Alcohol-Relateddiseasesand Carcinogenesis'

[CANCER RESEARCH 39, 2863-2886, July 1979] 0008-5472/79/0039-0000$O2.OO Alcohol-relatedDiseasesand Carcinogenesis'

Charles S. Lieber, Helmut K. Seitz, Anthony J. Garro, and Theresa M. Worner

Alcoholism Research and Treatment Center, Bronx V.A. Medical Center, and Mount Sinai School of Medicine, New York, New York 10468

Abstract on the generally held belief, supported by experimental and clinical evidence, that repeated cell injury may eventually pro Possible mechanisms whereby alcohol abuse and alcohol mote neoplastic transformation, possibly through inappropriate related diseases may promote the development of cancer are regenerative responses. The interaction of alcohol, nutrition, analyzed. The mechanisms discussed include: (a) contact-re and cancer will also be discussed. lated local effects on the upper gastrointestinal tract; (b) the presence of low levels of carcinogens in alcoholic beverages; Cancer of the Alimentary Tract and Local Effects of Ethanol (c) induction of microsomal enzymes involved in carcinogen metabolism; (d) various types of cellular injury produced by Clinically, an association between heavy drinking and certain ethanol and its metabolites and their relationship to cancer, types of cancer has been observed for many years. One of the particularly in the liver; (e) the nutritional disturbances fre first suggestions that there might be a relationship between quently associated with alcohol abuse. The relationship be alcohol and cancer was made in France when Lamu (194) tween alcohol-induced cirrhosis and hepatocellular carcinoma showed that the chronic intake of absinthe (a beverage con is also discussed, and case histories of patients seen at the taming wormwood as well as alcohol) was associated with Bronx Veterans Administration Medical Center with hepatocel carcinoma of the esophagus. In 1950, the relationship between lular carcinoma in the absence of cirrhosis are reviewed. Data cigarette smoking and lung cancer was widely recognized, and are presented demonstrating the induction, by chronic ethanol this led investigators to take a closer look at personal habits in consumption, of microsomal enzymes which convert procarcin relation to the development of cancer. In 1964, the World ogens to carcinogens. These data were derived from experi Health Organization surveyed the research on alcoholism and ments in which the ability of microsomes isolated from liver, cancer and concluded that an association existed between intestine, and lung tissues of ethanol-fed and control rats to excessive drinking of alcoholic beverages and cancer of the activate several test carcinogens was examined in the Ames mouth, the larynx, and the esophagus (400). In a series of Sa!monella-mutagenicity test. The hypothesis is presented that studies (405â€”408),heavy drinkers were found to have roughly ethanol-mediated induction of enzyme systems which activate a 10-fold increased risk of developing cancer of the mouth. procarcinogens to carcinogens in various tissues contributes Subjects who drink heavily often also smoke heavily. This latter to the enhanced incidence of cancer in the alcoholic. fact was first taken into account by Flamant et a!. (103), who assessed both factors and reported that there was a â€˜â€˜strongâ€• or â€˜â€˜verystrong'â€˜associationof alcohol intake with cancer of Introduction those sites that come most directly in contact with alcohol The epidemiology of the link between cancer and alcoholism (tongue, hypopharynx, larynx, esophagus). More recent stud has been discussed in detail in another paper of this workshop ies such as those of Keller and Terris (170), Rothman and (371 ). Detailed evidence has been presented of the increased Keller (31 7), and Martinez (244), which were included in the incidence of cancer of the upper alimentary tract and the colon Third National Cancer Survey (394), substantiate this associ in the alcoholic. This aspect therefore will not be reviewed in ation. All these studies provided evidence for a cooperative detail here. Hepatocellular carcinoma also has been associated role between tobacco and alcohol in tumor formation of the with alcoholism. However, this has usually been attributed not oral cavity, the larynx, and the esophagus. It was calculated to ethanol but rather to the ensuing cirrhosis. Recently, some that 76% of these cancers in males could be eliminated if clinical evidence has accumulated that, even in the absence of exposure to alcohol and tobacco were avoided (31 7). More cirrhosis, hepatocellular carcinoma of the liver is linked to recent studies in North America and Canada have confirmed alcoholism. Some observations will be presented to illustrate those findings. The risk for a heavy drinker who smoked to this possibility. The thrust of this paper, however, will be the develop oral cancer was 6 to 15 times greater than for non discussion of possible mechanisms whereby alcohol abuse drinkers and nonsmokers (98, 108). Also, women who drink may promote the development of cancers. Our considerations and smoke heavily develop cancer of the tongue and buccal will focus on: (a) local effects of ethanol in the gastrointestinal cavity some 15 years earlier than do women abstaining from tract; (b) induction, by chronic ethanol consumption, of micro both (5). It appears that alcohol plays a more important role somal enzymes which convert procarcinogens to carcinogens; than smoking with respect to cancer of the esophagus whereas and (c) general mechanisms of cellular injury produced by smoking seems to be more strongly associated with cancer of ethanol. The relationship of the latter effect to cancer is based the mouth and pharynx (103). Recently, additional sites of cancer associated with alcohol were detected in the pancreas (39, 138, 149), the cardia of the stomach (237), and the colon

I Presented at the Alcohol and Cancer Workshop, October 23 and 24, 1978, (394). Bethesda, Md. Original studies were supported in part by the Medical Research How cancers of the gastrointestinaltract relate to alcoholis Service of the Veterans Administration and USPHS Grants AA03508, AA00224, AM1251 1, and CA22354; by American Cancer Society Grant BC 200A, and by not known. One mechanism that has been suggested is the the Deutsche Forschungsgemeinschaft (Se 333/2). decreased salivary secretion which might result in both higher

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local concentrations of carcinogens and lesser rinsing of the is mediated by the release of antral gastrin (25, 399), an mucosal surfaces (180). Another mechanism may be related to increased vagal tone (120), and enhanced synthesis or release the tissue damage produced by the high local ethanol concen of gastric histamine (80, 81). Chronic administration of ethanol tration to which, some of these tissues are subjected. Indeed, increases the gastrin response to a meat meal (368) and, at ethanol has been shown to have various local effects on the least initially, increases the maximal acid output of dogs (49). upper gastrointestinal tract. In Ontario, Canada (4), an oral In the latter study, the maximal acid output returned to normal cytology service identified dyskaryotic cells in 3.0% of the levels after approximately 6 months, suggesting that the pan 3445 smears of dental patients examined; there were twice as etal cell response to endogenous gastrin might be gradually many such lesions (6.5%) in 276 smears from alcoholics. In impaired by injury. the esophagus, functional abnormalities have been described Other work has focused on the direct effects of alcohol on in the alcoholic. Changes in peristalsis, without clinical com gastric permeability and on active transport of various ions. plaints of dysphagia, have been detected in a majority of Davenport (70) demonstrated that the direct application of alcoholics who presented with peripheral neuropathy (102, ethanol to the Heidenhain pouches of dogs resulted in the 395). Prominent changes included a decrease in frequency of insorption of hydrogen ions and the exsorption of sodium ions. primary peristaltic waves in the distal two-thirds of the esoph This observation was confirmed and extended by Dinoso et a!. agus as well as an increase in tertiary, nonpropulsive activity (78), who found that these ion fluxes and the accompanying after swallowing; there was no change in lower esophageal mucosal injury could be increased or decreased by the addition sphincter pressure. Little is known about the direct effects of of acid or buffer, respectively, to the alcohol perfusate. Using alcohol on esophageal functions, although ethanol apparently lithium as an ionic analog of H@,the same conclusion was does not promote luminal loss of hydrogen ions and does not reached in a human study (348). On the basis of these results, alter the electrical resistance of the mucosa (113). Thus, the it was concluded that alcohol alters the permeability of the changes documented by Winship et a!. (395) may represent gastric mucosal â€˜â€˜barrier'â€˜andthat the subsequent ingress of either direct neuropathic or myopathic effects of alcohol. The hydrogen is potentially injurious. More recently, others have role played by malnutrition has not been defined. suggested that the deleterious effects of alcohol are mediated Alcohol is also known to exert striking effects on the stom only in part by a change in mucosal permeability (113, 338, ach. The occurrence of gastritis after the acute administration 340). It was found that topical ethanol increases electrical of alcohol has been clinically well established since William resistance across the mucosa and diminishes the transmucosal Beaumont's observations (179). These classic studies were electrical potential. Such findings are more consistent with a confirmed more recently by endoscopy in binge drinkers. Hem change in active transport of ions, particularly Cr , rather than orrhagic lesions also involve the duodenum. Although blind a simple increase in permeability (which should decrease elec peroral biopsies of the duodenum had failed to reveal duoden tnical resistance), although the net ionic changes, namely, an itis during acute episodes of chronic alcoholism (303), fiber increase in luminal Na@and a decrease in luminal H@,would optic duodenoscopy detected gross and microscopic duoden be the same. A Mg2@andMg2@+ HCO3-ATPase is postulated itis in a majority of subjects in a more recent study (118). to link the active transport of Cl' and HC03 . It has been The incidence of chronic gastritis in alcoholics is a more shown that ethanol may reduce active transport of chloride by controversial issue. Whereas some studies detected no rela inhibiting this ATPase activity (359). tionship between alcohol intake and histological evidence of In summary, alcohol both stimulates and inhibits gastric acid atrophic gastritis (287, 398), other investigators have sug production by different mechanisms. In the dog, topical ethanol gested a connection (77, 163, 304, 312). In the study by causes stimulation of acid secretion (70). In humans, however, Dinoso et a!. (77), 50% of the alcoholics examined had either while i.v. ethanol stimulates acid secretion (139), p.o. ethanol superficial or atrophic gastritis which was present even after 2 produces no change or perhaps mild inhibition of acid secretion months of abstinence. Maximal acid output in these patients (59, 60). Thus, the importance of H@ in ethanol-mediated tended to increase with improvement in histology. Patient se gastric injury is not known. lection (nutritional status, definitions of alcoholism), biopsy Ultrastructural changes have been described in the gastric techniques (blind versus directed), and length of abstinence mucosa after topical ethanol administration, including disrup before examination may, at least in part, explain the lack of tion of tight cell junctions (83). Changes, however, were most agreement in the literature regarding the relationship between marked in the cytoplasm and nuclei. Dinoso et a!. (80) studied alcoholism and chronic gastritis. An increased incidence of the effect of ethanol in buffered solution on canine gastric atrophic gastritis would be of particular importance since this mucosa and found that with ethanol concentrations greater lesion appears to be a precursor of human gastric carcinoma. than 20% v/v there was disruption of the apical cell membrane The acute and chronic effects of alcohol on gastric acid and focal separation of tight junctions. Underlying atrophic secretion and gastric mucosal integrity have been studied gastnitis in humans causes increased blood loss during ethanol extensively both in vivo and in vitro. In the intact animal, the ingestion which suggests an injurious effect of ethanol (79), acute i.g.2 administration of ethanol results in an augmented but electron microscopic studies have not been done in this secretion of hydrochloric acid (81). It appears that this effect situation; therefore, the underlying lesion is not well defined. Furthermore, the relationship, if any, between cancer of the

