Jofre Tenorio-Laranga

Pekka T Männistö

Maria Karayiorgou

Joseph A Gogos

J Arturo García-Horsman

J Arturo García-Horsman

Sex-dependent compensated oxidative stress in the mouse liver upon deletion of catechol O-methyltransferase

Keywords: Pulmonary, Renal and Hepatic COMT, oxidative stress, dopamine, Parkinson's disease, proteomics

. Here we perform a proteomic based analysis of the livers of COMT-KO mice in search for potential compensatory mechanisms developed to cope with the effects of disrupted catechol metabolism. We found sex specific changes in proteins connected to stress response. Our results show that alterations in protein levels contribute to the homeostatic regulation in the liver of COMT deficient mice.

Introduction

Catechol-O-methyltransferase [COMT; EC 2.1. 1.6] catalyses the transfer of the methyl group of S-adenosyl-L-methionine into one of the phenolic groups of the catechol substrate in the presence of Mg 2+ . COMT is found in most mammalian tissues, with highest activity in the liver [START_REF] Männistö | Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors[END_REF]. Females have lower COMT activities than males, at least in part due to estrogen down regulation of the promoter function [START_REF] Jiang | Human catechol-O-methyltransferase down-regulation by estradiol[END_REF][START_REF] Xie | Characterization and implications of estrogenic down-regulation of human catechol-O-methyltransferase gene transcription[END_REF]. COMT deficient mice line has been used to evaluate the importance of COMT in catecholamine metabolism. In this animal model, there are dramatic changes in dopamine metabolism; the levels of 3,4-dihydroxyphenylacetic acid (DOPAC) are increased several fold while homovanillic acid (HVA) is undetectable [START_REF] Gogos | Catechol-Omethyltransferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels and behavior[END_REF][START_REF] Huotari | Brain catecholamine metabolism in catechol-O-methyltransferase (COMT)-deficient mice[END_REF][START_REF] Huotari | Effect of dopamine uptake inhibition on brain catecholamine levels and locomotion in catechol-O-methyltransferase-disrupted mice[END_REF]. In spite of that, tissue and extracellular dopamine levels have generally not been increased with the exception of the prefrontal cortex a modest increase of dopamine level (20-25%) was seen in male mice [START_REF] Gogos | Catechol-Omethyltransferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels and behavior[END_REF]. More importantly, a 2-fold slower decay of dopamine has been observed in the frontal cortex but not in the striatum [START_REF] Yavich | Site-specific role of catechol-O-methyltransferase in dopamine overflow within prefrontal cortex and dorsal striatum[END_REF]. Also, in young hairless puppies the skin tyrosinase activity was increased in the mice lacking COMT [START_REF] Forsberg | Lack of increased oxidative stress in catechol-O-methyltransferase (COMT)deficient mice[END_REF].

COMT deficiency results in a number of behavioural deficits, often in a sexually dimorphic and gene dosage dependent manner. COMT-deficient mice show an increase of aggressive behaviour and altered response in anxiety tests [START_REF] Gogos | Catechol-Omethyltransferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels and behavior[END_REF]; altered d-amphetamine induced locomotion induction [START_REF] Huotari | Damphetamine responses in catechol-O-methyltransferase (COMT) disrupted mice[END_REF] altered exploratory phenotypes [START_REF] Babovic | Exploratory and habituation phenotype of heterozygous and homozygous COMT knockout mice[END_REF] as well as a number of cognitive deficits [START_REF] Babovic | Phenotypic characterization of cognition and social behavior in mice with heterozygous versus homozygous deletion of catechol-O-methyltransferase[END_REF]12]. Additional phenotypes have also been described, such as blunted hypertensive response to sodium loading [START_REF] Helkamaa | Resistance to salt-induced hypertension in catechol-O-methyltransferase-genedisrupted mice[END_REF], enhanced voluntary ethanol drinking [START_REF] Tammimäki | Increase in free choice oral ethanol self-administration in male mice with catechol-Omethyltransferase gene disruption[END_REF] and altered pain perception [START_REF] Kambur | Stress-Induced Analgesia and Morphine Responses Are Changed in Catechol-Omethyltransferase-Deficient Male Mice[END_REF].

COMT inhibitors are being used as adjuncts to L-dopa decarboxylase inhibitor therapy in Parkinson's disease (PD). It has been suggested that L-dopa and dopamine are toxic to dopaminergic cells, and therefore the dopamine substitution therapy would A c c e p t e d M a n u s c r i p t 4 enhance oxidative stress in PD patients and in this way accelerate the progression of the disease [START_REF] Fahn | Levodopa-induced neurotoxicity -Does it represent a problem for the treatment of Parkinson's disease?[END_REF][START_REF] Olanow | Oxidation Reactions in Parkinsons-Disease[END_REF]. Several in vitro results indicate that, at high micromolar concentrations, L-dopa and dopamine are indeed toxic to dopaminergic cells and this effect can be linked to increased formation of ROS [START_REF] Basma | L-Dopa Cytotoxicity to Pc12 Cells in Culture Is Via Its Autoxidation[END_REF][START_REF] Mena | Neurotoxicity of Levodopa on Catecholamine-Rich Neurons[END_REF][START_REF] Michel | Toxicity of 6-Hydroxydopamine and Dopamine for Dopaminergic-Neurons in Culture[END_REF]. However, the in vivo evidence of any neuronal damage of L-dopa is conflicting and even a cell protecting role has been reported (reviewed in [START_REF] Fahn | Levodopa-induced neurotoxicity -Does it represent a problem for the treatment of Parkinson's disease?[END_REF]).

