ALTERATION OF ACETALDEHYDE METABOLISM IN CARBON TETRACHLORIDE-INTOXICATED RAT LIVER: ANALYSIS USING LIVER PERFUSION SYSTEM

The hepatic metabolism of acetaldehyde in carbon tetrachloride(CCl₄)-intoxicated rats was studied using a non-recirculatinghaemoglobin-free liver-perfusion system. Acetaldehyde uptakeby the liver from acutely CCl₄-treated animals (4.16 mmol/kg,i.p.) at 24 hr after the treatment was not significantly altered,whereas that by the liver from chronically CCl₄-treated animals(2.08 mmol/kg,i.p., twice a week, for 8–12 weeks) wasdecreased by approximately 50% when it was determined in thepresence of 0.01–5 mM acetaldehyde. In liver from ratschronically intoxicated with CCl₄, the following important biochemicalchanges were observed: (1) The activity of low K_m aldehyde dehydrogenase(ALDH) in hepatic mitochondria was decreased by approximately75%. (2) The basal levels of the lactate/pyruvate (cytosolic[NADH]/[NAD⁺]) ratio as well as the ß-hydroxybutyrate/acetoacetate(mitochondrial [NADH]/[NAD⁺]) ratio were elevated by more than2-fold. (3) Mitochondrial NADH oxidation was also reduced byapproximately 35% of the control level. (4) The basal levelof hepatic oxygen uptake was attenuated by approximately 50%,and the infusion of acetaldehyde (0.01–5.0 mM) causeda further decrease in the uptake. (5) The rate of ethanol productionfrom acetaldehyde by the catalytic action of alcohol dehydrogenasewas found to be unaltered when low concentrations of acetaldehyde(0.01–0.2 mM) were used, whereas a significant suppressionof the rate of ethanol production was detected in the presenceof high concentrations of acetaldehyde (0.6–5 mM). Thesedata suggest that the changes in activity of the lowK_m mitochondrialacetaldehyde dehydrogenase and those in mitochondrial NADH oxidationcoupled with mitochondrial respiration may, at least in part,play important roles in the decreased hepatic acetaldehyde metabolismobserved in chronically CCl₄-treated rats.     

EFFECTS OF ETHANOL ON TESTICULAR STEROIDOGENESIS IN THE RAT

The effects of ethanol and NAD⁺ on the pregnenolone-to-testosteronepathway of testicular steroidogenesis in lysed Leydig cell preparationswere investigated. Testosterone and four precursor steroidswere determined by gas chromatography/mass spectrometry afterincubation of the cell preparations with 100 µM of pregnenoloneand appropriate concentrations of ethanol (1–100mM). Concentrationsof all 4-ene-steroids measured were significantly decreasedeven at the lowest concentration of ethanol. When NAD⁺ (0.1mM) was added to the incubation medium, the levels of progesteroneand 17-hydroxyprogesterone returned to control values, whereasthose of androstenedione and testosterone remained decreased.These results suggest that ethanol may inhibit testicular steroidogenesisby suppressing at least two steps in the pregnenolone-to-testosteronepathway, the pregnenolone-to-progesterone step catalysed byNAD⁺-dependent 5-ene-3ß-hydroxysteriod dehydrogenase/isomeraseand the 17-hydroxyprogesterone-to-androstenedione step catalysedby the NAD⁺-independent C_17–20 lyase.     