2 The abbreviations used are: i.g. , intragastric; BP, benzo(a)pyrene; DMN, stomach and local injurious effects of ethanol is not known. dimethylnitrosamine; SEA, smooth endoplasmic reticulum; ADH, alcohol dehy In addition to the local effects of ethanol itself, some conge drogenase; MEOS, microsomal ethanol-oxidizing system: PB, phenobarbital: 3- MC, 3-methylcholanthrene: BP hydroxylase, benzo(a)pyrene hydroxylase: DMN ners in alcoholic beverages may play an etiological role in the N-demethylase, dimethylnitrosamine N-demethylase; i.r., intrarectally. development of cancer. Indeed, esophageal cancer has been

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Alcohol-related Diseases and Carcinogenesis produced in animals by administering relatively large amounts what is probably most critical with respect to the relationship of nitrosamines which may be congeners in some alcoholic between induction of microsomal enzyme activity and carci beverages (227, 235). Furthermore, the presence of ethanol nogenesis is the relative effect of the inducer on carcinogen seems to catalyze the production of nitrosamines from nitrites activation and inactivation. and secondary amines in vitro (301 ). An increased incidence Reactions which are cytochrome P-450 dependent and are of esophageal cancer has been described in the alcoholic (58, known to play a role in chemical carcinogenesis include: (a) N- 369). In France, the risk of esophageal neoplasia is particularly hydroxylation of aromatic amines [such as the potent procar pronounced in alcoholics who drink apple brandy whisky but cinogens 2-aminofluorene and 2-acetylaminofluorene (265)], is less apparent in those consuming beer or wine (369). Simi hydrazine compounds [such as 1,2-dimethylhydrazine which larly, in eastern and southern Africa, beverages derived from produces tumors of the colon and rectum (391 )], and some maize have been implicated as risk factors (58). A variety of substituted hydrazines (including isoniazid) which produce pul polycyclic hydrocarbons (phenanthrene, fluoranthrene, benz monary tumors in specific strains of mice (172, 339, 366); (b) anthracene, BP, chrysene) have been found in alcoholic bev dealkylation of alkylnitrosamines, e.g. , N-demethylation of erages (248), and asbestos fibers (derived from filters) have DMN (66, 1 25, 1 26, 265); (c) epoxide formation from poly been found in beer, wine, sherry, and vermouth (31 , 32, 65, cyclic hydrocarbons (69, 347), aflatoxins (265), and furosem 390). ide (396). In humans, microsomal biotransforming activity and/or cy Induction of Enzyme Activities by Ethanol in Microsomes of tochrome P-450 have been found in the liver (33, 71, 192, Liver, Intestine, and Lungs and Its Possible Link to 333), lymphocytes (393), skin (206), placenta (57), intestine Carcinogenesis (140), colon (12), and lungs (354). Although the liver is the principal organ of xenobiotic metabolism, metabolism in other Relationship of the Microsomal Cytochrome P-450 System to tissues, especially in those which have direct contact with the Carcinogenesis environment (intestine, lung, skin), is also important since such It is well established that the microsomal cytochrome P-450- metabolism can influence absorption, biological reactivity, and dependent biotransformation system is involved in the meta systemic distribution of these compounds. Many procarcino bolic conversion of many structurally diverse chemicals to gens do not produce tumors at the site of application (391). electrophiles capable of reacting with nucleic acids, proteins, They may be activated in tissues other than where they finally and lipids (392). It is these electrophilic metabolites that are act with activated metabolites being released into the blood generally responsible for the carcinogenic effects of the parent stream and affecting special target organs (22, 391). compounds. Activation of many procarcinogens by microsomal Effect of Alcohol Consumption on Hepatic Microsomal enzymesis an obligatoryeventfor mutagenesisinmicrobiolog Enzymes ical assays and cell transformation in tissue culture (391). Induction of microsomal enzyme activity increases the muta Changes in Ethanol and Drug Metabolism. The first mdi genic effect of many compounds in the Ames Salmonella cation of an interaction of ethanol with the microsomal fraction mutagenesis assay (3,1 25, 305). Since the extent of metabolic of the hepatocyte was provided by the morphological obser activation of various secondary carcinogens can be correlated vation that in rats ethanol feeding resulted in a proliferation of with microsomal enzyme activity (57, 67, 82, 99, 114, 126), the SER (151 , 152). This increase in SER resembled that seen factors such as environmental pollutants, drugs, and diet which after the administration of a wide variety of xenobiotic corn can influence the activity of this enzyme system are also pounds including known hepatotoxins (257), numerous thera expected to affect tumor formation in animals exposed to peutic agents (56), and food additives (197). Most of the carcinogens (189, 246, 256, 298, 316, 389). Although there substances which induce a proliferation of the SER are metab are experiments which appear to contradict this expectation, olized, at least in part, in the rnicrosornal fraction of the hepa there are a number of studies which have shown a positive tocyte which comprises the SER. The observation that ethanol correlation between induction of microsomal enzyme activity produced proliferation of the SER raised the possibility that, in and chemical carcinogenesis (90, 171, 189). There are several addition to its oxidation by ADH in the cytosol, ethanol may possible explanations for these seemingly contradictory obser also be metabolized by the microsornes. A microsomal system vations. Since many inducers of microsomal enzymes are also capable of methanol oxidation had been described (285), but substrates for these enzymes, the simultaneous presence of its capacity for ethanol oxidation was extremely low. Further both inducer and carcinogen may result in competition for more, this system could not oxidize long-chain aliphatic alco enzyme-binding sites. Furthermore, the components of the hols such as butanol and was sensitive to catalase inhibitors, cytochrome P-450-dependent system and associated enzymes azide, and cyanide. Therefore, Ziegler (413) concluded that such as epoxide hydrase and glutathione transferases also are this system is clearly different from the cytochrome P.450- involved in the detoxification of many of the same chemicals dependent system and involves the H2O2-mediated ethanol which require activation for carcinogenesis. These enzymes peroxidation by catalase. However, a MEOS with a rate of not only respond differently to various inducers but also vary ethanol oxidation 10 times higher than reported by Orme with respect to their relative basal concentrationsfrom tissue Johnson and Ziegler (285) has been described (21 2, 214). to tissue and may vary with respect to their relative inducibility The system requires NADPH and 02 and is relatively insensitive in different tissues. This latter point may prove to be particularly to catalase inhibition. Furthermore, the MEOS was differen important with respect to ethanol as a microsomal inducer, as tiated from the system reported by Orme-Johnson (285) and will be illustrated by data presented subsequently. Ultimately, from catalase by its ability to oxidize long-chain aliphatic al

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cohols (362) which are not substrates for catalase (48). The of hepatic cytochrome P-450. Although components of micro striking increase in the non-ADH fraction of ethanol metabolism somal mixed-function oxidases have previously resisted many with increasing ethanol concentrations (123, 252, 363) is attempts at purification, recently, several groups (129, 135, consistent with the known Kmfor MEOS and ADH. Whereas 147, 326) succeeded in purifying multiple forms of cytochrome the former has a value of 8 to 10 m@i(214), the latter has a Km P-45O from rat and rabbit hepatic microsomes. One form, for varying from 0.26 to 2 mM (100, 230, 241 , 310). In perfused example, is inducible by PB and another is inducible by poly liver, the Km of ADH is even lower (230). The in vitro Kmof cyclic aromatic compounds such as $-naphthoflavone and 3- MEOS agrees well with in vivoKmmeasurementsfromexperi MC. These forms have different molecular weights as deter ments utilizing the ADH inhibitor pyrazole and from experiments mined by sodium dodecyl sulfate-polyacrylamide gel electro with isolated hepatocytes (254), suggesting that MEOS may phoresis and different spectral, immunological, and catalytic play a significant role in ethanol metabolism. properties. The PB-inducible form is relatively more active for Reconstitution of MEOS from the 3 microsomal components benzphetamine N-demethylation than is the other, whereas the (cytochrome P.450, NADPH-cytochrome c reductase, lecithin) 3-MG-inducible form is more active for benzopyrene hydrox (Chart 1) has been demonstrated by Ohnishi and Lieber (277) ylation. We have observed at least 3 forms of cytochrome P and confirmed by Miwa et a!. (266). The activity of the recon 450 (determined by continuous sodium dodecyl sulfate-poly stituted MEOS showed a dependency upon cytochrome P-450 acrylamide gel electrophoresis with heme staining) in chronic and the reductase and required synthetic phospholipids (such ethanol-fed rats. One of these forms is distinct from those as lecithin) for its maximal activity. The involvement of reduced induced by PB or 3-MG treatment. Partially purified cytochrome cytochromeP.450 in MEOS activitywas further shownby the P-450 from ethanol-fed rats was more active for alcohol oxi action spectra (93). How cytochrome P.450 mediates ethanol dation than was the control preparation in the reconstituted oxidation has not been clarified. It could contribute to the system (276). generation of a hydroxyl radical which in turn might be â€˜â€˜scav In addition to MEOS activity, repeated ethanol administration enged' â€˜byethanol since other compounds that interact with also results in increased activities of a variety of microsomal hydroxyl radicals inhibit MEOS activity (43, 278). drug-detoxifying enzymes (8, 42, 159, 264, 322). Some effects Following chronic ethanol consumption, there is a significant are readily detectable after a single ethanol dose (306). The increase in MEOS activity (212, 214). This is associated with increase in the activity of hepatic microsomal drug-detoxifying an increase in various constituents of the smooth fraction of enzymes and in the content of cytochrome P.450 induced by the membranes involved in drug metabolism, such as phospho ethanol ingestion offers a likely explanation for the observation lipids, cytochrome P.450 reductase, and cytochrome P-450 that ethanol consumption enhances the rate of drug clearance (153, 159). The increase of cytochrome P-450 was associated in vivo. The tolerance of the alcoholic to various drugs has with the appearance of a distinct form of cytochrome P.450 been generally attributed to central nervous system adaptation which exhibited a high affinity for cyanide (54, 133, 158). (164). However, there is occasionally a dissociation in the time Further evidence in favor of an increase of a distinct species of course of the decreased drug sensitivity of the animals and the cytochrome P.450 after ethanol treatment was derived from occurrence of central nervous system tolerance, the former inhibitor studies (375). More direct proof was obtained from preceding the latter (308). Thus, in addition to central nervous studies of microsomal proteins (277) which indicated that the system adaptation, metabolic adaptation must be considered. rise in cytochrome P-450 involved a hemoprotein different from Indeed, it has been shown that the rate of drug clearance from those induced by PB or 3-MG. Moreover, sodium dodecyl the blood is enhanced in alcoholics (166). Of course, this could sulfate-polyacrylamide gel electrophoresis showed an induc be due to a variety of factors other than ethanol, such as the tion of microsomal protein with a molecular weight of 53,400 congeners present in alcoholic beverages and the use of other which was presumed to be an apoprotein of cytochrome P drugs so commonly associated with alcoholism. Controlled 450. The partially purified cytochrome P.450 from ethanol-fed studies have shown, however, that administration of pure rats was more active for alcohol oxidation than was the control ethanol with nondeficient diets either to rats or humans (under preparation in the presence of an excess of NADPH-cyto metabolic ward conditions) results in a striking increase in the chrome c reductase and L-a-dioleoyl lecithin. These findings rate of blood clearance of meprobamate and pentobarbital are in keeping with recent demonstrations of the heterogenicity (264) (Chart 2). Similarly, increases in the metabolism of ami nopyrine (379), tolbutamide (42) and rifamycin (119) were I.0 found. Furthermore, the capacity of liver slices from animals @ IETHANOL 1 @ w I PROPANOLI fed ethanol to metabolize meprobamate was also increased

