Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 221–226

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Progress in Neuro-Psychopharmacology & Biological Psychiatry

Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver Steven Tran a,⁎, Magda Nowicki b, Diptendu Chatterjee b, Robert Gerlai a,b a b

Department of Cell and Systems Biology, University of Toronto Mississauga, Canada Department of Psychology, University of Toronto Mississauga, Canada

a r t i c l e

i n f o

Article history: Received 13 August 2014 Received in revised form 16 September 2014 Accepted 29 September 2014 Available online 5 October 2014 Keywords: Alcohol dehydrogenase Aldehyde dehydrogenase Chronic ethanol Tolerance Zebrafish

a b s t r a c t Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Ethanol (alcohol or ethyl alcohol) is primarily metabolized in the liver in a two-step process by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). In humans, ethanol is converted into acetaldehyde by a class 1 alcohol dehydrogenase (ADH1), a class of enzymes responsible for the oxidation of alcohols to aldehydes (Hoog and Ostberg, 2011). Class III alcohol dehydrogenases have also been reported to contribute to ethanol metabolism but to a lesser extent (Haseba et al., 2003, 2006). The catalytic oxidation of ethanol to acetaldehyde by alcohol dehydrogenase requires nicotinamide adenine dinucleotide

Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ADH1, class 1 alcohol dehydrogenase; ADH3, class 3 alcohol dehydrogenase; NAD+, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide reduced; cDNA, complementary deoxyribonucleic acid; ALDH1, aldehyde hydrogenase 1; ALDH2, aldehyde dehydrogenase 2; km, Michaelis constant; ZFIN, Zebrafish Information Network; ANOVA, analysis of variance; HSD, Honestly Significant Difference; S.E.M., standard error of the mean. ⁎ Corresponding author at: Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road North, Rm 1022D, Mississauga, Ontario L5L 1C6, Canada. Tel.: +905 569 4277 (office), +905 569 4257 (lab). E-mail address: [email protected] (S. Tran).

http://dx.doi.org/10.1016/j.pnpbp.2014.09.011 0278-5846/© 2014 Elsevier Inc. All rights reserved.

(NAD+), a co-enzyme which is reduced to NADH. In the second step, acetaldehyde is converted into acetic acid by aldehyde dehydrogenase 2 (ALDH2), the primary enzyme in the liver responsible for the oxidation of aldehydes to its corresponding carboxylic acids also by reducing NAD+ to NADH (Hoog and Ostberg, 2011). Current evidence suggests that zebrafish metabolize ethanol in a similar manner. In zebrafish, 3 different genes encoding alcohol dehydrogenases have been identified. The first cDNA cloned was ADH3, a class III alcohol dehydrogenase with a predicted amino acid sequence with 81% similarity to the human sequence (Dasmahapatra et al., 2001). ADH3 mRNA was detected in the adult zebrafish liver and may encode a protein that metabolizes ethanol at a lower rate compared to class I alcohol dehydrogenases similar to humans (Dasmahapatra et al., 2001). Subsequently, two class I alcohol dehydrogenases were identified in zebrafish. The two cDNA sequences were cloned (ADH8A and ADH8B) and their predicted amino acid sequence was 72 and 68%, respectively, similar to the human ADH1 (Reimers et al., 2004). Notably, the ADH8B isoform was unable to metabolize ethanol, whereas the ADH8A metabolized ethanol at a similar rate to that of the human ADH1 (Reimers et al., 2004). In addition, the gene encoding the zebrafish aldehyde dehydrogenase was also recently identified. cDNA for the zebrafish ALDH2 gene was cloned and the