Normally, the toxicity of ROS can be handled by defence mechanisms that are readily induced in vivo (see [START_REF] Morel | Repression of gene expression by oxidative stress[END_REF]). For example, the following pathways are activated: a) degradation of H 2 O 2 by catalase and glutathione peroxidase; b) synthesis of reduced glutathione (GSH) by γ-glutamylcysteine synthetase and reduction of oxidized glutathione (GSSG) by glutathione reductase; c) the activity of glutathione transferase and quinone reductase. Simultaneously, some endogenous ROS-generating systems, like liver cytochrome P450 mono-oxygenases, may be repressed. There is evidence from post-mortem studies that GSH levels are decreased [START_REF] Riederer | Transition-Metals, Ferritin, Glutathione, and Ascorbic-Acid in Parkinsonian Brains[END_REF][START_REF] Sian | Alterations in Glutathione Levels in Parkinsons-Disease and Other Neurodegenerative Disorders Affecting Basal Ganglia[END_REF][START_REF] Sian | Glutathione-Related Enzymes in Brain in Parkinsons-Disease[END_REF] and iron levels increased in the substantia nigra in PD [START_REF] Riederer | Transition-Metals, Ferritin, Glutathione, and Ascorbic-Acid in Parkinsonian Brains[END_REF][START_REF] Dexter | Increased Nigral Iron Content and Alterations in Other Metal-Ions Occurring in Brain in Parkinsons-Disease[END_REF][START_REF] Sofic | Increased Iron(Iii) and Total Iron Content in Post-Mortem Substantia Nigra of Parkinsonian Brain[END_REF]. However, the activities of γglutamylcysteine synthetase, glutathione peroxidase and glutathione transferase were not changed [START_REF] Sofic | Reduced and Oxidized Glutathione in the Substantia-Nigra of Patients with Parkinsons-Disease[END_REF]. A GSH deficiency may decrease the capacity of brain tissue to reduce H 2 O 2 and an increased reactive iron may promote the formation of potentially cytotoxic [START_REF] Fahn | Levodopa-induced neurotoxicity -Does it represent a problem for the treatment of Parkinson's disease?[END_REF][START_REF] Jenner | Oxidative stress and the pathogenesis of Parkinson's disease[END_REF][START_REF] Olanow | Etiology and pathogenesis of Parkinson's disease[END_REF]. L-dopa, endogenous catecholamines and their hydroxylated metabolites are all substrates of COMT (see [START_REF] Männistö | Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors[END_REF][START_REF] Kopin | Catecholamine Metabolism -Basic Aspects and Clinical-Significance[END_REF]) and the reaction products (3-O-methyldopa, 3methoxytyramine and homovanillic acid) may be less susceptible to auto-oxidation and even have some antioxidative properties [START_REF] Miller | Oxidative damage caused by free radicals produced during catecholamine autoxidation: Protective effects of O-methylation and melatonin[END_REF][START_REF] Nappi | Hydroxyl radical formation via iron-mediated Fenton chemistry is inhibited by methylated catechols[END_REF]. Since second-generation COMT inhibitors are being used as adjuncts to L-dopa decarboxylase inhibitor therapy in PD, it is important to determine whether deficient COMT activity would modify oxidative A c c e p t e d M a n u s c r i p t 5 stress in the brain, but also in the peripheral tissues. To our knowledge, there are only two conflicting reports dealing with this issue [START_REF] Gerlach | The central catechol-O-methyltransferase inhibitor tolcapone increases striatal hydroxyl radical production in L-DOPA/carbidopa treated rats[END_REF][START_REF] Lyras | Chronic high dose L-DOPA alone or in combination with the COMT inhibitor entacapone does not increase oxidative damage or impair the function of the nigro-striatal pathway in normal cynomologus monkeys[END_REF]. In our previous studies on COMT deficient mouse liver, only mild changes were seen in glutathione S-transferase and quinone reductase [START_REF] Forsberg | Lack of increased oxidative stress in catechol-O-methyltransferase (COMT)deficient mice[END_REF], which were not robust enough to indicate the presence of oxidative stress in these animals. Our preliminary proteomic studies had indicated that glutathione related enzymes and protein kinase C were dysregulated in a sex-dependent manner [START_REF] García-Horsman | Analysis of the proteome of COMT knockout mice[END_REF]. We have now performed a comprehensive proteomic analysis of the liver from COMT-deficient mouse, in search of compensatory mechanisms developed to deal with dramatic changes on dopamine-related metabolite levels and a potential increase of a number of environmental COMT substrates (e.g. dietary polyphenols), some of which may be harmful. Also, activities of enzymes related to oxidative stress were measured in order to determine stress and stress defence indices in the livers of COMT-deficient mice.

˙OH radical from H 2 O 2

Materials and methods.

Animals

Comt gene disrupted mouse strain was originally generated by Gogos and co-workers [START_REF] Gogos | Catechol-Omethyltransferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels and behavior[END_REF], the mutated COMT allele was originally introduced to mixed 129Sv/C57Bl/6J genetic background and by six generation backcrossing the mutation was introduced into a more homogeneous C57Bl/6J genetic background. To collect homozygous mice and their wild type littermates, heterozygous males and females were bred. Comt gene disrupted homozygous (COMT(-/-)) mice as well as their wild-type littermates, were six to eight months old at the time the samples were taken. The procedures with animals

Genotyping

Genomic DNA was isolated from tail biopsies as described in [START_REF] Laird | Simplified mammalian DNA isolation procedure[END_REF]. For genotyping, a PCR method was developed using 5'-ACCATGGAGATTAACCCTGACTACG-3' (sense), 5'-GTGTGTCTGGAAGGTA GCGGTC-3' (antisense) primer set to detect COMT gene (comt) alleles, and 5'-GTGTTCCGGCTGTCAGCGCA-3' (sense), 5'-GTCCTGATAGC-GGTCCGCCA-3' (antisense) primer set to detect mutant alleles containing the neomycin gene (neo) cassette that replaces the exons 2-4 of the COMT gene. Genomic PCR was completed using the Fail Safe PCR system (Epicentre Technologies, Madison, WI, USA) using Fail Safe buffer B and the following thermal cycles: an initial denaturing at 98°C for 1 min and 35 cycles consisting of denaturing temperature of 94°C for 30 s, annealing temperature of 65°C for 1 min, and extension at 72°C for 3 min with a final extension of 70°C for 10 min. The amplified fragments were visualized by ethidium bromide staining under UV light after electrophoresis in a 1.7 % agarose gel.