@ U. â€˜@ (264), which clearly showed that ethanol consumption affects @ â€” 0.5 drug metabolism in the liver itself, independent of drug excre >. < tion or distribution. Failure to verify such an effect (150) was probably due to the very low dosage of ethanol administered. @ 0 The stimulation of microsomal enzyme activities also applies p@45O REDUCTASE P'450 + + + LECITHIN LECIT@N REDUCTASE to those enzymes which convert exogenous substrates to toxic

LECITH1I compounds. For instance, CCL exerts its toxicity only after cYTOcHR0MEP.450(0.30,@,ol/mII NADPH'CYTOCHROMEc REDUCTASEI2OO@t./'td) conversion in the microsomes. Alcohol pretreatment dramati Lc(-OICL(OYL LECiTHIN (Ilpg/mII cally stimulates the toxicity of CCL (131 , 242). The experiments Chart 1. Ethanol, propanol, and butanol oxidation by the reconstituted MEOS. of Hasumura et a!. (131) were carried out at a time when the Active oxidation of alcohols is achieved by the combination of the 3 components. Acetaldehyde, propionaldehyde, and butyraldehyde in incubates were measured ethanol had disappeared from the blood to rule out the increase directly by gas-liquid chromatography (276). of the toxicity of CCI4 due to the presence of ethanol (367).

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.,-@ DURING CONTROL PEROD with a Kmof 0.2 mM, which is inhibited by the â€˜â€˜inducers'â€˜used â€˜â€”â€˜AFTER I MONTH OF 00 ETHANOL FEEDNG ow by Czygan et a!. (66). It was shown by Maling et a!. (242) that administration of an 4 w w acute dose of isopropyl alcohol or ethanol to rats enhances the activity of DMN N-demethylase at a DMN concentration of 10 mM. This increase of enzyme activity was accompanied with a @ w potentiation of DMN-induced hepatotoxicity. We have found (336) that chronic ethanol administration in a liquid diet to ox Sprague-Dawley rats results in a significant enhancement of 0 4 12 6 20 24 DMN N-demethylase activity measured at both high (1 00 mM) TIME IN HOURS and low DMN concentrations (5 mM) regardless of the sex of Chart 2. Effect of ethanol consumption on clearance of meprobamate from the animals. blood. Four volunteer alcoholics were tested before and after 1 month of ethanol ingestion: half-lives are shown by the dotted lines on x and y axes (264). Effect of Ethanol Consumption on Intestinal Microsoma! Enzymes and Associated Enhanced Mutagenicity The potentiation of the CCL toxicity by ethanol pretreatment may be accounted for by the increased production of toxic In the last decade, the importance of intestinal xenobiotic compounds of CCL since the conversion of 14CC14toâ€˜4CO2and metabolism has been recognized, although it has been realized covalent binding of CCL metabolites to protein were signifi that its activity is relatively small compared to that of the liver. cantly accelerated in microsomes of ethanol-pretreated rats Because of the relatively high concentrations of environmental (131). It is likely that a larger number of other toxic agents will dietary carcinogens in the small bowel and because of the be found to display a selective injurious action in the alcoholic. great variety of those compounds in the intestine, their intes Hepatic Activation of Procarcinogens after Alcohol. It has tinal activation may be of obvious clinical importance, espe been shown that there is a good relationship between BP cially since this activation may be linked to absorption and hydroxylase activity and tumor formation in vivo (90, 171). It is biological availability. of interest to note therefore that, after administration of ade Cytochrome P-450, NADPH-cytochrome P-450 reductase, quate liquid diets containing either 36% of total calories as and mixed-function oxidase activities have been demonstrated ethanol or isocaloric carbohydrates to female Sprague-Dawley in the gastrointestinal tract of several species (50, 352, 353, rats for 15 days, BP activity was elevated by 57% in the 387). Recent studies also have shown that this system may ethanol-fed animals (319). Recently, we extended these results mediate the intestinal metabolism of drugs and carcinogens on the effects of ethanol on BP metabolism in the liver (336) to and can be induced by dietary constituents (142, 288, 386). include hepatic microsomal activation of BP to its mutagenic For example, the intestinal metabolism of phenacetin is greatly derivatives using the Salmonella typhimurium His strain TA increased in rats fed a diet containing charcoal-broiled ground 100 to assay mutagenic activity (3). Over the entire range of beef (288). Moreover, in the rat, a number of vegetables (e.g., BP tested, the mutagenic effect of BP was enhanced when spinach, broccoli, and cabbage) enhance the intestinal metab hepatic microsomes from ethanol-fed female rats were used olism of BP (387), and dietary iron has prominent effects on for activation. The difference in the extent of BP activation was other drug-metabolizing activities (141 , 142). Finally, dietary similar to the difference in BP hydroxylase activity. Hepatic constituents such as polycyclic hydrocarbons and flavones microsomes from ethanol-fed and control male Sprague-Daw contained in animal chow (387) are capable of inducing the ley rats which did not show any significant difference in BP intestinal enzymes involved in the oxidative metabolism of hydroxylase activity also did not differ with respect to BP xenobiotics. The oxidative enzymes involved in drug metabo mediated mutagenesis. lism have been localized in the rat small intestine (142). Two A decrease in hepatic cytochrome P-450, benzo(a)pyrene gradients exist for the content of P-450 and activities of BP monooxygenase activity, and DNA binding of BP after acute hydroxylase, p-nitroanisole O-demethylase, and NADPH-cyto injection of different ethanol concentrations (0.7 to 1.8 mg/kg chrome P-450 reductase. Along the length of the small bowel, body weight) into mice was reported by Gapel et a!. (41), who the activity and content of these drug-metabolizing components also reported a decrease in BP hydroxylase activity and DNA are highest in the upper part. Within an individual villous binding of BP after chronic ethanol administration for 2 to 8 structure, these components are highest in the upper villous weeks. However,intheselatterexperiments,ethanolwasgiven tip cells and decrease progressively toward the crypt areas together with the drinking water (10% v/v); therefore, alcohol (141, 142). consumption must have been relatively low and nutrient intake Since ethanol is a common dietary constituent and produces must have been uncontrolled. ultrastructural changes in intestinal mucosa (e.g. , proliferation In addition to polycyclic hydrocarbons (248), a number of of the endoplasmic reticulum and mitochondrial distortion) nitrosamines have been detected in alcoholic beverages (227). similar to those in the liver (324), it was reasonable to expect It is generally agreed that the initial step in the bioactivation of that the intestinal metabolism of xenobiotics may also be al DMN to a methylating agent is microsomal N-demethylation. tered by ethanol consumption. Indeed, we have reported ele There are different isoenzymes of DMN demethylase. Czygan vated cytochrome P-450 levels and microsomal enzyme activ et a!. (66) characterized a DMN N-demethylase which has a Km ities in the upper small intestine after chronic ethanol ingestion of 40 mM and is inducible by PB, 3-MG, or Aroclor 1254 as a in rats (335, 337), a finding that has been confirmed recently cytochrome P.450-dependent enzyme system. On the other (157). As shown in Charts 3 and 4, chronic ethanol ingestion hand, Venkatesan et a!. (378) described a DMN N-demethylase increases BP hydroxylase activity by 200% and also enhances

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CYTOCHROME P-450 BENZO (@) PYRENE HYDROxYLASE ACTIVITY when activated by microsomes, have been shown to be muta râ€”ETHANOL-FEDRATSI genic (329). L PAIR@FEDCONTROLS@ 30 Tobacco pyrolysate was prepared from commercially avail I able cigarette tobacco which was heated under vacuum at z @:20 5000 for 1 mm (250). No difference was detected in 20 benzo(a)pyrene monooxygenase activity between alcohol-fed and control animals [22.4 Â±3.2 (S.E.) versus 21.5 Â±4.2 pmol 3-hydroxybenzo(a)pyrene per mg microsomal protein per 2 Iâ€” )-@ 10 l0 â€” T @2 mm]; lung microsomes from both groups of animals activated I BP to the same degree in the S. typhimuriumtest. However, when lung microsomes from ethanol-fed rats were incubated E 0 _ L@J@0 with tobacco pyrolysate, an enhanced activation to mutagenic p'0.0I derivatives was observed (Chart 5). Since no difference was Chart 3. Microsomal cytochrome P-450 content and BP hydroxylase activity seen in BP activation, compounds in tobacco pyrolysate other in the mucosa of the proximal small intestine after 25 days of ethanol administra than BP must be incriminated for the difference in mutagenicity. tion in 7 pairs of rats (335). The active compounds have not been identified but may include pyrolysis products of peptides (251 ) or of some amino acids (250) or nitrosamines which have been detected in trace

