Addiction B iolog y (1998) 3, 405 ± 412

INVITE D R E VIEW

Ethanol m etabolism by m acrophages: possible role in organ dam age S. N. W ICKR AM ASINGH E Departm ent of Haematolog y, Imperial College School of M edicine, London, UK

Abstract M acrophages and hepatocytes oxidize etha nol to acetate in vitro at com parable rates but by di Œerent biochem ical pathways. Eth anol metabolism by m acrophages is largely ADH -independ ent and m ainly based on cytochrom e P450 and on the extracellula r release of superoxide anion radicals. There is also evidence that during eth anol m eta bolism , m acrophages release m ore acetaldehyde extracellula rly than hepatocytes; the high concentrations of aceta ldehyde around m acrophages m ay dam age surrounding tissue cells. Some of this acetaldehyde for ms unsta ble cytotoxic complexes with ser um album in and with er yth rocytes. The superoxide anion radicals released by macrophages may not only oxidize ethanol to aceta ldehyde but also react with and damage cells in their im mediate vicinity. A fter exposure to eth anol, m acrophage-depleted rodents show markedly reduced levels of cytotoxic acetaldehyde± albumin com plexes in the blood and reduced levels of hydroxyethyl radicals in the bile com pared to control anim als, indicating that the generation of such potentia lly pathogenic molecules is, to a large extent, depend ent on macrophage activity. M acrophage-depleted animals also show less early liver dam age than control animals. The reduction in etha nol-ind uced liver dam age in macrophage-depleted m ice and rats m ay be due to a reduction or elim ination of the generation of various Kup Œer-cell-derived hepatotox ic substances, including acetald ehyde and reactive oxygen radicals, in such anim als. These data suggest that eth anol m etabolism by tissue m acrophages may play an important role in mediating etha nol-related tissue dam age.

Introdu ction Despite the eþ orts of many investigators, the mechanisms underlying ethanol-mediated tissue damage remain uncertain. The proposed mediators of tissue damage include (a) acetaldehyde produced by hepatocytes, (b) oxygen-derived free radicals and hydroxyethyl radicals produced by hepatocytes, (c) tumour necrosis factor and various cytokines produced by Kupþ er cells, (d) an antibody- or cell-mediated immune response to adducts between acetaldehyde and proteins or between hydroxyethyl radicals and proteins, (e)

acetaldehyde, superoxide anion radicals or hydroxyethyl radicals generated by m acrophages and (f ) circulating unstable acetaldehyde± albumin complexes. More than one cytotoxic mechanism may operate either together or in sequence to cause the observed damage and the importance of the various mechanisms m ay vary from tissue to tissue. Some reported alcohol-mediated biochemical disturbances are likely to be reversible and others may be irreversible or cumulative or both. This paper reviews the evidence on the extent and biochemical basis of ethanol m etabo-

Correspondence to: Prof. S. N. W ickramasinghe, Department of Haem atology, Imperial College School of Medicine, St Mary’s Hospital, London W 2 1PG , UK . Tel: 0171 594 3995; fax: 0171 262 5418. Received for publication 11th August 1997. Accepted 10th November 1997. 1355 ± 6215/98/040405 ± 08 $9.50 € Society for the Study of Addiction to Alcohol and Other Drugs Carfax Publishing Limited

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lism by human and m urine m acrophages and the role of macrophages in the generation of cytotoxic acetaldehyde± albumin complexes that appear in the blood after exposure to ethanol. It also includes a discussion of the possible importance of ethanol m etabolism by macrophages in the pathogenesis of alcohol-related organ damage.

rates similar to those shown by two human hepatoma cell lines, PL C /PRF /5 and Hep G2. 8 Kupþ er cells account for about 15% of liver cells, 2.5% of liver protein and 20% of the m acrophages in the body. Therefore, despite their impressive ability to oxidize ethanol, m acrophages can be responsible for only a small percentage of total ethanol metabolism. 9