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predicted amino acid sequence was found to be 75% similar to that of the mammalian ALDH2 (Lassen et al., 2005). The gene was then expressed using the baculovirus expression system and the purified protein was found to break down acetaldehyde at a rate comparable to the human ALDH2 (Lassen et al., 2005). Since the primary enzymes responsible for the breakdown of ethanol and its metabolite are present in zebrafish and are functionally similar to the mammalian enzymes, ethanol metabolism is likely to occur in a similar manner in these species. Zebrafish have become a popular animal model for examining the mechanisms underlying ethanol's actions in vivo (Dlugos and Rabin, 2003; Damodaran et al., 2006; Rico et al., 2007). It is a small vertebrate with similar physiology and neurochemistry compared to mammals including humans (Rico et al., 2011b). A number of practical advantages including small size and high fecundity make it an attractive animal model for high-throughput research (Gerlai et al., 2000). However, the greatest advantage (in our opinion) is the method of drug delivery. Water soluble drugs such as ethanol can be mixed directly with the tank water and is subsequently taken up by the immersed zebrafish (Dlugos and Rabin, 2003; Rosemberg et al., 2012; Tran et al., in press). Drug immersion is less invasive compared to injection or inhalation– vaporization based procedures used in rodents and have been successfully utilized in both larvae (Lockwood et al., 2004; Ramcharitar and Ibrahim, 2013) and adult zebrafish (Dlugos and Rabin, 2003; Kily et al., 2008; Mathur and Guo, 2011). Studies have shown that acute ethanol exposure alters a number of behavioral responses in adult zebrafish including locomotor activity, preference for the bottom, and erratic movement (Pannia et al., 2014; Rosemberg et al., 2012; Tran and Gerlai, 2013). These behavioral responses are accompanied by changes in the levels of neurotransmitters (Chatterjee et al., 2014; Gerlai et al., 2009; Tran et al., in press), enzyme activity and gene expression in the zebrafish brain (Rico et al., 2007; Rosemberg et al., 2010). Chronic (longterm) exposure to ethanol has also been shown to alter behavioral and neurochemical responses in zebrafish (Chatterjee et al., 2014; Dlugos and Rabin, 2003; Dlugos et al., 2011; Gerlai et al., 2009; Rico et al., 2011a; Tran et al., in press). Although chronic alcohol exposure has not been operationally defined in zebrafish, current studies suggest that as few as 7 days of continuous or 8 days of repeated ethanol exposure may be enough to induce observable changes in zebrafish behavior (Egan et al., 2009; Mathur and Guo, 2011). For example, zebrafish continuously housed in ethanol for 3 weeks respond to a subsequent acute ethanol challenge with an attenuated locomotor and neurochemical response (Chatterjee et al., 2014; Tran et al., in press). Long-term continuous ethanol exposure alters neurochemical levels, enzymatic activity, gene and protein expression in the adult zebrafish brain (Chatterjee et al., 2014; Damodaran et al., 2006; Pan et al., 2011; Rico et al., 2011a; Tran et al., in press). Repeated intermittent exposure to ethanol has also been shown to alter behavioral responses and gene expression (Blaser et al., 2010; Kily et al., 2008; Mathur and Guo, 2011). Changes within these systems suggest the development of tolerance at the level of the central nervous system. However, following chronic ethanol exposure genes encoding liver enzymes responsible for the breakdown of ethanol such as alcohol dehydrogenase and aldehyde dehydrogenase have been shown to be upregulated in the zebrafish brain (Pan et al., 2011). Although these changes were detected in the brain, it may suggest adaptation in the periphery as well (i.e. the liver). It is currently unknown whether chronic ethanol exposure alters the activity of alcohol dehydrogenase and aldehyde dehydrogenase in the zebrafish liver. In the current study we first examine the effect of acute ethanol exposure on the locomotor responses and total ADH and ALDH activity in the zebrafish liver. Subsequently, we determine whether chronic ethanol exposure (repeated and continuous) alters locomotor responses and the activity of both enzymes in a subsequent acute ethanol challenge.

2. Methods 2.1. Animals and housing 9 and 24 month old zebrafish (50% males and females) of the AB strain (progenitors obtained from the ZFIN Center (Eugene, Oregon, USA)) were used for the current study (24 month old zebrafish were used for experiment 1 and 9 month old zebrafish were used for experiment 2). The AB strain was chosen based on previous studies demonstrating robust behavioral and neurochemical changes in response to acute and chronic ethanol exposure (Chatterjee et al., 2014; Tran et al., in press). Zebrafish were raised and housed in 37 L glass tanks containing system water supplemented with 60 mg/L of instant ocean sea salt (Big Al's Aquarium) with biological filtration prior to ethanol exposure. Water quality parameters were monitored on a weekly basis and maintained at optimal conditions (Conductivity: 100–300 μS, Temperature: 26–28 °C, pH: 6.8–7.2). Additional details on housing conditions and maintenance are described elsewhere (Tran and Gerlai, 2013). 2.2. Experimental design and procedure 2.2.1. Experiment 1 Ethanol naïve zebrafish were netted from their group housing tanks and were individually exposed to 0.0%, 0.25%, 0.50%, or 1.0% v/v ethanol in a 37 L tank containing 28 L water (n = 16 per group) for 30 min. The duration of exposure is based on prior studies demonstrating the biphasic effect of ethanol, with maximal locomotor stimulation at 30 min in 1.0% v/v ethanol (Tran and Gerlai, 2013). The behavioral tests were conducted in 37 L glass tanks with white corrugated plastic sheets on the back and sides of these experimental tanks, which obscured external cues and provided a uniform testing environment. Water quality parameters in the experimental tanks matched those of the housing tanks with the exception of the addition of ethanol. Zebrafish motor responses were recorded using a video camera from the front view during acute exposure. Following the 30 min acute ethanol exposure, zebrafish were immediately netted and decapitated with the body stored at −80 °C until processing. 2.2.2. Experiment 2 A group of zebrafish (n = 20) was continuously housed in ethanol to induce ethanol tolerance using a previously established chronic ethanol exposure paradigm that utilized a dose escalation procedure (Tran and Gerlai, 2013, in press). Briefly, zebrafish were initially housed in 37 L tanks containing 0.125% v/v ethanol for 4 days, followed by 0.25% v/v for 4 days and 0.375% for 4 days, for a total of 12 days. Starting on day 13, zebrafish were then housed in 0.50% v/v ethanol for 10 days, i.e. the experimental subjects were continuously exposed to ethanol for a total of 22 days. During continuous ethanol exposure, biological filtration was turned off to prevent the death of bacterial fauna in the filters. Water changes occurred every other day to remove biological waste and ensure appropriate ethanol concentrations. On day 13, a second group of zebrafish (n = 22) received intermittent exposure to ethanol (fluctuating concentration) on a daily basis for 10 consecutive days, an exposure regimen that more closely resembled human binge drinking. Briefly, zebrafish were exposed to 1.0% ethanol for 1 h every day from 11:00 to 12:00 for 10 consecutive days. The concentration, exposure duration and number of exposures were based on a number of previous studies showing behavioral and gene expression related changes following repeated intermittent exposure to ethanol (Blaser et al., 2010; Kily et al., 2008; Mathur and Guo, 2011). Finally, a third group (n = 20) was handled in an identical manner as group 2 but without ethanol to serve as controls (i.e. repeated exposure to freshwater for 10 consecutive days). All tanks were aerated using aeration stones to ensure maximal oxygen levels, specifically during the 1 h acute exposures. On day 23, all groups were challenged with an acute dose of 1.0% v/v ethanol in a 37 L tank for 30 min. The