CHARACTERISTICS OF ALDEHYDE DEHYDROGENASES OF CERTAIN AEROBIC BACTERIA REPRESENTING HUMAN COLONIC FLORA

We have proposed the existence of a bacteriocolonic pathwayfor ethanol oxidation resulting in high intracolonic levelsof toxic and carcinogenic acetaldehyde. This study was aimedat determining the ability of the aldehyde dehydrogenases (ALDH)of aerobic bacteria representing human colonic flora to metabolizeintracolonically derived acetaldehyde. The apparent Michaelisconstant (K_m) values for acetaldehyde were determined in crudeextracts of five aerobic bacterial strains, alcohol dehydrogenase(ADH) and ALDH activities of these bacteria at conditions prevailingin the human large intestine after moderate drinking were thencompared. The effect of cyanamide, a potent inhibitor of mammalianALDH, on bacterial ALDH activity was also studied. The apparentK_m for acetaldehyde varied from 6.8 (NADP⁺ -linked ALDH of Escherichiacoli IH 13369) to 205 µM (NAD⁺ -linked ALDH of Pseudomonasaeruginosa IH 35342), and maximal velocity varied from 6 nmol/min/mg(NAD⁺ -linked ALDH of Klebsiella pneumoniae IH 35385) to 39nmol/min/mg (NAD⁺ -linked ALDH of Pseudomonas aeruginosa IH35342). At pH 7.4, and at ethanol and acetaldehyde concentrationsthat may be prevalent in the human colon after moderate drinking,ADH activity in four out of five bacterial strains were 10–50times higher than their ALDH activity. Cyanamide inhibited onlyNAD⁺ -linked ALDH activity of Pseudomonas aeruginosa IH 35342at concentrations starting from 0.1 mM. We conclude that ALDHsof the colonic aerobic bacteria are able to metabolize endogenicacetaldehyde. However, the ability of ALDHs to metabolize intracolonicacetaldehyde levels associated with alcohol drinking is ratherlow. Large differences between ADH and ALDH activities of thebacteria found in this study may contribute to the accumulationof acetaldehyde in the large intestine after moderate drinking.ALDH activities of colonic bacteria were poorly inhibited bycyanamide. This study supports the crucial role of intestinalbacteria in the accumulation of intracolonic acetaldehyde afterdrinking alcohol. Individual variations in human colonic floramay contribute to the risk of alcohol-related gastrointestinalmorbidity.     

THE EFFECTS OF DISULFIRAM ON EQUINE HEPATIC ALCOHOL DEHYDROGENASE AND ITS EFFICIENCY AGAINST ALCOHOLISM: VINEGAR EFFECT

The effects of disulfiram, its metabolite diethyldithiocarbamateand dithiodipyridine on alcohol metabolism of equine hepaticalcohol dehydrogenase (EC.1.1.1.1.) have been investigated.They were found to form enzyme-NAD⁺-inhibitor complexes whichwere competitive inhibitors of alcohol metabolism with dissociationconstants (K_EO,I) at pH 7.0 of 50 µM, 1.3 mM and 260 µM,respectively. Acetate and vinegar behaved similarity in formingan inhibitory enzyme-NAD⁺-acetate ternary complex competitivewith ethanol, with at pH 7.0 essentially identical dissociationconstants of 4.0 mM and 3.8 mM, respectively. Disulfiram, diethyldithiocarbamateand dithiodipyridine were also found to exhibit affinity-labellingkinetics with liver alcohol dehydrogenase. The liver enzymeis chemically modified and inactivated in a similar manner byall three reagents via binary enzyme complexes with dissociationconstants of 30 µM, 200 µM and 50 µM, respectively.Used as a protector against enzyme inactivation by DL-     

EFFECT OF CHRONIC ETHANOL ADMINISTRATION ON THIAMINE TRANSPORT IN MICROVILLOUS VESICLES OF RAT SMALL INTESTINE

The effect of long-term ethanol administration on the membranemechanism of thiamine entry in rat enterocytes was investigatedby using microvillous vesicles of small intestine. Experimentswere carried out in three groups of Wistar albino rats of bothsexes (290–400 g of initial body wt). Group I did notreceive any treatment, group II received 4.7 g of ethanol/kgbody wt (as a 50% hydroalcoholic solution) daily by gastricgavage for 35 days and group III (pair-fed controls) receiveda daily solution of saccharose (isoenergetic with the dose ofethanol administered to group II) by gastric gavage for 35 days.Ethanol or saccharose were administered in the morning and astandard diet was given throughout the treatment period. Allanimals were killed by decapitation 24 hr after the last administration,when the blood level of ethanol was virtually zero. Microvilloussmall intestinal vesicles were incubated with ³H-thiamine (³H-T)at 25°C and the amount of ³H-T taken up was measured bya rapid filtration method. Compared with data obtained in groupsI and III, chronic ethanol administration was found to inducea statistically significant decrease in ³H-T vesicular uptakeat 4 sec (determined at ³H-T concentrations ranging from 0.12to 7.5 µM) and a decrease in the apparent J_max (maximaltransport rate) value of the saturable component, without affectingthe apparent K_m (half-saturation concentration) value. These results indicate that in rats chronic ethanol administrationmay impair the intestinal absorption of thiamine by reducingthiamine entry into the enterocyte.     

