysis. MATERIALS AND METHODS: The effects of the catalase inhibitor sodium azide (SA; 0 or 10 mg/kg, IP) on ethanol-induced anxiolysis (0.0, 0.5, or 1.0 g/kg, IP) were evaluated in CD1 mice in two anxiety paradigms, the elevated plus maze and the dark/light box. Additional studies assessed the effect of the noncompetitive catalase inhibitor 3-amino-1,2,4-triazole (AT; 0.5 g/kg, IP) and the acetaldehyde inactivation agent D: -penicillamine (50 mg/kg, IP) on the plus maze. RESULTS: SA reduced the anxiolytic effects of ethanol on several parameters evaluated in the elevated plus maze and in the dark/light box. In the plus maze, AT completely blocked and D-penicillamine significantly reduced the anxiolytic properties of ethanol. CONCLUSIONS: Thus, when cerebral metabolism of ethanol into acetaldehyde is blocked by catalase inhibitors, or acetaldehyde is inactivated, there is a suppressive effect on the anxiolytic actions of ethanol. These data provide further support for the idea that centrally formed or administered acetaldehyde can contribute to some of the psychopharmacological actions of ethanol, including its anxiolytic properties.

Lead-induced catalase activity differentially modulates behaviors induced by short-chain alcohols.

Acute lead administration produces a transient increase in brain catalase activity. This effect of lead has been used to assess the involvement of brain ethanol metabolism, and therefore centrally formed acetaldehyde, in the behavioral actions of ethanol. In mice, catalase is involved in ethanol and methanol metabolism, but not in the metabolism of other alcohols such as 1-propanol or tert-butanol. In the present study, we assessed the specificity of the effects of lead acetate on catalase-mediated metabolism of alcohols, and the ability of lead to modulate the locomotion and loss of the righting reflex (LRR) induced by 4 different short-chain alcohols. Animals were pretreated i.p. with lead acetate (100 mg/kg) or saline, and 7 days later were injected i.p. with ethanol (2.5 or 4.5 g/kg), methanol (2.5 or 6.0 g/kg), 1-propanol (0.5 or 2.5 g/kg) or tert-butanol (0.5 or 2.0 g/kg) for locomotion and LRR, respectively. Locomotion induced by ethanol was significantly potentiated in lead-treated mice, while methanol-induced locomotion was reduced by lead treatment. The loss of righting reflex induced by ethanol was shorter in lead-treated mice, and lead produced the opposite effect in methanol-treated mice. There was no effect of lead on 1-propanol or tert-butanol-induced behaviors. Lead treatment was effective in inducing catalase activity and protein both in liver and brain. These results support the hypothesis that the effects of lead treatment on ethanol-induced behaviors are related to changes in catalase activity, rather than some nonspecific effect that generalizes to all alcohols.

Stress-ethanol interaction: involvement of endogenous opioid mechanisms.

Stress effects vary with different environmental situations or stress intensities. The effects of restraint stress on locomotion and or corticosterone were examined. Rats were restrained for 0, 15, 30, 60, 90 or 120 min, subsequent locomotion was measured for 10 min. Rats were also sacrificed for corticosterone determinations. Restraint stress affects both variables. Locomotion was recorded in rats pretreated with naltrexone or vehicle prior to restraint of 15 or 60 min. Naltrexone influenced the effects of stress differentially. It did not affect the results following 15 min of restraint but suppressed locomotion after 60-min restraint to a level comparable to that found after 15 min. Treatment with ethanol (1.0, 1.5, 2.0 g/kg) prior to 15 or 60 min of restraint resulted in the prevention of hypomotility induced by 15-min stress. It also interacted nonadditively with 15-min stress on corticosterone release. No such interaction occurred with 60 min stress. Also, naltrexone made it possible to block the effect of ethanol on restraint-induced hypomotility. Results describe stress as a nonunitary concept. Its effects tend to vary with its duration. The differential interaction of stress with naltrexone and ethanol depending on its duration supports the above notion. Results further suggest recruitment of opioid systems in long duration stress (60 min).