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dose used for the ethanol challenge was based on prior studies showing robust behavioral and neurochemical changes (Tran et al., in press). Behavioral responses during the challenge were recorded using a video camera from the front view. Immediately following the 30 min of exposure to ethanol, zebrafish were decapitated and the bodies were stored at −80 °C until processing. 2.3. Behavioral quantification Video recordings were subsequently quantified using the automated video tracking software EthoVision XT 8.0 (Noldus Information Technology, Wageningen, The Netherlands). The primary behavioral variable quantified was total distance traveled (cm), a response which is robustly altered by acute and chronic ethanol exposure (Tran and Gerlai, 2013, in press). Total distance traveled for 21–30 min was calculated, a time period during which the stimulant effect of ethanol was shown to be at its highest in prior studies using experimental conditions similar to those of the current study (Tran and Gerlai, 2013; Pannia et al., 2014). 2.4. Sample preparation and protein assay Samples were prepared according to Koivula et al. (1975) and modified for zebrafish tissue. Liver samples first were freeze–thawed twice to disrupt mitochondrial membranes. Whole livers were then suspended and sonicated in 25 μL of 0.24 M sucrose containing 10 mM Na–phosphate buffer, pH 7.4. Subsequently, 1 μL of the sonicate was used for protein measurement using the BioRad protein assay reagent (BioRad, Hercules, CA, United States) which employed bovine serum albumin as the standard. The sonicate was then centrifuged at 10,000 rpm for 20 min and the supernatant extracted for ADH and ALDH activity assays. 2.5. Alcohol dehydrogenase activity assay Measurement of whole liver alcohol dehydrogenase activity was adapted from a protocol by Kim et al. (2001) and modified for zebrafish tissue. The final reaction mixture was 500 μL and consisted of 0.5 M Tris–HCl, pH 7.8, 2.8 mM NAD+, 340 mM ethanol and 10 μL of the sample. The formation of NADH was measured at 340 nm every minute for a total of 5 min using a Genesys 10 UV–vis spectrophotometer. Total ADH activity was expressed as moles of NADH produced (nmol/min/mg protein) using NADH as a standard. All chemicals were obtained from Sigma Aldrich (Oakville, ON, CA).

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3. Results 3.1. Experiment 1 Zebrafish acutely exposed to ethanol exhibited a dose-dependent increase in locomotor activity (Fig. 1A) (F(3, 60) = 9.779, p b 0.001). Tukey's HSD posthoc multiple comparison test determined that zebrafish exposed to 1.0% ethanol swam significantly further compared to controls (p b 0.01). We found an inverted U-shaped dose–response curve for alcohol dehydrogenase activity (Fig. 2A). There was a dosedependent increase in ADH activity in response to low doses of ethanol (0.25% and 0.50% v/v). However, exposure to the highest concentration of ethanol (1.0% v/v) showed a reduction in ADH activity. ANOVA found a significant main effect of acute ethanol exposure on ADH activity (F(3, 58) = 5.050, p = 0.004). Tukey's post-hoc HSD determined that zebrafish exposed to 0.5% ethanol had significantly higher ADH activity compared to controls and to zebrafish exposed to 1.0% v/v (p b 0.05). Acute ethanol exposure decreased liver aldehyde dehydrogenase activity (Fig. 3A). ANOVA found a significant main effect of acute ethanol exposure (F(3, 54) = 4.822, p = 0.005). Tukey's post-hoc HSD determined that zebrafish of all ethanol concentration groups exhibited significantly lower ALDH activity compared to control zebrafish (p b 0.05). 3.2. Experiment 2 Chronic ethanol treatment differentially altered locomotor responses in the subsequent acute ethanol challenge (Fig. 1B). ANOVA found a significant main effect of chronic ethanol treatment (F(2, 53) = 14.372, p b 0.001). Tukey's post-hoc HSD determined that control zebrafish previously exposed to system water swam significantly further during the acute ethanol challenge compared to zebrafish that were continuously or repeatedly exposed to ethanol (p b 0.01). However, total ADH activity in the liver did not match these behavioral responses. Only zebrafish that were continuously exposed to ethanol showed altered (increased) ADH activity (Fig. 2B). ANOVA found a significant main effect of chronic ethanol exposure (F(2, 53) = 10.714, p b 0.01). Tukey's post-hoc determined that zebrafish that were continuously exposed to ethanol exhibited significantly higher ADH activity levels during the acute ethanol challenged compared to controls and zebrafish that received the repeated intermittent ethanol exposure (p b 0.01). ADH activity of repeated ethanol exposed fish did not differ from freshwater exposed fish in the acute ethanol challenge (p N 0.05). Finally, examination of total ALDH activity in the liver determined that continuous and repeated ethanol exposed fish did not differ from freshwater controls (Fig. 3B) (F(2, 44) = 0.489, p = 0.617).

