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in vivo 28: 1175-1180 (2014)

Total Nitric and Inducible Synthase in -dependent Individuals During Detoxification Therapy

MARGARITA ZOGA1, ELIAS TZAVELLAS2, ANASTASIOS IOANNIDIS3, DIMITRIOS KARAISKOS2, THOMAS PAPARRIGOPOULOS2, IOANNIS LIAPPAS2, PARASKEVI PLIATSIKA1, CHRYSSOULA NIKOLAOU1 and STYLIANOS CHATZIPANAGIOTOU1

Departments of 1Medical Biopathology, and 2Psychiatry, Eginition Hospital, National and Kapodistrian University of Athens, Medical School, Athens, Greece; 3Department of Nursing, Faculty of Human Movement and Quality of Sciences, University of Peloponnese, Sparta, Greece

Abstract. Background: The alterations of total nitric oxide NO is produced via the reaction of L-, NADPH (NO) (through total /) and inducible nitric oxide and , catalyzed by the oxide synthase (NOS), synthase (iNOS) concentrations were determined in a producing NO and (4). In mammals, there are three population of alcohol-dependent individuals without liver distinct genes encoding an equal number of NOS isozymes disease upon admission for detoxification, two weeks later or isoforms: neuronal (nNOS or NOS1) found in neuronal and after completion of detoxification (4-6 weeks in total). cells, endothelial (eNOS or NOS3) found in endothelial cells Materials and Methods: Thirty-eight men and nine women and the inducible (iNOS or NOS2) found in , were included in the study. Endogenous nitrite and total chondrocytes and hepatocytes (5). iNOS and nNOS are nitrite/nitrate concentrations were measured colorimetrically soluble found predominantly in the cytosole, and iNOS concentration was measured by enzyme-linked while eNOS is membrane-associated. immunosorbent assay (ELISA). Results: Endogenous and The iNOS isoform is induced by cytokines and endotoxin, total nitrite concentrations were found to be diagnostically producing large amounts of NO as a defense mechanism equally conclusive, whereas iNOS values were not correlated mainly against parasites, bacterial infection and tumor with the other two parameters. All three parameters were growth. It is also involved in the pathogenesis of septic significantly higher in alcohol-dependent individuals shock, as well as in autoimmune diseases and ectopic compared to controls at all time points. Conclusion: The pregnancy (5-7). Altered concentrations of NO have been preventive therapeutic use of iNOS inhibitors in alcohol- found to be associated with inflammatory processes related dependent individuals might avoid the injurious effects of to , reproduction, infection, hypertension, exercise, chronic alcohol abuse, and should be a matter of further type 2 diabetes, hypoxia, and cancer (8-10). investigation. In chronic alcohol abuse, alcoholic liver disease (ALD) is caused by endotoxemia associated with intestinal barrier Nitric oxide (NO) is an important intra- and intercellular leakiness and increased intestinal permeability, through mediator. Its was first identified as the endothelial- disruption of tight junctional proteins and damage of the derived relaxing factor, the principal signal for vascular microtubuli, a result of oxidative injury based on cell relaxation (1). In addition, NO regulates stimulation of the iNOS. However, the exact mechanism of diverse biological activities, such as neurotransmission, tumor alcohol- induced iNOS-mediated disruption is unknown (11- cell killing, immunity and inflammatory processes (2, 3). 13). It has been shown that a -sensitive transcription factor (SNAIL), stimulating epithelial mesenchymal transition (EMT), is activated by iNOS in gut leakiness triggered by alcohol use. EMT is a process by which Correspondence to: Professor Dr. med. Stylianos Chatzipanagiotou, epithelial cells lose their cell polarity and cell–cell adhesion, Ave. Vassilissis Sofias 72, 11526, Athens, Greece. E-mail: and gain migratory and invasive properties to become [email protected], [email protected] mesenchymal stem cells. It is characterized by increased Key Words: Alcohol abuse, NO, iNOS, nitrite, nitrate, alcoholic liver expression of mesenchymal cell markers such as vimentin, disease. as well as by decreased expression of epithelial cell markers,