1â€”OETHA$OL-FED RATS I amounts in tobacco (227). At the present time, further obser I@''.REm.FEDcoin@ou] 200 A vations are needed to assess the clinical relevance of our findings of enhanced pulmonary activation of tobacco derivates to mutagenic compounds after chronic ethanol consumption. Similar microsomal studies should also be carried out in other tissues in which an association of cancer and alcoholism has been described. For instance, in addition to cancer of the colon @ ISO and rectum, cancer of breast and thyroid gland have been C associated by Breslow and Enstrom (37) with beer consump 0 0 00 tion and they also showed an association between kidney and + U) bladder cancer and alcohol. Some investigators (296, 357) I 50 have found an association between alcohol and prostatic can cer, but others (334, 41 1) have not. Still others have noted an association between alcohol consumption and melanoma l'o I'S 20 25 5 0 5 20 (394). A summary of epidemiological data is presented in jig BENZO(O()PYRENE/PLATE various reviews on alcohol and cancer (1, 180, 370) and Chart 4. Metabolic activation of BP to a mutagen by intestinal microsomes isolated from ethanol-fed and control rats. Each point is an average of the number elsewhere in this issue (371). of Hisâ€˜coloniesof S. typhimurium strain TA 100 present on duplicate plates. The inductive effect of chronic ethanol ingestion on the The levels of spontaneous His â€˜revertantshave been subtracted. In each case, the microsomes were from pools of 3 animals pair fed either the ethanol capacity of isolated liver, intestine, and lung microsomes to containing or the control diet. The microsomal protein concentrations per plate activate procarcinogens to mutagens suggests a possible link were:A, 0.5 mg: B. 0.5 mg: C. 1.0mg: D. 1.4mg (335). between this effect of ethanol and the enhanced incidence of cancer in the alcoholic. These speculations are particularly the capacity of intestinal microsomes to activate BP to a mu relevant since in various animal experiments ethanol has been tagen in the S. typhimurium test system. In the same test shown to have syncarcinogenic effects. The carcinogenic ef system, 2 other compounds, namely, 2-aminofluorene and fect of 7,12-dimethylbenzanthracene applied to either the tryptophan pyrolysate, both of which need microsomal activa cheek epithelium of young Syrian hamsters (88, 89) or the skin tion to become mutagenic, were studied (336). The capacity of of mice (351 ) was enhanced by ethanol. Numerous papillomas @ intestinal microsomes to activate both compounds to mutagens r ET$ANOL@FEDRATS @ also was significantly enhanced after chronic alcohol ingestion. 8 I.â€”PAIR'FED t4 500 A.0.7 mg m@crpro$W@/pIati ,0 Changes in Microsomes of Lungs and Possible Relationship @L&J @@Between Cancer of Lungs and Other Tissues and Alcoholism 400 @ Although an association between alcoholism and lung cancer E 300 has not been established, xenobiotic metabolism in the lungs >.z @ Iâ€” 200 seems to be important since, as in the case of the intestine, 40 lungs represent a major site of entry into the body. Since 100 alcohol and tobacco play a synergistic role with respect to carcinogenesis in the oropharynx, larynx, and esophagus

(41 2), we wondered whether chronic ethanol administration to mg TOBACCO/PLATE rats may increase pulmonary activation of procarcinogens con Chart 5. Metabolic activation of tobacco pyrolysate to mutagens by lung tamed in tobacco pyrolysates, which may be inhaled by smok microsomes (micr.) isolated from ethanol-fed and control rats. Assays were ing. Indeed, tobacco pyrolysates and smoke condensates, performed as described in Chart 4.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Alcoho!-re!ated Diseases and Carcinogenesis and 2 carcinomas of the esophagus were obtained by the exclusively nutritional origin of alcoholic liver disease arose administration of BP to mice in dilute alcohol (145), while no from an improvement in the method of alcohol feeding to carcinomas were detected when ethanol was given alone or experimental animals. Indeed, when the conventional alcohol when BP was given in other solvents. Furthermore, the simul feeding procedure is used, namely, when ethanol is given as taneous administration of ethanol and DMN in rats has resulted part of the drinking water, rats usually refuse to take a sufficient in a significantly elevated number of papillomas and epidermoid amount of ethanol to develop liver injury, if the diet is adequate. carcinomas (1 16). It should be noted, however, that in another This aversion of rats to ethanol was counteracted by the study the simultaneous administration of ethanol (25 and 38% introduction of the new technique of feeding ethanol as part of in drinking water) did not modify the hepatocarcinogenesis a nutritionally adequate, totally liquid diet (72, 219, 220). With effect of diethylnitrosamine (330, 331 ). The results of this latter this procedure, ethanol intake was sufficient to produce a fatty study must be questioned, however, since when administered liver despite an adequate diet. This technique is now widely in drinking water ethanol is consumed in only small amounts adopted for the study of the pathogenesis of the fatty liver in by rodents. the rat. In addition to the fatty liver, ethanol dependence It should also be pointed out that acetaldehyde, the product developed in these rats, as witnessed by typical withdrawal of ethanol metabolism, was also found to induce sister chro seizures after cessation of alcohol intake (215). matid exchange and cross-links in DNA in human cells (31 1). Having established an etiological role for ethanol in the Consistent with this observation is the finding that ethanol pathogenesis of the experimental fatty liver, the question of its causes chromosomal changes in vivo but not in vitro (275). importance for the development of human pathology remained. To determine whether ingestion of alcohol in amounts compa Hepatocellular Carcinoma and Mechanisms of Ethanol induced Liver Injury rable to those consumed by chronic alcoholics is capable of injuring the liver even in the absence of dietary deficiencies, Among the various theories of hepatocellular cancer, it has volunteers (with and without a history of alcoholism) were given been proposed that repeated cell injury promotes the devel a variety of nondeficient diets under metabolic ward conditions, opment of tumors. Experimentally, liver cancer can indeed be with ethanol either as a supplement to the diet or as an induced more readily if superimposed on a regenerating liver isocaloric substitution for carbohydrate (219, 220, 223). In all (62); clinically, as discussed before, a majority of hepatocel these individuals, ethanol administration resulted in fatty liver lular tumors are associated with cirrhosis of the liver. It is development which was evident both on morphological exam therefore germane to review and examine mechanisms ination and by direct measurement of the lipid content of the whereby alcohol injures the liver, including the pathogenesis liver biopsies, which revealed a rise in triglyceride concentra ofcirrhosis. tion up to 15-fold. Origin and Mechanism of Fat Deposition In the Liver. Lipids Pathogenesis of Alcoholic Fatty Liver which accumulate in the liver can originate from 3 main sources: dietary lipids (which reach the blood stream as chy In its milder form, alcoholic liver disease is characterized by lomicrons); adipose tissue lipids (which are transported to the accumulation of excess fat in the liver, so-called fatty liver. liver as free fatty acids; and lipids synthesized in the liver itself Alcohol as a Direct Cause of the Fatty Liver. Each g of (Chart 6). These fatty acids of various sources can accumulate ethanol provides 7.1 calories, which means that 20 oz (or 586 in the liver as a result of a large number of metabolic disturb ml) of 86 proof (43%, v/v) beverage (not an unusual intake for ances, primarily (a) decreased lipid oxidation in the liver, (b) the alcoholic) represent about 1500 calories or one-half to two enhanced hepatic lipogenesis, (c) decreased hepatic release thirds of the normal daily caloric requirement. Therefore, the of lipoproteins, (d) increased mobilization of peripheral fat, and alcoholic has a much reduced demand for food to fulfill his (e) enhanced hepatic uptake of circulating lipids. Depending caloric needs. Since alcoholic beverages do not contain sig On the experimental conditions, any of the 3 sources and the nificant amounts of protein, vitamins, and minerals, the intake various mechanisms can be implicated. of these nutrients may become readily borderline or insufficient. During consumption of ethanol with lipid-containing diets, Economic factors may also reduce the consumption of nutrient rich food by the alcoholic. In addition to acting as â€˜â€˜empty'â€˜or