The effect of 3-amino-1,2,4-triazole on voluntary ethanol consumption: evidence for brain catalase involvement in the mechanism of action.

The effects of the catalase inhibitor, 3-amino-1,2,4-triazole (AT), on maintenance of voluntary consumption of ethanol was tested in male Long-Evans rats. AT produced a dose-dependent reduction in ethanol intake but did not affect total fluid consumption. AT also produced a dose-dependent inhibition of brain catalase lasting throughout the drinking period. These results suggest a role for brain catalase in determining the level of ethanol intake in rats.

Acetaldehyde may mediate reinforcement and aversion produced by ethanol. An examination using a conditioned taste-aversion paradigm.

Groups of water-deprived rats were exposed to acetaldehyde, ethanol or vehicle control. On the conditioning day, the animals were first presented with a solution of saccharin after which the animals that were exposed to acetaldehyde received ethanol and those exposed to ethanol received acetaldehyde. Saccharin was again presented on three more occasions (testing days) without injection of drug. Using the percentage change in saccharin consumed from the first presentation as a measure of aversion, it was found that exposure to acetaldehyde blocked the taste aversion conditioned by ethanol. Animals exposed to ethanol showed no aversion to the saccharin which was paired with a small dose of acetaldehyde, indicating a symmetrical relationship between ethanol and acetaldehyde at this dose. However, exposure with ethanol did not block the aversion produced by conditioning with larger doses of acetaldehyde. These results suggest that the mechanism underlying the smaller dose of the taste aversion conditioned with acetaldehyde may be central while the mechanism underlying the larger dose is probably peripheral.

A time-dependent biphasic effect of an acute ethanol injection on 3-methoxy 4-hydroxyphenylethylene glycol sulfate in rat brain.

The present experiment demonstrated that acute administration of ethanol appeared to have a biphasic effect on the accumulation of 3-methoxy-4-hydroxyphenylethylene glycol sulfate (MHPG-SO4) in rat brain. The magnitude of these alterations in MHPG-SO4 levels was also observed to be highly correlated with peripheral blood ethanol levels. Since levels of MHPG-SO4 are considered to be an index of noradrenergic activity, the findings suggest that ethanol may affect norepinephrine activity in a specific dose- and time-dependent manner. These results are discussed in reference to previous reports describing apparent divergent effects on norepinephrine. Possible mechanisms for the biphasic actions are also suggested.

Ethanol metabolism in rat brain homogenates by a catalase-H2O2 system.

Homogenates of perfused rat brains incubated in the presence of ethanol (50-100 mM) and glucose (10 mM) were found to oxidize ethanol to acetaldehyde. The addition of glucose oxidase, a known hydrogen peroxide generator, to the incubation medium, significantly (P less than 0.05) increased the generation of acetaldehyde. The presence in the incubation medium of metyrapone, an inhibitor of cytochrome P450, or pyrazole, an alcohol dehydrogenase inhibitor, did not affect the levels of acetaldehyde obtained. Conversely, the presence of 3-amino-1,2,4-triazole, a known catalase inhibitor, induced a concentration-dependent reduction of the amount of acetaldehyde generated after incubation, even in the presence of glucose oxidase. Homogenates of perfused brains of rats treated with 3-amino-1,2,4-triazole or cyanamide (another H2O2-dependent catalase blocker) also showed a dose-dependent reduction of the acetaldehyde obtained. These findings support the notion that a catalase-mediated oxidation of ethanol is present in rat brain homogenates. It is suggested that this local oxidation of ethanol may have important biological implications. The data of both studies increase support for the notion that acetaldehyde is produced directly in the brain and that it may be the agent mediating some of the psychopharmacological properties of ethanol and be one of the factors determining the propensity of an animal to voluntarily consume ethanol.