Two-dimensional difference gel electrophoresis (2D-DIGE)

For proteomics analysis tissues were homogenised with 7 M urea, 4 M thiourea and 4% CHAPS (GE Healthcare, Uppsala, Sweden) with a ratio of 3 ml per mg of tissue. After homogenisation samples were spun at 100,000 x g for 30 min, and the supernatant was The protein standard, labelled with Cy2, consisted of a protein-pool of all homogenates used in the analysis at the same proportions. This Cy2-labelled protein-standard and the Cy3, or Cy5, labelled liver homogenate from wild type, or COMT KO, were combined for IEF and SDS-PAGE. The differentially labelled 2D-gels were serially imaged by laser scanning using Typhoon 9400 (GE Healthcare, Piscataway, USA). Images were normalized against the protein standard and quantitatively analysed using DeCyder software (GE Healthcare Piscataway, USA). Spots which showed statistically relevant density changes were localized in gels, cut out, reduced, alkylated, trypsinized and analysed by LC-MS/MS, using Qstar XL instrument (Applied Biosystems, Foster City, USA). The peptides resulted from digestion were applied in a reverse phase HPLC PepMap C-18 (75 µm x 150 mm, LC-Packing) at 200 nl/min flow, and separated by a 14-47 % acetonitrile gradient in 0.1% formic acid. Eluted fractions were directly applied to a nanospray source of the mass spectrometer, and IDA (informationdependent acquisition) analysis was carried out with acquisition cycles in MS and MS/MS mode along all the chromatogram. Resulting MS/MS spectra were sent to MASCOT server (www.matrixscience.com) using Mascot Daemon programme (Matrix Science Ltd London, UK). with Pro-Q Diamond (Molecular Probe, Leiden, the Netherlands ) for detection of phosphate groups attached to tyrosine, serine or threonine residues. 2D-gels were imaged by laser scanning using Typhoon 9400 (GE Healthcare, Uppsala, Sweden).

Phospho-protein detection in 2D-PAGE

Tissue preparation

Liver was homogenized in 1 ml of buffer (sucrose 250 mM, 10 mM Tris-HCl pH 7.4 and PMSF 0.1 mM) per 100 mg of tissue with a Teflon homogenizer (5 strokes) and centrifuged at 1000 x g for 10 min.

Glutathione determination

Total glutathione was determined by the increase of absorbance at 405 nm due to 5-thio-2-nitrobenzoic (TNB) formation in presence of glutathione reductase and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), using glutathione assay kit (Cayman chemical, Ann Arbor, USA) following manufacturer's instructions. To determinate the oxidized glutathione, the same kit was used but derivatizing reduced glutathione was blocked with 2vinylpyridine before the assay.

Glutathione peroxidase

A c c e p t e d M a n u s c r i p t 9 Glutathione peroxidase activity was determined by measuring the variation of absorbance at 340 nm in presence of NADPH, glutathione, glutathione reductase and ter-butyl hydroperoxide, using BIOXYTECH GPx-340 kit (Oxiresearch, Foster city, USA).

Glutathione reductase

Glutathione reductase activity was determined by measuring the variation of absorbance at 340 nm in presence of GSSG and NADPH, as described in the glutathione reductase assay kit (TREVIGEN Gaithersburg, USA).

Lipid peroxidase

To measure the lipid peroxidase activity, 50 µl of liver tissue samples were diluted to 500 µl with 250 mM sucrose, 10 mM Tris-HCl pH 7.4 and incubated at 37 ºC for 2 hours. After incubation, 10 % ice cold trichloroacetic acid (TCA) was added and spun at 1000 X g for 10 min. From the resulting supernatant, 0.5 ml were separated and mixed with thiobarbutiric acid (TBA) 0.67% and the mixture was boiled for 10 min. After boiling and cooling of the sample, 1 ml of Milli-Q water was added and measured the absorbance at 532 nm.

SDS-PAGE and Western-blot

A c c e p t e d M a n u s c r i p t 10 For PKC-β-I detection, samples were diluted 1:1 with Laemmli loading buffer (Bio-Rad, Hercules CA; 100 mM Tris-HCl pH 6.8, 70 % glycerol, 2 % SDS, 0.005 % bromophenol blue, 10 mM β-mercaptoethanol). The samples were boiled for 5 min, and for PKC-α detection, samples were diluted 1:1 with Laemmli loading buffer and incubated at 30 ºC for 2 h. The samples were loaded to standard 10 % discontinuous gels. Then, the protein was transferred to nitrocellulose by standard procedure (Mini Trans-blot cell, Bio-Rad, Hercules CA). Western blotting was performed under standard conditions using anti-PKC-α or anti-PKC-β-I (Santa Cruz, Santa Cruz,,USA) as primary antibodies, at 1:500 and 1:400 dilution respectively, and anti-rabbit-horseradishperoxidase complex (Pierce, Rockford, USA) diluted to 1:30,000 as secondary antibody. Anti-GADPH primary antibodies were used as control (CalBiochem, San Diego, USA) at 1:3,000, using anti-mouse-horseradish-peroxidase complex (Pierce, Rockford, USA) diluted to 1:30,000 as secondary antibody. Protein visualization was performed using ECL-kit (Amersham-Bioscience, Little Chalfont, UK).

Results

Proteomic analysis

A c c e p t e d M a n u s c r i p t 11 Preliminary analysis of the liver of female COMT-KO mice using traditional 2D proteomics techniques showed altered expression of thrombopoietin, glutathione-Stransferase, glutathione peroxidase, protein kinase C-α and α-actin [START_REF] García-Horsman | Analysis of the proteome of COMT knockout mice[END_REF]. Based on these finding, a larger scale 2D-DIGE analysis was carried out comparing liver samples from COMT-KO and wild type mice of both sexes (Fig. 1). We detected intensity changes in several hundreds of spots. Changes in approximately 100 spots showed a significant pvalue. Out of them, we were able to identify around 50 spots from the gels. The majority of them likely reflects compensatory mechanisms developed in the liver of COMT deficient mice, In COMT-KO female liver, we were able to identify 7 up-and 7 down-regulated proteins. Notably, several are involved in the energy production, such as ATP synthase subunit β and succinyl Coa ligase (which were up-regulated in COMT-KO ~ 2-and ~1.4-fold, respectively, in comparison with their wild type littermates, see Table 1), as well as fructose 1,6-diphosphate aldolase B and aldehyde dehydrogenase (both downregulated ~1.5-fold). Pyruvate carboxylase, an anabolic enzyme from the gluconeogenesis pathway, was also down-regulated ~2-fold. Of interest was that two key enzymes of the urea cycle, agrinase 1 and carbamoyl phosphate synthase 1 were upand down-regulated (1.4 and 1.6-fold) respectively.

