Active immunization against serum alcohol dehydrogenase normalizes brain dopamine metabolism disturbed during chronic alcohol consumption

Accepted Manuscript Active immunization against serum alcohol dehydrogenase normalizes brain dopamine metabolism disturbed during chronic alcohol consumption Nikita A. Mitkin, Petr K. Anokhin, Maria V. Belopolskaya, Olga Y. Frolova, Ekaterina A. Kushnir, Maxim L. Lovat, Vsevolod V. Pavshintsev PII:

S0741-8329(18)30358-6

DOI:

https://doi.org/10.1016/j.alcohol.2019.06.006

Reference:

ALC 6924

To appear in:

Alcohol

Received Date: 30 December 2018 Revised Date:

17 June 2019

Accepted Date: 25 June 2019

Please cite this article as: Mitkin N.A, Anokhin P.K, Belopolskaya M.V, Frolova O.Y, Kushnir E.A, Lovat M.L & Pavshintsev V.V, Active immunization against serum alcohol dehydrogenase normalizes brain dopamine metabolism disturbed during chronic alcohol consumption, Alcohol (2019), doi: https:// doi.org/10.1016/j.alcohol.2019.06.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Active immunization against serum alcohol dehydrogenase normalizes brain

dopamine metabolism disturbed during chronic alcohol consumption Nikita A Mitkin1, Petr K Anokhin2, Maria V Belopolskaya3, Olga Y Frolova3, Ekaterina A

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Kushnir4, Maxim L Lovat4, Vsevolod V Pavshintsev5,* Laboratory of Intracellular Signaling in Health and Disease, Engelhardt Institute of Molecular

Biology, Russian Academy of Sciences, Vavilov Str. 32, 119991 Moscow, Russia

V. P. Serbsky National Medical Research Center for Psychiatry and Narcology, the Ministry of

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Health of the Russian Federation, Kropotkinsky lane 23, 119034 Moscow, Russia 3

Institute of Mitoengineering MSU, Leninskiye Gory 1, 119192 Moscow, Russia.

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Faculty of Biology, Lomonosov Moscow State University, Leninskye Gory 1-12, 119192

Moscow, Russia

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

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Leninskiye Gory 1-40, 119234 Moscow, Russia; - Corresponding author. Belozersky Institute of Physico-Chemical Biology, Lomonosov

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Moscow State University, Leninskye gory 1-40, 119234 Moscow, Russia. Phone: +7-919-990-

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1400; E-mail: [email protected]

Abstract

Chronic ethanol consumption in high doses is associated with constitutively elevated activity of the serum alcohol dehydrogenase I (ADH I) isoform, which demonstrates a high affinity not only for ethanol but also for a number of bioamine metabolites. Such excessive ADH activity is probably associated with disruptions in the metabolism of neurotransmitters (dopamine, 1

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serotonin and norepinephrine) and subsequent long-term changes in the activity of their receptors. Ultimately, a stable depressive-like condition contributes to the development of patients' craving for ethanol intake, frequent disruptions during therapy, and low efficiency of treatment.

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We applied active immunization against ADH to investigate its efficiency in the reduction of excessive serum ADH activity and regulation of ethanol consumption by chronically ethanol-fed Wistar rats (15% ethanol, 4 months, free-choice method), and we analyzed its ability to influence

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the levels of bioamines in the brain. Immunization (2 injections, 2-week intervals) was performed using a combination of recombinant horse ADH isozyme as an antigen and 2%

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aluminum hydroxide-based adjuvant.

The efficiency of immunization was demonstrated by the production of high titers of ADHspecific antibodies, which was consistent with the significantly reduced ADH activity in the serum of chronically ethanol-fed rats. On the 26th day after the first vaccine injection, we registered significantly lower levels of alcohol consumption compared to ethanol-fed control

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animals, and the difference reached 16% on the 49th day of the experiment. These observations were accompanied by data that showed reduced levels of ethanol preference in immunized rats.

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Chronic alcohol drinking led to a decrease in dopamine and DOPAL (a direct dopamine metabolite and a high-affinity ADH substrate) levels in the striatum, while immunization

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neutralized this effect. Additionally, we observed that inhibition of serum ADH activity caused a decrease in peak dopamine levels during acute alcohol intake in chronically ethanol-fed rats during ethanol withdrawal that was associated with reduced tyrosine hydroxylase activity in the striatum.

The obtained data suggest a significant contribution of ADH to the changes in neurotransmitter systems during chronic alcohol consumption and makes available new prospects for developing innovative strategies for treatment of excessive alcohol intake.

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ACCEPTED MANUSCRIPT Keywords: Alcohol dehydrogenase; ADH; Alcohol consumption; Immunization; Dopamine; DOPAL; DOPET

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Introduction Despite the significance of alcoholism as a disease, the mechanisms of its development and formation of persistent alcohol addiction remain unclear. The key pathway of utilization of exogenous ethanol is provided by the family of alcohol dehydrogenase (ADH) enzymes that

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perform its oxidation to acetaldehyde. In addition, different forms of ADH are known to

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participate in the metabolism of a number of endogenous neurotransmitters (steroid hormones, GABA, catecholamines, serotonin) and their catabolites with high affinity (Huang, 2010; Mårdh, Dingley, Auld, & Vallee, 1986). Chronic alcohol consumption causes increased expression and activity of ADH I in the liver (Crabb, Matsumoto, Chang, & You, 2004) and ADH III in the brain (Raskin & Sokoloff, 1972), as well as a significant constitutive increase in serum ADH I

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levels (Kravos, Malešič, & Levanič, 2005; Meier-Tackmann, Agarwal, & Goedde, 1984). Nonspecific ADH activity could hypothetically influence the neurotransmitters balance in

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the brain. Specifically, ethanol intake leads to significant activation of dopamine receptors in brain areas responsible for reinforcement and reward activities: the nucleus accumbens and

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ventral tegmental area. Euphoria and subsequent dependence on alcohol could be triggered by the response of these areas to ethanol-induced dopamine release (Alaux-Cantin et al., 2013). ADH is known to participate in catecholamine metabolism (Fig. 1), catalyzing conversion of DOPAL (a direct dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde) to DOPET (3,4dihydroxyphenylethanol) (Meiser, Weindl, & Hiller, 2013). Constitutively increased liver and serum ADH activity could be associated with intense conversion of DOPAL located in the serum, exhaustion of its general pool and become the reason for subsequent decreases in dopamine levels (Meiser et al., 2013). It is also known that DOPAL is able to condense with 3

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dopamine resulting in the generation of tetrahydropapaveriloline (THP) (Marchitti, Deitrich, & Vasiliou, 2007). THP, in its turn, appears to be an inhibitor of tyrosine hydroxylase (Kim, Kim, Lee, & Lee, 2005), one of the key enzymes of the dopamine synthesis pathway and an inhibitor of the dopamine transporter (Okada et al., 1998), which is responsible for dopamine reuptake.