Rates of ethanol m etabolism by m acrophage s During investigations into the m echanisms underlying ethanol-induced bone marrow damage, suspensions of human bone marrow cells were found to have a modest capacity to oxidize 14 C-ethanol to 1 4 C-acetate.1 ,2 The amount of acetate produced was proportional to the ethanol concentration in the incubation mixture in the range 2.6 m M (0.1 mg /ml) to 79 m M (3 m g /ml). When marrow cell suspensions were depleted of cells able to adhere to the culture ¯ ask their ethanol m etabolizing capacity was considerably reduced, suggesting that much of this metabolism was m ediated by m acrophages.3 That macrophages had an impressive ability to oxidize ethanol was directly con® rm ed by studies of human macrophage cultures derived from the blood, bone marrow and spleen,3 ± 5 and murine macrophage cultures derived from the liver, bone mar6 row, spleen and thymus. The average rates of oxidation of ethanol (26 m M ; 1 m g /ml) to acetate by human m acrophages varied between 2140 and 2960 nmol/10 7 cells /hour and by murine macro7 phages between 1850 and 2320 nmol/10 cells / hour. The values for human m acrophages are about one-® fth that of the 11790 nmol/10 7 cells / hour found in isolated human hepatocyte suspensions prepared from a wedge biopsy of the liver.7 Similarly, when expressed in the same units, mouse m acrophages metabolized ethanol at about one-® fth of the rate found in mouse hepatocytes.6 The higher rates of metabolism of ethanol by macrophages are related to the fact that hepatocytes are much larger than macrophages and when this is taken into account by expressing the data per g wet weight per hour, murine macrophages (including Kupþ er cells) metabolized ethanol at about twice the rate shown by murine hepatocytes.6 When the oxidation of ethanol to acetate is expressed in nmol ethanol per m g cell protein per hour, human blood monocyte derived m acrophages metabolize ethanol at

Biochem ical basis of ethan ol m etabolism The enzymes known to have a capacity to m etabolize ethanol are alcohol dehydrogenases (ADH), catalase and cytochrome P450 2E1 (CYP2E1). 1 0 Class I ADH is found especially in the liver and the gut, has a low K m for ethanol (0.2 ± 2 m M ) and is responsible for m ost of the in vivo ethanol metabolism at low ethanol concentrations; ADH catalyses the oxidation of ethanol to acetaldehyde. Low levels of m RNA for class I ADH are detectable in several human tissues other than the liver and gut but not in the brain. 1 1 Catalase has a high K m for ethanol and is therefore thought not to m etabolize appreciable amounts of ethanol in vivo ; it is found in various tissues including the brain and accounts for part of the acetaldehyde generated by colonic bacteria. CYP2E 1 is found in hepatocytes, has a moderately high K m for ethanol (8 ± 10 m M ) and is thought to contribute to total ethanol metabolism at high blood alcohol levels. It is induced up to ® ve-fold in 75% of subjects who abuse alcohol chronically. CYP2E1mediated ethanol m etabolism generates reactive oxygen species such as superoxide anion radicals, H 2 O 2 and hydroxyl radicals as well as hydroxyethyl radicals.1 2 CYP2E1 is not con® ned to the liver but is also found in other tissues including the brain and gastrointestinal tract. Acetaldehyde molecules that are generated from the oxidation of ethanol by ADH are further oxidized to acetate, mainly through the action of NAD-dependent aldehyde dehydrogenase (ALDH) found in the mitochondria and cytosol of hepatocytes. Studies with rat liver slices suggest that a low K m mitochondrial ALDH (ALDH 2) accounts for 60% of acetaldehyde metabolism and a high K m cytosolic ALDH (ALDH1) for 20% . 1 3 Recent evidence indicates that there is also an NADPH -dependent microsomal acetaldehydeoxidizing system which is largely based on CYP2E 1 (CYP1A2 and 4A2 may also be involved to a small extent). In liver homogenates of control rats and rats treated with ethanol, respectively,