Proteins related to the redox regulation and oxidative stress protection, such as carbonic anhydrase and glutathione S-transferase, were up-regulated in the mutant female liver, while 3-hydroxy anthranylate 3,4 dioxygenase, the last enzyme within the kynurenin pathway, was down regulated by a factor of 2. Additionally, regucalcin, a protein which regulates Ca 2+ signalling, was down-regulated 1.7-fold.

Structural proteins were also altered in female mice. Actin 1 decreased by 1.4fold and levels of cytokeratin 8 and cytokeratin 18 isoforms, main partners in In livers from KO male animals, the structural cytokeratins (-8, and -18) were also changed upon COMT disruption, but the enzymatic changes were different and more dramatic than those found in females. We identified three forms of selenium binding protein 2 (SBP2) that were differentially changed in COMT KO mouse liver (spots 5, 6 and 8 in Fig. 1; Table 2). The more acidic isoform (protein 5) was downregulated (-2.2 fold), but those SBP2 isoforms with higher pI (protein 6 and 8) were substantially up-regulated: +2.4, +3.3, respectively. Proteins 1 and 15, identified as an elongation factor 2 and transferrin, were also substantially up-regulated (2.5-and 2-fold, respectively). Down-regulation of proteins 12, 9/18 and 6/16/17, identified as glutathione S-transferase (protein 12), cytokeratine 8 (proteins 9 and 18) and cytokeratine 18 (proteins 6, 16, and 17) was also observed (Table 1).

Biochemical indicators of oxidative stress

Many proteins found to be dysregulated by the disruption of COMT are related to hepatic stress. The possibility that COMT deficiency results in oxidative stress [START_REF] Forsberg | Lack of increased oxidative stress in catechol-O-methyltransferase (COMT)deficient mice[END_REF], prompted us to re-examine a number of liver redox homeostasis markers. GSH:GSSG ratios of around 100 were found in for both genotypes irrespective of sex (Fig. 2A), confirming a redox balance consistent with previous reports [START_REF] Forsberg | Lack of increased oxidative stress in catechol-O-methyltransferase (COMT)deficient mice[END_REF].

Apparently, the levels of GSSG were increased in COMT KO samples but without statistic significance (Fig. 2B). The activities of lipid peroxidase were also higher in A c c e p t e d M a n u s c r i p t 13 females than males. However, only homozygous males showed a slight but consistent increase over the wild type controls (Fig. 2C).

These findings, as well as the identification of changes in a number of glutathione-related enzymes in our proteome analysis, prompted us to assay also the activities of glutathione peroxidase and glutathione reductase (Fig. 3). We found that COMT deficient female mice showed a consistent 25 % increase on glutathione peroxidase activity compared with their wild type littermates, while samples from males showed no significant difference (Fig. 3A). These activities were always slightly higher in males than females. Sex differences were also observed for glutathione reductase: males had considerably lower levels than females but there was no difference between the genotypes (Fig. 3B).

Western blot analysis of protein kinase system

Our proteomic analysis identified variations in different isoforms of either cytokeratin-8 or cytokeratin-18 in both sexes. These proteins are regulated by multiple phosphorylation [START_REF] Omary | Heads and tails" of intermediate filament phosphorylation: multiple sites and functional insights[END_REF] which has a net effect on the isoelectric point observed in 2-D electrophoresis. Additionally, since phosphorylation is a predicted modification for SBP2 [START_REF] Mattow | Selenium-binding protein 2, the major hepatic target for acetaminophen, shows sex differences in protein abundance[END_REF], the different forms of SBP2 found to be modified by COMT disruption, may indicate changes in phosphorylation level as well. These hypotheses were verified by phosphoprotein detection in a 2-D gel electrophoresis, where both unphosphorylated and phosphorylated versions of SBP2 and cytokeratin 8 were detected (Fig. 4).

The changes in phosphorylation levels, in turn, may indicate changes in the phosphatases/kinases balance in the cell. Our preliminary proteome analysis [START_REF] García-Horsman | Analysis of the proteome of COMT knockout mice[END_REF] suggested that the expression of PKC-α may be altered by COMT deficiency and A c c e p t e d M a n u s c r i p t 14 provided indications that other PKC isoforms may be altered as well. We were not able to detect any statistically significant change in PKC levels in our expanded proteomic analysis. However, using western blot analysis to probe directly the levels of two main PKC isoforms from COMT-KO mice livers we found that both liver PKC isoforms levels in COMT KO in males were decreased by about 50 % (PKC-α, Fig. 5A) and 75 % (PKC-β-I, Fig. 6A) compared to wild type littermates. We found no differences in PKC-α(Fig. 5B) and β-I isoforms (Fig. 6B) in the female mice.

Discussion

The main finding of this report is that mice with a disruption of the Comt gene, which causes a complete lack of COMT activity, display multiple and sex dependent metabolic changes, as shown by proteomic, biochemical or immunological assays. Some of these changes probably reflect compensatory responses. Some of these probably reflect compensatory responses. In that regard, it is notable that no changes in the machinery of synthesis or metabolism of dopamine, nor its receptor levels, have been reported previously for these mice [START_REF] Männistö | Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors[END_REF][START_REF] Huotari | Brain catecholamine metabolism in catechol-O-methyltransferase (COMT)-deficient mice[END_REF]. It is important to mention that as a result of similarities of molecular weight and isoelectric point of among many proteins in the proteome, some changes have gone undetected in the present study. Nevertheless, a considerable number of changes could be identified in our proteomic study (see Tables 1 and2).

One general conclusion from the enzymatic assays is that there is no remarkable oxidative stress in COMT deficient mouse liver, supporting our earlier results [START_REF] Forsberg | Lack of increased oxidative stress in catechol-O-methyltransferase (COMT)deficient mice[END_REF].