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Thus, low DOPAL levels are associated with high peak dopamine release during alcohol intake that is provided by elevated intensity of its synthesis and re-uptake, and functionally results in a high level of positive reinforcement that forms a strong alcoholic motivation (Banerjee, 2014).

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ADH of classes I and II are also involved in serotonin metabolism by inducing the conversion of 5-HIAL (5-hydroxyindole-3-acetaldehyde) to 5-HTOL (5-hydroxytryptophol)

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(Svensson et al., 1999). Acute alcohol intake is characterized by significant induction of serotonin levels, whereas in cases of chronic ethanol consumption serotonin levels are decreased due to strengthened catabolic pathways (Sari, Johnson, & Weedman, 2011). Changes in serotonin transporter 5-HTT and serotonin 5-HT3, 5-HT1B, 5-HT1A receptor activities that are observed during chronic ethanol intake play an important role in the formation of alcoholic

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motivation and, most likely, appear to be compensatory responses to disorders in serotonin metabolism (Sari et al., 2011).

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Similarly, a role of ADH in the exchange of epinephrine and norepinephrine has been shown. ADH demonstrates high affinity to its main metabolite MHPG (3-methoxy-4-

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hydroxyphenylglycol) (Reuster, Buechler, Winiecki, & Oehler, 2003). In cases of increased serum ADH activity, MHPG could be immediately metabolized in the blood (Eisenhofer, Kopin, & Goldstein, 2004), which then leads to a reduction of epinephrine/norepinephrine levels in the brain. In a number of experiments on laboratory animals it has been reported that chronic ethanol intake modulates the levels of norepinephrine in the brain, which in a feedback loop stimulates ethanol consumption. It has been proposed that the postsynaptic alpha-1 adrenergic receptor, present in neurons of the cerebral cortex, plays a key role in this process. Alpha-1 adrenergic receptor blockade results in reduced alcohol consumption (Becker, 2012). 4

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In principle, suppression of excessive ADH activity during chronic alcohol consumption could stabilize the levels of dopamine, serotonin and epinephrine/norepinephrine as well as decrease their peak emission during acute ethanol intake. In our previous study we demonstrated that inhibition of ADH activity with the use of the nonselective blocker 4-methylpyrazole

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influences the levels of dopamine and its metabolites in the striatum of Wistar rats (Pavshintsev et al., 2017). According to this concept, long-term decreases in ADH activity appears to be a perspective therapeutic strategy. Earlier, we have reported that active immunization of

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chronically ethanol-fed Wistar rats using recombinant horse ADH leads to generation of high levels of cross-specific anti-ADH antibodies that is accompanied by a 30% reduction in 15%

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ethanol consumption and normalization of the range of behavioral parameters (Pavshintsev et al., 2017). However, the mechanism of these effects, including the influence of immunization on neurotransmitter metabolism, still appears to be a subject for comprehensive study. Thereby, the main aim of this study was investigation of the changes in functioning of brain neurotransmitters systems as a result of ADH-specific immunization of chronically

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ethanol-fed Wistar rats that could underlie the effect of reduced ethanol consumption.

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Materials and methods

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Experimental rats, groups and the design of the study In the study, we used 100 male Wistar SPF rats (7–8 weeks old) that were received from

the EMBI SPF animal facility of the Russian Laboratory Animal Science Association (EMBI LLC, Novosibirsk, Russia), and they were divided into 10 equal groups according to their weight. All experiments were performed in the animal facility of the Mitoengineering Institute MSU (Moscow, Russia). The animals were housed 3–5 rats per cage under standard conditions: 12/12 light cycle, 10 rph ventilation rate, 19–24 °С (daily temperature variance not more than 2 °С), humidity 30–70%, and standard diet. 5

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The model of chronic alcohol consumption was carried out in 110 rats using a two-bottle free choice method (water and 15% ethanol) for 4 months (Green & Grahame, 2008). The animals had unlimited 24-hour access to 15% ethanol and water. For the first 3.5 months the rats were group housed and the levels of ethanol consumption were measured to control the general

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efficiency of the drinking procedure, but we did not use them for subsequent animal evaluations. Then, 15 days prior to the immunization, the rats were housed in individual cages, and their levels of ethanol consumption were measured daily by weighing the drinking bottles. We

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selected 70 rats that consumed more than 4.7 g of pure ethanol per kg body weight per day based on the 15 days of consumption (the other 40 animals were excluded from subsequent

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experiments). Experimental animals were divided into 7 equal alcohol drinking groups. This type of chronic drinking model was verified and approved in our previous study (Pavshintsev et al., 2017): alcohol consuming rats demonstrated decreased motor activity in exploratory closed plusmaze tests and increased immobility time in the Porsolt forced swim test, suggesting an increase in depressive-like behaviors. We supposed that the rats after the same drinking procedure and

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consuming comparable ethanol quantities in the same two-bottle free-choice method should have similar behavioral features and could be considered as an appropriate model system.

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In addition, 30 control rats were housed in the same conditions as the alcohol-drinking animals (including the individual cages stage) but were not provided with ethanol

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supplementation.

The comprehensive scheme of the experiment is represented in Fig. 2.

Description of the experimental groups are as follows:

1. Control (n=10) – saline injection without ethanol supplementation 2. Control+AlumVax (n=10) – AlumVax adjuvant injection without ethanol supplementation 3. Control immun. (n=10) – immunization with ADH (150 µg/rat) + AlumVax without ethanol supplementation 4. Alc. control (n=10) – saline injection, administration of 15% ethanol, free choice method 6

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5. Alc. control, withdr. (n=10) – saline injection, administration of 15% ethanol, free choice method, alcohol withdrawal 6. Alc. control, withdr., acute EtOH (n=10) – saline injection, administration of 15% ethanol, free choice method, then alcohol withdrawal, and 15% ethanol 8 hours before euthanasia

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7. Alc.+AlumVax (n=10) – AlumVax adjuvant injection, administration of 15% ethanol, free choice method

8. Alc. immun. (n=10) – immunization with ADH (150 µg/rat) + AlumVax, administration of

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15% ethanol, free choice method

9. Alc. Immun, withdr., (n=10) – immunization with ADH (150 µg/rat) + AlumVax,

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administration of 15% ethanol, free choice method, alcohol withdrawal

10. Alc. Immun, withdr., acute EtOH (n=10) – immunization with ADH (150 µg/rat) + AlumVax, administration of 15% ethanol, free choice method, alcohol withdrawal, and 15% ethanol 8 hours before euthanasia

Euthanasia of experimental animals was performed by decapitation on the 49th day of the

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study (counting from the beginning date of immunization) between 2 PM and 4 PM Moscow Russia time. Some of the experimental animals (groups 4, 7, 8) received 15% ethanol up until

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euthanasia, while animals from the 5, 6, 9 and 10 groups experienced a period of ethanol withdrawal for 14 days prior to euthanasia. We provided 15% ethanol to animals from the 6 and

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10 groups 8 hours before euthanasia to estimate the influence of acute alcohol intake on the range of parameters. This kind of experimental design allowed us to evaluate the efficiency of our immunization approach both in chronic alcohol drinking and alcohol withdrawal models. Ethical approval

All in vivo studies were approved by the Official Bioethical Commission of the Mitoengineering Institute, MSU, Moscow (official approval №75). Immunization against ADH

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Immunization was performed in animals of the 3, 8, 9 and 10 groups by subcutaneous injection of the vaccine at four abdominal points on the 1st and 21st days of the experiment. The immunization regime was optimized during preliminary studies (unpublished data). Two-time vaccine administration was necessary for accumulation of high and stable titers of ADH-specific

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antibodies. The vaccine included recombinant horse ADH (Sigma-Aldrich, USA) as an antigen (dose: 150 µg per injection) and 2% aluminum hydroxide-based adjuvant AlumVax (OZ Biosciences, France), and it was adjusted with saline to a final volume of 800 µl per injection.