Eth anol m eta bolism by macrophages 14

NADPH -dependent acetate production from Cacetaldehyde accounted for 12 and 34% of NADdependent acetate production.1 4 The metabolism of ethanol by human and murine m acrophages is only slightly inhibited by pyrazole (an inhibitor of ADH), 4-iodopyrazole (an inhibitor of p - ADH) and 3-amino-1, 2, 4triazole (an inhibitor of catalase) and is, therefore, 4 ,6 largely independent of ADH and catalase. By contrast, three inhibitors of cytochrome P450, namely carbon m onoxide, m etyrapone and tetrahydrofurane, suppressed ethanol metabolism in human and m urine macrophages by 50 ± 75% indicating that this metabolic activity is dependent to a large degree on cytochrome P450. 4 ,6 In addition, superoxide dismutase causes a 50% inhibition of ethanol metabolism in bloodmonocyte-derived human macrophages, pointing to the participation of superoxide anion radicals in the oxidation of ethanol.1 5 Since superoxide dismutase is a large molecule and is therefore unlikely to penetrate cells, most of the superoxide-anion-radical-dependent ethanol oxidation probably occurs extracellularly. Evidently, macrophages diþ er from hepatocytes in that the latter metabolize ethanol mainly via the action of ADH and only to a small extent (usually less than 5%) via ethanol-inducible CYP2E 1. The cytochrome P450 superfamily includes several members involved in the metabolism of xenobiotics (CYP1-CYP4) and a number involved in the biosynthesis of various endogenous molecules (CYP5-CYP27). 1 6 Using the techniques of reverse transcription± polymerase chain reaction RT± PCR and of immunocytochemistry (employing a rabbit polyclonal antibody against rat CYP2E 1), we have demonstrated recently that cultured human blood-monocyte-derived macrophages contain mRNA for CYP2E1 as well as CYP2E1 protein ( J. Hutson & S. N. Wickramsinghe, unpublished). Although it has not yet been shown that the species of CYP450 involved in the metabolism of ethanol by human and murine tissue macrophages consists of CYP2E1, this is a possibility. There is evidence that rat Kupþ er cells contain low levels of imm unochemically detectable CYP2E1 and that the levels are increased more than 10-fold in acetone-treated animals.1 7 In addition, CYP2E1 and CYP1A1 (an enzyme responsible for the bioactivation of many polycyclic hydrocarbons and aromatic amines) have been shown to be present constitutively in rabbit bone marrow microsomal prepara-

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tions on the basis of imm unoblot analysis and catalytic activities. CYP2E1 was induced 12.9fold by acetone treatment and CYP1A1 15.7-fold 18 by Aroclor 1254 treatment. Another study has demonstrated both the constitutive and induced expression of mRNA for CYP1A1 in human 19 pulmonary alveolar macrophages.

M acro phage-dependent generatio n of u nstable ac etaldehy de± alb um in com plexes and acetaldehyde± red-cell com plexes The administration of 60 ± 95 g ethanol to healthy adults over a period of 20 ± 45 min is followed by the appearance of a non-dialysable cytotoxic activity in the serum, 2 0 ± 2 2 the generation of which is largely based on macrophage activity. 2 3 The non-dialysable cytotoxic activity peaks at 8 hours after alcohol consumption begins, at a time when blood alcohol levels have returned to normal or near-normal and is just detectable at 24 hours. This cytotoxicity was initially discovered by its eþ ect of causing detachment of A9 cells 2 0 and was subsequently demonstrated using three other techniques, namely: (a) inhibition of 2 H-thymidine incorporation in various human cell lines,2 1 (b) impairment of mitogen-stimulated human lymphocyte transform ation 2 4 and (c) reduction in the generation of coloured dye in the colorimetric M TT (tetrazolium) assay (which detects a reduction in mitochondrial dehydrogenase activity).2 3 Since the word cytotoxic is used frequently to describe substances or cells causing cell death and there is as yet no evidence as to whether or not the above-mentioned in vitro eþ ects do lead to cell death, it m ay be preferable to describe these eþ ects of dialysed serum as cytoinhibitory rather than cytotoxic. In this review the word cytotoxic is used to indicate inhibition of cell function rather than mediation of cell death. Three lines of evidence indicate that the non-dialysable cytotoxic activity largely resides in unstable acetaldehyde± albumin complexes (Schiþ bases). First, Sephacryl S-300 gel ® ltration of post-alcohol serum revealed that the cytotoxic activity was in the albumin fraction. 2 0 Secondly, complexes formed by the reaction of acetaldehyde with human serum albumin were also cytotoxic.2 0 Thirdly, the cytotoxicity of both post-alcohol serum and arti® cially produced acetaldehyde± albumin complexes fell sharply after treatment with the reducing agent sodium borohydride, presumably due to stabilization of