However, since high levels of L-dopa and dopamine are expected to be toxic, possibly through formation of ROS [START_REF] Basma | L-Dopa Cytotoxicity to Pc12 Cells in Culture Is Via Its Autoxidation[END_REF][START_REF] Mena | Neurotoxicity of Levodopa on Catecholamine-Rich Neurons[END_REF][START_REF] Michel | Toxicity of 6-Hydroxydopamine and Dopamine for Dopaminergic-Neurons in Culture[END_REF], we looked for compensatory mechanisms that could explain the lack of distinct oxidative stress. In this context, the changes described here A c c e p t e d M a n u s c r i p t 15 can be described according to number of different perspectives. First, a number of the observed changes such as increases of proteins involved in detoxification or ROS metabolism appear to reflect compensatory mechanisms to cope with an apparent harsher environment in COMT disrupted liver,. Second, most of the proteins found dysregulated in the COMT disrupted livers, which are not necessarily connected to detoxification routes, are dysregulated in the same way in liver disease, toxicant response, or oxidative stress. Finally, our analysis indicates that several of the changes are sexually dimorphic. Our findings are summarized in Fig. 7. Female mice had milder changes in the proteome expression, but in several diverse pathways. On the other hand, the changes found in the male liver upon COMT disruption, were more dramatic but affected only a small subset of proteins, which were changed in females.

It is important to note, that a direct link between the changes discussed below, the lack of COMT and the changes in catecholamine metabolite levels previously reported [START_REF] Männistö | Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors[END_REF][START_REF] Huotari | Brain catecholamine metabolism in catechol-O-methyltransferase (COMT)-deficient mice[END_REF], especially regarding to the observed sexually dimorphic responses, remains to be demonstrated. Understanding the basis of the observed sexual dimorphisms and the contribution of oestrogens is particularly interesting, since oestrogen catabolism in the liver occurs through a COMT-mediated pathway [START_REF] Männistö | Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors[END_REF].

COMT inhibitors are currently used along with L-dopa therapy in PD. Although COMT KO has been a model for COMT inhibition, the changes reported here, in a COMT-deficient animal model, may not necessarily reflect accurately the ones emerging as a result of acute pharmacological inhibition of COMT activity. There are indeed numerous developmental factors that might determine the outcome in the COMT KO mice, which are certainly not operative during therapy treatment. However, our results might help determine not only unaccounted side effects of COMT inhibitors, but

Proteome of female COMT knockout liver

One remarkable finding in the proteome of the female COMT KO mice was the alteration in the hepatic levels of proteins involved in energy metabolism. We found evidence for dysregulation of several central enzymes in this pathway, such as ATP synthase, succinyl-CoA synthase, and fructose-bisphosphate aldolase. These changes might reflect differences in energy demand between genotypes. These enzymes have also in common that they are related to peroxisome proliferator activated receptor signalling (PPAR), and their expression in hepatic tissues is modified by tumour producing compounds which have peroxisome proliferation activity [see 39, 40 and 41].

In this respect COMT deficiency seems to mimic, in some fashion, carcinogenic activity.

Among the proteins dysregulated in liver from female knockout mice, carbonic anhydrase 3 and GST have documented connection to oxidative stress. Aryl hydrocarbon receptor (AHr) ligands, like PCB126, cause a dramatic reduction of carbonic anhydrase 3 in rat liver which could account for the suppression of the defence system for oxidative stress [START_REF] Ishii | Suppression of carbonic anhydrase III mRNA level by an aryl hydrocarbon receptor ligand in primary cultured hepatocytes of rat[END_REF]. In addition, carbonic anhydrase 3 is significantly increased, in a sex dependent way, in alcohol intoxicated rats where the oxidative stress is evident [START_REF] Parkkila | Expression of testosterone-dependent enzyme, carbonic anhydrase III, and oxidative stress in experimental alcoholic liver disease[END_REF]. GST's, conserved enzymes which detoxify several xenobiotic compounds, are reported to detoxify some of the secondary ROS [START_REF] Gallagher | Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria[END_REF][START_REF] Ravichandran | Sthiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the phagocytosisassociated respiratory burst in blood monocytes[END_REF]. Both enzymes, along with glutathione peroxidase, are substantially increased in COMT disrupted female liver. This is in accordance with the relative increase of oxidized 3-hydroxy-anthranilate 3,4-dioxygenase, which catalyzes the last step before the formation of quinolinic acid (QA), was decreased in COMT disrupted females. QA is a neurotoxin that induces neuronal calcium uptake leading to ROS generation through NMDA-receptor stimulation [START_REF] Jacquard | Brain mitochondrial defects amplify intracellular [Ca2+] rise and neurodegeneration but not Ca2+ entry during NMDA receptor activation[END_REF]. There are indications, that there are NMDA-receptor independent routes for ROS generation operating in the liver [47]. In aged rats, 3hydroxy-anthranilate 3,4-dioxygenase and other kynurenine pathway enzymes are progressively decreased [START_REF] Comai | The effect of age on the enzyme activities of tryptophan metabolism along the kynurenine pathway in rats[END_REF]. The fact that 3-hydroxy-anthranilate 3,4-dioxygenase is decreased in COMT disrupted liver may lead to low QA levels and, in turn, a decreased risk of oxidative damage. It is of interest that carbamoyl-phosphate synthase I and arginase 1, enzymes within the urea cycle and found dysregulated in the liver of COMT disrupted female mice, have been found to be reduced in hepatocellular carcinoma [START_REF] Kinoshita | Underexpression of mRNA in human hepatocellular carcinoma focusing on eight loci[END_REF][START_REF] Yokoyama | Proteomic profiling of proteins decreased in hepatocellular carcinoma from patients infected with hepatitis C virus[END_REF]. Three structural proteins, actin, and cytokeratin 8 and 18, are reported here to be dysregulated in female COMT knockout mice liver. Actin 1 (β-actin, or ACTB), was down-regulated. This protein has been considered a housekeeping enzyme. However, it has been reported dysregulated in cancer cells [START_REF] Khimani | Housekeeping genes in cancer: normalization of array data[END_REF], liver metastases [START_REF] Blanquicett | Housekeeping gene variability in normal and carcinomatous colorectal and liver tissues: applications in pharmacogenomic gene expression studies[END_REF], and in aged rats [START_REF] Chen | Identification of valid housekeeping genes and antioxidant enzyme gene expression change in the aging rat liver[END_REF]. In COMT deleted mice, the changes in actin 1 might also be related to the response to stress in liver.