Dose- and time-dependent effect of an acute 3-amino-1,2,4-triazole injection on rat brain catalase activity.

The results presented in this study demonstrate a progressive inhibition of rat brain catalase activity by AT in vivo. Furthermore, the inhibition of brain catalase by AT demonstrates the presence of hydrogen peroxide in brain, since AT inhibits catalase in the presence of this compound. The rate of inhibition of catalase seems to be dependent upon the rate by which H2O2 is generated. A time course study showed slower onset of the inhibition of brain as compared to liver catalase, possibly reflecting tissue hydrogen peroxide levels or, alternatively, a rate-limiting penetration of AT into brain and into the catalase compartment. The presence of AT in brain was confirmed over the time period of the observed inhibition of brain catalase. Catalase inhibitors are of particular interest in the study of the physiological role of catalase. This study further supports the use of AT in investigations designed to further understand the role of brain catalase.

Catalase activity measured in rats naive to ethanol correlates with later voluntary ethanol consumption: possible evidence for a biological marker system of ethanol intake.

Catalase activity in blood collected from young rats naive to ethanol (65 days) was significantly and positively correlated with later voluntary consumption of ethanol. Catalase activity levels were also correlated with catalase activity in brain and blood sampled after exposure to ethanol. The results obtained in the present study extend and confirm earlier findings (Aragon et al. 1985c) that brain catalase activity and voluntary ethanol intake are unidirectionally and causally related. The results also suggest that brain catalase activity may be part of an enzymatic system controlling the production and elimination of acetaldehyde in brain. This system may be a biological marker system mediating the affinity of organisms to ingest ethanol.

Involvement of endogenous opioid mechanisms in the interaction between stress and ethanol.

The involvement of endogenous opioid mechanisms in the interaction between stress and ethanol was investigated in the rat. Animals were pretreated with naltrexone (10 mg/kg) or saline 3 h before a second injection consisting of ethanol (1.0 g/kg) or saline. They were then restrained for 15 or 60 min or left in home cages for an equivalent amount of time. After restraint, animals were either subjected to an open-field test or decapitated to collect blood for corticosterone determinations. Locomotor depression was found to be induced by 15 but not 60 min restraint. In naltrexone-treated animals, however, 60 min restraint was also found to induce locomotor depression. Ethanol pretreatment was found to block the locomotor depression induced by 15 min restraint. Such an interaction was in turn antagonized by naltrexone. In the 15 min condition, stress and ethanol were also found to interact in their effects on plasma levels of corticosterone. Naltrexone did not alter any effects of the stressors on corticosterone levels. These results provide support for the involvement of endogenous opioid mechanisms in the interaction of stress and ethanol at a behavioural level.

Ethanol-induced CTA mediated by acetaldehyde through central catecholamine activity.

The possible involvement of catecholamines (CA) in the mediation of acetaldehyde's conditioned taste aversion (CTA) was examined by testing the effects of alpha-methyl-para-tyrosine (AMPT, a tyrosine hydroxylase inhibitor) on the CTAs produced by acetaldehyde. AMPT blocked the acquisition of the CTA normally produced by a low dose of acetaldehyde (0.2 g/kg), but had no significant effect on CTA produced by a high dose of acetaldehyde (0.3 g/kg). In a second study, acetaldehyde's role in the CTA produced by ethanol was investigated using the pre-exposure conditioned taste aversion paradigm. Pre-exposure to acetaldehyde (both doses) blocked the ethanol CTAs but when pre-exposure with acetaldehyde was coupled with AMPT, only the larger dose of acetaldehyde blocked the ethanol aversion. These results suggest that while the CTA to the low dose of acetaldehyde may be primarily central and catecholamine-mediated, the mechanism underlying the high dose CTA is probably peripheral and emetic in nature. These findings support the conclusion that acetaldehyde may be mediating many of the actions of ethanol.