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the unstable Schiþ bases by addition of hydrogen across the double bond, C 5 N. 2 1 Cytotoxicity develops in the albumin fraction not only in humans consuming alcohol but also in mice exposed to ethanol vapour for 1 ± 7 days.2 5 Using double-labelled arti® cially generated acetaldehyde± albumin complexes, it has been con® rm ed that the cytotoxicity results from the release of acetaldehyde from such complexes and the preferential binding of the free acetaldehyde to target cells.2 1 There are also data indicating that after the administration of ethanol, red cells of humans and baboons contain reversibly-bound acetaldehyde.2 6 ,2 7 This acetaldehyde may be transferred to target tissue cells.2 6 ± 2 8 Two lines of evidence indicate that the macrophage is an important source of acetaldehyde in the acetaldehyde± protein complexes. The ® rst comes from studies of supernatants from ethanolcontaining cultures of blood-monocyte-derived human macrophages and two human hepatom a cell lines in which the cell numbers were adjusted such that the rates of oxidation of ethanol to acetate were comparable. Albumin fractions prepared from such supernatants induced a much larger percentage reduction of adherent A9 cells (i.e. were more cytotoxic) when derived from macrophage cultures than from hepatoma cell cultures.8 Therefore, for a given quantity of acetate generated from ethanol in vitro , the macrophages released m ore acetaldehyde extracellularly than the hepatom a cell lines. This phenomenon may be related to the observation that some of the oxidation of ethanol by bloodmonocyte-derived human m acrophages is mediated through superoxide anion radicals and that at least part of this superoxide-dependent oxida15 tion m ay take place extracellularly. It is also likely that m acrophages contain lower levels of acetaldehyde dehydrogenase than hepatocytes and are, therefore, unable to oxidize intracellular acetaldehyde to acetate as eþ ectively as hepatocytes. If the in vitro data on acetaldehyde release by hepatom a cell lines outlined above apply to normal human hepatocytes in vivo , macrophages would be an important source of ethanol-derived acetaldehyde. The second line of evidence indicating that macrophages have an important role in the generation of acetaldehyde± albumin complexes comes from studies of mice in which macrophages were depleted by an intravenous injection of liposome-encapsulated dichloromethylene

diphosphonate (DMDP). A single injection results in a near-complete elimination of Kupþ er cells and a 90% depletion of splenic macrophages between 1 and 5 days after the injection. Following exposure to ethanol for 4 days (average serum ethanol concentration, 150 mg /dl), the albumin fraction prepared from the sera of DMDP-treated mice displayed m uch less cytotoxicity than that prepared from uninjected mice or mice injected 23 with empty liposomes. Since macrophages release acetaldehyde extracellularly and appear to be a m ajor source of acetaldehyde in the blood, it is likely that after alcohol consumption, much higher concentrations of acetaldehyde are found imm ediately adjacent to macrophages than in circulating blood.9 Therefore, in m acrophage-rich tissues such as the liver, bone marrow, spleen and lymph nodes, macrophage-derived acetaldehyde may play an important role in damaging surrounding cells. It is noteworthy that macrophages protrude long thin cytoplasmic processes at their surface and that these are in intimate contact with surounding cells. Consequently, a single ethanol-metabolizing macrophage may adversely aþ ect several neighbouring cells. The possibility that circulating unstable acetaldehyde± albumin complexes may contribute to alcohol-related tissue damage is suggested but not proven by the demonstration, in a group of heavy drinkers, of a statistically signi® cant correlation between an index of cytotoxicity in dialysed serum on the one hand and serum aspartate aminotransferase (AST) or total serum 29 creatine kinase on the other hand. Such complexes may be much m ore important as a mediator of tissue dysfunction and damage in tissues and organs that do not have a signi® cant capacity to generate acetaldehyde in situ or convert acetaldehyde to acetate than in those that do.