It should be noted that a number of the observed changes are not consistent with the idea of being compensatory in nature. This is the case, for example, for aldehyde dehydrogenase (ALDH2) and regucalcin, which are down regulated in the liver of female COMT KO mice. It has been established that ALDH2 plays a major role in A c c e p t e d M a n u s c r i p t 18 acetaldehyde detoxification, and its deficiency increases vulnerability to oxidative stress [START_REF] Ohsawa | Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells[END_REF]. In iron loaded hepatic cells, a decrease in ALDH2 activity [START_REF] Britton | Hepatic mitochondrial malondialdehyde metabolism in rats with chronic iron overload[END_REF] and expression [START_REF] Petrak | Proteomic analysis of iron overload in human hepatoma cells[END_REF] have been reported. On the other hand Pastorelli et al. [START_REF] Pastorelli | Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses[END_REF] reported overexpression of ALDH2 in the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated liver in several rat strains. With reference to regucalcin, it was observed in this latter study that it was considerably more abundant in TCDD-resistant rats than in those sensitive to the toxicant. Regucalcin is an intracellular Ca 2+ regulator., which attenuates cell death caused by intracellular Ca 2+ accumulation by enhancing plasma membrane Ca 2+ pumping activity [START_REF] Yamaguchi | Role of regucalcin in calcium signaling[END_REF]. Pastorelli and co-workers [START_REF] Pastorelli | Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses[END_REF] also speculate that the elevated basal levels of regucalcin may confer to the TCDD-resistant strain a protection against disruption of calcium homeostasis as might be mediated by TCDD. Therefore, the decrease of the levels of ALDH2 and regucalcin in the COMT disrupted liver may not be compensatory in nature, but on the contrary, these changes might make the liver more vulnerable to the cellular environment under COMT disruption..

Status of the proteome on male liver from COMT disrupted animals

The main changes found in male COMT-KO mice liver were down-regulation of selenium binding protein 2 (SBP2), and dysregulation of the hepatic specific cytokeratins 8/18 (CK8/18). Transferrin, albumin, and an elongation factor 2 were also found up-regulated. SBP2 is associated with detoxification of chemical compounds. In the mouse, multiple forms of SBP2 are most abundantly expressed in the liver [START_REF] Lanfear | Different patterns of regulation of the genes encoding the closely related 56 kDa selenium-and acetaminophen-binding proteins in normal tissues and during carcinogenesis[END_REF]. We found three protein spots of full-size SBP2, having different pI-values, to be differentially expressed I). These forms are differentially phosphorylated (Fig. 4). We found that SBP2a was down regulated (-2.2-fold), but SBP2b and SBP2c were up-regulated (+2.4-and +3.3-fold, respectively). SBP2 is the toxicity target for acetaminophen in the mouse liver [START_REF] Hoivik | Evidence suggesting the 58-kDa acetaminophen binding protein is a preferential target for acetaminophen electrophile[END_REF][START_REF] Pumford | Immunoblot Analysis of Protein Containing 3-(Cystein-S-Yl)Acetaminophen Adducts in Serum and Subcellular Liver Fractions from Acetaminophen-Treated Mice[END_REF][62]. SBP2 was reduced in the livers of the mice under atherogenic diet [START_REF] Park | Proteomic analysis of diet-induced hypercholesterolemic mice[END_REF], in mice treated with peroxisome proliferators [START_REF] Giometti | Mouse liver selenium-binding protein decreased in abundance by peroxisome proliferators[END_REF] and as a response of activators of peroxisome proliferator-activated receptor-α (PPARα). PPARα activation is important for the induction of cell specific pleiotropic responses, including liver tumours [START_REF] Chu | Protein profiling of mouse livers with peroxisome proliferator-activated receptor alpha activation[END_REF]. In addition, SBP2 may play a role in fibrosis [START_REF] Henkel | Identification of fibrosis-relevant proteins using DIGE (Difference in gel electrophoresis) in different models of hepatic fibrosis[END_REF]. Despite these findings, the precise biological function of SBP2 is scarcely known and its role in catecholamine metabolism is unidentified. It is remarkable that some changes in COMT-KO females were also in proteins known to be modified by peroxisome proliferators, like pyruvate carboxylase, the β subunit of the ATP synthase, and aldehyde dehydrogenase. CK8 (type II) and CK18 (type I) are the only CKs expressed in normal hepatocytes. Changes in CK intermediate filament expression are associated with an abnormal cellular behaviour [START_REF] Ku | Effect of mutation and phosphorylation of type I keratins on their caspase-mediated degradation[END_REF][START_REF] Stumptner | Hepatocyte cytokeratins are hyperphosphorylated at multiple sites in human alcoholic hepatitis and in a Mallory body mouse model[END_REF]. There is a relationship between CK8/18 filaments and the formation of Mallory bodies, hyaline and keratin containing inclusion particles associated with hepatic damage [START_REF] Stumptner | Hepatocyte cytokeratins are hyperphosphorylated at multiple sites in human alcoholic hepatitis and in a Mallory body mouse model[END_REF]. The CK intermediate filament network is important for the structural integrity of hepatocytes [START_REF] Omary | Intermediate filament proteins of the liver: emerging disease association and functions[END_REF], but CK proteins seem to have important non-structural functions as well, including protection from toxic stress in a number of complicated situations [START_REF] Omary | Intermediate filament proteins of the liver: emerging disease association and functions[END_REF][START_REF] Denk | Mallory bodies revisited[END_REF][START_REF] Ku | Mutation of a major keratin phosphorylation site predisposes to hepatotoxic injury in transgenic mice[END_REF][START_REF] Zatloukal | Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form mallory bodies[END_REF]. Our results show that in the male COMT KO liver, the amounts of CK8 and CK18 are reduced to half and one third, respectively, compared with these levels detected in the wild type controls (Table I). Interestingly, we identified several protein spots, such as CK8 (proteins 7, 16 and 17) and CK18 (proteins in the cellular stress response and seems to play a role in the protection from hepatotoxicity [START_REF] Omary | Intermediate filament proteins of the liver: emerging disease association and functions[END_REF][START_REF] Ku | Implications of intermediate filament protein phosphorylation[END_REF]. In human alcoholic hepatitis, phosphorylation of CKs at multiple sites and accumulation of phosphorylated CKs in Mallory bodies, have been reported [START_REF] Chu | Protein profiling of mouse livers with peroxisome proliferator-activated receptor alpha activation[END_REF]. Griseofulvin-or 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated animals (alcoholic hepatitis models) showed an increase of the more acidic isoelectric CK variants due to increased phosphorylation [START_REF] Denk | Mallory bodies revisited[END_REF][START_REF] Cadrin | Modifications in Cytokeratin and Actin in Cultured Liver-Cells Derived from Griseofulvin-Fed Mice[END_REF][START_REF] Salmhofer | Posttranslational Events Involved in Griseofulvin-Induced Keratin Cytoskeleton Alterations[END_REF]. The relation of these findings to the lack of COMT is currently poorly understood, but it seems that the decrease in these cytokeratins might be a sign of predisposition to hepatotoxic injury and increase ability to form Mallory bodies in the liver of COMT disrupted male mice. However, an opposite pattern is evident in female COMT KO mice, where the levels of these CK's, with the exception of one isoform of CK18 (which is slightly decreased, spot 37 in Table 1), where found up-regulated. The mechanistic basis of this difference is not understood, but it might be due to different response to oxidative between sexes (see below)..