ETHANOL AND PHOSPHATIDYLETHANOL REDUCE THE BINDING OF [3H]INOSITOL 1,4,5-TRISPHOSPHATE TO RAT CEREBELLAR MEMBRANES

In this study, we have analysed the effect of ethanol and phosphatidylethanol,a unique phospholipid formed only in the presence of ethanol,on the binding of [³H]inositol 1,4,5-trisphosphate to rat cerebellarmembranes. Rats were intraperitoneally injected daily with 3g of ethanol/kg body weight for different periods of time. Repeatedadministration of ethanol induced a reduction in the bindingcapacity (B_max) without affecting the affinity constant (K_d).A significant 32% reduction was observed after 21 days of exposure(from control B_max values of 25±3 pmol/mg and K_d valuesof 9±2 nM). In an in-vitro assay, phosphatidylethanol(500 µM) and phosphatidic acid (500 µM), but noother phospholipids tested, induced a reduction in B_max (39%and 43%, respectively). The observed effect displayed by phosphatidylethanolwas not due to its degradation to phosphatidic acid or otherphospholipids. The results emphasize the importance of examiningphosphatidylethanol (PEth) as a possible mediator of the effectsof ethanol on cellular processes. However, the role of PEthin the observed effect of long-term ethanol exposure still needsfurther consideration.     

TIME COURSE AND GENETIC VARIATION IN THE REGULATION OF CALCIUM CHANNEL ANTAGONIST BINDING SITES IN RODENT TISSUES DURING THE INDUCTION OF ETHANOL PHYSICAL DEPENDENCE AND WITHDRAWAL

Physical dependence was induced by ethanol inhalation in maleSprague-Dawley rats and, in parallel experiments, in two linesof mice (WSR and WSP) genetically selected for differentialseverity of ethanol withdrawal. In dependent rats [³H]nitrendipinebinding sites were significantly increased in cerebral cortex,cardiac and smooth muscle (vas deferens). Cerebral corticalmembranes were the first to show an increase, the B_max for nitrendipinebinding rising sharply after 3–4 days of ethanol administration,whereas binding sites in the other tissues increased after 5–6days. Nitrendipine binding affinity in all tissues was consistentlyreduced immediately preceding the rise in B_max to a new steadystate, but then returned to control values. Between 6 and 10days of ethanol exposure there was no further increase in theB_max for nitrendipine binding, and on removal of ethanol, thenumbers of nitrendipine binding sites fell precipitously tocontrol levels within 24 h of withdrawal. In the geneticallyselected mice, the up-regulation of nitrendipine binding sitesin cardiac membranes was significantly greater in the WSP line.This correlates with severity of physical signs of withdrawaland parallels previous results obtained in brain. The resultsare consistent with an increase in the synthesis and membraneinsertion of dihydropyridine sensitive calcium channel proteinsin several tissues during the induction of ethanol dependenceand suggest that in the brain this change may play a role inthe ethanol withdrawal syndrome.     

EFFECT OF ACUTE AND CHRONIC ETHANOL ADMINISTRATION ON THE TRANSPORT OF THIAMINE FROM PLASMA TO DIFFERENT BRAIN REGIONS OF THE RAT

The effect of ethanol (4.7 g/kg body wt intragastrically asa single dose or once daily for 35 days) on the transport ofthiamine from plasma to four brain regions (cerebellum, cerebralcortex, pons and medulla) was studied in albino rats. Animalswere given an intravenous injection of labelled thiamine witha sampling procedure which allowed the determination of regionalblood flow and tissue thiamine uptake. Regional blood flow wasfound to be enhanced after acute, but non chronic, ethanol administration.The magnitude of increase ranged from 13 to 35% depending onthe brain region being considered. Thiamine was transferredfrom plasma to cerebral tissue by a saturable process with anon-saturable component prevailing at thiamine concentrationsabove 10–15 µM. Three main modifications in thethiamine transport were found as a result of ethanol treatment:a reduction in affinity for the carrier (K_m increased), an increasein maximal transport rate (J_max and an increase in non-saturablediffusion (K_D constant increased). The effects were more pronouncedafter acute ethanol administration. As a consequence of thesemodifications both acute and chronic ethanol treatment causedan increase in thiamine transport rate at high plasma concentrations.On the contrary, at low (physiological) plasma concentrations,thiamine transport was little increased by acute ethanol administrationand virtually unaffected by chronic ethanol intoxication.     