0258-851X/2014 $2.00+.40 1175 in vivo 28: 1175-1180 (2014) such as the cell–cell junctional proteins E-cadherin, occludin inpatient treatment program with short-term psychotherapy of and the claudins (14-17). cognitive-behavioral orientation. NO is a gaseous free radical with a short in vivo half-life Fasting blood was obtained within 24 h upon admission for detoxification, two weeks later and after completion of the of a few seconds, which is converted to the very stable detoxification protocol (4-6 weeks). Hepatic serum metabolites nitrite and nitrate. Thus, plasma nitrite and glutamic oxaloacetic transaminase (SGOT), serum glutamic pymvic nitrate concentrations are used as markers for activity of transaminase (SGPT), Gamma-glutamyl transferase (γGT), glucose, NOS and the total production of NO radicals. In laboratory Thyroid-stimulating hormone TSH and mean corpuscular volume, practice, plasma nitrite determination is meaningless because or "mean cell volume" (MCV) were measured using conventional nitrite is rapidly oxidized to nitrate. Thus, first endogenous automated laboratory diagnostic methods. plasma nitrite is measured, then nitrate is enzymatically Endogenous nitrite and total nitrite/nitrate concentrations were measured by means of a commercial Total Nitric Oxide kit (R & D converted to nitrite, and the total nitrite concentration is Systems, Minneapolis, USA, Supplied by Anti-cell, Athens, measured (18). Greece). Firstly, endogenous nitrite was measured colorimetrically The aim of the present study was to investigate the by detection of an azo dye product of the Griess reaction. Secondly, alterations of total NO (through total nitrite/nitrate) nitrate was converted to nitrite by and the total concentration and iNOS activity in a population of alcohol- nitrite concentration was determined. Nitrate concentration was dependent individuals without liver disease upon admission to calculated by subtraction of endogenous nitrite from total nitrite. an inpatient alcohol detoxification program, two weeks later, iNOS concentration in plasma was measured by means of a commercial enzyme-linked immunoassay (ELISA) kit following and after the completion of detoxification (4-6 weeks in total), the instructions of the manufacturer (CUSABIO BIOTECH CO., as well as to assess their correlations at these time points. LTD, Hubei Province, PRC). The method was based on the incubation of diluted plasma samples and standards on anti-iNOS Patients and Methods antibody pre-coated microplate wells, and the subsequent detection of bound antigen - antibody complexes by a biotin- conjugated antibody specific for iNOS (sandwich ELISA). For Forty-seven (n=47) alcohol-dependent individuals (38 men and nine visualization of the reaction, an avidin-conjugated horseradish women) were included in the study. Participants were enrolled over peroxidase was added, converting a chromogenic substrate to a a one-year period and had consecutively contacted the Drug and photometrically measurable product, determined using a Alcohol Addiction Clinic of the Eginition University Hospital in microplate reader set to 450 nm with wavelength correction set Athens, Greece. All patients fulfilled the Diagnostic and Statistical to 570 nm. The minimum detectable dose of human iNOS was Manual of Mental Disorders DSM-IV-TR diagnostic criteria for typically less than 0.225 IU/ml. alcohol abuse/dependence and were admitted to this specialized unit Statistical analysis was conducted using SPSS version 15.0 for inpatient alcohol detoxification (19). Patients had abstained from (SPSS Inc., Chicago IL, USA) and Stata version 9.2 (Stata Corp. alcohol for a mean±SD of 24.0±12.2h prior to their admission. LP, College Station, TX, USA). Extremely skewed parameters were Written informed consent was obtained from participants; log-transformed to achieve normality. Fisher’s exact test was used permission for the study was obtained from the Ethics Scientific for comparisons of categorical data. ANOVA was used, with post- Committee of the Eginition Hospital, and the procedures followed hoc Bonferroni-corrected method for multiple comparisons when were in accordance with the ethical standards of the Helsinki needed, to univariately compare mean values between (controls, Declaration of 1975, as revised in 1983. Inclusion criteria were: (i) patients) and within (three discrete time-points of measurement) 22-76 years of age; (ii) absence of serious physical illnesses, as groups, including all participants. All statistical inferences were assessed through physical examination and routine laboratory based on two-tail probabilities. Statistical significance was set at the screening; (iii) absence of another pre- or coexisting DSM-IV-TR p-value of less than 0.05. axis I major mental disorder; however, if symptoms of disturbed mood were present concurrently with alcohol abuse, patients were not excluded from the study, and (iv) absence of other substance Results abuse, with one exception: nicotine. The participants were diagnosed by the Schedules for Clinical Endogenous nitrite, total nitrite and iNOS concentrations. Assessment in Neuropsychiatry (SCAN) and assessed through the Composite International Diagnostic Interview (CIDI) for their The mean values for all three parameters were significantly pattern of alcohol abuse, potential major life problems related to higher in alcohol-dependent individuals compared to controls alcohol consumption and the occurrence of withdrawal symptoms at all times of measurement (p<0.001, Table I). Significantly in the past (20-23). Sociodemographic and psychiatric history data higher endogenous nitrite value was only found at discharge were also recorded. Details on patient assessment are provided in a . admission (p=0.031). previous study published by our group (24). A control group Endogenous and total nitrite concentrations were found to consisting of 160 healthy blood donors (138 men and 22 women) be positively correlated (p<0.001, results not shown), being was used for comparisons. Alcohol detoxification lasted approximately for a week (range=7- diagnostically equally conclusive. iNOS values were not 10 days). supplementation (B complex, C, E) and oral correlated with the other two study parameters (iNOS vs. (30-60 mg daily in divided doses), with gradual tapering endogenous nitrite p=0.311; iNOS vs. total nitrite p=0.384, off over 1-2 weeks, were given, followed by a four- to five-week results not shown).