2.6. Aldehyde dehydrogenase activity assay 4. Discussion Measurement of acetaldehyde dehydrogenase activity was adapted from a protocol by Koivula et al. (1975) and modified for zebrafish tissue. The final reaction mixture was 500 μL and consisted of 70 mM sodium pyrophosphate buffer, pH 8, 1.33 mM NAD+, 1.67 mM pyrazole, 100 mM acetaldehyde, and 10 μL of the sample. The formation of NADH was measured at 340 nm every minute for a total of 5 min using a Genesys 10 UV–vis spectrophotometer using NADH as a standard. Total ALDH activity was expressed as moles of NADH produced (nmol/min/mg protein) using NADH as a standard. All chemicals were obtained from Sigma Aldrich (Oakville, ON, CA). 2.7. Statistical analysis A one-way ANOVA was conducted for experiment 1 with acute ethanol concentration as the between-subject factor (4 levels). A one-way ANOVA was also conducted for experiment 2 with chronic ethanol treatment as the between-subject factor (3 levels). Tukey's Honestly Significant Difference tests were performed when a significant main effect was detected in either case with significance reported at p b 0.05.

Long term ethanol exposure paradigms have been successfully used with zebrafish in the past to demonstrate the development of tolerance (Blaser et al., 2010; Dlugos and Rabin, 2003; Mathur and Guo, 2011; Rico et al., 2011a; Tran and Gerlai, 2013). Two of the most common chronic ethanol exposure paradigms used with zebrafish employed either repeated intermittent or continuous ethanol exposure (see Tran and Gerlai, in press for review). Here we report that continuous ethanol exposure induces tolerance at both the behavioral and physiological levels examined. However, repeated intermittent exposure to ethanol for 10 consecutive days only induced mild tolerance detected at the behavioral level but not at the physiological level when examined during an acute ethanol challenge. In contrast with the literature, Blaser et al. (2010) demonstrated that repeated exposure to ethanol under similar conditions we used in the current study induced sensitization in wildtype zebrafish, resulting in an increase in locomotor response during a subsequent acute ethanol challenge. The discrepancies, i.e. the development of mild tolerance rather than sensitization in zebrafish repeatedly exposed to ethanol, may result from the different genetic background of

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Fig. 1. Total distance traveled for 21–30 min is shown during the acute ethanol challenge for ethanol naïve zebrafish (panel A) and chronic ethanol or freshwater pre-treated zebrafish (panel B). Mean + S.E.M. are shown. Note the dose-dependent increase in total distance traveled in ethanol naïve zebrafish exposed acutely to different concentrations of ethanol. Zebrafish continuously pre-exposed to ethanol exhibited a significantly attenuated locomotor response during the acute 1% ethanol challenge (third bar, panel B), whereas zebrafish that received the intermittent repeated ethanol pre-exposure exhibited a mild attenuation of the locomotor response (second bar, panel B).

the strains used in the current versus in the previously published study. The AB strain of zebrafish used in the current study has been reported to be more sensitive to the effects of ethanol compared to the wild-type (outbred) population of fish at both the behavioral and neurochemical levels (Chatterjee et al., 2014; Gerlai et al., 2009). In the current study we found that acute exposure to ethanol in zebrafish differentially altered the total activity of ADH and ALDH in the zebrafish liver. At low doses of ethanol (0.25 and 0.5%) there was a dose-dependent increase in ADH activity suggesting that ethanol metabolism increased as ethanol levels in the liver increased. However, at the highest concentration of ethanol (1.0%) there was a decrease in ADH activity. The pattern of ADH activity we found in response to acute ethanol exposure is similar to those seen in mice and rats with inhibition of total ADH activity at high ethanol doses (Braggins and Crow, 1981; Haseba et al., 2003, 2012; Sharkawi, 1984). Two liver ADH isozymes have been identified for their contribution to ethanol metabolism, class I ADH (ADH1) and class III ADH (ADH3) (Haseba and Ohno, 2010; Hoog and Ostberg, 2011). ADH1 has a very low Km (substrate concentration required to reach half of maximum reaction velocity) and metabolizes ethanol at lower concentrations. ADH3 has a much higher Km and contributes to ethanol metabolism only when the ethanol concentration increases due to its low affinity. The inverted U-shaped curve of total ADH activity can be explained by the two-ADH model which suggests that ethanol metabolism is regulated by both enzymes