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Rats in the control groups were injected with saline of an equal volume. To evaluate any nonspecific action of the adjuvant, we injected animals in the 2 and 7 groups with AlumVax

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without the antigen.

Evaluation of anti-ADH antibodies levels in the serum after immunization We collected blood samples from the tail vein of the rats on the 0, 21, 35 and 49 days of the experiment, between 2 PM and 4 PM Moscow Russia time. Preparation of serum samples for

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analysis was performed as described earlier (Pavshintsev et al., 2017). These blood samples were used for all subsequent experiments.

Antibodies levels were estimated in blood serums by enzyme-linked immunosorbent assay

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(ELISA) using HRP-conjugated secondary rabbit anti-rat antibodies (IMTEK, Russia) in a

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1:10,000 dilution and ready to use substrate solution containing 3,3′,5,5′-Tetramethylbenzidine and hydrogen peroxide (Immunotek, Russia). Color intensity was measured at 450 nm using plate spectrophotometer (MaxiSorp, Nunc). As a specific antigen for ELISA we applied recombinant horse ADH, the same that we used for immunization. Also, we used BSA as an antigen that served as a control of antibodies specificity. Measurement of enzymatic serum ADH activity We estimated ADH enzymatic activity in the serum using a modified version (LaniewskaDunaj et al., 2013) of the protocol originally described by Skurský et al. (Skurský, Kovář, & 8

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Štachová, 1979). We mixed 0.1 ml of filtered serum and 1.8 ml of 26 µM pnitrosodimethylaniline (NDMA, Sigma-Aldrich, USA) solution in 0.1 M sodium phosphate buffer (pH 8.5) and 0.1 ml of a mixture containing 0.25 M n-butanol (Sigma-Aldrich, USA) and 5 mM NAD+. NDMA served as a substrate, and a Shimadzu UV-240 spectrophotometer

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(wavelength 440 nm) was used to measure ADH activity using by NDMA discoloration as the result of its reduction by NADH2 produced during n-butanol oxidation by ADH. The obtained values were normalized to protein concentrations in the samples, which were estimated by a

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Bradford assay (Coomassie (Bradford) Protein Assay Kit, Pierce, USA).

Evaluation of enzymatic serum ADH activity towards dopamine metabolites

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We estimated serum ADH activity towards dopamine metabolites DOPAL (3,4dihydroxyphenylacetaldehyde) and DOPET (3,4-dihydroxyphenylethanol). The methodology was the same as described in the previous section, except that we used 0.6 M DOPAL (SigmaAldrich, USA) or 0.6 M of DOPET (Sigma-Aldrich, USA) as a substrate. In addition, we

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performed the same measurements in the presence of 1 mM 4-methylpyrazole (Acros Organics, USA), a nonselective competitive inhibitor of ADH (Chen, McAlhany Jr, & West, 1995), to validate the specificity of ADH action on DOPAL and DOPET processing. We used different

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aliquots of the same samples for the analysis of ADH activity in the absence and presence of 4-

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methylpyrazole.

Analysis of bioamines brain levels in the experimental rats Striatum samples were isolated and homogenized according to the protocol described in

our previous study (Pavshintsev et al., 2017). Measurement of dopamine, serotonin, norepinephrine, DOPAL, DOPET, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) levels in the striatum samples were assessed using high-performance liquid chromatography (HPLC) with the following parameters: electrochemical detection, electrode oxidation potential +0.85 В, mobile phase: 70 mM sodium hydrogen phosphate, 1.5 mM sodium 9

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octyl sulfonate and 40 mM chloroacetic acid, sorbent: silica gel, elution: acetonitrile gradient (0– 60%), elution time: 6–15 min, sensitivity: 0.4 ng/ml. Calibration was performed using commercially available standards (Sigma-Aldrich, USA). Measurement of tyrosine hydroxylase activity in striatum samples

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Measurement of enzymatic tyrosine hydroxylase activity in striatum samples was performed according to the classical methodology (Naoi, Takahashi, & Nagatsu, 1988). Briefly, striatum samples were isolated from the rats 8 hours after acute ethanol intake, homogenized at

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10x volume of 10 mM potassium phosphate buffer (pH 7.4) and centrifuged at 1000g for 60 minutes. We isolated supernatants, measured their total protein content using Bradford assay

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(Coomassie (Bradford) Protein Assay Kit, Pierce, USA) and stored them at -80oC until use. Supernatants (700 µg of total protein per sample) were incubated with 200 µM L-tyrosine (Sigma-Aldrich, USA) as a substrate, 1 mM tetrahydropterin (6R-BH4; Sigma-Aldrich, USA) as a cofactor, 10 µg of catalase (Sigma-Aldrich, USA), 1 mM NSD-1055 (an inhibitor of amino

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acid decarboxylase, BOC Sciences, USA) and 2 mM ferrous ammonium sulfate (Sigma-Aldrich, USA) in 100 mM sodium acetate-acetic acid buffer (pH 6.0). After incubation for 10 minutes at 37oC we stopped the reaction by addition of the equal volume of 0.1 M perchloric acid (Sigma-

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Aldrich, USA), containing 0.1 mM disodium EDTA. The solution was centrifuged for 10

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minutes at 1000g, and supernatant was used for measurement of generated L-DOPA levels by reverse phase HPLC with pulsed amperometric detection according to previously published protocol (Naoi et al., 1988). The mobile phase included 35 mM citric acid, 90 mM sodium acetate, 130 µM EDTA, 230 µM sodium n-octanesulphonate and 10.5% methanol. Elution was carried out at room temperature, flow rate 0.8 ml/min. We performed L-DOPA quantitation by comparison of the peak area with that of standard. Measurement of ethanol levels in blood samples using gas chromatography

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EtOH levels were measured using a Kristall 5000 chromatograph (Chromatek, Russia) under the following conditions: 30 M X 0.25 MM SolGel-Wax 0.25 UM column; injector temperature: 220°C; injection volume: 1 µl; oven temperature: 50°C for 1 min, followed by an increase at 20°C/min to 200°C; flame ionization detection.