â€˜â€˜naked' â€˜calories, alcohol can result in malnutrition by interfer ing with the normal processes of food digestion and absorption (229). For all these reasons, deficiency diseases readily de velop in the alcoholic. In rodents, severely deficient diets result in liver damage even in the absence of alcohol. Extrapolation from these animal results to humans led to the belief that in alcoholics the liver disease is due not to ethanol but solely to the nutritional deficiencies and that, given an adequate diet, alcohol is merely acting by its caloric contribution and is not more toxic than a similar caloric load derived from fats or starches (30). This opinion prevailed despite some statistical evidence gathered both in France (297) and in Germany (203) which indicated that the incidence of liver disease correlated with the amount of alcohol consumed rather than with deficien Chart 6. Possible mechanismsof fatty liver production through either increase cies in the diet. A major challenge to the concept of the (_4) or decrease (â€˜â€”ii--') of lipid transport and metabolism. FFA. free fatty acids.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. C. S. Lieber et a!. the fatty acids which accumulate in the liver are derived pri mobilization from adipose tissue was found to be mediated by manly from dietary fatty acids; whereas when ethanol is given acetate (63). Acetate is the end product of ethanol metabolism with a low-fat diet, endogenously synthesized fatty acids are in the liver (Chart 6) and is released into the blood stream. deposited in the liver (224â€”226).Someof these effects can be Since stressful doses of ethanol probably both stimulate fatty considered as consequences of the metabolism of ethanol in acid mobilization (via catecholamine release) and depress it the liver (Chart 7). Depending on the metabolic state of the (via the acetate produced), the net effect may depend upon animal, both decreased lipid oxidation and enhanced lipo the particular experimental conditions. This may account for genesis can be linked to ethanol oxidation and the associated some of the apparent contradictions of the literature. increased generation of NADH as discussed elsewhere (208). Thus, the main events leading to the development of the In addition to the functional changes which are a direct con alcoholic fatty liver can be summarized as follows. Ethanol, sequence of the metabolism of ethanol, chronic ethanol abuse which has an almost â€˜â€˜obligatory'â€˜hepaticmetabolism, replaces results in more persistent changes in the mitochondria (209). the fatty acids as a normal fuel for the hepatic mitochondria. The striking structural changes of the mitochondria are asso This results in fatty acid accumulation, directly because of ciated with corresponding functional abnormalities including a decreased lipid oxidation and indirectly because one way for decreased capacity to oxidize fatty acid (253). Thus, de the liver to dispose of excess hydrogen generated by ethanol creased fatty acid oxidation, whether as a function of the oxidation is to synthesize more lipids. Fatty acids derived from reduced citric acid cycle activity (secondary to the altered adipose tissue accumulate in the liver only when very large oxidation-reduction potential) or as a consequence of perma amounts of ethanol are given. The lipids increase in the liver nent changes in mitochondrial structure, offers the most likely despite the fact that the transport mechanism via release of explanation for the deposition of fat in the liver, especially fat lipoproteins from the liver into the blood stream is stimulated derived from the diet. Contrasting with fed rats, in which there by ethanol, at least during the initial state of intoxication. is evidence for both accelerated lipogenesis and increased With progressing liver injury, as discussed subsequently, the utilization of dietary fat (7), ethanol did not stimulate fatty acid hypersecretion of lipoprotein fades away and may actually fall synthesis in fasted rats (283). Moreover, in adequately fed rats below normal levels. At that stage, instead of offsetting, in part, given one large, sublethal dose of ethanol, it was observed that the lipid accumulation, altered lipoprotein secretion may ac fatty acids resembling those of adipose tissue accumulate in tually contribute to it and thereby aggravate liver injury even the liver (130, 225). Experimental procedures or agents which further (see below). reduce the normal rate of peripheral fat mobilization, i.e., The Influence of Dietary Factors. Role of Dietary Fat. As adrenalectomy, spinal cord transection, or ganglioplegic drugs, discussed before, alcohol ingestion leads to deposition in the prevented or decreased this type of hepatic fat accumulation liver of dietary fat. This observation prompted an investigation (38, 243, 309). More direct approaches, however, such as into the role of the amount and kind of dietary fat in the studies in rats with prelabeled epididymal fat pads, yielded pathogenesis of alcohol-induced liver injury. Rats were given conflicting information, with evidence for increased (173) or liquid diets containing a normal amount of protein for rodents unchanged (160) fatty acid mobilization. Similarly, in rats, one (18% of total calories), with varying amounts of fat. Reduction large dose of ethanol has been reported to result in increased in dietary fat to a level of 25% (or less) of total calories was (38, 243) or unchanged (87) circulating levels of free fatty accompanied by a significant decrease in the steatosis induced acids. In humans, even with amounts of ethanol as large as by ethanol (21 3). The importance of dietary fat was confirmed 300 g/day, the concentration of circulating free fatty acids did in volunteers. For a given alcohol intake, much more steatosis not increase; it rose only after ingestion of very large doses of developed with diets of normal fat content than with a low-fat ethanol (400 g/day) (220). In short-term studies, ethanol ad diet (224). In addition to the amount, the chain length of the ministration produced a fall in the level of circulating free fatty dietary fatty acid is also important for the degree of fat depo acids in humans (160, 219) with reduced peripheral venous sition in the liver after alcohol feeding. Replacement of dietary arterial differences in free fatty acids (221 ), decreased free triglycerides containing long-chain fatty acids by fat containing fatty acid turnover (161), and concomitant reduction in circu medium-chain fatty acids markedly reduced the capacity of lating glycerol (96). This effect of ethanol upon free fatty acid alcohol to produce a fatty liver in rats (222). The propensity of medium-chain fatty acids to oxidation rather than to esterifi cation is the most likely explanation of the reduction in alcoholic steatosis upon replacement of dietary long-chain fatty acids by medium-chain fatty acids. Role of Protein and Lipotropic Factors (Choline and Methio nine). In perfused liver, ethanol was shown to increase choline uptake (16), but this was found to be unrelated to lipid accu mulation (259). In growing rats, deficiencies in dietary protein and lipotropic factors (choline and methionine) can produce fatty liver (30), but primates are far less susceptible to protein and lipotrope deficiency than are rodents (143). Clinically, treatment with choline of patients suffering from alcoholic liver injury has been found to be ineffective in the face of continued Chart 7. Oxidation of ethanol in the hepatocyte and its link to fatty liver, alcohol abuse (284); experimentally, massive supplementation hyperlipemia, hyperuricemia, hyperlactacidemia, ketosis, hypoglycemia, and drug and acetaldehyde metabolism. â€”â€”â€”â€”,pathwaysthat are depressed by with choline failed to prevent fatty liver produced by alcohol in ethanol (210); a Glycerophos., a-glycerophosphate. volunteer subjects (323). Unlike rat liver, primate livers contain

2870 CANCER RESEARCH VOL. 39

very little choline oxidase activity, which may explain the dif alcoholic liver disease; alcohol-induced injury in other organs; ference with regard to choline deficiency. The phospholipid and associated disease states. The frequent concurrence of content of the liver represents another key difference between chronic alcoholism and nutritional deficiency makes the sepa the ethanol- and choline-deficient fatty liver. After the admin ration of these variables especially difficult. However, whereas istration of ethanol, hepatic phospholipids increase (219); branched-chain amino acids and a-amino-n-butyric acid were whereas in the fatty liver produced by choline deficiency, they found to be increased 2- to 3-fold and 7-fold, respectively, in decrease (10). Moreover, orotic acid, which prevents the phos the plasma of baboons fed alcohol as 50% of total calories pholipid decrease and the development of fatty liver due to (341 ), these amino acids are all depressed when measured in choline deficiency, had no such effects after ethanol (86). protein deficiency. Following intestinal bypass for obesity, de Furthermore, hepatic ATP decreased after chronic ethanol creased absorption of dietary protein is observed (271 ). In feeding (29, 112, 385) but was unaffected in choline deficiency such patients, plasma a-amino-n-butyric and branched-chain (346). Conversely, hepatic carnitine is decreased by choline amino acids, phenylalanine, threonine, and lysine are de deficiency (61) but increased after ethanol feeding (185). creased while plasma serine and glycine, both nonessential Moreover, ethanol-induced fatty liver is associated with in amino acids, are increased. By contrast, relatively increased creased circulatory lipoproteins and enhanced incorporation of a-amino-n-butyric acid was considered to be characteristic of [14G]Iysineinto lipoproteins (19), whereas the opposite occurs changes of plasma amino acids in alcoholics (344). with choline deficiency (232). Ultrastructurally, the lesions also Transitional Lesions from Reversible Alcoholic Fatty Liver to differ (152, 325). Thus, hepatic injury induced by choline Irreversible Liver Injury deficiency appears to be primarily an experimental disease of rats with little, if any, relevance to alcoholic liver injury in At the 2 extremes in the spectrum of alcoholic liver disease, humans. Even in the rats, massive choline supplementation one has the fully reversible fat accumulation which can occur failed to prevent fully the ethanol-induced lesion, whether al after a few days of alcohol abuse and the irreversible cirrhosis cohol was administered acutely (76) or chronically (21 1). Al characteristic of end-stage alcoholic liver disease. In humans, cohol has been reported either to aggravate (360) or attenuate the evolution from fatty liver to cirrhosis may take 5 to 20 years. (292) choline-induced liver injury. In the sequence of events, a number of lesions occur, which Protein deficiency may affect the liver, but this has not yet can be considered as transitional stages between the alcoholic been clearly delineated in human adults. In children, protein fatty liver and cirrhosis. When these lesions are particularly deficiency leads to steatosis, one of the manifestations of florid, with manifest extensive necrosis and polymorphonuclear kwashiorkor. In adolescent baboons, however, protein restric inflammation, they are sometimes considered to be character tion from 20% to 7% of total calories (as part of a low-fat diet, istic of a stage of â€˜â€ãlcoholichepatitis'â€˜whichis believed by 14% of calories) did not result in liver injury on either biochem many to be â€˜â€˜the'â€˜precursorlesion of cirrhosis, intermediate ical analysis or light and electron microscopic examination between the fatty liver stage and the end-stage fibrosis. In even after 19 months (21 7). Conversely, an excess of protein practice, delineation of such a hepatitis stage is not always was not capable of preventing ethanol from producing fat easy. In fact, extensive hepatitis is relatively uncommon in an accumulation in human volunteers (223). In that study, dietary alcoholic population, as compared to the frequency of fatty protein represented 25% of total calories, or 2.5 times the liver and cirrhosis. Fatty liver, however, is also associated with recommended amount. Thus, even in the absence of protein milder forms of necrosis and inflammation, in which case the deficiency, ethanol is capable, in humans, of producing striking term â€˜â€˜severe'â€˜fattyliver or â€˜â€˜mild'â€ãlcoholichepatitis is being changes in the liver. Severe protein deficiency (4% of total used. In fact, at the early so-called simple or uncomplicated calories) also produced steatosis in the baboon (21 7). Similar fatty liver stage, as discussed before, striking lesions in the lesions were reported in the rhesus monkey (191). When mitochondria and the rough endoplasmic reticulum can be protein deficiency is present, it could potentiate the effect of detected. These in a sense might also be considered as tran @ ethanol. Indeed, administration of ethanol with a diet deficient sition lesions between â€˜simple'â€˜fataccumulation and more in protein and lipotropic factors had more pronounced effects severe liver injury. In fact, the injurious effects of alcohol in the than that of either factor alone (182, 226), at least in rodents. liver are multiple, and the classic distinction between â€˜â€˜simple Possible interactions of ethanol and protein nutrition were also fatty liver, â€˜â€˜â€˜â€ãlcoholichepatitis,â€˜â€ãndâ€˜â€˜cirrhosis'â€˜isarbitrary. suggested by the observation that ethanol feeding increases In reality, there is a continuum of lesions, and the more sensitive the activities of hepatic cystathionine synthetase and 5-meth the mode of investigation, the earlier signs of such injury can yltetrahydrofolate-homocysteine methyltransferase, which may be found. However, 2 types of lesions stand out as key stages impair mechanisms for methionine conservation in protein de in the evolution of alcoholic liver disease, namely, the swelling ficiency (101). To what extent such abnormalities contribute to or â€˜â€˜ballooning'â€õfthe hepatocyte and the necrosis with asso the ethanol-induced liver lesions remains to be assessed. In ciated inflammation. any event, the ultrastructural abnormalities produced by protein â€œBallooningâ€•ofthe Hepatocyte and Associated Impair deficiency (91 , 294) differ from those resulting from alcohol ment of Protein and Lipoprotein Export. In addition to fat (152, 196). Furthermore, clinical protein malnutrition is asso accumulation, the alcoholic fatty liver is characterized by strik ciated with characteristic plasma amino acid abnormalities, ing deposition of protein (17, 18). In the early stages of fatty including a depression of branched-chain amino acids, liver development, this protein accumulation was found to be whereasthe alcohol-inducedliverinjuryin the well-fed baboon as important quantitatively as that of fat and contributed to a was associated with opposite amino acid changes (341 ). Of similar extent to the hepatomegaly which developed after course, in alcoholic patients, plasma amino acid abnormalities chronic alcoholism. Although enhanced organelle proteins may reflect a complex interaction of many factors: nutrition; (mitochondria and microsomes) do contribute to the total in