Effect of taurine on ethanol-induced changes in open-field locomotor activity.

In the present investigation we questioned whether taurine antagonized the effects of ethanol on motor activity measured in the open field. Ten minutes following simultaneous administration (IP) of ethanol (1.0, 1.5, 2.0 and 2.5 g/kg) or saline and taurine (30, 45 and 60 mg/kg) or saline, mice were placed in open field chambers and locomotor activity was measured during a 10 min testing period. A significant interaction was found between taurine and ethanol. Taurine-treated mice displayed lower motor excitation with the 1.0 g/kg dose of ethanol than the saline group treated with the same dose of ethanol. However at the 2.0 g/kg ethanol dose, taurine-treated mice demonstrated higher motor activity than the saline treated mice, once again, treated with the same dose of ethanol. No differences in blood ethanol levels were observed between the two groups. In a second study, taurine administration (30, 45 and 60 mg/kg) did not show any effect on d-amphetamine-induced enhancement of locomotor activity (1, 2, and 3 mg/kg). Data from this study demonstrated an interaction between taurine and ethanol in their effect on locomotor activity in the open field.

Higher correlation of ethanol consumption with brain than liver aldehyde dehydrogenase in three strains of rats.

Voluntary ethanol consumption and high Km (mM range) brain and liver aldehyde dehydrogenase (ALDH) activity were measured in male rats of the Long-Evans, Wistar and Sprague-Dawley strains. The total amounts of ethanol consumed by the three strains did not differ significantly, nor did the levels of cerebral ALDH activity. Levels of brain ALDH did not differ as a function of ethanol exposure and across strains. Levels of ethanol consumption correlated better with levels of brain than liver aldehyde-oxidizing capacity, which were tested separately for each strain and also combining all the animals. Inherent variation in brain ALDH may be a biochemical counterpart of observed differences in voluntary ethanol intake within strains.

Cyanamide reduces brain catalase and ethanol-induced locomotor activity: is there a functional link?

The present study was designed in an attempt to assess a previously suggested role of brain catalase activity in ethanol-induced behaviour by examining ethanol-induced locomotor activity in cyanamide-treated mice. Mice were pretreated with IP injections of the catalase inhibitor cyanamide (3.75, 7.5, 15, 30 or 45 mg/kg) or saline. Following this treatment, animals in each group received IP injections of ethanol (0.0, 1.6, 2.4 or 3.2 g/kg) and locomotion was recorded. Several time intervals (0, 5, 10, 15, 20 or 25 h) between the two treatments were also evaluated. Results indicated that cyanamide administration produced a dose-dependent decrease in ethanol-induced locomotor activity that depends on the time between treatments. However, cyanamide did not change spontaneous or d-amphetamine-induced locomotor activity. Moreover, an additive effect of cyanamide and another brain catalase inhibitor, 3-amino-1,2,4-triazole (AT), on the reduction of ethanol-induced locomotor activity was observed. Perfused brain homogenates of mice treated with cyanamide, AT or cyanamide+AT showed a significant reduction of brain catalase activity. The dose and time patterns of both effects were closely related and a significant correlation between them was obtained. These results suggest that cyanamide could reduce locomotor activity through its inhibition of brain catalase, giving further support to the notion that brain catalase may be an important regulator of some ethanol-induced behavioural effects.

Lession on the hypothalamic arcuate nucleus by estradiol valerate results in a blockade of ethanol-induced locomotion.