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depending on the concentration of ethanol. Recent studies support this and have shown that the contribution of ADH1 decreases and ADH3 increases in a dose-dependent manner following acute ethanol exposure in mice (Haseba et al., 2012). Specifically, ADH1 liver content was shown to exhibit the same inverted U-shaped curve in response to increasing concentrations of ethanol in mice (Haseba et al., 2012). Furthermore, substrate inhibition of ADH1 further decreasing ADH1 activity at higher ethanol concentrations has also been reported (Bosron et al., 1983). Although relative ADH3 content was shown to increase in a dose-dependent manner in mice to compensate for the decrease in ADH1 activity, total ADH activity was found to still remain low at higher ethanol concentrations (Haseba et al., 2012). Since zebrafish have been reported to express both class I (ADH8A) and class III (ADH3) isozymes (Dasmahapatra et al., 2001; Reimers et al., 2004), the initial increase and subsequent decrease in ADH activity observed at the highest ethanol dose (1%) may occur in a similar manner, a pattern of responses that suggests evolutionary conservation across fish and mammals not only at the amino-acid sequence of the enzymes involved but also in their biological function. We found that continuous but not intermittent repeated ethanol exposure increased total ADH activity in a subsequent acute ethanol challenge compared to controls. The result of continuous ethanol exposure in zebrafish is similar to those obtained for long-term ethanol exposure in rodents as well as humans. For example, prolonged ethanol exposure

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Fig. 2. Total alcohol dehydrogenase activity is shown following the acute ethanol exposure in ethanol naïve zebrafish (panel A) and chronic ethanol or freshwater pre-treated zebrafish (panel B). Mean + S.E.M. are shown. Note the inverted U-shaped dose–response curve in ethanol naïve zebrafish. Zebrafish continuously pre-exposed to ethanol exhibited a significantly higher level of total ADH activity in response to the acute 1% ethanol challenge compared to zebrafish that received freshwater pre-treatment or intermittent repeated exposure to 1% ethanol on a daily basis prior to the challenge.

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Fig. 3. Total aldehyde dehydrogenase activity in response to the acute ethanol challenge in ethanol naïve zebrafish (panel A) and chronic ethanol or freshwater pre-treated zebrafish (panel B). Mean + S.E.M. are shown. Note the decrease in activity following acute exposure in ethanol naïve zebrafish. Chronic ethanol treatment (continuous and repeated) did not significantly alter activity levels compared to controls in the subsequently 1% acute ethanol challenge.

has been shown to increase total ADH activity in mice (Kishimoto et al., 1995) and rats (Buris et al., 1985; Matsuzaki et al., 1981). Although total ADH activity was found to increase, certain studies suggested that this was primarily due to an increase in the activity of the high Km isozyme (Kishimoto et al., 1995; Matsuzaki et al., 1981). Chronic ethanol exposure has been shown to increase lipid accumulation in the liver leading to increased fat content commonly referred to as steatosis (Romero et al., in press). The increase in liver hydrophobicity due to lipid accumulation following chronic ethanol exposure has been suggested to induce a shift in the main ethanol metabolizing enzyme from the low Km ADH1 to the high Km ADH3 (Haseba and Ohno, 2010). Although the substrate binding pocket of ADH3 has a larger volume than that of ADH1 (i.e. lower affinity for ethanol), it has been previously shown that hydrophobic substances increase the catalytic efficiency (kcat/Km) of mouse ADH3 by decreasing Km of the enzyme. Binding of hydrophobic ligands in the substrate binding pocket of ADH3 is suggested to induce a structural change to increase the enzyme's affinity for ethanol (Haseba et al., 2006). In adult zebrafish, fatty liver can be induced by exposing adults to 1.2% ethanol for just 9 h a day for 7 days (Jang et al., 2012). The 22 days of continuous ethanol exposure used in the current study increased total ADH activity which may reflect increased ADH3 activity due to the development of a fatty liver. In the case where our fish did not develop fatty livers during continuous ethanol exposure, total ADH activity may be attributed to increased liver ADH1 content as an adaptive response to chronic high ethanol concentrations. This has been shown in mice, whose rate of ethanol metabolism increased due to an increase in ADH1 liver content after one week of chronic ethanol feeding (Kurosu et al., 1988). The question whether the increase in total ADH activity we observed reflects increased ADH1 or ADH3 content will be addressed in future studies. In contrast to ADH activity, acute ethanol exposure in ethanol naïve zebrafish decreased ALDH activity at all doses. Despite the counterintuitive nature of this finding, the result is in good agreement with those obtained using rodents (Tomita et al., 1990). Similar to liver ADH, there are 2 ALDH isozymes, one with a high Km and one with a low Km for acetaldehyde (Klyosov et al., 1996). Acute ethanol exposure in mice has been shown to inhibit ALDH activity with a more drastic decrease in the activity of the low Km isozyme (Tomita et al., 1990). Ethanol induced inhibition of acetaldehyde dehydrogenase may occur through the production of reactive oxygen species (ROS). For example, acute ethanol exposure increases the levels of ROS in rat liver cells (Bailey and Cunningham, 1998). These reactive oxygen species induced by ethanol exposure have been shown to inhibit ALDH1 and ALDH2 in rat liver cells via S-nitrosylation (Moon et al., 2006, 2007). Acute ethanol exposure has also been shown to induce oxidative stress in zebrafish (Rosemberg et al., 2010) which may subsequently inhibit ALDH activity in a similar manner.

