Hydrogen peroxide (H2O2) is the cosubstrate used by catalase to form the catalase-H2O2 system (known as Compound I), which is the major pathway of brain ethanol (EtOH) metabolism (Zimatkin et al., 2006). It has been postulated that central EtOH metabolism by the activity of this enzymatic complex mediates some of the psychopharmacological effects produced by this alcohol (Deng and Deitrich, 2008). Previous research has demonstrated that individual differences in the activity of brain catalase in rats of the same strain correlate positively with their voluntary EtOH intake (Amit and Aragon, 1988; Aragon et al., 1985). Moreover, it has been shown that strains of rats with higher brain catalase activity drink more EtOH than other strains with a lower activity of this enzyme in the central nervous system (CNS) (Gill et al., 1996a). In another set of reports, it has been observed that the administration of the brain catalase inhibitor 3-amino-1,2,4-triazole (AT) prevents voluntary EtOH consumption in rats (Aragon and Amit, 1992; Tampier et al., 1995). Existing data also showed that rats treated with an anticatalase shRNA lentiviral vector, which inhibits catalase synthesis and expression in the CNS, consume lower amounts of EtOH than their control mates (Karahanian et al., 2011). Therefore, it has been proposed that differences in brain catalase activity correlate positively with the amount of EtOH intake and determine EtOH consumption in rats.
On the other side, it has been reported that strains of mice with a lower activity of this enzyme exhibited an increased preference to voluntarily consume EtOH. Thus, acatalasemic mice, which have lower levels of catalase (50%) in the brain, displayed a greater EtOH intake compared with normal mice (Aragon and Amit, 1993). Similarly, the C57BL/6J strain of inbred mice, which have a significantly lower activity of this enzyme in the brain, showed an increment in the amount of alcohol intake with regard to other strains with higher levels of catalase, thus suggesting a negative correlation between the expression of the enzyme catalase in the CNS of mice and EtOH self-administration (Correa et al., 2004; Gill et al., 1996b). However, other studies showed that mice treated with AT displayed a dose-dependent reduction in their volitional EtOH intake (Koechling and Amit, 1994). Overall, although the direction of the relationship between catalase activity and EtOH ingestion is still not clear, given all these data, it has been postulated that differences in brain catalase may be one of the factors determining EtOH consumption in alcohol naïve animals.
In addition to EtOH intake, there are many other behaviors that could be modulated by alterations or differences in brain catalase. It has been shown that strains of mice with a lower activity of this enzyme in the CNS exhibited a decreased response to the locomotor-stimulating effects induced by EtOH (Aragon and Amit, 1993; Correa et al., 2004). Moreover, both rats and mice treated with the catalase inhibitor AT or with the anticatalase shRNA lentiviral vector displayed an attenuation of a wide range of EtOH-elicited behaviors such as locomotor stimulation (Escarabajal et al., 2000), conditioned place preference (Font et al., 2008), and EtOH-induced reward and relapse (Quintanilla et al., 2012) compared with their normal control mates. These results indicate that this enzyme participates in the mediation of some of the psychopharmacological effects caused by alcohol.
Nonetheless, the exact contribution of the catalase-H2O2 system to EtOH-induced behaviors has yet to be investigated in detail. In this respect, recent studies suggest that in addition to catalase, H2O2 could be a factor that modulates EtOH behavioral effects. Interestingly, it has been demonstrated that different treatments that boosted H2O2 central levels produced an increase in EtOH-induced locomotor stimulation in mice (Manrique et al., 2006; Pastor et al., 2002). In contrast, administration of H2O2 scavengers, which decrease H2O2 availability in the brain, reduced the locomotor stimulation evoked by an EtOH challenge (Ledesma and Aragon, 2012; Ledesma et al., 2012) and the acquisition and reconditioning of EtOH-induced conditioned place preference in mice (Ledesma and Aragon, 2013).
The aim of the present work was to evaluate the role of H2O2 on EtOH binge drinking following a drinking-in-the-dark (DID) procedure. Hence, we selected alpha-lipoic acid (LA) and ebselen (Ebs) as H2O2 scavengers. These compounds have been demonstrated to modulate brain H2O2 levels when administered systemically (Ledesma and Aragon, 2012, 2013; Ledesma et al., 2012). We hypothesized that the reduction in endogenous H2O2 levels by LA and Ebs would decrease the activity of the brain catalase-H2O2 system, and therefore voluntary EtOH consumption. We have also assessed whether these pharmacological tools were selective in reducing EtOH intake when compared to a sweetened solution. Moreover, the present investigation was also extended to study the ability of these agents to reduce the H2O2 levels in the brain. The results derived from the present work highlight the relevance of central H2O2 availability and thereby the functional role of catalase in volitional EtOH intake.