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Statistical analyses We performed statistical analysis using one-way analysis of variance (ANOVA) with post hoc Fisher's LSD test or two-way ANOVA followed by Bonferroni’s multiple comparison post hoc

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test applying STATISTICA 10 (TIBCO Software, USA) and Microsoft Excel (Microsoft, USA)

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software. All results are expressed as the mean ± SEM.

Results

Active immunization of Wistar rats causes production of high levels of ADH-specific antibodies

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We used recombinant horse ADH (Sigma-Aldrich, USA) as an antigen during immunization because it is highly conserved in the mammals (Jörnvall & Markovič, 1972) and should cause production of cross-reactive antibodies specific to rat serum ADH. The second

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administration of the vaccine (consisting of horse ADH and AlumVax adjuvant), performed on

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the 21st day of the experiment, caused a significant 5-fold induction of serum anti-ADH antibody titer compared to the animals of the Control and ethanol-fed control groups (Fig. 3) and provided maintenance of the antibodies at high levels until euthanasia (the 49th day of the study) (twoway ANOVA with Bonferroni’s multiple comparison post hoc test; F (7, 288) = 39.362, p < 0.0001 for the experimental group effect; F (3, 288) = 38.419, p < 0.0001 for the time effect; F (21, 288) = 11.282, p < 0.0001 for both factors effect). Post hoc analysis showed that ethanol withdrawal starting from the 35th day of the study did not influence the levels of ADH-specific antibodies in the serum of immunized Wistar rats. Administration of AlumVax adjuvant alone did not cause any changes in the titers of ADH-specific antibodies, which indicates the optimal 11

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immunization conditions. Also, it should be mentioned that the immunization demonstrates high specificity to ADH verified by application of BSA as a control antigen during enzyme-linked immunosorbent assays (Fig. 3 BSA). These data fully agree with our previous results

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(Pavshintsev et al., 2017) and demonstrate reproducibility of our immunization approach.

Chronic alcohol drinking is associated with increased ADH enzymatic activity in the serum of the rats that is neutralized by immunization against ADH

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In our previous study we demonstrated that immunization against ADH in Wistar rats leads

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to normalization of the range of behavioral parameters, including alcohol consumption, which is disturbed during chronic alcohol drinking (Pavshintsev et al., 2017); however, we could not provide any functional explanation of these effects. To evaluate the role of the serum ADH isoform in these processes we estimated its enzymatic activity in both ethanol-fed and immunized groups of animals (Fig. 4) (two-way ANOVA with Bonferroni’s multiple

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comparison post hoc test; F (7, 288) = 101.88, p < 0.0001 for the experimental group effect; F (3, 288) = 16.611, p < 0.0001 for the time effect; F (21, 288) = 4.7941, p < 0.0001 for both factors

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effect). Chronic alcohol drinking led to significantly elevated levels of ADH activity in the serum, in agreement with previously reported data (Kravos et al., 2005; Meier-Tackmann et al.,

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1984). Importantly, elevated serum ADH activity is observed in chronically ethanol-fed animals even after 2 weeks of ethanol withdrawal, which indicates stable physiological changes that could underlie the depression-like condition. Immunization against ADH leads to normalization of its enzymatic activity in the serum of both chronically ethanol-fed rats and the rats after the period of ethanol withdrawal. Furthermore, the decrease in ADH activity observed in response to high titers of anti-ADH antibodies in the serum (Fig. 3) indicates the high specificity of these antibodies against rat ADH.

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Immunization against ADH leads to lower levels of 15% ethanol consumption by chronically ethanol-fed Wistar rats compared to the Alc. control starting from the 26th day after the first vaccine administration A two-way ANOVA of 15% ethanol consumption by the experimental groups showed that

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they were significantly different (F (2, 1323) = 11.95, p < 0.0001 for the experimental group effect; F (48, 1323) = 17.231, p < 0.0001 for the time effect; F (96, 1323) = 21.474, p < 0.0001 for both factors effect). A Bonferroni’s post hoc test showed that administration of the vaccine

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on the 1st and 21st days of the experiment was associated with significantly lower levels of 15% ethanol consumption by chronically ethanol-fed Wistar rats compared to the Alc. control starting

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from the 26th day (p < 0.05; 5.17±0.084 g of pure ethanol/ kg body weight against 5.52±0.079 g of pure ethanol/ kg body weight in the Alc. Control) (Fig. 5A). The effect was stable and was observed until euthanasia and it reached its maximum (16%) on the 49th day of the experiment (4.84±0.212 g of pure ethanol/ kg body weight in the Alc. Immune. group against 5.81±0.059 g

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of pure ethanol/ kg body weight in the Alc. Control). There were no statistically significant differences between the Alc. control and Alc.+Alumvax groups (F (1, 882) = 1.741, p = 0.1874 for the experimental group effect).

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In addition, we calculated the ratio between 15% ethanol intake and total fluid intake (Fig.

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5B) that serves as an indicator of ethanol preference (Li et al., 2014). The rats demonstrated lower ethanol preference after the second vaccine administration, which confirms the functional effect of immunization on alcohol consumption (two-way ANOVA with Bonferroni’s multiple comparison post hoc test; F (2, 1323) = 17.34, p < 0.0001 for the experimental group effect; F (48, 1323) = 21.541, p < 0.0001 for the time effect; F (96, 1323) = 18.352, p < 0.0001 for both factors effect). We observed no differences in either ethanol consumption or ethanol preference between the alcohol drinking control group and the Alc+AlumVax comparison group, which indicates the insufficiency of adjuvant administration to produce a functional effect.

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Immunization against ADH does not influence blood ethanol levels either during chronic alcohol consumption or after acute ethanol intake We measured ethanol levels in the blood of all experimental rats to estimate the influence of immunization against ADH on ethanol metabolism (Fig. 6). Chronic alcohol consumption

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(rats of Alc. control, Alc.+AlumVax and Alc. immun. groups) was associated with stable moderate blood ethanol levels, while they were significantly induced after acute ethanol intake (one-way ANOVA with post hoc analyses Fisher LSD; F (9, 90) = 24.751, p < 0.0001). We did

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not detect ethanol in the blood of either alcohol naïve animals or the rats after the ethanol withdrawal period. It is important to add that immunization against ADH did not influence blood

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ethanol levels during chronic alcohol consumption and after acute ethanol intake. These data are in agreement with our proposition that the produced antibodies could not affect the main pools of ADH located in the liver and the stomach and do not influence ethanol metabolism in general. Chronic alcohol drinking disturbs dopamine metabolism in the brain of Wistar rats, while

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immunization against ADH partially reverses this effect

To evaluate the possible mechanism of the influence of immunization on ethanol consumption, we used HPLC to measure the levels of dopamine, serotonin and norepinephrine in

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the striatum of Wistar rats on the 49th of the experiment (one-way ANOVA with post hoc

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analyses Fisher LSD; dopamine: F (7, 72) = 4.1396, p = 0.0007; DOPAL: F (7, 72) = 4.5509, p = 0.0004; and DOPAC: F (7, 72) = 5.056, p < 0.0001). Despite the fact that a number of researchers have reported the possibility of serotonin

involvement in the mechanism of the development of alcohol dependence (Huang, 2010), in our study serotonin levels did not significantly differ in any of the studied groups (one-way ANOVA with post hoc analyses Fisher LSD; F (7, 72) = 1.4527, p = 0.1981). Similar results were obtained for norepinephrine (data are presented in Supplementary Table 1). In the case of dopamine, prolonged alcohol drinking reduces its concentration in the striatum by almost 2 times 14

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compared to control rats that did not receive ethanol (Fig. 7). Immunization of both ethanol-fed rats and the animals during alcohol withdrawal against ADH leads to normalization of the dopamine level, while immunization by itself without ethanol administration does not affect the values of this parameter.