1176 Zoga et al: Nitric Oxide and iNOS in Alcohol Abuse

Table I. Endogenous nitrite, total nitrite and inducible (iNOS) concentrations, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pymvic transaminase (SGPT), Gamma-glutamyl transferase (γGT) activities and mean corpuscular volume (MCV) values in alcohol -dependent individuals during detoxification therapy and in healthy controls. For alI parameters, the mean and SD (in parentheses) are given.

Alcohol-dependent patients (N=47)

Baseline p-Value* Intermediate p-Value* Final p-Value* p-Value Parameter Controls Intermediate Final Final (N=160) vs. vs. vs. baseline baseline intermediate

Endogenous nitriteLN (μmol/l) 7.42 (8.596) 16.35 (12.034) <0.001 22.02 (17.096) <0.001 20.66 (16.602) <0.001 0.921 0.031 0.164

Total nitriteLN (μmol/l) 8.36 (8.488) 17.06 (12.285) <0.001 21.38 (18.244) <0.001 20.41 (17.010) <0.001 0.343 0.219 0.601 iNOSLN (IU/ml) 25.05 (23.521) 52.20 (63.204) <0.001 44.64 (34.314) <0.001 51.56 (50.308) <0.001 0.874 0.668 0.894

TSH - 1.83 (1.095) - 1.34 (0.991) - 2.07 (1.107) - 0.315 0.489 0.985

MCV - 93.50 (9.249) - 92.42 (7.904) - 91.58 (7.028) - 0.046 0.061 0.260

GlucoseLN - 104.15 (26.822) - 102.32 (22.701) - 106.33 (22.121) - 0.469 0.963 0.141

SGOTLN (U/l) - 62.40 (43.086) - 42.38 (32.654) - 35.02 (25.564) - 0.003 <0.001 0.003

SGPTLN (U/l) - 62.75 (58.293) - 50.78 (53.133) - 43.76 (47.489) - 0.309 0.022 0.002

γGTLN (U/l) - 217.68 (258.584) - 131.70 (210.021) - 105.48 (236.226) - <0.001 <0.001 <0.001

Baseline: on admission; intermediate: 2 weeks after admission; final: on discharge, 4-5 weeks after admission; *vs. controls. LN: Original mean values shown in table, but log-transformed variables used for statistical analysis due to skewness.

Finally, in the control group, total nitrite and iNOS important role in initiating and promoting ALD. concentrations were significantly higher in men than in Concurrently, anti-inflammatory and hepatoprotective women (for total nitrite p=0.048 and for iNOS p<0.001, cytokines, such as interleukin-6 (IL6), IL10, IL22 and results not shown). adiponectin are up-regulated, as a compensatory mechanism protecting against alcoholic injury and (27-30). Routine laboratory examinations. A significant decline of Ethanol consumption inhibits the function of natural killer MCV, SGOT, SGPT and γGT values, which routinely serve cells, which play key roles in anti-viral, antitumor and as indices of the patient’s alcohol misuse, was observed; this antifibrotic defense in the liver (31, 32). is consistent with what is usually expected during alcohol In vitro studies have shown that alcohol-induced intestinal detoxification. There were no significant differences in barrier disruption is caused by the action of iNOS, through glucose and TSH concentrations between patients and the NO overproduction, leading to oxidative tissue damage, gut control group (Table I). leakiness, endotoxemia and liver injury (11). iNOS is also responsible for SNAIL activation, stimulating EMT, resulting Discussion in the disruption of intestinal cell permeability (15). In alcohol abuse, on the clinical level, iNOS determination, ALD is an inflammatory condition resulting from a variety either directly or through endogenous nitrite/total nitrite of factors, including ethanol-induced metabolic-associated determination, might serve as a prognostic marker for NO- depletion, abnormal methionine dependent gut-associated ALD pathogenesis. , malnutrition and activation of innate immunity The population of the present study comprised of (25, 26). , complement, and tumor individuals without liver disease, who were hospitalized for necrosis factor-α, a pro-inflammatory cytokine, play an a four- to six-week detoxification therapy. Determination of