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- Materials and Methods
In the present study, a systemic injection of LA and Ebs diminished voluntary EtOH intake in mice. Furthermore, these compounds did not modify saccharin ingestion, at least at the times and doses tested in our experiments. In addition, we have found that these treatments were able to reduce brain H2O2 levels, as measured by the H2O2-dependent inactivation of endogenous brain catalase activity following an injection of cyanamide. Therefore, we suggest that a reduction in central H2O2 levels could alter the motivational properties of EtOH that lead to alcohol binge drinking.
In the DID experiments, LA and Ebs administration dose-dependently decreased voluntary EtOH consumption. Mice treated with LA (25 or 50 mg/kg) or Ebs (12.5 or 25 mg/kg) displayed a significant reduction in their voluntary EtOH intake. The DID method represents a model of animal EtOH binge drinking that could be used to explore the mechanisms underlying high rates of alcohol drinking and to screen new compounds capable of limiting its excessive intake (Kamdar et al., 2007; Rhodes et al., 2005). This behavioral procedure captures important features of alcoholism, such as drinking to the point of intoxication and rapid initiation to high levels of drinking (Rhodes et al., 2005). In our experiments, DID EtOH intakes were consistent with the available literature demonstrating that the DID model consistently yields high EtOH consumption over a relatively short time period (Kamdar et al., 2007; Rhodes et al., 2005). Considering that binge drinking is a substantial risk factor predicting the development of alcohol use disorders (Crabbe et al., 2011), our data are significantly relevant as they represent a validation of 2 new pharmacological tools to impair EtOH binge drinking.
Both LA and Ebs are H2O2 scavengers that are able to decrease the production and the concentrations of reactive oxygen species (ROS) in the CNS, including H2O2. LA is an essential cofactor for mitochondrial enzymes that has been described as a novel biological antioxidant and a potent free radical scavenger (Somani et al., 2000). This agent participates in the recycling of vitamins C and E, increases levels of glutathione, and suppresses nonenzymatic glycation (Suzuki et al., 1992). Acute administration of LA enhances glutathione peroxidase activity in different regions of the rat brain and decreases the concentration of ROS, especially H2O2 (Bilska et al., 2007). Similarly, Ebs is a seleno–organic compound with radical-scavenging activity (Müller et al., 1984). This compound has been shown to be involved in a number of antioxidant activities both in vitro and in vivo (Moretto et al., 2004). Ebs is able to increase the rate of elimination of H2O2 because it catalyzes the thiol-peroxidase reaction in a mechanism that appears to be kinetically identical to the mechanism of glutathione peroxidase (Parnham and Sies, 2000). In the second set of experiments, we focused on the effect of LA and Ebs treatment on the inhibitory effect that sodium cyanamide causes in brain catalase. Cyanamide is a suicide inhibitor of the enzyme catalase (DeMaster et al., 1986; Sanchis-Segura et al., 1999). Interestingly, this drug only inhibits catalase activity when this enzyme reacts to H2O2 by forming Compound I (Cederbaum and Dicker, 1985; DeMaster et al., 1986).Therefore, the effect of cyanamide on catalase activity may be used as an indirect measure of the capacity of this enzyme to form Compound I, because the cyanamide-mediated inactivation of brain catalase is H2O2-dependent. Thus, based on the suggestion that both LA and Ebs are capable of reducing endogenous H2O2 in rodents, in Experiments 5a and 5b, we hypothesized that the inhibition of cerebral catalase induced by cyanamide will be decreased in mice treated with these compounds. In agreement with this idea, our results showed that brain homogenates of mice treated with LA (50 mg/kg)-Cyanamide (40 mg/kg) and Ebs (25 mg/kg)-Cyanamide (40 mg/kg) have a higher catalase activity than Vh-Cyanamide (40 mg/kg)-treated mice. This indicates that, by reducing H2O2 availability in the CNS, both LA and Ebs treatments could reduce the formation of Compound I in the brain. These findings are in agreement with previous data obtained in our laboratory in which it has been also showed that both LA and Ebs reduced the amount of H2O2 available for brain catalase, as measured by the H2O2-dependent inactivation of central catalase activity following an injection of AT (Ledesma and Aragon, 2012, 2013; Ledesma et al., 2012).