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Additionally, we estimated the levels of key dopamine metabolites in the striatum: DOPAL (3,4-dihydroxyphenylacetaldehyde), DOPAC (3,4-dihydroxyphenylacetic acid), DOPET (3,4dihydroxyphenylethanol) and HVA (homovanillic acid). Alcohol drinking is associated with a

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strong decrease in the level of DOPAL (0.26±0.128 ng/mg) when compared to the control (0.75±0.105 ng/mg), while immunization normalizes its concentration (0.72±0.108 ng/mg). A

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similar effect was obtained by analyzing the concentration of DOPAC, which is generated from DOPAL through further oxidation by acetaldehyde dehydrogenase (ALDH); alcohol drinking caused a significant reduction in DOPAC concentration from 1.78±0.367 ng/mg (Control) to 0.68±0.292 ng/mg (Alc. control), which is in agreement with previously published data (Woods & Druse, 1996). Immunization compensates for this effect both in ethanol-fed rats and in the

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animals experiencing the period of ethanol withdrawal. DOPET, the product of DOPAL oxidation, demonstrates no variations in all groups of animals. The absence of differences in

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HVA levels could be explained by insufficient sensitivity of the analytical method used and, as a result, high variations in the obtained values.

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Immunization results in changes in the enzymatic activity of serum ADH in chronically ethanol-fed rats towards dopamine metabolites We suggested a mechanism of the influence of the serum ADH isoform on dopamine

levels in the brain. Despite the fact that the metabolic conversion by ADH of DOPAL to DOPET is described as minor (Meiser et al., 2013), ethanol consumption leads to its intense induction as shown in an elevated DOPET/DOPAC ratio (Tank & Weiner, 1979). We estimated the enzymatic activities in the serum collected on the 49th day of the experiment towards DOPAL

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and DOPET (Fig. 8). According to our results, chronic alcohol drinking is associated with elevated serum enzymatic activity towards DOPAL, while both immunization against ADH and inhibition of ADH using 4-methylpyrazole, a nonselective competitive inhibitor, neutralize this effect (one-way ANOVA with post hoc analyses Fisher LSD; F (4, 90) = 10.071, p <0.0001 for

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the experimental group effect; F (1, 90) = 54.693, p <0.0001 for the 4-MP effect; F (4, 90) = 9.7408, p <0.0001 for both factors effect). These data indicate that serum ADH is highly involved in processing of DOPAL, the main dopamine metabolite and that immunization could

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alter this process. Serum enzymatic activity towards DOPET is constitutively insignificant and demonstrates no changes in response to alcohol administration or immunization, which indicates

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low specificity of both ADH and serum in general to this substrate (one-way ANOVA with post hoc analyses Fisher LSD; F (4, 90) = 1.5568, p = 0.19271 for the experimental group effect; F (1, 90) = 0.71796, p = 0.39906 for the 4-MP effect; F (4, 90) =0.18212, p = 0.1821 for both factors effect).

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Immunization against ADH normalizes peak dopamine levels during acute ethanol intake in chronically alcohol drinking Wistar rats experiencing the period of ethanol withdrawal To estimate the influence of alcohol drinking and immunization on the dopamine system,

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we measured peak dopamine levels in the brain during acute ethanol intake (Fig. 9A). In the

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chronically alcohol drinking animals, after the period of ethanol withdrawal followed by acute ethanol intake, there was a significant induction of dopamine level that reached the values of the Intact control, while immunization against ADH neutralized this effect (for the ethanol withdrawal groups: two-way ANOVA with Bonferroni’s multiple comparison post hoc test; F (1, 36) = 16.758, p < 0.0001 for the acute EtOH effect; F (1, 36) = 0.48725, p = 0.4864 for the group effect; F (1, 36) = 6.4083, p = 0.01587 for both factors effect). We proposed that such an outcome could be triggered by the system of dopamine synthesis that is mainly represented by tyrosine hydroxylase (Daubner, Le, & Wang, 2011). We compared the levels of enzymatic tyrosine hydroxylase activity in the striatum of the rats after the period of ethanol withdrawal and 16

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the rats after acute ethanol intake (Fig. 9B). Ethanol intake leads to a significant induction of tyrosine hydroxylase activity in the animals during ethanol withdrawal, while during ADHspecific immunization this effect is much more moderate (two-way ANOVA with Bonferroni’s multiple comparison post hoc test; F (1, 36) = 14.748, p = 0.0005 for the acute EtOH effect; F (1,

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36) = 18.368, p < 0.0001 for the group effect; F (1, 36) = 5.2701, p = 0.02763 for both factors effect). These data indicate that immunization against ADH leads to inhibition of tyrosine hydroxylase activity, which results in reduced peak dopamine levels during acute ethanol intake.

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Discussion

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Active immunization against regulatory self-proteins is now considered to be a powerful approach that enables long-term modulation of physiological processes. A number of autovaccines successfully passed the complex of preclinical trials (Jia, Pan, Li, & Wang, 2013)(Durez et al., 2014)(Krishnamurthy, Selck, Chee, Jhala, & Kay, 2016) and demonstrated both effectiveness and safety. In our previous study we showed that immunization against

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alcohol dehydrogenase (ADH) leads to production of specific antibodies that were associated with reduced ethanol intake and normalization of some behavioral parameters in chronically ethanol-fed Wistar rats (Pavshintsev et al., 2017). Immunization itself did not lead to any

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behavioral impairments or changes in biochemical blood parameters, which indirectly indicates

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the safety of the process, but this undoubtedly requires confirmation in a series of preclinical trials. The main fundamental problem of our approach is the unknown mechanism of action and the lack of information on the link between serum ADH inhibition and specific targets in the brain. In this study, we tried to reveal the role of anti-ADH immunization in metabolism of the key neurotransmitters. Immunization of the Wistar rats with the vaccine, containing horse ADH as an antigen and aluminum hydroxide adjuvant AlumVax, demonstrated reproducible results; it led to stable production of specific anti-ADH antibodies (Fig. 3), significantly lowered levels of ethanol 17