INVOLVEMENT OF GENETIC POLYMORPHISM OF ALCOHOL AND ALDEHYDE DEHYDROGENASES IN INDIVIDUAL VARIATION OF ALCOHOL METABOLISM

The involvement of genetic polymorphism at the alcohol dehydrogenase2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci in determiningblood acetaldehyde levels and the rate of ethanol eliminationafter ethanol intake was investigated. Sixty-eight healthy subjectsingested 0.4 g of ethanol per kg of body weight over 10 min.Blood acetaldehyde levels scarcely increased in the subjectshomozygous for ALDH2^*I, regardless of their ADH2 genotypes (ADH2^*1/^*1,ADH2^*1/^*2 and ADH2^*2/^*2). The acetaldehyde levels in the subjectswith the ALDH2^*1/^*2 heterozygote increased to 23.4 µMon average, and no significant differences were observed betweenthe three ADH2 genotype groups. Subjects homozygous for ALDH2^*2showed very high levels of blood acetaldehyde, and the averagevalue was 79.3 µM. The values of Widmark's ß₆₀(mg/ml/hr)and ethanol elimination rate (mg/kg/br) showed significant differencesamong the three ALDH2 genotypes, and in decreasing order thevalues were ALDH2^*1/^*1, ALDH2^*1/^*2, ALDH2^*2 However, no significantdifferences were seen among the ADH2 genotypes.     

PLATELET AFFINITY FOR SEROTONIN IS INCREASED IN ALCOHOLICS AND FORMER ALCOHOLICS: A BIOLOGICAL MARKER FOR DEPENDENCE?

The kinetics of ³H serotonin platelet uptake were studied inalcoholics and former alcoholics to see whether differencesfound between alcohol-preferring and non-preferring rats couldbe reproduced in man. Three groups of patients were studied:10 dependent alcoholics on admission for treatment; 10 dependentalcoholics after 20 days of treatment; 8 former dependent alcoholics,abstinent for 1–11 years. Controls were non-alcoholics,matched for age and sex. The K_m for ³H serotonin uptake in platelets was lower in patientsfrom all three groups compared to 15 controls. This phenomenon could be congenital or induced by the previousexcessive intake of alcohol. We believe that this increased platelet affinity for serotonin,in the absence of cirrhosis of the liver and/or depression couldbe a marker for alcohol dependence, enabling the therapeuticeffort to be focussed on these patients.     

ERRATUM

In the article entitled ‘Lead Absorption in Police Small-armsInstructors’, Journal of the Society of Occupational Medicine,26, 139–140, the threshold limit value for lead in airwas given as 0.5 mg/m³. This is incorrect and the figure shouldread 0.15 mg/m³. The author believes that this does not alterin any way the main conclusions drawn in the article but infact reinforces them.     

AGE-RELATED DIFFERENCES IN BLOOD ETHANOL PARAMETERS AND SUBJECTIVE FEELINGS OF INTOXICATION IN HEALTHY MEN

Healthy men within 4 age groups, 20–29, 30–39, 40–49and 50–59 years (N = 12 per group), drank 0.68 g/kg ethanolas neat whisky after fasting overnight. The drink was finishedwithin 20 min and the concentrations of ethanol in capillaryblood were determined at 30/60 min intervals for 7 hr. At thetime of blood sampling, the men were asked to estimate theirfeelings of intoxication according to an arbitrary scale onwhich the score 10 indicated ‘tipsy’ or a ‘Littlehigh’. The time course of blood ethanol concentrationwas similar in all 4 groups with the curve for 50–59-year-oldmen on the highest level and 20–29-year-old men lowest.Subjective intoxication scores were significantly less in theyoungest age group and the maximum score for each subject wascorrelated with age (r = 0.45, P << 0.01). Total bodywater (TBW) was not correlated with age directly (r = 0.17,P > 0.05) but when it was expressed as percent of body weighta strong negative correlation was found (r = –0.77, P> 0.001). The mean total body water estimated from ethanoldilution was 46.7 ± 4.11. (+ SD, N = 48) and this correspondsto 58% of mean body weight of the men. The distribution volumeof ethanol/kg body weight (V_d) decreased with ageing (r = –0.59,P < 0.001) being 0.72, 0.71, 0.68 and 0.66 l/kg on averagein the 20–29, 30–39, 40–49 and 50–59-year-oldage groups respectively. The reduction of V_d corresponds toan age-related decrease in the percentage of body water andan increase in the percentage of body fat. This results in elevatedblood ethanol concentrations in older individuals for a constantdose/kg body weight. The absorption of ethanol from the gutand its rate of disappearance from blood were not markedly influencedby age. The lower subjective feelings of intoxication reportedby younger men may indicate that age-related differences inacute functional tolerance to ethanol or different thresholdvalues of blood ethanol may exist before an effect is felt.     

