C.M.G. Aragon

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.

Impact of TLR4 on behavioral and cognitive dysfunctions associated with alcohol-induced neuroinflammatory damage

Toll-like receptors (TLRs) play an important role in the innate immune response, and emerging evidence indicates their role in brain injury and neurodegeneration. Our recent results have demonstrated that ethanol is capable of activating glial TLR4 receptors and that the elimination of these receptors in mice protects against ethanol-induced glial activation, induction of inflammatory mediators and apoptosis. This study was designed to assess whether ethanol-induced inflammatory damage causes behavioral and cognitive consequences, and if behavioral alterations are dependent of TLR4 functions. Here we show in mice drinking alcohol for 5months, followed by a 15-day withdrawal period, that activation of the astroglial and microglial cells in frontal cortex and striatum is maintained and that these events are associated with cognitive and anxiety-related behavioral impairments in wild-type (WT) mice, as demonstrated by testing the animals with object memory recognition, conditioned taste aversion and dark and light box anxiety tasks. Mice lacking TLR4 receptors are protected against ethanol-induced inflammatory damage, and behavioral associated effects. We further assess the possibility of the epigenetic modifications participating in short- or long-term behavioral effects associated with neuroinflammatory damage. We show that chronic alcohol treatment decreases H4 histone acetylation and histone acetyltransferases activity in frontal cortex, striatum and hippocampus of WT mice. Alterations in chromatin structure were not observed in TLR4(-/-) mice. These results provide the first evidence of the role that TLR4 functions play in the behavioral consequences of alcohol-induced inflammatory damage and suggest that the epigenetic modifications mediated by TLR4 could contribute to short- or long-term alcohol-induced behavioral or cognitive dysfunctions.

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.

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 CTAs 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.

Catalase and the production of brain acetaldehyde: a possible mediator of the psychopharmacological effects of ethanol

This review represents an attempt to assess the available data on the role of catalase in the mediation of the behavioral actions of ethanol and the regulation of voluntary ethanol consumption. It is argued that acetaldehyde may be formed in brain through the peroxidatic activity of catalase. Furthermore, acetaldehyde formed centrally through the activity of this enzyme, may be responsible, at least in part, for some of the motivational, behavioral and neurotoxic effects of ethanol.

Differences in ethanol-induced behaviors in normal and acatalasemic mice: Systematic examination using a biobehavioral approach

In studies designed to further examine the previously reported involvement of catalase in ethanol-induced effects, we attempted to confirm earlier observations by using normal (C3H-N) and acatalasemic (C3H-A) mice. These mice are identical in every respect and differ only in their catalase activity. Data suggested that the application of 3-amino-1,2,4-triazole (AT), a catalase inhibitor, to both substrains of mice resulted in a proportional decrease in motor activity, thus supporting our earlier observations. We also showed that this effect was specific to ethanol because AT did not have any effect on cocaine-induced motor activity in both substrains. Contrary to the effects of ethanol, these substrains did not differ in motor activity in response to cocaine. In an additional study, we observed that acatalasemic mice differed from the normals in their pattern of voluntary ethanol consumption. Acatalasemic mice consumed more ethanol but only when it was presented in the range of concentrations between 12 and 18%. Finally, we also obtained data suggesting that acatalasemic mice have longer duration of sleep time following ethanol administration compared to normals. Catalase activity was measured in both substrains. Results, once again, confirmed earlier data that the substrains differ in this activity and that AT further decreases brain catalase activity in both mice. Finally, when brain homogenates derived from both substrains were incubated with ethanol significant differences in the amount of generated acetaldehyde were found between the two mice strains. Together, these results provide strong support for the involvement of brain catalase in a variety of ethanol-induced behavioral effects.

Cytokines and chemokines as biomarkers of ethanol-induced neuroinflammation and anxiety-related behavior: role of TLR4 and TLR2.

Recent evidence supports the influence of neuroimmune system activation on behavior. We have demonstrated that ethanol activates the innate immune system by stimulating toll-like receptor 4 (TLR4) signaling in glial cells, which triggers the release of inflammatory mediators and causes neuroinflammation. The present study aimed to evaluate whether the ethanol-induced up-regulation of cytokines and chemokines is associated with anxiety-related behavior, 24 h after ethanol removal, and if TLR4 or TLR2 is involved in these effects. We used WT, TLR4-KO and TLR2-KO mice treated with alcohol for 5 months to show that chronic ethanol consumption increases the levels of cytokines (IL-1β, IL-17, TNF-α) and chemokines (MCP-1, MIP-1α, CX3CL1) in the striatum and serum (MCP-1, MIP-1α, CX3CL1) of WT mice. Alcohol deprivation for 24 h induces IFN-γ levels in the striatum and maintains high levels of some cytokines (IL-1β, IL-17) and chemokines (MIP-1α, CX3CL1) in this brain region. The latter events were associated with an increase in anxiogenic-related behavior, as evaluated by the dark and light box and the elevated plus maze tests. Notably, mice lacking TLR4 or TLR2 receptors are largely protected against ethanol-induced cytokine and chemokine release, and behavioral associated effects during alcohol abstinence. These data support the role of TLR4 and TLR2 responses in neuroinflammation and in anxiogenic-related behavior effects during ethanol deprivation, and also provide evidence that chemokines and cytokines can be biomarkers of ethanol-induced neuroimmune response.

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 ond-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.

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.

Ethanol-induced motor activity in normal and acatalasemic mice

The role of brain catalase in modulating the psychopharmacological effects of ethanol was investigated by examining ethanol induced motor activity in normal, C3H-N, and a corresponding group of acatalasemic C3H-A, mice. Following administration of one of three doses of ethanol (0.8, 1.6, and 3.2 g/kg) or saline, mice were placed in open field chambers and locomotor and rearing activity was measured during a 10-min testing period. A significant increase in locomotor activity was recorded in both groups of mice at lower doses of ethanol, while the higher dose produced a marked depression. Normal mice demonstrated more locomotor activity than acatalasemic mice at all ethanol doses. No differences between both groups of mice were observed in rearing activity. Also, no differences in blood ethanol levels were observed between the two substrains. Brain and liver residual catalase activity in the acatalasemic mice was found to be 40% and 50%, respectively, of normal mice. Furthermore, evidence for possible involvement of the peroxidatic activity in ethanol-induced motor activity is presented. These results suggest a role for centrally formed acetaldehyde as a factor mediating some of ethanols psychopharmacological effects.