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consumption compared to the Alc. control and decreased levels of alcohol preference in chronically ethanol-fed animals (Fig. 5), which are results similar to what is described in our previous work (Pavshintsev et al., 2017). We suppose that two-time vaccine administration was required for accumulation of high titers of specific antibodies sufficient to obtain the observed

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effects. We considered serum ADH as the target of immunization by virtue of it being the only extracellular ADH form available for the antibodies to bind to, since they cannot pass through intercellular membranes and influence ADH activity in the tissues. This assumption was

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confirmed by the fact that immunization did not influence blood ethanol levels both during chronic alcohol consumption and after acute ethanol intake (Fig. 6), which indicates the inability

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of the produced antibodies to influence ethanol metabolism and to affect the main ADH pools. Apart from this, we did not consider brain ADH to be a possible target because in our previous study its local inhibition did not influence the levels of ethanol intake in alcohol drinking rats (Pavshintsev et al., 2017). Serum ADH activity demonstrated a significant increase as a result of chronic alcohol consumption (Fig. 4) which is in agreement with the existing data (Kravos et al.,

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2005; Meier-Tackmann et al., 1984). This effect was fully neutralized by immunization, which indicates the high specificity of the generated antibodies against the rat serum ADH isoform.

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Due to the described ability of ADH to use the key metabolites of dopamine (Meiser et al., 2013), serotonin (Svensson et al., 1999) and epinephrine/norepinephrine (Reuster et al., 2003) as

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substrates, we proposed that transfer of these metabolites to the bloodstream could be accompanied by their immediate processing by elevated serum ADH, resulting in a reduction in their levels and disturbed neurotransmitters metabolism in the brain. Although serotonin (Huang, 2010) and norepinephrine (Patkar, Belmer, & Bartlett, 2016) are postulated as inducers of alcohol dependence development, in our study their levels demonstrated no changes, neither after chronic alcohol consumption nor as a result of immunization (data are represented in Supplementary Table 1). These results, however, do not allow us to completely exclude the proposition of serotonin and norepinephrine participation in the studied mechanisms: in the 18

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literature much attention is paid to the role of receptors and transporters of these neurotransmitters in the modulation of alcohol addiction (Becker, 2012; Sari et al., 2011). Striatal dopamine level demonstrated a 2-fold decrease during chronic alcohol consumption that was compensated by immunization (Fig. 7). It is important to mention that 2

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weeks of ethanol withdrawal itself did not lead to normalization of the dopamine level, while immunization against ADH was associated with this effect. The obtained data could indicate the primary role of immunization but not the levels of ethanol intake in stabilization of striatal

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dopamine levels. These observations lead us to consider dopamine metabolism pathways. DOPAL, the direct dopamine derivative, can either be oxidized by acetaldehyde dehydrogenase

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(ALDH) to DOPAC (the major pathway) or reduced by ADH/ aldehyde reductase to DOPET (the minor pathway) (Delcambre, Nonnenmacher, & Hiller, 2016; Eisenhofer et al., 2004). Chronic alcohol drinking led to a reduction in DOPAL and DOPAC levels in the striatum (Fig. 7), while during immunization these alterations disappeared. A decrease in DOPAC during longterm ethanol consumption is described in the literature (Woods & Druse, 1996) and could be the

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result of switching of DOPAL processing to the minor pathway (Pérez-Mañá et al., 2015). Although we could not detect any alterations in DOPET striatal levels (Fig. 7), its induction by

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alcohol in other tissues is postulated in some instances (Schröder et al., 2009). We suppose that elevated serum ADH could be involved in excessive DOPAL processing that results in depletion

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of this component from metabolic pathways and disruption of dopamine metabolism in the brain. In support of this assumption, the serum of chronically ethanol-fed Wistar rats demonstrated high activity towards DOPAL, which was reduced both by immunization against ADH and the use of 4-methylpyrazole, a nonselective competitive inhibitor (Fig. 8). These data indicate that serum ADH is highly involved in processing of DOPAL and that immunization could alter this process.

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Importantly, all ethanol-induced impairments in dopamine metabolism were stable during the 2-week ethanol withdrawal and were neutralized by immunization in a similar way as in the chronically ethanol-fed group. In addition, we estimated the influence of alcohol consumption and immunization on the

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dopamine system during acute ethanol intake. For this reason we used chronically alcohol drinking rats after the period of ethanol withdrawal, which seemed to be the most appropriate model because they were accustomed to ethanol consumption compared to alcohol-naïve control

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animals (in which the observed effect could be masked by the differences in ethanol

consumption). In these rats, acute ethanol intake led to a significant increase in dopamine levels

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(Fig. 9A), which is believed to play an important role in ethanol-induced euphoria (Hui & Gang, 2014). Although the induced dopamine levels reached the values of the Intact control, we suppose that this effect should be considered as stimulatory and not as a return to control values because short-term dopamine induction in the striatum caused by acute ethanol intake has been previously reported in many instances (Adermark et al., 2011). Immunization against ADH

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altered this effect. We propose that such an outcome could be triggered by the system of dopamine synthesis that is mainly represented by tyrosine hydroxylase (Daubner et al., 2011).

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Tyrosine hydroxylase activity can be inhibited by tetrahydropapaverinoline, the product of DOPAL and dopamine condensation, that appears to be a reverse regulatory loop (Kim et al.,

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2005). As we have shown (Fig. 5), DOPAL levels are significantly decreased as a result of chronic ethanol consumption, which could be the reason for the elevated tyrosine hydroxylase activity. Testing of enzymatic tyrosine hydroxylase activity in the striatum of the rats experiencing the period of ethanol withdrawal demonstrated that acute ethanol intake is associated with significantly elevated tyrosine hydroxylase activity, but after immunization this effect is not observed. We understand that this result could be explained by normalization of DOPAL levels in the brain (disturbed during chronic alcohol consumption) as a result of immunization, which leads to inhibited tyrosine hydroxylase activity and reduced peak dopamine 20

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levels during acute ethanol intake. Evidently, this mechanism could clarify the influence of immunization against ADH on dopamine levels and its effect on ethanol consumption in general. Thereby, in the current study, we demonstrated the influence of immunization against serum ADH on dopamine metabolism in the brain, but there are still some challenges in the

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evaluation of the role of the neurotransmitters’ receptors and transporters in the described functional effects. In recent studies, a significant role of compounds in the signaling systems of dopamine (receptors D1 and D2, dopamine transporter) (Cheng et al., 2017), serotonin (receptors

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5-HT3, 5-HT1B, 5-HT1A and transporter 5-HTT) (Sari et al., 2011) and

epinephrine/norepinephrine (adrenergic receptor Alpha-1) (Becker, 2012) in the induction of

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alcohol dependence has been extensively discussed. Investigation of the influence of immunization on the functioning of these systems could be a good goal for further studies.