As outlined above, we propose that both LA and Ebs reduce the formation of Compound I by decreasing H2O2 levels. As a result, when animals are exposed to EtOH, the rate of brain-EtOH metabolism would be decreased, thus preventing voluntary EtOH drinking. As noted earlier, the first product coming from brain-EtOH metabolism by the activity of Compound I is acetaldehyde. Acetaldehyde is a psychoactive molecule that, when administered centrally, exerts a broad range of behavioral (Arizzi-LaFrance et al., 2006; Rodd-Henricks et al., 2002; Smith et al., 1984) and neurochemical (Diana et al., 2008; Enrico et al., 2009; Foddai et al., 2004; Sirca et al., 2011) effects that are similar to those induced by EtOH in rodents. For this reason, it has been proposed that brain-EtOH-derived acetaldehyde could play a role in some of the psychopharmacological effects caused by EtOH. In agreement to this, it has been observed that administration of acetaldehyde chelating agents, such as d-penicillamine and l-cysteine, prevents a wide range of EtOH-elicited behaviors, including EtOH intake (Font et al., 2005, 2006; Martí-Prats et al., 2010; Pautassi et al., 2010; Peana et al., 2010). The results obtained in this study indicated that H2O2 levels are critical to EtOH intake. We suggest that in addition to the levels of catalase present in the organisms, another factor that modulates EtOH-induced behaviors might be the availability of H2O2, which would determine the rate of acetaldehyde production at the time of EtOH administration.
It has been suggested that the mesocorticolimbic dopamine (Da) is involved in EtOH drinking. Various reports have demonstrated that manipulations that reduce the activity of this neurotransmitter system decrease voluntary EtOH intake in rodents (Nguyen et al., 2007; Thanos et al., 2005). Moreover, acetaldehyde has been shown to be capable of stimulating the transmission of mesolimbic Da (Deehan et al., 2013; Deng and Deitrich, 2008; Enrico et al., 2009; Foddai et al., 2004; Sirca et al., 2011). Several authors have proposed that acetaldehyde mediates the EtOH-induced stimulation of mesolimbic Da transmission. It has been reported that both acetaldehyde sequestration with d-penicillamine and l-cysteine, or the inhibition of brain catalase activity by AT prevent the activation of this brain circuitry evoked by EtOH (Deng and Deitrich, 2008; Enrico et al., 2009; Sirca et al., 2011). Brain catalase has been shown to be highly active in aminergic neurons and along the mesolimbic DA pathway (Deng and Deitrich, 2008; Zimatkin and Lindros, 1996). Therefore, given the above studies, we suggest that the reduction in EtOH self-administration observed in our experiments could be due to a decreased activation of this neurotransmitter in the mesolimbic DA pathway system.
Alternative explanations should also be considered. That is, this reduction in volitional EtOH consumption could be interpreted as being produced by an unspecific alteration of these pharmacological tools in sensitivity to taste, or an impairment of some brain areas or neurotransmitter circuitry related to reinforcement and motivation. However, here, we showed that voluntary saccharin intake was not affected by these treatments. Therefore, if the reduction in EtOH drinking was only the result of a general alteration of these compounds in motivated behavior, we should expect these drugs to have similar consequences on saccharin ingestion. Our results demonstrated that these treatments selectively affected EtOH consumption because saccharin intake remained unaltered after LA and Ebs administration, thus ruling out that possibility. As support to this notion, previous data also showed that the effects of these compounds are specifically selective for other EtOH-induced behaviors (Ledesma and Aragon, 2012, 2013; Ledesma et al., 2012).
Another possibility that should be contemplated is that the capacity of these H2O2 scavengers to diminish volitional EtOH drinking might be mediated by a modulation in spontaneous locomotion or in blood EtOH levels after its administration. Nonetheless, data obtained in other studies demonstrated that these compounds did not modify the baseline motor activity or blood EtOH concentrations in mice (Ledesma and Aragon, 2012, 2013; Ledesma et al., 2012).
In conclusion, we propose here a reduction in the activity of the catalase-H2O2 system as the main mechanism responsible for the interaction between LA and Ebs in modulating voluntary EtOH self-administration. The current findings represent an extension of previous research in which it has also been suggested that modifications of H2O2 levels in the CNS could alter some EtOH-induced behaviors (Ledesma and Aragon, 2012, 2013; Ledesma et al., 2012; Manrique et al., 2006; Pastor et al., 2002). What is most important here is that the brain peroxidative status of the organism could modulate the acute psychopharmacological effects of EtOH. It seems that variations in the physiological conditions, that is, in the levels of H2O2 present in the CNS in a particular moment in the same organism, could determine some effects induced by EtOH. These data are particularly relevant to be able to understand the mechanisms underlying the causes of EtOH binge drinking. Because EtOH binge drinking is a feature of alcoholism, further research is needed to test the implication of the catalase-H2O2 system in the EtOH-dependent brain, for example, by performing the same experiments described here in EtOH-dependent rats. This should open up the way to the development of new pharmacological tools to reduce chronic alcohol use.