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. C. S. Lieber et a!. crease, the major fraction of the proteins is deposited in the Ultrastructura! Changes of Mitochondria. In humans chronic cytosol. The nature of all the proteins which accumulate in the alcohol consumption results in striking mitochondrial altera cytosol has not been elucidated, but up to now increases have tions which include swelling and disfiguration of mitochondria, been found in export proteins such as albumin and transferrin disorientation of the cristae, and intramitochondrial crystalline (1 8) but not in constituent proteins of the cytosol. This obser inclusions (178, 358). Similarly, in the rat, isocaloric substitu vation led to the hypothesis that one of the early lesions tion of ethanol for carbohydrate in otherwise adequate diets induced by chronic alcoholism may be the interference with the leads to enlargement and alterations of the configuration of the capacity of the liver to export proteins. Consistent with this mitochondria (152), indicating that ethanol itself or one of its concept was the finding of a decrease in hepatic microtubulin metabolites causes the alterations rather than dietary deficien believed to be implicated in the export of proteins from the liver cies. Mitochondrial changes similar to those seen in chronic (18) and delayed serum albumin labeling after administration alcoholics were also produced by isocaloric substitution of of labeled amino acids (18). Metabolites of ethanol may be ethanol for carbohydrate in baboons (21 7), and in humans, responsible for this effect (249), particularly acetaldehyde, a both in alcoholics (196, 223) and in nonalcoholics (323). known hepatotoxin (see below). The retention of protein may Degenerated mitochondria were conspicuous, and the debris contribute to the â€˜â€˜ballooning'â€˜ofthe hepatocytes, a common of these degraded organelles was also found within autophagic morphological alteration found in alcohol liver injury, and the vacuoles and residual vacuolated bodies (321 ). The striking decrease in plasma transferrin in alcoholics with or without structural changes of the mitochondria are associated with cirrhosis (195). corresponding functional abnormalities. In addition to protein release, lipoprotein secretion is also Alterations of Mitochondria! Functions. These injured mito affected. Contrasting with the hyperlipemia, which is commonly chondria have a reduction in cytochrome a and b content (320) associated with the administration of moderate to large and in succinic dehydrogenase activity (286, 320), although in amounts of ethanol (21 , 233, 253), an extremely high dose has one study (380) succinic dehydrogenase activity measured in been reported to decrease serum triglycerides (68), very-low total liver homogenates was reported to be increased in density lipoproteins (240), high-density lipoproteins (184), and ethanol-fed rats. The respiratory capacity of the mitochondria the incorporation of glucosamine into the carbohydrate moiety was found to be depressed (117, 132, 177) using pyruvate of serum lipoproteins (267) in the rat. succinate and acetaldehyde as substrates. Oxidation of other In volunteers, chronic ethanol administration resulted in initial substrates was also found to be reduced in mitochondria of hyperlipemia (220). However, blood lipid content declined after ethanol-fed rats, except for a-glycerophosphate, the oxidation 2 to 3 weeks, implying that lipoprotein output falls with pro of which was reported by some to be increased (176) or gressing alcoholic liver injury. This concept is supported by the unchanged (302), whereas others found it to be decreased study of Marzo et a!. (247), who correlated serum lipids with (134). various histological stages in 90 alcoholics. Peak serum lipid Oxidative phosphorylation was found to be selectively altered values were found during the stage of fatty metamorphosis. at Site 1 (46). Since the structural changes of the mitochondria During the succeeding stages of steatosis and interstitial persist, the question arose as to whether these in turn could be chronic hepatitis, a progressive decrease in serum lipids oc responsible for some alterations in lipid metabolism beyond curred. The decrease was predominantly in the triglyceride and those which were attributed to the altered oxidation-reduction cholesterol fractions. In well-established cirrhosis, circulating change. The first indication that ethanol consumption may lipoproteins are generally low (24, 40). In addition, ct-lipopro result in more persistent metabolic changes came from the teins are absent by electrophoresis in sera from patients with observation that alcohol ingestion is associated with a progres some alcoholic liver injury (289, 327), associated with the sive increase in ketonemia and ketonuria, which was most appearance of abnormal lipoproteins (13, 327). Thus, after the pronounced in the fasting state (202). The ketonemia may initial development of fatty liver accompanied by hyperlipemia, aggravate the acidosis of the hyperlactacidemia (220) and on the blood lipids return toward normal and even decrease (36). occasion may lead to severe alcoholic ketoacidosis (156, 207). Progressive deterioration of liver function, including lipoprotein The capacity for ethanol to produce ketonemia was found to production and secretion, could be responsible and may sec be greater than that of fat itself, provided, however, that fat was ondarily aggravate fat accumulation in the liver and the swelling present in the diet. Thus, fat seems to play a permissive role of the hepatocyte. It is reasonable to postulate that excessive (202). Mitochondria obtained from ethanol-fed rats, when in swelling in turn leads to cell death or necrosis, a key feature of cubated in vitro, even in the absence of ethanol, display de alcoholic hepatitis. creased capacity to oxidize fatty acids but enhanced fl-oxida Development of Mitochondrial Injury. In additionto altera tion, which is possibly responsible for the increased ketogen tions of the rough endoplasmic reticulum, as previously noted, esis (44). Decreased fatty acid oxidation, whether as a function alcoholics are known to have profound hepatic mitochondrial of the reduced citric acid cycle activity (secondary to the changes (358) which are associated with increased serum altered oxidation-reduction potential) (208) or as a conse activity of the intramitochondrial enzyme glutamate dehydro quence of permanent changes in mitochondrial structure (as genase (186). From these clinical observations, however, it emphasized in this paragraph), offers the most likely explana was impossible to assess whether the mitochondrial changes tion for the deposition of fat in the liver after chronic alcohol were a direct result of chronic ethanol intake or were secondary ingestion, especially fat derived from the diet (224â€”226,258). to other factors such as dietary deficiencies. Recent studies It is noteworthy that high concentrations of acetaldehyde, the have incriminated alcohol itself as the responsible agent and product of ethanol metabolism, mimic the defects produced by have clarified some functional counterparts of the ultrastruc chronic ethanol consumption on oxidative phosphorylation at tural lesions. Site 1 (45). One may wonder to what extent chronic exposure

2872 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Alcohol-related Diseases and Carcinogenesis to acetaldehyde is the cause for the defects observed after phosphatase, and high bilirubin may mimic extrahepatic ob chronic ethanol consumption, especially since it was found struction (15, 262). The differentiation of alcoholic hepatitis, a recently that, after chronic ethanol consumption, mitochondria medical condition, and extrahepatic obstruction, a surgical become more susceptible to the toxic effects of acetaldehyde. entity, is important since an increased mortality has been These toxic effects occur even at 0.2 m@iacetaldehyde (253), observed in open liver biopsies of patients with alcoholic hep a concentration reported in the liver in vivo after ethanol ad atitis (122). ministration. Alcoholics may exhibit higher acetaldehyde levels Thus, it appears that clinical symptoms and biochemical than do nonalcoholics for a given ethanol load and blood level tests, although sometimes suggestive, do not permit a definitive (188). It is therefore not unreasonable to speculate that expo diagnosis and that correct staging depends on morphological sure to high acetaldehyde levels may in turn affect mitochon criteria. drial function and result in a vicious cycle, especially since Although liver biopsy is the most accurate way of determining mitochondria of alcohol-fed animals were found to have a the degree of liver cell necrosis, this is obviously not a practical reduced capacity to oxidize acetaldehyde (134). tool for routine follow-up of a patient or for mass screening Alcoholic Hepatitis. Alcoholic hepatitis is a morphological purposes. Therefore, spillover in the blood of liver enzymes, entity characterized by hepatocellular necrosis and an inflam especially transaminases, is commonly used as a marker of matory reaction of polymorphonuclear leukocytes. The lesions liver cell necrosis. However, blood transaminase values are a are characteristically localized in the centrolobular area but poor reflection of liver cell necrosis as revealed on biopsy may be more diffusely distributed in more severe cases. Intra (165), especially in alcohol-induced liver cell injury. In alcoholic cellular irregular clumps of hyalin, so-called Mallory bodies, hepatitis, for example, levels of transaminases are only mod may be present and, although helpful in establishing an etio erately elevated, and normal values can occasionally be found. logical diagnosis, are not pathognomonic for the disease. In â€˜y-Glutamyltranspeptidaseisno more reliable, although some deed, similar structures have occasionally been described in correlation with liver cell necrosis exists (401 ). Elevation from primary biliary cirrhosis (115, 238), in hepatocellular carci nonhepatic origin is common, and in some alcoholics elevated noma (168), in Indian childhood cirrhosis (318), after griseo levels may only reflect microsomal induction (1 11, 154, 361), fulvin treatment (75), in patients who underwent bypass oper although some have attributed this effect to lack of carbohy ation for morbid obesity (127), and in tissue cultures of cellsâ€¢drate (270). However, more recent studies showed this not to derived from rats treated with diethylnitrosamine, a chemical be the case (343). In contrast with transaminases and â€˜y-gluta carcinogen (35). Although the severity of the lesions is usually myltranspeptidase, which show a considerable overlap be somewhat greater in cases with Mallory bodies (51 , 299), no tween patients with and without significant cell necrosis on significantly worse prognosis was associated with their pres biopsy, in a recent study it was found that glutamate dehydro ence. genase reflected more accurately the degree of underlying cell The exact nature and pathogenesis of alcoholic hyalin has necrosis (376)(Chart 8). The high liver specificity ofthe enzyme not been elucidated, but the fact that, experimentally, hyalin (332), its solely mitochondrial origin (mitochondrial lesions bodies can be produced by agents which interfere with protein associated with alcoholic liver disease have been discussed secretion supports the concept that these bodies may be before), and its predominantly centrolobular localization (124) sequela of the impaired protein secretion discussed before (the area which suffers the major impact of alcoholic liver (74).Immune responseshavebeen attributedtohyalin.The injury) could explain the superiority of the enzyme as an index role of this and other â€˜â€˜autoimmune'â€˜reactionsin the pathogen of liver cell necrosis in alcoholic patients. esis of alcoholic liver injury are discussed in detail in another The mechanisms whereby alcohol abuse leads to necrosis review (109). The clinical picture of alcoholic hepatitis was of the hepatocyte have not been fully elucidated. It is likely that originally restricted to severely ill patients with jaundice, fever, a variety of mechanisms are involved, such as toxic effects of ascites, right upper quadrant pain, and hepatomegaly,some acetaldehyde; consequences of injury to the endoplasmic re times progressing to coma and death (300). However, it soon ticulum, mitochondria, and microtubules; as well as the physical became obvious that the typical morphological lesions also damage resulting from the accumulation of fat and protein and could be observed in patients with minimal and nonspecific the distention of the hepatocyte. complaints of anorexia, weight loss, or nausea (26, 121). Thus, As mentionedbefore, there is a continuumof graduationsof the clinical expression of alcoholic hepatitis may range from liver necrosis and inflammation ranging from its virtual absence nearly asymptomatic to a fulminant fatal disease (231 ). Severe in the initial fatty liver stage to extensive necrosis and inflam clinical symptoms are usually associated with severe histolog mation in the case of florid hepatitis. In the absence of liver ical lesions (181), but the reverse does not hold true, and biopsy, a determination of glutamate dehydrogenase may help extensive necrosis may be seen in patients with minimal corn define the evolution of the patient from one stage of alcoholic plaints (300). Characteristically, serum transaminases are only liver injury to another. moderately elevated in alcoholic hepatitis in contrast to viral Collagen Accumulation, Fibrosis, and Cirrhosis hepatitis, and there is a considerable overlap between the values in patients with and without hepatitis (376). Serum There is not only a wide spectrum of inflammation and alkaline phosphatase is often elevated, and leukocytosis may necrosis in alcoholic liver injury, but there is also a great be present. Other tests reflectthe degree of liverinsufficiency, variability in the magnitude of collagen deposition. At the earlier and elevated bilirubin, low serum albumin, and prolonged pro stages in the so-called simple or uncomplicatedfatty liver, time may be present to a variable degree. The combination of collagen is detectable by chemical means only (97, 293). When fever, jaundice, and right upper quadrant pain together with collagen deposition is sufficient to become visible by light moderately elevated transaminases, leukocytosis, high alkaline microscopy, usually it appears first around the central vein,

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C. S. Lieber et a!.