Author Contributions

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N.A.M. designed the study, carried out most of the experiments, analyzed the data and drafted the manuscript. P.K.A. provided bioamines level analysis using HPLC method. M.V.B. participated in substances administration and samples collection. O.Y.F. provided biochemical

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analysis. E.A.K. provided important help with study design and data analysis. M.L.L. participated in data analysis and revised the manuscript. V.V.P. supervised the study, drafted the

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manuscript.

Funding

All experiments were supported by grant 18-75-00057 from Russian Science Foundation.

Conflicts of Interest The authors declare no conflict of interest.

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Crabb, D. W., Matsumoto, M., Chang, D., & You, M. (2004). Overview of the role of alcohol dehydrogenase and aldehyde dehydrogenase and their variants in the genesis of alcohol-related pathology. Proceedings of the Nutrition Society, 63(1), 49–63. Daubner, S. C., Le, T., & Wang, S. (2011). Tyrosine hydroxylase and regulation of dopamine synthesis. Archives of Biochemistry and Biophysics, 508(1), 1–12.

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Jörnvall, H., & Markovič, O. (1972). Structural studies of alcohol dehydrogenase from rat liver. European Journal of Biochemistry, 29(1), 167–174. Kim, Y. M., Kim, M. N., Lee, J. J., & Lee, M. K. (2005). Inhibition of dopamine biosynthesis by tetrahydropapaveroline. Neuroscience Letters, 386(1), 1–4. Kravos, M., Malešič, I., & Levanič, S. (2005). Serum alcohol dehydrogenase levels in patients with mental disorders. Clinica Chimica Acta, 361(1–2), 86–94.

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Laniewska-Dunaj, M., Jelski, W., Orywal, K., Kochanowicz, J., Rutkowski, R., & Szmitkowski, M. (2013). The activity of class I, II, III and IV of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in brain cancer. Neurochemical Research, 38(7), 1517– 1521.

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Li, Y. L., Liu, Q., Gong, Q., Li, J. X., Wei, S. P., Wang, Y. T., … Liang, J. H. (2014). Brucine suppresses ethanol intake and preference in alcohol-preferring Fawn-Hooded rats. Acta Pharmacologica Sinica, 35(7), 853–861. https://doi.org/10.1038/aps.2014.28 Marchitti, S. A., Deitrich, R. A., & Vasiliou, V. (2007). Neurotoxicity and metabolism of the catecholamine-derived 3, 4-dihydroxyphenylacetaldehyde and 3, 4dihydroxyphenylglycolaldehyde: the role of aldehyde dehydrogenase. Pharmacological Reviews, 59(2), 125–150.

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Mårdh, G., Dingley, A. L., Auld, D. S., & Vallee, B. L. (1986). Human class II (pi) alcohol dehydrogenase has a redox-specific function in norepinephrine metabolism. Proceedings of the National Academy of Sciences, 83(23), 8908–8912. Meier-Tackmann, D., Agarwal, D. P., & Goedde, H. W. (1984). Plasma alcohol dehydrogenase in normal and alcoholic individuals. Alcohol and Alcoholism, 19(1), 7–12.

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Meiser, J., Weindl, D., & Hiller, K. (2013). Complexity of dopamine metabolism. Cell Communication and Signaling, 11(1), 34.

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Naoi, M., Takahashi, T., & Nagatsu, T. (1988). Simple assay procedure for tyrosine hydroxylase activity by high-performance liquid chromatography employing coulometric detection with minimal sample preparation. Journal of Chromatography B: Biomedical Sciences and Applications, 427, 229–238. Okada, T., Shimada, S., Sato, K., Kotake, Y., Kawai, H., Ohta, S., … Nishimura, T. (1998). Tetrahydropapaveroline and its derivatives inhibit dopamine uptake through dopamine transporter expressed in HEK293 cells. Neuroscience Research, 30(1), 87–90. Patkar, O. L., Belmer, A., & Bartlett, S. E. (2016). Contribution of noradrenaline, serotonin, and the basolateral amygdala to alcohol addiction: Implications for novel pharmacotherapies for AUDs. In Recent Advances in Drug Addiction Research and Clinical Applications (pp. 115– 141). InTech. Pavshintsev, V. V., Mitkin, N. A., Frolova, O. Y., Kushnir, E. A., Averina, O. A., & Lovat, M. L. (2017). Individual roles of brain and serum alcohol dehydrogenase isoforms in regulation of alcohol consumption in SPF Wistar rats. Physiology and Behavior, 179, 458–466. https://doi.org/10.1016/j.physbeh.2017.07.022 23

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Pérez-Mañá, C., Farré, M., Pujadas, M., Mustata, C., Menoyo, E., Pastor, A., … de la Torre, R. (2015). Ethanol induces hydroxytyrosol formation in humans. Pharmacological Research, 95, 27–33. Raskin, N. H., & Sokoloff, L. (1972). Ethanol-induced adaptation of alcohol dehydrogenase activity in rat brain. Nature New Biology, 236(66), 138–140.

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Reuster, T., Buechler, J., Winiecki, P., & Oehler, J. (2003). Influence of reboxetine on salivary MHPG concentration and cognitive symptoms among patients with alcohol-related Korsakoff’s syndrome. Neuropsychopharmacology, 28(5), 974. Sari, Y., Johnson, V. R., & Weedman, J. M. (2011). Role of the serotonergic system in alcohol dependence: from animal models to clinics. In Progress in molecular biology and translational science (Vol. 98, pp. 401–443). Elsevier.

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Schröder, H., de la Torre, R., Estruch, R., Corella, D., Martínez-González, M. A., Salas-Salvadó, J., … Covas, M. (2009). Alcohol consumption is associated with high concentrations of urinary hydroxytyrosol. The American Journal of Clinical Nutrition, 90(5), 1329–1335. Skurský, L., Kovář, J., & Štachová, M. (1979). A sensitive photometric assay for alcohol dehydrogenase activity in blood serum. Analytical Biochemistry, 99(1), 65–71.

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Svensson, S., Some, M., Lundsjö, A., Helander, A., Cronholm, T., & Höög, J. (1999). Activities of human alcohol dehydrogenases in the metabolic pathways of ethanol and serotonin. European Journal of Biochemistry, 262(2), 324–329. Tank, A. W., & Weiner, H. (1979). Ethanol-induced alteration of dopamine metabolism in rat liver. Biochemical Pharmacology, 28(20), 3139–3147.

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Figure legends

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Woods, J. M., & Druse, M. J. (1996). Effects of Chronic Ethanol Consumption and Aging on Dopamine, Serotonin, and Metabolites. Journal of Neurochemistry, 66(5), 2168–2178. https://doi.org/10.1046/j.1471-4159.1996.66052168.x

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Figure 1. Schematic illustration of ADH participation in metabolic pathways of dopamine, norepinephrine and serotonin ADH – alcohol dehydrogenase; MAO - monoamine oxidase; COMT – catechol-Omethyltransferase; ALDH - aldehyde dehydrogenase; DOPAC - 3,4-dihydroxyphenylacetic acid; DOPET - 3,4-dihydroxyphenylethanol; DOPAL - 3,4-dihydroxyphenylacetaldehyde; MHPG - 3methoxy-4-hydroxyphenylglycol; VMA – vanillylmandelic acid; HVA - homovanillic acid; MOPEGAL - 3-methoxy-4-hydroxyphenylglycoladehyde; MHPE - 3-methoxy-4hydroxyphenylethanol; 5-HIAL - 5-hydroxyindole-3-acetaldehyde; 5-HIAA - 5-hydroxyindole3-acetic acid; 5-HTOL - 5-hydroxytryptophol.