The kinase connection

Protein kinases have a central role in practically all functions of the cell, including the response to stress. Considering our biochemical results with sex selective changes in PKC isoforms in COMT KO mice, some explanations seem feasible. It has been reported that PKC-α and -β are activated by ROS, by means of cysteine oxidation of the zinc in the regulatory domain [START_REF] Gopalakrishna | Protein kinase C signaling and oxidative stress[END_REF][START_REF] Kass | Activation of Hepatocyte Protein Kinase-C by Redox-Cycling Quinones[END_REF]. The modification is translated to interactions between the regulatory and catalytic domains, resulting in enzyme activation. In oxidative stress [START_REF] Ohshiro | Reduction of diabetes-induced oxidative stress, fibrotic cytokine expression, and renal dysfunction in protein kinase C beta -Null mice[END_REF]. As a supporting argument, it can be mentioned that PKC-β deficient mice show a reduction of the diabetes-induced oxidative stress [START_REF] Ohshiro | Reduction of diabetes-induced oxidative stress, fibrotic cytokine expression, and renal dysfunction in protein kinase C beta -Null mice[END_REF]. These effects are due, in part, to the decrease of NADPH oxidase activity [START_REF] Dekker | Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils[END_REF]. PKC-β deficient mice show unaffected levels of other PKC isoforms, but the basal and the NADPH stimulated levels of oxygen consumption were reduced. Such results link directly the decrease of PKC-β and the reduction of oxidative stress. Our results suggest a taming of oxidative stress in COMT deficient mice, among other possibilities, through a decrease on PKC-α and PKC-β expression. This might be reflected in the changes on the relative proportions of protein isoforms with certain phosphorylation level.

COMT disruption and sexual dimorphism

The liver redox metabolism is known to be sexually dimorphic. In mitochondria, there is a higher antioxidant gene expression in females than males, and the former sex have a 40 % higher peroxide formation. Our enzymatic and immunoblotting data show that some indicators of cell redox balance in the liver are indeed changed in a sex specific manner due to lack of COMT. Homozygous males showed an slight increase of oxidised glutathione an also o lipid peroxidation was somehow increased. COMT (-/-) homozygous females showed a modest increase in glutathione peroxidase activity, while this change was absent in males. Changes associated with COMT deficiency were not observed when assaying glutathione reductase. Other studies have reported, in agreement with ours, higher importance of glutathione metabolism and general antioxidants defences in females than in males [START_REF] Forsberg | Lack of increased oxidative stress in catechol-O-methyltransferase (COMT)deficient mice[END_REF][START_REF] Mitchell | Quantitative profiling of tissue-and gender-related expression of glutathione S-transferase isoenzymes in the mouse[END_REF]. In females, the shift of redox balance caused by COMT deletion is visualized as an up-regulation of glutathione S-transferase and carbonic anhydrase III, and possibly as an increase of total GSH. This is in agreement with previous studies [START_REF] Jornot | Hyperoxia, unlike phorbol ester, induces glutathione peroxidase through a protein kinase C-independent mechanism[END_REF][START_REF] Pinkus | Role of Quinone-Mediated Generation of Hydroxyl Radicals in the Induction of Glutathione-S-Transferase Gene-Expression[END_REF][START_REF] Raza | Multiple isoforms of mitochondrial glutathione S-transferases and their differential induction under oxidative stress[END_REF], pointing to a general glutathione mobilization as an adaptation to a new environment. Unexpectedly indeed, we observed that in COMT KO males, glutathione S-transferase was downregulated. The meaning of this change remains elusive, but it might account for a higher sensitivity of male than female liver to toxic damage.

The sexually dimorphic changes in the content of hepatic intermediate filament components, CK-8 and CK-18, in the COMT KO mice are more difficult to interpret. While, on average, the levels of these proteins are decreased in males, they are increased in females. The series of events linking the lack of COMT and the CK's regulatory elements are unknown. Notably, changes in these particular cytoskeletal proteins have been linked to conditions which distress liver homeostasis, and this is probably the case in this mouse line. The sex-related difference might be indicative of the different strategies employed by the female organism to cope with stress compared to males.

Sex differences in the responses of Comt gene disruption make the importance of the threshold marker of oxidative stress relevant. During oxidation and excessive thiol consumption, the aromatic amino acids may become the key targets of toxicity. Ldopa is a major product of tyrosine targeted radical reactions [START_REF] Gieseg | Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins[END_REF]. L-dopa itself is a natural cellular constituent and a dopamine precursor, but its levels are very low compared to tyrosine levels. Recently, it has been reported that under conditions where the ratio L-dopa/tyrosine is increased, like in the treatment of PD [START_REF] Rodgers | Evidence for L-dopa incorporation into cell proteins in patients treated with levodopa[END_REF], L-dopa is able to be incorporated in proteins instead of tyrosine [START_REF] Ozawa | Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins[END_REF][START_REF] Rodgers | Biosynthesis and turnover of DOPA-containing proteins by human cells[END_REF]. The increase of L-dopa A c c e p t e d M a n u s c r i p t 23 containing proteins has been proposed to be a trigger for antioxidant defence [START_REF] Nelson | Protein-bound 3,4-dihydroxyphenylanine (DOPA), a redox-active product of protein oxidation, as a trigger for antioxidant defences[END_REF] which might be operative in COMT-KO livers.