We report that chronic ethanol treatment (continuous and repeated) did not significantly alter total ALDH activity in the liver during the acute ethanol challenge, a finding similar to those seen in humans. For example, total liver ALDH activity was found not to be significantly different between alcoholic and non-alcoholic patients (Vidal et al., 1998). However, other reports of chronic ethanol exposure have found increases in ALDH activity in mice suggesting physiological tolerance (Aoki and Itoh, 1989). Inconsistencies regarding changes in ALDH activity following chronic ethanol exposure may reflect the time of measurement. For example, mice chronically treated with ethanol for one week showed an initial decrease in ALDH activity with levels recovered by 4 weeks, and increased by 10 weeks (Tomita et al., 1992). Similar rebounding effects have also been reported for zebrafish behavioral responses during chronic treatment. For example, Damodaran et al. (2006) reported the development of behavioral tolerance to ethanol to manifest after 2 weeks of continuous treatment. However, the effects appeared to rebound back to disruptive levels beyond 6 weeks. In addition to the time of measurement, mitochondrial and cytosolic ALDH have been found to exhibit different Kms for acetaldehyde. ALDH from different subcellular fractions may be differentially altered by acute and chronic ethanol treatments and may account for some of the inconsistencies reported (Greenfield et al., 1976). Although the zebrafish liver is small, future studies may be able to differentiate mitochondrial and cytosolic fractions of ALDH to determine their specific activity. Notably, perusal of Fig. 3 suggests that ALDH activity in ethanol naïve zebrafish exposed to 1% ethanol in experiment 1 (Panel A, black bar) is lower compared to zebrafish that were repeatedly exposed to freshwater and subsequently challenged with 1% in experiment 2 (Panel B, white bar). It is unlikely that repeated exposure to freshwater in experiment 2 accounts for the apparent difference in ALDH activity. The difference in ALDH activity is likely attributed to the use of older zebrafish (24 months old) in experiment 1, and younger zebrafish in experiment 2 (9 months old). For example, the activity of enzymes metabolizing ethanol including ADH and ALDH has been reported to decrease with age in humans (Meier and Seitz, 2008). Although directly comparing experiments 1 and 2 was not the goal of this paper, future studies may want to characterize age related changes in ADH and ALDH activity in the zebrafish liver. Overall, we have identified two chronic ethanol treatment protocols which differentially altered behavior and ethanol metabolism in zebrafish. Continuous ethanol exposure led to the development of tolerance detected at both the behavioral and physiological levels, whereas repeated ethanol exposure was found to lead to changes detected at only the behavioral level. It is often assumed (and experimentally found too) that the CNS is more prone to damage and is more responsive to external insults than most other organs. The ethanol exposure induced changes we report here suggest that central nervous system