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Figure 2. Design of the experiment

Figure 3. Immunization of Wistar rats against ADH leads to production of high levels of anti-ADH antibodies Immunization against ADH leads to elevated levels of anti-ADH antibodies in the serums of Wistar rats on the 21, 35 and the 49 days of the experiment evaluated by enzyme-linked

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immunosorbent assay (ELISA). Injection of AlumVax adjuvant in the absence of antigen do not cause production of anti-ADH antibodies. Immunization demonstrates high specificity to ADH verified by application of BSA as a control antigen during ELISA. N = 10 in each group, * - p< 0.05 in respect to intact control; # - р< 0.05 in respect to alcohol drinking control. The data are

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represented as mean values ± SEM.

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Figure 4. Chronic alcohol drinking is associated with increased ADH enzymatic activity in the serum of Wistar rats that is neutralized by immunization against ADH Serum ADH activity was evaluated by spectrophotometric assay. Significantly elevated level of serum ADH activity is observed both in chronically ethanol-fed Wistar rats and the animals after the period of ethanol withdrawal, that is fully neutralized by immunization against ADH. AlumVax adjuvant administration in the absence of antigen does not influence serum ADH

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activity. Immunization of intact Wistar rats does not influence the level of background serum ADH activity. Each experimental group included 10 rats, * - p< 0.05 in respect to intact control; # - р< 0.05 in respect to alcohol drinking control. The data are represented as mean values ±

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SEM.

Figure 5. Immunization against ADH leads to lower levels of 15% ethanol consumption by

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chronically ethanol-fed Wistar rats compared to the Alc. control starting from the 26th day after the first vaccine administration (A) The difference in ethanol consumption (evaluated by two-bottle choice method) between control groups and immunized animals was 16% on the 49th day of the experiment. The data are represented as g (grams) of pure ethanol intake per kg body weight per day. Two-way ANOVA with Bonferroni’s post hoc test indicates that alcohol consumption in group Alc. Immun. was significantly lower in respect to alcohol drinking control starting from the 26th day of the experiment, * - р< 0.05 in respect to Alc. control. Animals injected with AlumVax adjuvant without antigen showed no significant differences comparing to alcohol drinking control. (B) The rats demonstrated lower ethanol preference (the ratio between 15% ethanol intake and total fluid intake) after the second vaccine administration. AlumVax administration did not influence 25

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this parameter. N = 10 in each group, * - р< 0.05 in respect to Alc. control. The data are represented as mean values ± SEM.

Figure 6. Immunization against ADH does not influence blood ethanol levels both during chronic alcohol consumption and after acute ethanol intake Blood ethanol levels were measured using gas chromatography. Chronic alcohol consumption

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was associated with stable moderate blood ethanol levels, while they were significantly induced after acute ethanol intake. Immunization against ADH did not influence blood ethanol levels in both cases. Ethanol was not detected in the blood of both alcohol naïve animals and the rats after ethanol withdrawal period. N = 10 in each group, * - p< 0.05 in respect to intact control; # - р<

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0.05 in respect to alcohol drinking control. The data are represented as mean values ± SEM.

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Figure 7. Immunization against ADH normalizes dopamine metabolism in striatum of chronically ethanol-fed Wistar rats

Dopamine, DOPAL, DOPET, DOPAC and HVA levels were measured in striatum samples gained from the rats on the 49th day after the first vaccine administration using high performance liquid chromatography (HPLC). Chronic ethanol consumption leads to a decrease in dopamine levels in striatum of Wistar rats. Immunization of both ethanol-fed rats and the animals during

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alcohol withdrawal against ADH leads to normalization of dopamine, DOPAL and DOPAC levels, while immunization by itself without ethanol administration does not affect the values of these parameters. Administration of adjuvant AlumVax in the absence of the antigen does not influence the levels of dopamine and its metabolites both in ethanol-fed and intact rats. DOPET,

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the product of DOPAL oxidation, demonstrates no variations in all groups of animals. The absence of differences in HVA levels could be explained by insufficient sensitivity of the

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analytical method used and, as a result, high variations in the obtained values. All groups are N = 10, * - p< 0.05 in respect to intact control; # - р< 0.05 in respect to alcohol drinking control. The data are represented as mean values ± SEM.

Figure 8. Chronic alcohol drinking induces serum ADH activity towards DOPAL, the key dopamine metabolite, while immunization neutralizes this effect We estimated the enzymatic activities in the serums collected on the 49th day of the experiment by spectrophotometric assay using the key dopamine metabolites, DOPAL and DOPET, as substrates. Chronic alcohol drinking is associated with elevated serum enzymatic activity towards DOPAL, while both immunization against ADH and inhibition of ADH using 1 mM 4methylpyrazole, a nonselective competitive inhibitor, neutralize this effect. Serum enzymatic 26

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activity towards DOPET is constitutively insignificant and demonstrates no changes in response to alcohol administration or immunization, which indicates low specificity of serum ADH to this substrate. N = 10 in each group, * - p< 0.05 in respect to intact control; # - р< 0.05 in respect to alcohol drinking control. The data are represented as mean values ± SEM.

Figure 9. Immunization against ADH normalizes peak dopamine levels and striatum

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tyrosine hydroxylase activity during acute ethanol intake in chronically ethanol-fed Wistar rats after the period of alcohol withdrawal

(A) In the chronically alcohol drinking animals, after the period of ethanol withdrawal followed by acute ethanol intake, there is a significant induction of dopamine level that reaches the values

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of the Intact control, while immunization against ADH neutralizes this effect. Dopamine levels are measured in striatum samples gained from the rats on the 49th day after the first vaccine

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administration using high performance liquid chromatography (HPLC). (B) Ethanol intake leads to significant induction of tyrosine hydroxylase activity in the animals during ethanol withdrawal, while during ADH-specific immunization this effect is much more moderate. Enzymatic tyrosine hydroxylase activity is measured in collected striatum samples by quantitation of generated L-DOPA. N = 10 in each group, * - p< 0.05 in respect to Alc. control,

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withdr. The data are represented as mean values ± SEM.

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Highlights

- Immunization against ADH reduces ethanol consumption by chronically ethanol-fed rats - Increased ADH activity in serum of alcohol drinking rats is neutralized by immunization - Immunization normalizes levels of dopamine in the brain of alcohol drinking rats

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- Immunization normalizes peak dopamine levels during acute ethanol intake