1177 in vivo 28: 1175-1180 (2014) endogenous nitrite, total nitrate and iNOS concentrations at 4 Marletta MA: Nitric oxide synthase: aspects concerning structure three time points, i.e. upon admission, two weeks after, and and . Cell 78: 927-930, 1994. before discharge, showed that all three parameters were 5 Knowles RG and Moncada S: Nitric oxide synthases in significantly higher than in the control population during the mammals. The Biochemical J 298(Pt 2): 249-258, 1994. 6 Mungrue IN, Husain M and Stewart DJ: The role of NOS in entire treatment. No trend for a decrease was observed, even heart failure: lessons from murine genetic models. Heart Failure for those with concentrations higher than of the controls Reviews 7: 407-422, 2002. between admission and discharge, in contrast to the other 7 Al-Azemi M, Refaat B, Amer S, Ola B, Chapman N and Ledger routine laboratory examinations. In a previous report, W: The expression of inducible nitric oxide synthase in the specific markers for alcohol overconsumption, such as human fallopian tube during the menstrual cycle and in ectopic carbohydrate-deficient transferrin and IL6, were found to pregnancy. Fertility Sterility 94: 833-840, 2010. drop significantly following this four- to six-week 8 Lyamina NP, Dolotovskaya PV, Lyamina SV, Malyshev IY and Manukhina EB: Nitric oxide production and intensity of free detoxification program (24). radical processes in young men with high normal and hypertensive Obviously, it is practically difficult to follow-up the course blood pressure. Medical Science Monitor: International Medical J of iNOS changes after completion of detoxification and Experimental Clinical Res 9: CR304-310, 2003. patient discharge; thus, it is unknown whether and when the 9 Maeda S, Tanabe T, Otsuki T, Sugawara J, Iemitsu M, Miyauchi enzyme concentrations may reach normal levels. Taking into T, Kuno S, Ajisaka R and Matsuda M: Moderate regular exercise consideration the pathogenetic involvement of iNOS in ALD, increases basal production of nitric oxide in elderly women. we may hypothesize that the increase in enzyme Hypertension research : official journal of the Japanese Society of Hypertension 27: 947-953, 2004. concentration and activity are already established in alcohol 10 Yugar-Toledo JC, Tanus-Santos JE, Sabha M, Sousa MG, Cittadino dependence before the onset of ALD. Although alcohol- M, Tacito LH and Moreno H Jr.: Uncontrolled hypertension, dependent individuals without liver disease could be uncompensated type II diabetes, and smoking have different considered as a group at high-risk for development of ALD if patterns of vascular dysfunction. Chest 125: 823-830, 2004. untreated, it is problematic for ethical reasons to run studies 11 Tang Y, Forsyth CB, Farhadi A, Rangan J, Jakate S, Shaikh M, in order to acquire data regarding the association of alcohol Banan A, Fields JZ and Keshavarzian A: Nitric oxide-mediated abuse and onset of ALD. intestinal injury is required for alcohol-induced gut leakiness and The present clinical study is in agreement with previous liver damage. Alcoholism, Clinical Experimental Research 33: 1220-1230, 2009. experimental results showing the positive pathogenetic 12 Banan A, Choudhary S, Zhang Y, Fields JZ and Keshavarzian A: relationship between iNOS and alcohol abuse (11, 12, 15). The Ethanol-induced barrier dysfunction and its prevention by main challenge arising would be the preventive therapeutic use growth factors in human intestinal monolayers: evidence for of iNOS inhibitors in the present population in order to avoid oxidative and cytoskeletal mechanisms. J Pharmacology the injurious effects of chronic alcohol abuse. In experimental Experimental Therapeutics 291: 1075-1085, 1999. animal models, the non-selective L-arginine analog NOS 13 Banan A, Fields JZ, Decker H, Zhang Y and Keshavarzian A: inhibitor NG-nitro-L-arginine methyl ester (L-NAME), which Nitric oxide and its metabolites mediate ethanol-induced microtubule disruption and intestinal barrier dysfunction. The J is active against all NOS isoforms, and the selective iNOS Pharmacology Experimental Therapeutics 294: 997-1008, 2000. inhibitor L-N6-(1-iminoethyl)- (L-NIL) have been 14 Yang Z, Rayala S, Nguyen D, Vadlamudi RK, Chen S, and Kumar effective in preventing the manifestation of ALD (11, 12). In R: Pak1 phosphorylation of snail, a master regulator of epithelial- previous reports, both agents have been clinically implemented to-mesenchyme transition, modulates snail’s subcellular in humans, L-NIL for the treatment of asthmatic patients and localization and functions. 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