It has been suggested that the endogenous opioid system, especially b-endorphins (b-ep), can play a key role in the behavioral effects of ethanol. A single injection of estradiol valerate (EV) produces a neurotoxic effect on the b-endorphin cell population of the hypothalamic arcuate nucleus. In the present study we questioned whether mice pretreated with EV, exhibit any alterations in ethanol-induced behavioral effects. Female Swiss mice were pretreated with EV (2 mg/0.2 ml per mice) or vehicle and, 8 weeks later, these animals were challenged with ethanol (0.0-3.2 g/kg). Immediately after ethanol injection, mice were placed in the open field chambers and locomotor activity was assessed. EV administration did not produce any change in spontaneous locomotor activity but, conversely, blocked the locomotor activity induced by low (0.8 g/kg) and moderate (1.6 or 2.4 g/kg) doses of ethanol. Interestingly, the behavioral effects of higher doses of ethanol on locomotor activity as well as on the duration of the loss of righting reflex were unaffected by EV. Moreover, neither rota-rod performance or blood ethanol levels were affected by EV. In a second study, the effects of EV pre-treatment on caffeine- and 1-propanol-induced locomotor activity was tested. No differences were observed between groups in caffeine- or 1-propanol-induced locomotion. The results of the present study indicate that EV blocks ethanol-induced locomotor activity and that this effect can not be related with any difference in ethanol levels or nonspecific motor impairment. Furthermore, they suggest that b-ep containing neurons of the hypothalamic arcuate nucleus may play a role in some, but not all, behavioral effects of ethanol.

Influence of brain catalase on ethanol-induced loss of righting reflex in mice.

The effect of lead acetate and 3-amino-1, 2, 4-triazole (AT) on ethanol-induced loss of righting reflex (LORR) and brain catalase activity was studied in an attempt to confirm earlier observations on the involvement of catalase in ethanol-induced effects. Lead acetate (0 or 100 mg/kg) or AT (0 or 500 mg/kg) was injected (acutely) into mice 7 days or 5 h before testing. Other mice were exposed to drinking fluid containing 500 ppm lead acetate for 60 days. On the test day, mice received an intraperitoneal injection of ethanol (4.0 or 4.5 g/kg) and the duration of LORR was recorded. Acute lead-treated animals demonstrated a reduction in the duration of the LORR. However, both chronic administration of lead acetate and AT treatment increased the duration of ethanol-produced LORR. Furthermore, brain catalase activity in acute lead pretreated animals showed a significant induction, whereas it was reduced in chronic lead and AT treated mice. These results suggest that brain catalase activity, and by implication centrally formed acetaldehyde, may modulate ethanol-induced LORR.

Blockade of ethanol induced conditioned taste aversion by 3-amino-1,2,4-triazole: evidence for catalase mediated synthesis of acetaldehyde in rat brain.

This investigation seeks to present evidence for the oxidation of ethanol in the brain via the peroxidatic activity of catalase and simultaneously provide evidence for the role of central acetaldehyde (ACH) in the mediation of an ethanol-induced conditioned taste aversion (CTA). Ethanol is capable of inducing a conditioned taste aversion. Pretreatment with the catalase inhibitor, 3-amino-1,2,4-triazole (AT), shows an attenuation of this ethanol-induced CTA. Animals receiving ethanol injections showed a CTA to a novel solution paired with a drug administration, while ethanol injected animals pretreated with AT did not show a CTA to ethanol administration. This effect of AT appears to be specific to the effects of ethanol as CTA's to morphine and lithium chloride were not affected by AT pretreatment. Peripheral levels of ethanol were the same in all animals regardless of pretreatment indicating that AT had no effect on peripheral levels of ethanol. These data increase support for the notion that acetaldehyde is produced directly in the brain and that it may be the agent mediating some of the psychopharmacological properties of ethanol.

The nitric oxide synthase inhibitor NW-nitro-L-arginine methylester attenuates brain catalase activity in vitro.