700 SGOT SGPT 500 300

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DEGREE OF INFLAPAMATaN/NECROSIS

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0.3 0 If 2+ 3+ 0 I. 5+ 3+ 1,.37) 5..31) I@IS) 1@44) @ wst@ ,..s) I..i., 0 1+ 2+ 3+ (P.33) 1.'lS) 1.4S) (..H)

OtSREE OF E4@LAM$.ATV'JN/NtCROSiS Chart 8. Correlation of serum glutamate dehydrogenase (GDH), serum glutamic-oxaloacetic transarriinase(SGOT), serum glutamic-pyruvic transaminase (SGPT). y-glutamyltranspeptidase (y-GTP), and ornithine carbamyl transferase (OCT) with degree of liver cell necrosis in 100 alcoholics (376).

resulting in so-called â€˜â€˜perivenular'â€˜sclerosis,(previously called out a mandatory stage of full-blown hepatitis but with only

â€˜â€˜pericentral' â€˜ sclerosis). This lesion is usually described in moderate inflammation and necrosis (216, 218). Moreover, association with full-blown alcoholic hepatitis. Less well rec there is a relative paucity of cases of alcoholic hepatitis as ognized is the fact that this perivenular sclerosis frequently compared to fatty liver and cirrhosis in many populations. It may be seen in the absence of overt hepatitis (84). In a appears now that collagen deposition, perivenular sclerosis, retrospective study of liver biopsies from alcoholic patients that and even cirrhosis can develop in the presence of minimal had been read by the pathologists as simple fatty liver, appro inflammation and necrosis, which may suffice to trigger the priate staining disclosed perivenular sclerosis in 40% (377). fibrosis (209). On the other hand, it is also possible that alcohol Experimental studies in the baboon model for alcoholic liver may have some direct effect on collagen metabolism independ injury have shown that, in animals that progressed to cirrhosis, ent of the necrosis and inflammation. In support of this possi perivenular sclerosis invariably occurred at the fatty liver stage bility is the observation of possible enhanced collagen produc in the absence of alcoholic hepatitis or preceding it, before the tion after alcohol (97, 260). Theoretically, interference by al development of the irreversible lesions of fibrosis and cirrhosis; cohol of collagen breakdown could also contribute to collagen by contrast, animals that did not develop the lesion did not accumulation. Thus far, however, there is no experimental progress beyond the stage of fatty liver. These experimental evidence to support such a mechanism. There is actually data suggest that, at least in the baboon, perivenular sclerosis evidence for enhanced rather than decreased collagen degra in the absence of hepatitis is a common and early warning sign dation (261 , 279). of impending cirrhosis, ifdrinking continues (377). The concept In the discussion of the pathogenesis of the alcoholic fatty of perivenular sclerosis as a precirrhotic lesion in alcoholics is liver, reference was made to the debate of the respective role further strengthened by similar observations in patients with of malnutrition and alcohol itself as an etiological factor. A fatty livers after bypass operation for morbid obesity (245) and similar debate has been raging with regard to the development in diabetic individuals (94). of alcoholic cirrhosis. Recently, this long-standing discussion In view of these observations, one must now reconsider the whether the liver injury observed in the alcoholic is due to classic concept according to which a major precursor lesion malnutrition rather than alcohol per se was rekindled by a for cirrhosis is the extensive inflammation and necrosis of report of Patek et a!. (292). In this epidemiological study of 304 fulminant alcoholic hepatitis, which in turn triggers the scarring. alcoholic patients, alcohol intake and dietary habits were eval Indeed, collagen deposition occurs already prior to the devel uated. There were 195 patients with hepatic cirrhosis, 40 opment of full-blown alcoholic hepatitis. Furthermore, in ex precirrhotics, and 69 noncirrhotics. By history, alcohol contrib periments in the baboons, full-blown cirrhosis developed with uted 50 to 59% of total calories. Two-thirds of the patients

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Alcohol-related Diseases and Carcinogenesis drank for more than 20 years. Duration and degree of alcohol ment of cirrhosis in humans is less clear. Epidemiological abuse was comparable in all 3 groups. Dietary intake, however, studies of Lelbach (203, 204) did not detect dietary insuffi differed. Over the 2-year period preceding the presenting ciency as a precondition for alcoholic cirrhosis. The incidence illness, noncirrhotics had higher food calorie intake and higher of cirrhosis correlated with the amount of alcohol consumed, protein intake than did the cirrhotics. This type of retrospective not with history of dietary deficiencies; similar results were study, however, is complicated by the difficulty in differentiating observed in France by Pequignot (297). Some have even cause and effect. It is well known that complications of severe suggested a lowered incidence of cirrhosis with dietary insuf liver disease, particularly cirrhosis, can by themselves be rea ficiency (193), whereas others as discussed before reached sons for poor dietary intake. It is therefore not clear whether the opposite conclusion (292). Of course, regardless of the the difference in dietary intake between cirrhotics and noncir controversy surrounding the role of malnutrition in the patho rhotics is the cause rather than a consequence of that disease. genesis of liver injury in the alcoholic, in our present state of The etiological role of alcohol per se (in the absence of knowledge, one is still justified in stressing the importance of dietary deficiency) in the pathogenesis of alcoholic liver injury correcting nutrient deficiencies when present, especially in was assessed in experimental studies carried out in the ba view of the general interaction of alcoholism, nutrition, and boon. The dose of alcohol given was comparable to that of cancer to be discussed in a subsequent section. alcoholics, namely, 50% of the total calories, taking advantage Hepatoce!lu!ar Carcinoma in the Alcoholic of the liquid diet technique first developed in the rat and now applied to the baboon (216). With this diet, alcohol intake was Hepatocellular carcinoma in alcoholics is commonly thought sufficient to result in periods of obvious inebriation. Upon to occur in association with cirrhosis of the liver. Indeed, the interruption of alcohol administration, some withdrawal symp incidence of cirrhosis in patients with hepatocellular carcinoma toms (such as seizures) were observed. Twenty-three baboons has been reported to vary between 16 and 80% (47), with most fed the isocaloric control diet retained normal livers, whereas reports indicating a 55 to 80% association. Numerous etiolo the animals given ethanol all developed excessive fat accu gies have been proposed for carcinoma in cirrhotic patients, mulation. In addition, 5 showed mild inflammation, 9 developed especially in conjunction with the regeneration associated with fibrosis, and in 6 baboons studied for 2 to 5 years cirrhosis cirrhosis. Of special interest concerning the possible role of evolved (Fig. 1). alcohol in carcinogenesis is the fact that hepatocellular carci Not all baboons fed ethanol progressed to cirrhosis; similarly, noma can also occur in noncirrhotic alcoholics. The occurrence not all alcoholics develop this complication: the incidence of primary hepatocellular carcinoma is world-wide (6, 47, 64, varies depending on duration and dose of alcohol intake (204). 85, 95, 136, 144, 174, 187, 190, 198, 239, 268, 269, 280â€” Regardless of possible predisposing factors, the baboon study 282, 290, 295, 307, 345, 350, 364, 365), but reports specif clarifies the respective role of malnutrition and alcohol itself in ically characterizing hepatocellular carcinoma in noncirrhotic the pathogenesis of cirrhosis. Thus, one may conclude from patients in Western populations are scarce. Because little our studies that, although earlier evidence suggested that information is available for this noncirrhotic group of patients, malnutrition can cause liver damage, alcohol itself can result in we have retrospectively undertaken a review of autopsy cases the development of the typical complications observed in the seen at the Bronx Veterans Administration Medical Center alcoholic. An important corollary of this finding is the fact that since 1947. adequate diet did not prevent the development of the alcoholic Personal Series of Hepatocellular Carcinoma at the Bronx lesions. The therapeutic implication of this observation is that Veterans Administration Medical Center. All autopsy files alcoholics cannot fully prevent the development or the aggra were reviewed, and clinical records were obtained for all cases vation of liver injury by maintaining an adequate diet unless of hepatocellular carcinoma in patients who did not manifest they also control the degree of alcohol intake. It has been hepatic cirrhosis in the nontumorous liver. shown in the past by others and our own group that alcohol Since 1947, there have been 48 cases of hepatocellular ingestion results in impaired digestion and malabsorption and carcinoma, and of these 15 (31 %) have been in patients without that it produces intestinal injury (20). It is unlikely, however, cirrhosis in the nontumorous liver. The age range of this group that the effects described are of sufficient magnitude to offset of noncirrhotic patients was 22 to 79 years; the average age the large excess of nutrients present in our baboon diet. was 53.4years. Moreover, under our experimental conditions in the baboon, Alcoholism was diagnosed in 7 patients, but quantitation of there was absence of protein and fat malabsorption.3 Further intake and duration were seldom recorded. Three patients were more, as discussed before, plasma amino acid changes ob considered â€˜â€˜social'â€˜drinkers.Of the remaining 5 patients, 4 served in these baboons were the opposite of those produced patients had no statement recorded concerning alcohol con by protein malnutrition (enhanced as opposed to depressed sumption, and one patient is reported to have consumed no branched-chain amino acids) (341 ). The possibility that nutri alcohol. tional deficiencies may potentiate the effect of alcohol is pres No subject had a prior history of hepatitis or jaundice, ently being investigated in the baboon, since such a phenom although 3 patients had undergone previous surgical proce enon was observed for the fatty liver in the rat (226). Experi dures. The presence of Australia antigen was not recorded in mentally, drastic dietary alterations (such as cholesterol over any patient. No patient had a history of parasitic infestation. load) may favor the development of cirrhosis (291 ). Whether Results of stool examinations for parasites were negative in the this applies to clinical conditions and particularly the develop one patient tested. Four patients had 5 associated neoplasms (2 with colonic lesions, 1 with an oropharyngeal lesion, and 1