Involvem ent of Kupþ er cells in ethano lrelated liver dam age W hen m ale Wistar rats were continuously administered ethanol for 4 weeks via an infusion pump attached to an intragastric cannula tunnelled subcutaneously to the dorsal aspect of the neck, the serum AST activity increased and the liver showed fatty change, necrosis and in¯ ammation. In these animals, the urine alcohol concentration varied m arkedly from near zero to m ore than 300 mg /dl. If in this model of alcohol-induced

Eth anol m eta bolism by macrophages

liver damage, Kupþ er cells are inactivated by intravenous injections of gadolinium chloride (GdCl3 ) twice a week, there is a dram atic reduction in the serum AST levels and in hepatic steatosis, in¯ ammation and necrosis. 3 0 These data clearly show that Kupþ er cell inactivation and depletion m inimises alcohol-induced early liver damage. Adachi et al. 3 0 suggested that this eþ ect may be explained on the basis that in animals exposed to ethanol, Kupþ er cells become activated, 3 1 ,3 2 possibly as a consequence of elevated plasma endotoxin levels,3 3 and cause liver damage by (a) increasing oxygen consumption in hepatocytes, especially in the centrilobular zone,3 4 ± 3 7 and /or (b) releasing hepatotoxic substances such as TN F, interleukin (IL)-1, IL-6, IL-8, prostaglandins and oxygen radicals.3 8 The authors did not consider the additional possibility that the hepatotoxic substance may be macrophagederived acetaldehyde. In a subsequent paper, 39 Knecht et al. demonstrated that in rats subjected to the continuous intragastic administration of an ethanol-containing high-fat diet for 2 or more weeks, there is severe hepatic damage and a hydroxyethyl radicals are found in the bile. These authors also showed a statistically signi® cant correlation between liver injury and radical form ation. Furthermore, depletion of Kupþ er cells with GdCl 3 resulted in a 50% decrease of radicals in the bile, indicating that the Kupþ er cell was an important source of hydroxyethyl radicals. As mentioned earlier, reactive oxygen species and hydroxyethyl radicals are known to result from CYP2E1-mediated ethanol m etabolism by hepatocytes. The involvement of Kupþ er cells in ethanolmediated hepatic damage was independently 40 demonstrated by Goldin et al. using C57 BL / 10 mice. When such mice were kept within an inhalation chamber through which a mixture of air and ethanol vapour was pumped continuously so that the serum ethanol concentration was 48 ± 190 mg /dl, fatty change developed after exposure to ethanol for 2 days and in¯ ammatory foci and hepatocyte necrosis after exposure for 5 or more days.2 5 This histological evidence of liver damage was accompanied by abnormalities in liver function test results. W hen m ice were depleted of macrophages by two intravenous injections of liposomes containing DM DP given on day 1 and day 5 and exposed to ethanol vapour from day 2 for 9 days, the in¯ amm atory and necrotic changes in the liver and the extent

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of elevation of serum ALT activity were all signi® cantly less than in uninjected m ice or mice injected with empty liposomes that were exposed 40 to ethanol vapour for the same duration. Thus in this m odel, too, macrophage depletion was associated with signi® cantly reduced ethanolmediated liver damage. The possibility that in ethanol-treated macrophage-depleted mice, one mechanism of reduced liver damage may be via a reduction of local acetaldehyde generation from the oxidation of ethanol by Kupþ er cells gains circumstantial support from the ® nding that such mice show a marked reduction in circulating cytotoxic acetaldehyde± albumin complexes.2 3

Biolog ical eþ ects of m acrop h age-derived ac etaldehyde Acetaldehyde is a highly reactive molecule and it is likely that any that may be found in relatively high concentrations around macrophages and the smaller amounts delivered to tissue cells via circulating unstable acetaldehyde± albumin and acetaldehyde± red cell complexes contribute to alcohol-related tissue damage. A number of investigators have shown that acetaldehyde adversely aþ ects several cell functions and cell types. The adverse eþ ects include impairment of rat liver mitochondrial function, inhibition of guinea pig cardiac microsomal protein synthesis, depression of protein synthesis and glycoprotein secretion by rat liver slices, impairment of mitogen-stimulated human lymphocyte transformation and prolongation of the doubling time and increase in the 41 modal volume of human cell lines (reviewed in ). Acetaldehyde has also been shown to increase procollagen type I and ® bronectin gene transcription in fat-storing cells of the rat via a protein synthesis dependent m echanism. 4 2 Apart from forming adducts with albumin and red cell constituents, acetaldehyde is known to bind to other proteins. Thus, in biopsy specimens from alcoholic patients, immunohistochem ical studies have demonstrated intracellular acetaldehyde± protein adducts in hepatocytes, with a more intense staining in zone 3 hepatocytes, and extracellular adducts in areas of active ® brogenesis. 4 3 In addition, increased levels of acetaldehyde± collagen adducts have been demonstrated by Western blotting in livers of patients with alcoholic liver disease. 4 4 Acetaldehyde may exer t its pathogenic eþ ects by forming adducts with various enzymes and thereby altering their func-