COMT is involved in estrogen metabolism that has obvious sex related differences [START_REF] Männistö | Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors[END_REF]. Furthermore, it is of interest that AHr antagonists, like TCDD, increase the hepatic activity of COMT for 2-and 4-hydroxyl 17-β-estradiol methylation, by an unknown mechanism, and that there are studies showing that a cross-talk between the AhR system and the peroxisome proliferator activation receptor system, as well as with other nuclear receptor systems, is operative in normal and stressed conditions, see [START_REF] Hanlon | AhR-and ERKdependent pathways function synergistically to mediate 2,3,7,8-tetrachlorodibenzo-pdioxin suppression of peroxisome proliferator-activated receptor-gamma1 expression and subsequent adipocyte differentiation[END_REF] Therefore it is logical that most of the changes recorded in our present study reflect changes in AhR and peroxisome proliferator activation receptor systems.

We conclude, on one hand, that the livers of COMT deficient mice do not express obvious stress because of the prevalence of effective compensatory mechanisms. These defences involve detoxification of related proteins and protein kinases in a sex dependent manner. That the induction of compensatory mechanism is mostly seen in males, may be related to the fact that the normal levels of liver COMT are higher in males than females. Induction of compensatory mechanism may explain different phenotype-gene-dosage depending phenomena. This hypothesis points to a threshold necessary to surpass in order to active the compensatory machinery.

According with this hypothesis we suggest that the loss of COMT activity in males is sufficient to induce the compensatory mechanism while, in females, which naturally express lower COMT activity levels, the disruption of the gene does not overcome the threshold. On the other hand, and due to the fact that the protein expression changes found in our study mimic those found in hepatic stress in general, it appears that the liver of COMT KO mice is more vulnerable. This might due to increase in catecholamine levels, but also due to the accumulation of other environmental A c c e p t e d M a n u s c r i p t 24 compounds, normally metabolized by COMT, or endogenous catechol oestrogens also affected by the inactivation of the enzyme. Indeed, increased levels of 2methylestradiol, a product of COMT,is thought to have an impact on ovarian and uterine processes [START_REF] Kanasaki | Deficiency in catechol-Omethyltransferase and 2-methoxyoestradiol is associated with pre-eclampsia[END_REF] where oxidative stress could play a role [START_REF] Banerjee | Mouse models for preeclampsia: disruption of redox-regulated signaling[END_REF]. Additional studies are necessary to unravel potential connections along these lines.

  to European Community Guidelines for the use of experimental animals and reviewed by the Animal Ethics Committees at the University of Helsinki in conformance with current legislation.

  2-D DIGE experiments were carried out by the proteomics facility of the Centro de Investigación Príncipe Felipe, Valencia, Spain. Each gel was loaded with a mixture of differentially labelled protein from wild type and COMT KO livers. Duplicates of a total number of 4 gels per gender were run using samples from 4 different COMT KO and 4 different wild type animals. Samples were differentially labelled with the fluorescent dyes Cy2, Cy3, or Cy5 (GE Healthcare, Piscataway, USA).

A c c e p t e d M a n u s c r i p t 8 Phosphoproteins

 8 detection experiments were carried out by the proteomics facility of Centro de Investigación Principe Felipe, Valencia, Spain. Liver homogenate from wild type mice were separated by IEF and SDS-PAGE. The 2D-PAGE were stained for total protein detection with Sypro Ruby (Molecular Probe, Leiden, the Netherlands ) and

  A c c e p t e d M a n u s c r i p t 12 hepatocyte cytoskeleton, were modified. While cytokeratine 8 was up-regulated (~1.5 fold) cytokeratine 18 isoforms were either up-(spot 6, -1.9 fold) or down regulated (spot 37, -1.6-fold).

  explain the decay over time of the effectiveness of this therapeutic regime.

  in KO female liver, since both glutathione peroxidase and Stransferase have a common substrate: reduced glutathione. However, the overall balance of GSH:GSSH, an index of cellular oxidative stress, remains practically unchanged in the KO liver.

  of Comt gene disrupted male mice (Table

Page 21 of 50 A

 50 c c e p t e d M a n u s c r i p t 20 Phosphorylation is one of the most important regulatory protein modifications, particularly in response to external stimuli. Hyper-phosphorylation of CKs is involved

  activation of PKC-α and -β-I isoforms has been reported in diabetic

Figure 1 .

 1 Figure 1. Representative 2-D-DIGE gels showing differentially expressed protein spots in COMT deficient mice with respect to wild type. A. Females, B. Males. Green (Cy3 dye) wild type proteins; Red (Cy5 dye) KO proteins; blue (Cy2) standard proteins (WT:KO mix 50:50%).Numbers refer to the specific spots in tables 1 and 2.

Figure 2 .

 2 Figure 2. Glutathione (A and B) and lipid peroxidase (C) levels in female (♀) and male (♂) liver samples from COMT (-/-) homozygotes (slashed bars) compared with wild type (clear bars) mice. p-values < 0.01, **.

Figure 3 .

 3 Figure 3. Glutathione peroxidase (A), and glutathione reductase (B), and lipid peroxidase (c) activities in liver homogenates from females (first pair) and males (second pair) , comparing COMT knockout (slashed bars) and wild type (clear bars) mice. p-values < 0.001, ***.

Figure 4 .

 4 Figure 4. 2-D-DIGE of liver total protein from male wt stained for protein (Spyro red)

Figure 5 .

 5 Figure 5. Levels of protein kinase C-alpha, determined by western-blot band density, in

Figure 6 .

 6 Figure 6. Levels of protein kinase C-beta I, determined by western-blot band density, in

Figure 7 .

 7 Figure 7. Summary of protein expression changes in mouse liver from COMT-KO

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Figure 2 A

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 6 Figure 6

Figure 7

 7 Figure 7

Acknowledgments

We thank Ms. Marjo Vaha and Ms. Anna Niemi for excellent technical support, and to the Proteomic facility of the Centro de Investigación Príncipe Felipe, member of the Spanish ProteoRed, for outstanding proteomic analysis and support. This work was supported by the Sigrid Juselius Foundation and the Finnish Academy (grant No. 210758 to PTM), and by grants from the Fundación Centro de Investigación Príncipe Felipe (JT-L and JAG-H), and the Ministerio de Educación y Ciencia Ramon y Cajal Programme (JAG-H).