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changes do contribute, at least partially, to the development of ethanol tolerance in zebrafish. The employed chronic ethanol treatment paradigms may be useful in understanding ethanol addiction and ethanol's mechanism in vivo. For example, in addition to being able to dissociate some brain-specific functional changes from peripheral mechanisms, repeated intermittent ethanol exposure may be useful in modeling ethanol consumption in clinical populations (binge drinking). Although continuous exposure may not resemble alcohol consumption in humans, it may be useful in uncovering the mechanisms of ethanol tolerance without the complicating aspects of potential confounds resulting from repeated withdrawal from ethanol. In conclusion, we assert that chronic ethanol exposure paradigms can be successfully utilized in zebrafish, a translationally relevant model that may enable the investigator to study behavioral and/or physiological tolerance to ethanol in a highly controlled and cost effective manner in the laboratory. Acknowledgments This project was supported by an NSERC Discovery grant (#311637) issued to R.G. We would like to thank Ana Carolina Luchiari for her help with liver dissections. Reference Aoki T, Itoh H. Effects of alcohol consumption on mitochondrial aldehyde dehydrogenase isoenzymes in rat liver. Enzyme 1989;41:151–8. Bailey SM, Cunningham CC. Acute and chronic ethanol increases reactive oxygen species generation and decreases viability in fresh, isolated rat hepatocytes. Hepatology 1998;28:1318–26. Blaser RE, Koid A, Poliner RM. Context-dependent sensitization to ethanol in zebrafish Danio rerio. Pharmacol Biochem Behav 2010;95:278–84. Bosron WF, Crabb DW, Li TK. Relationship between kinetics of liver alcohol dehydrogenase and alcohol metabolism. Pharmacol Biochem Behav 1983;18:223–7. Braggins TJ, Crow KE. The effects of high ethanol doses on rates of ethanol oxidation in rats. A reassessment of factors controlling rates of ethanol oxidation in vivo. Eur J Biochem 1981;119:633–40. Buris L, Csabai G, Fodor M, Varga M. Increase of alcohol dehydrogenase and protein content of liver following chronic ethanol administration. FEBS Lett 1985;183:143–4. Chatterjee D, Shams S, Gerlai R. Chronic and acute alcohol administration induced neurochemical changes in the brain: comparison of distinct zebrafish populations. Amino Acids 2014;46:921–30. Damodaran S, Dlugos CA, Wood TD, Rabin RA. Effects of chronic ethanol administration on brain protein levels: a proteomic investigation using 2-D DIGE system. Eur J Pharmacol 2006;547:75–82. Dasmahapatra AK, Doucet HL, Bhattacharyya C, Carvan III MJ. Developmental expression of alcohol dehydrogenase (ADH3) in zebrafish (Danio rerio). Biochem Biophys Res Commun 2001;286(5):1082–6. Dlugos CA, Rabin RA. Ethanol effects on three strains of zebrafish: model system for genetic investigations. Pharmacol Biochem Behav 2003;74:471–80. Dlugos CA, Brown SJ, Rabin RA. Gender differences in ethanol-induced behavioral sensitivity in zebrafish. Alcohol 2011;45:11–8. Egan RJ, Bergner CL, Hart PC, Cachat JM, Canavello PR, et al. Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behav Brain Res 2009; 205:38–44. Gerlai R, Lahav M, Guo S, Rosenthal A. Drinks like a fish: zebrafish (Danio rerio) as a behavior genetic model to study alcohol effects. Pharmacol Biochem Behav 2000;67: 773–82. Gerlai R, Chatterjee D, Pereira T, Sawashima T, Krishnannair R. Acute and chronic alcohol dose: population differences in behaviour and neurochemistry of zebrafish. Genes Brain Behav 2009;8:586–99. Greenfield NJ, Pietruszko R, Lin G, Lester D. The effect of ethanol ingestion on the aldehyde dehydrogenases of rat liver. Biochim Biophys Acta 1976;428:627–32. Haseba T, Ohno Y. A new view of alcohol metabolism and alcoholism—role of the highKm Class III alcohol dehydrogenase (ADH3). Int J Environ Res Public Health 2010;7: 1076–92. Haseba T, Tomita Y, Kurosu M, Ohno Y. Dose and time changes in liver alcohol dehydrogenase (ADH) activity during acute alcohol intoxication involve not only class I but also class III ADH and govern elimination rate of blood ethanol. Leg Med 2003;5: 202–11. Haseba T, Duester G, Shimizu A, Yamamoto I, Kameyama K, Ohno Y. In vivo contribution of Class III alcohol dehydrogenase (ADH3) to alcohol metabolism through activation by cytoplasmic solution hydrophobicity. Biochim Biophys Acta 2006;1762:276–83. Haseba T, Kameyama K, Mashimo K, Ohno Y. Dose-dependent change in elimination kinetics of ethanol due to shift of dominant metabolizing enzyme from ADH 1 (Class 1) and ADH3 (Class III) in mouse. Int J Hepatol 2012;2012:408190. Hoog JO, Ostberg LJ. Mammalian alcohol dehydrogenases — a comparative investigation at gene and protein levels. Chem Biol Interact 2011;191:2–7.