Nitric oxide has been implicated in mediating the neurotoxic effects of ischemia in the brain. However, studies of the effects of nitric oxide inhibition with nitric oxide synthase inhibitors have provided controversial results. One of the reasons for the controversy may be related to the specificity of the nitric oxide synthase inhibitors, such as Nw-nitro-L-arginine methylester (L-NAME), which has recently been questioned. The present work investigated the possible interaction of L-NAME with the enzyme catalase in vitro. Catalase is an iron containing enzyme which could potentially interact with the iron-binding groups of L-NAME. Since the normal function of catalase in the brain is to remove excess hydrogen peroxide, the inhibition of this process could have potentially toxic effects. L-NAME was found to attenuate the catalase inhibiting effects of the known catalase inhibitor cyanamide in vitro, suggesting a competition between cyanamide and L-NAME for catalase. In addition, L-NAME by itself attenuated catalase activity in vitro. These results indicate that in addition to inhibiting nitric oxide synthase, L-NAME may have effects on catalase activity.

The ethanol-induced open-field activity in rodents treated with isethionic acid, a central metabolite of taurine.

The effect of isethionic acid, a central metabolite of taurine, on ethanol-induced locomotor activity was investigated in rodents. Ten minutes following an (i.p.) simultaneous administration of ethanol (0.0, 1.5, 2.0, 2.5, 3.0, 3.5 g/kg) and isethionic acid (0.0, 22.5, 45.0, 90.0, 180.0 mg/kg), mice were placed in the open-field chambers and locomotor activity was measured during a ten-minute testing period. A significant interaction was found between isethionic acid and ethanol. Isethionic acid pre-treated mice (45.0, 90.0 and 180.0 mg/kg) showed a higher locomotor activity than the saline group at 2.5 and 3 g/kg of ethanol. In a second study, isethionic acid (45 mg/kg) and ethanol (1 g/kg) were simultaneously injected to rats. Ten minutes after the two treatments, rats were placed in the open-field chamber for a 30-minute period. The depressant effects that ethanol produced on rat locomotion were amplified by the same dose of isethionic acid as it affected ethanol-induced locomotion in mice (45 mg/kg). However, isethionic acid did not change the spontaneous locomotion at any of the doses tested in mice or rats. Since no differences in blood ethanol levels were detected in both mice and rats, the interaction between isethionic acid's action and ethanol-related locomotion does not seem to be due to different rates of absorption of ethanol or any other pharmacokinetic process related to ethanol levels. The current study displayed that isethionic acid, administered intraperitoneally, behaves in a similar way to its immediate precursor, taurine, by amplifying ethanol-induction of the locomotor activity.

Brain catalase activity is highly correlated with ethanol-induced locomotor activity in mice.

It has been demonstrated that acute administration of lead to mice enhances brain catalase activity and ethanol-induced locomotion. These effects of lead seem to be related, since they show similar time courses and occur at similar doses. In the present study, in an attempt to further evaluate the relation between brain catalase activity and lead-induced changes in ethanol-stimulated locomotion, the interaction between lead acetate and 3-amino-1H,2,4-triazole (AT), a well-known catalase inhibitor, was assessed. In this study, lead acetate or saline was acutely injected intraperitoneally to Swiss mice at doses of 50 or 100 mg/kg 7 days before testing. On the test day, animals received an intraperitoneal injection of AT (0, 10, or 500 mg/kg). Five hours following AT treatment, ethanol (0.0 or 2.5 g/kg, ip) was injected and the animals were placed in open-field chambers, in which locomotion was measured for 10 min. Neither lead exposure nor AT administration, either alone or in combination, had any effect on spontaneous locomotor activity. AT treatment reduced ethanol-induced locomotion as well as brain catalase activity. On the other hand, ambulation and brain catalase activity were significantly increased by both doses of lead. Furthermore, AT significantly reduced the potentiation produced by lead acetate on brain catalase and on ethanol-induced locomotor activity in a dose-dependent manner. A significant correlation was found between locomotion and catalase activity across all test conditions. The results show that brain catalase activity is involved in the effects of lead acetate on ethanol-induced locomotion in mice. Thus, this study confirms the notion that brain catalase provides the molecular basis for understanding some of the mechanisms of the action of ethanol in the central nervous system.