The A1 allele of the D2 dopamine receptor gene is associated with high dopamine transporter density in detoxified alcoholics

The A1 allele of TaqI A restriction fragment length polymorphism(RFLP) in the D₂ receptor (DRD₂) gene locus has been suggestedto be associated with low D₂ receptor density in man. Striataldopamine transporter (DAT) densities were studied with [¹²³I]2-ß-carbometoxy-3ß(4-iodophenyl)tropaneand single-photon emission tomography in 29 detoxified alcoholics,who were also genotyped for the two alleles of TaqI A RFLP atthe DRD2 receptor gene locus. Alcoholics with the A1/A2 genotypes(n = 10) had statistically significantly higher DAT densitiesthan subjects with the A2/A2 genotypes [n = 19; 8.0 ±1.2 (mean ± SD) vs 6.9 ± 1.1, P = 0.035]. We suggestthat the TaqI A RFLP is in linkage disequilibrium with a genevariant modifying DAT density in alcoholics.     

PARAMETERS Vm'. AND Km FOR ELIMINATION OF ALCOHOL IN YOUNG MALE SUBJECTS FOLLOWING LOW DOSES OF ALCOHOL

Seventy-four (44 under fasting conditions and 30 following oralliquid meals) sets of post-absorptive human capillary bloodalcohol concentration-time data were computer-fitted to theintegrated form of the Michaelis-Menten equation by numericalintegration by nonlinear least squares to provide 74 pairs ofthe kinetic V_m' and K_m, parameter values. The parameters werehighly correlated (r = 0.915) by orthogonal least squares. Eightof the fasting subjects received four different oral doses ofalcohol and fourteen subjects each received three differentalcohol treatments. Intra-subject variances of V_m', K_m, andthe ratio V_m', K_m were calculated from the multiple treatments.Inter-subjed variances were calculated from the 22 mean valuesof each parameter. Each parameter and the intra-subject variancesof the parameters were found to be log normally distributed.The Liquid meals (carbohydrate, fat and protein separately)appeared not to affect the parameter values. The computer fittingswere all excellent as evidenced by the relatively small standarddeviations of the estimated parameters and other statisticalmeasures of fit.     

REGULATION OF RAT NEURONAL NITRIC OXIDE SYNTHASE ACTIVITY BY CHRONIC ALCOHOLIZATION

Rat neuronal nitric oxide (NO) synthase (nNOS) activity wasmeasured in frontal cortex, hippocampus, striatum and cerebellumusing the assay of [³H]citrulline, following chronic alcoholization.The K_m and V_max values were significantly increased in the frontalcortex and in the striatum, and were not affected in the cerebellumand hippocampus.     

INACTIVATION OF CEREBELLAR NITRIC OXIDE SYNTHASE BY ETHANOL IN VITRO

The N-methyl-D-aspartate receptor/nitric oxide synthase (NOS)/guanylatecyclase pathway, which plays a crucial role in synaptic plasticityin the brain, is modulated by ethanol. We studied the effectof ethanol in vitro on NOS in rat cerebellum and showed thatethanol (25–200 mM) inactivated NOS in a dose-dependentmanner. This inactivation was prevented by the biopterin cofactortetrahydrobiopterin (BH⁴) as well as by L-arginine, a NOS substrate,but not by NADPH. These results suggest that ethanol reducesNOS activity by modulating the conformation of the enzyme andthereby its stability, probably by interacting with the bindingsites of BH⁴ and/or of L-arginine. Our data also suggest thatinactivation of NOS may contribute to the decrease in the cGMPlevel, and thus may play a role in the pharmacological actionsof ethanol in vivo.     