Jang ZH, Chung HC, Ahn YG, Kwon YK, Kim JS, Ryu JH, Ryu DH, Kim CH, Hwang GS. Metabolic profiling of an alcoholic fatty liver in zebrafish (Danio rerio). Mol Biosyst 2012; 8:2001–9. Kily LJ, Cowe YC, Hussain O, Patel S, McElwaine S, Cotter FE, et al. Gene expression changes in a zebrafish model of drug dependency suggest conservation of neuroadaptation pathways. J Exp Biol 2008;211:1623–34. Kim DS, Lee CB, Park YS, Ahn YH, Kim TW, Kee CS, et al. Effect of thyroid hormone on the alcohol dehydrogenase activities in rat tissues. J Korean Med Sci 2001;16:313–6. Kishimoto R, Fujiwara I, Kitayama S, Goda K, Nakata Y. Changes in hepatic enzyme activities related to ethanol metabolism in mice following chronic ethanol administration. J Nutr Sci Vitaminol (Tokyo) 1995;41:527–43. Klyosov AA, Rashkovetsky LG, Tahir MK, Keung WM. Possible role of liver cytosolic and mitochondrial aldehyde dehydrogenases in acetaldehyde metabolism. Biochemistry 1996;35:4445–56. Koivula T, Koivusalo M, Lindros KO. Liver aldehyde and alcohol dehydrogenase activities in rat strains genetically selected for their ethanol preference. Biochem Pharmacol 1975;24:1807–11. Kurosu M, Haseba T, Tomita Y, Kamii H, Yamamoto I, Watanabe T. Effects of chronic ethanol feeding on alcohol dehydrogenase (ADH) isozymes in mouse liver. Alc Alc 1988; A62, The abstracts of 4th congress of ISBRA; 1988. Lassen N, Estey T, Tanguay RL, Pappa A, Reimers MJ, Vasiliou V. Molecular cloning, baculovirus expression, and tissue distribution of the zebrafish aldehyde dehydrogenase 2. Drug Metab Dispos 2005;33:649–56. Lockwood B, Bjerke S, Kobayashi K, Guo S. Acute effects of alcohol on larval zebrafish: a genetic system for large-scale screening. Pharmacol Biochem Behav 2004;77:647–54. Mathur P, Guo S. Differences of acute versus chronic exposure on anxiety-like behavioural response in zebrafish. Behav Brain Res 2011;219:234–9. Matsuzaki S, Gordan E, Lieber CS. Increased alcohol dehydrogenase independent ethanol oxidation at high ethanol concentrations in isolated rat hepatocytes: the effect of chronic ethanol feeding. J Pharmacol Exp Ther 1981;217:133–7. Meier P, Seitz HK. Age, alcohol metabolism and liver disease. Curr Opin Clin Nutr Metab Care 2008;11:21–6. Moon KH, Hood BL, Kim BK, Hardwick JP, Conrads TP, Veenstra TD, et al. Inactivation of oxidized and S-nitrosylated mitochondrial proteins in alcoholic fatty liver of rats. Hepatology 2006;44:1218–30. Moon KH, Abdelmegeed MA, Song BJ. Inactivation of cytosolic aldehyde dehydrogenase via S-nitrosylation in ethanol-exposed rat liver. FEBS J 2007;581:3967–72. Pan Y, Kaiguo M, Razak Z, Westwood JT, Gerlai R. Chronic alcohol exposure induced gene expression changes in the zebrafish brain. Behav Brain Res 2011;216:66–76. Pannia E, Tran S, Rampersad M, Gerlai R. Acute ethanol exposure induces behavioural differences in two zebrafish (Danio rerio) strains: a time course analysis. Behav Brain Res 2014;259:174–85. Ramcharitar J, Ibrahim RM. Ethanol modifies zebrafish responses to abrupt changes in light intensity. J Clin Neurosci 2013;20:476–7. Reimers MJ, Hahn ME, Tanguay RL. Two zebrafish alcohol dehydrogenases share common ancestry with mammalian class I, II, IV, and V alcohol dehydrogenase genes but have distinct functional characteristics. J Biol Chem 2004;279:38303–12. Rico EP, Rosemberg DB, Dias RD, Bogo MR, Bonan CD. Ethanol alters acetylcholinesterase activity and gene expression in zebrafish brain. Toxicol Lett 2007;174:25–30. Rico EP, Rosemberg DB, Langoni AS, Souto AA, Dias RD, Bogo MR, et al. Chronic ethanol treatment alters purine nucleotide hydrolysis and nucleotidase gene expression pattern in zebrafish brain. Neurotoxicology 2011a;32:871–8. Rico EP, Rosemberg DB, Seibt KJ, Capiotti KM, Da Silva RS, Bonan CD. Zebrafish neurotransmitter systems as potential pharmacological and toxicological targets. Neurotoxicol Teratol 2011b;33:608–17. Romero F, Shah D, Duong M, Stafstrom W, Hoek JB, Kallen CB, et al. Chronic alcohol ingestion in rats alters lung metabolism, promotes lipid accumulation, and impairs alveolar macrophage functions. Am J Respir Cell Mol Biol 2014. http://dx.doi.org/10. 1165/rcmb.2014-0127OC. (in press). Rosemberg DB, da Rocha RF, Rico EP, Zanotto-Filho A, Dias RD, Bogo MR, et al. Taurine prevents enhancement of acetylcholinesterase activity induced by acute ethanol exposure and decreases the level of markers of oxidative stress in zebrafish brain. Neuroscience 2010;171:683–92. Rosemberg DB, Braga MM, Rico EP, Loss CM, Cordova SD, Mussulini BHM, et al. Behavioral effects of taurine pretreatment in zebrafish acutely exposed to ethanol. Neuropharmacology 2012;63:613–23. Sharkawi M. In vivo inhibition of liver alcohol dehydrogenase by ethanol administration. Life Sci 1984;35:2353–7. Tomita Y, Haseba T, Kurosu M, Watanabe T. Effects of acute ethanol intoxication on aldehyde dehydrogenase in mouse liver. Arukoru Kenkyuto Yakubutsu Ison 1990;25: 116–28. Tomita Y, Haseba T, Kurosu M, Watanabe T. Effects of chronic ethanol intoxication on aldehyde dehydrogenase in mouse liver. Alcohol Alcohol 1992;27:171–80. Tran S, Gerlai R. Time-course of behavioural changes induced by ethanol in zebrafish (Danio rerio). Behav Brain Res 2013;252:204–13. Tran S, Gerlai R. Recent advances with a novel model organism: alcohol tolerance and sensitization in zebrafish (Danio rerio). Prog Neuropsychopharmacol Biol Psychiatry 2014;55:87–93. Tran S, Chatterjee D, Gerlai R. An integrative analysis of ethanol tolerance and withdrawal in zebrafish (Danio rerio). Behav Brain Res 2014. (in press). Vidal F, Toda R, Guitierrez C, Broch M, Fernandez-Muixi F, Lorenzo A, et al. Influence of chronic alcohol abuse and liver disease on hepatic aldehyde dehydrogenase activity. Alcohol 1998;15:3–8.