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tion. Alternatively, acetaldehyde± protein adducts may serve as neoantigens and provoke an immune response against cells containing such adducts. In this connection it is of interest that guinea pigs immunized with acetaldehyde± humanhaemoglobin adducts but not those imm unized with unmodi® ed haemoglobin developed hepatic ® brosis after being fed ethanol for 40 days.4 5

M ac rophag es as a source of superoxide and hydroxyethyl rad icals As has been mentioned, superoxide anion radicals appear to be involved in the extracellular oxidation of ethanol by human blood-monocytederived macrophages and these radicals may not only react with ethanol but also with various constituents of adjacent cells. It is not yet known whether human or murine tissue macrophages release superoxide anion radicals when exposed to ethanol. CYP2E1-mediated oxidation of ethanol by hepatocytes results in the generation of hydroxyethyl radicals that form immunogenic stable adducts with C YP2E1 on the surface of hepatocytes and with other m icrosomal proteins within hepatocytes. Alcoholics have increased titres of antibody reacting with such adducts 4 6 and cellmediated immunological damage to antibodycoated hepatocytes may play a role in the pathogenesis of alcoholic liver disease. Another possible pathogenetic m echanism is the alteration of protein (e.g. enzyme) function as a result of the formation of hydroxyethyl radical± protein adducts. In ethanol-fed rats, treatment with diallyl sulphide or phenylethyl isothiocyanate (which suppress the induction of CYP2E 1 by ethanol) reduces the production of hydroxyethyl radical-derived epitopes in the liver as well as both the pathological changes in this organ and the formation of antibodies against hydroxyethyl radical± protein adducts.4 7 Although it had been initially assumed that hepatocytes are the only source of hydroxyethyl radicals in the liver, the studies of Kupþ er-cell-depleted rats m entioned above3 9 indicate that Kupþ er cells are also involved in hydroxyethyl radical formation at least in the alcohol-fed rat.

C onc lusion The data reviewed here suggest that although macrophages account for only a small proportion

of total ethanol metabolism in the body, acetaldehyde generated locally as a consequence of ethanol metabolism by macrophages may contribute substantially to ethanol-related organ damage. This contribution may be especially important in those organs such as the bone marrow, which are rich in macrophages and in which other cell types have little or no capacity to generate acetaldehyde and release this extracellularly. In the case of the bone marrow, a possible role of m acrophages in the pathogenesis of marrow damage had been supported by in-vitro studies of adherent-celldepleted bone m arrow cells cultured on their own or co-cultured with m onolayers of macrophages, with and without ethanol.4 8 There is also evidence that Kupþ er-cell-derived hydroxyethyl radicals may be involved in ethanol-mediated liver damage in the rat.3 9 It is noteworthy that neuroglial cells belong to the same family of cells as m acrophagesÐ namely, the mononuclear phagocyte system. However, although neuroglial cells are known to be capable of oxidizing ethanol by an ADH-independent and 7 cytochrom e P450-dependent pathway, it is not yet known whether they resemble m acrophages in their capacity to release acetaldehyde extracellularly. If they do resemble macrophages in this respect, they m ay play a role in the pathogenesis of ethanol-related damage to the nervous system by generating acetaldehyde locally. Macrophages are also responsible for producing much of the acetaldehyde in circulating unstable acetaldehyde± albumin complexes and, presumably, also unstable acetaldehyde± red cell complexes. These complexes could be important in mediating functional disturbances particularly in organs without a substantial capacity to oxidize ethanol. Their generation extends the period of potential toxicity after alcohol consumption well beyond that during which blood alcohol levels are high.

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Ethanol metabolism by macrophages: possible role in organ damage.

Macrophages and hepatocytes oxidize ethanol to acetate in vitro at comparable rates but by different biochemical pathways. Ethanol metabolism by macro...
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