Ca2+-activated K+ channels involved in duodenal dismotility induced by ethanol

The purpose of this study was to investigate the role of K⁺channels in duodenal dismotility induced by ethanol in vitro.The amplitude of spontaneous contractions was reduced by ethanolin longitudinal and circular muscle, while frequency did notchange. Charybdotoxin antagonized ethanol-induced inhibitionof the amplitude of spontaneous contractions. Ethanol decreasedACh-induced contractions and this effect was cancelled out bycharybdotoxin. Ca²⁺-activated K⁺ channels may be involved induodenal dismotility induced by ethanol.     

Response to Giacaman: terror toll before Jenin

Having worked in joint Israeli–Palestinian projects inpublic health going back to the 1980's on lead poisoning,^2–5environmental determinants of asthma in Gaza children⁶, pesticides,and dioxins,^7–8, I have to comment on the context of Giacamanet al's report on the health, psychological and     

D2 DOPAMINE RECEPTOR TaqI ALLELES IN MEDICALLY ILL ALCOHOLIC AND NONALCOHOLIC PATIENTS

The prevalence of TaqI A alleles of the D₂ dopamine receptor(DRD2) gene was examined in two subgroups of medically ill nonalcoholics(more prevalent and less prevalent substance users, MPSU andLPSU, respectively) and in two subgroups of medically ill alcoholics(more severe and less severe alcoholics, MSA and LSA, respectively).The prevalence of the Al allele in the 80 nonalcoholic and 73alcoholic patients was 30.0% and 52.1%, respectively (P = 0.009).In the four subgroups of these patients, the prevalence of thisallele was: LPSU = 18.2%, MPSU = 34.5%, LSA = 44.4% and MSA= 58.3%. Linear trend analysis showed that as the use of substancesand severity of alcoholism increase, so does Al prevalence (P= 0.001). Specific, subgroup comparisons showed Al prevalencein MSA to be about 3-fold (P = 0.007) and 1.5-fold (P = 0.04)higher than in LPSU and MPSU subgroups, respectively. Similarly,in a combined analysis of independent studies, Al prevalencein MSA was higher when compared to LSA (P < 5 x 10^–3),MPSU (P < l0^–4 and LPSU (P < l0^–8) subgroups.There was virtually no difference in the prevalence of the Alallele between LSA and MPSU subgroups. None of the specificmedical or neuropsychiatric complications of alcoholism wasassociated with the Al allele. In conclusion, the severity ofalcohol dependence in alcoholics and of substance use behaviorsin controls are important variables in DRD2 allelic association.The present report and converging lines of evidence suggestthat the DRD2 locus could represent a prominent gene risk factorfor susceptibility to severe alcoholism. However, other genesand environmental factors, when combined, still play the largerrole.     

EFFECT OF CHRONIC ETHANOL CONSUMPTION ON IN VIVO PROTEIN SYNTHESIS IN LIVERS FROM FEMALE AND MALE RATS FED TWO DIFFERENT DIET REGIMENS

Hepatic protein synthesis was studied in male and female ratsfed ethanol-supplemented diets for 6–7 weeks. In one group(group 1), male rats were fed an all-liquid diet with 36% ofthe energy as ethanol. The controls were pair-fed with carbohydratereplacing ethanol isoenergetically. The second group of malerats (group 2) was given a mixture of solid and liquid diets.The solid food was given ad libitum and was supplemented witheither an ethanol-containing liquid (20–30% of energyas ethanol) or isoenergetic amounts of lipid. Female rats (group3) received the same diet regimen as group 2. Rates of hepaticprotein synthesis were measured after a 12–18 hr fastby a 32 min continuous infusion of ³H-valine. Specific precursorradioactivity (valyl tRNA) was calculated from valine specificradioactivities and concentrations in intra- and extracellularwater at 12, 22 and 32 min. The rates of protein synthesis were lower in all three groupsof ethanol-treated rats than in controls. In group 1, ethanolfeeding resulted in protein accumulation, and the plasma proteinconcentration was significantly lower at 20 and 32 min. In conclusion,female and male rats fed various diets were susceptible to thesame inhibitory effect of chronic ethanol consumption on hepaticprotein synthesis.