3 J. Lindenbaum and C. S. Lieber, unpublished observations. with an esophageal lesion and a meningioma). Only one patient,

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. C. S. Lieber et a!. a painter, had a known chemical exposure. was positive; one was negative. Two patients had been diagnosed at outside hospitals prior Two patients underwent chemotherapy. One patient, who to Bronx Veterans Administration Medical Center admission. received Gytoxan, succumbed 3 months after diagnosis. The Of the remaining 13 patients, the distribution of symptoms is other, who received methotrexate and vinblastine, succumbed shown in Table 1. Physical findings in these 13 patients are 8 months after diagnosis. Seven patients in whom the diagnosis listed in Table 2. The 2 patients who had been diagnosed was made at the time of admission survived an average of 2.5 previously at other hospitals were clinically severely ill at the months. One patient, misdiagnosed as having metastatic ad time of admission. enocarcinoma, survived 4 months. In 5 patients, the diagnosis In 5 patients, the diagnosis was completely unsuspected at of hepatocellular carcinoma had not been anticipated before the time of admission. Admission diagnoses included one each autopsy. Average survival of these 5 patients was 7 months. of inguinal hernia, psychiatric disease, oropharyngeal carci Characteristics of Hepatocellular Carcinoma in the Ab noma, metastatic colon carcinoma, and congestive heart fail sence of Cirrhosis. Althoughthe majorityof our patientshad ure. a heavy alcohol intake, quantitation was not possible from the Laboratory parameters for this group of patients are shown hospital records. Only a few patients had been overseas, and in Table 3. Of note is the mild anemia as well as elevation of there were no histories of a hepatitis-like syndrome or jaundice. serum glutamic-oxaloacetic transaminase and alkaline phos The role of viral infection alone or in conjunction with alcohol phatase. Only one patient had documented hypercalcemia. a- remains to be elucidated as a causative factor in this disease. Fetoprotein determinations were performed in 2 patients: one Presenting symptoms were suggestive of malignant disease, although these complaints may also occur in any patient with a heavy alcohol intake. The pain was predominantly in the right Table1 upper quadrant or epigastric, without any particular distinguish Presentingcarcinomawithout symptoms in 13 patients with hepatocellular cirrhosisatCenterSymptoms theBronx VAMedical ing characteristics, as has been noted previously (282). Ap patientsAbdominal No. of proximately 40% of patients had no symptoms directly related to carcinoma. 5Epigastricpain (2)Right Physical findings are not specific for hepatocellular carci (2)Retrosternalupper quadrant noma, since they may be found in any chronic debilitating (1)Wt loss4Anorexia disease. Hepatomegaly, however, was frequently found in 4Mass these patients, and a nodular liver was palpable in 31% of 2Right these patients at the time of admission. (1)Epigastricupper quadrant (1)Constipation Laboratory parameters were of no particular assistance in 2Low pointing toward the correct diagnosis. However, hypercalcemia 1Lethargyback pain in the presence of hepatomegaly and cachexia should suggest 1Leg 1Hematemesisedema a malignant process. 1Rectal The survival interval from time of diagnosis was remarkably 1Behavioralbleeding 1Nonreduciblechange brief, averaging 4.5 months overall. Chemotherapy did not hernia 1 enhance survival in 2 patients. Although cause of death was difficult to ascertain, the usual terminal event was abrupt and clinically unexplained. Table 2 Because of these dismal statistics, it is imperative to define Physicalcarcinoma- findings in 13 patients with hepatocellular this lesion earlier. Hopefully, this will be possible through patientsCachexia8Hepatomegaly6NodularSignsNo. of increased clinical awareness and application of recent diag nostic advances (23, 92, 107, 110, 167, 175). Because of the noncirrhotic nature of the uninvolved liver, it is possible that liver4Peripheral edema3Mass3Epigastric many of these patients will be candidates for newer treatment modalities, such as partial hepatectomy (282). (2)Right In any event, our findings, as well as those of others (169, (1)Ascites3Vascularupper quadrant 372), of hepatocellular carcinoma in the alcoholic in the ab (abdominal)2Icterus1Spiderprominence sence of cirrhosis are consistent with the possible role of alcohol as cocarcinogen. Further studies, however, are needed angiomata1 to determine whether there is a truly statistically significant increase and incidence of such liver tumors in the alcoholic in the absence of cirrhosis. Indeed, previous reports of enhanced Table 3 incidence of hepatic cancer in the alcoholic (200, 290) did not Laboratory testscarcinomaTestAv. in 13 patients with hepatocellular differentiate those with and without associated cirrhosis, which, valueTotal as discussed, may contribute to the development of cancer mlAlbumin3.8protein7.0 gIbe independently of alcohol. mlTotal g/100 mlSerumbilirubin1 .0 mg/ 100 Alcoholism, Nutrition, and Cancer mlinaseAlkalineglutamic-oxaloacetic acid transam 160 IU/100 General Interaction of Alcohol, Nutrition, and Cancer mlHematocrit33%phosphatase225 IU/100 In the foregoing sections, we have discussed how alcohol

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. Alcohol-related Diseases and Carcinogenesis abuse, independent of malnutrition, can affect the liver, alimen fourths of oropharyngeal cancer patients had subnormal vi tary tract, and other tissues and possibly influence carcinogen tamin A levels. Retinoid deficiency enhances the binding of BP esis. However, in addition to these direct effects of ethanol, metabolites to tracheal epithelial DNA. On the other hand, the carcinogenesis may also be influenced by some indirect con absence of vitamin A in the media of cultured rat colon resulted sequences of alcoholism resulting from the malnutrition corn in a lower binding of BP to DNA and protein and in a lower monly associated with alcohol abuse. Indeed, deficient intake activity of BP hydroxylase (1 1). This could be explained by the of thiamine, folic acid, magnesium, and iron are often present fact that vitamin A deficiency decreases mixed-function oxi in alcoholics and deficiency of pyridoxine, pantothenic acid, dase activity at least in the liver (27). However, i.r. administra riboflavin, and zinc may also occur (137). Alcohol also has tion of N-methyl-N'-nitro-N-nitrosoguanidine, a primary carcin been shown to affect the metabolism of various essential nutri ogen to vitamin A-deficient rats, also produced less tumors ents as well as the absorption, distribution, metabolism, acti than in vitamin A-supplemented rats (273). Thus, the effect of vation, storage, and excretion of many dietary components vitamin A on chemical carcinogenesis may depend on the type (342). Each of these deficiencies and their relation to cancer of carcinogen chosen and on the tissue studied. will be discussed individually. In general, it is well known that Vitamin B1.The alcoholicis commonlydeficientin thiamine, diet modifies tumor formation in laboratory animals (391 ). Ger but thiamine deficiency has not been implicated in carcinogen tam dietary habits also affect carcinogenesis in humans (402â€” esis and is not known to affect immunocompetency. 404). Development in the field of azo dye carcinogenesis drew Vitamin B2. Riboflavindeficiency is common among alco attention many years ago to the fact that diet can modify the holics especially in the lower socioeconomic groups. Both effectiveness of chemical carcinogenesis. Rats fed a rice diet riboflavin and iron play important roles in the activity of respi low in protein and riboflavin were highly sensitive to liver tumor ratory enzymes, and deficiencies of both produce similar din formation when treated with 4-dimethylaminoazobenzene, but ical features (glossitis, cheilosis, brittle nails). A Plummer-Vin a diet containing adequate amounts of protein and vitamin B2 son-like syndrome was observed among female patients with reduced and in some cases prevented the carcinogenicity. oral cancer (410). Wynder and Chan (409) found a significant These observations may have been related to alterations in the hyperplasia on the skin of riboflavin-deficient mice, and the level of azo dye reductase, which is involved in the degradation importance of riboflavin has been demonstrated by the fact of the carcinogen (263). Furthermore, the carcinogenicity to that the skin of riboflavin-deficient mice had increased suscep the liver of the mycotoxin aflatoxin B1was increased when rats tibility to tumor formation. The effect of riboflavin deficiency on were maintained on a low lipotrope diet (315). On the other the carcinogenicity of azo dye compounds has been mentioned hand, DMN was not carcinogenic when given with a protein before. free diet, presumably because of the severe reduction of mi Vitamin B6. Pyridoxine deficiency occurs in the alcoholic, crosomal activating enzymes in the liver (255). However, under and acetaldehyde has been incriminated in the accelerated these conditions, rats exhibited tumors in the kidney after a destruction of the vitamin (236). In addition to its key function fairly long latent period. in hematopoiesis, vitamin B6has been shown to play an impor Whatever its role in promoting cancer, malnutrition is very tant role in the production of antibody response to the admin common in cancer patients. The relationship of malnutrition in istration of various antigens (14); this may influence tumor the pathogenesis of oral cancer in Swedish women is well development indirectly. Wynder (403) has related pyridoxine established (410). It has been shown (180) that head and neck deficiency to enhanced liver tumor formation. cancer patients had significantly poorer nutrition than do con Vitamin E. Abnormally low blood levels of vitamin E in trols. Studies in Iran (183), in Puerto Rico (244), and in Sweden alcoholics have been found by Losowsky and Leonard (234) (155) showed a correlation of malnutrition with the development and Myerson (272). Vitamin E and other antioxidants such as of esophageal cancer in humans. Deficiencies reported were butylated hydroxytoluene, propyl gallate, and ethoxyquin have mainly those of iron,zinc, vitamin A, and vitamin B. In a clinical reduced the tumor induction with certain carcinogens in a study, Kissin and Kaley (180) determined blood levels of thia number of target organs (128, 374, 388). In some cases, the mine, riboflavin, and ascorbic acid in patients with head and effect was noted at low levels of those agents, and it can be neck cancer and found all levels to be significantly decreased. assumed that this effect depended indeed upon antioxidant Specific Nutritional Deficiencies in the Alcoholic and Their properties of the compound. Impact on Oncogenesis Vitamin C. Alcoholicshave a decreased urinaryexcretionof ascorbic acid, which may reflect a general deficiency (205). Vitamin A. Low serum carotene (148) and vitaminA (349) Folic Acid. It is well established that, in addition to a de levels have been reported in alcoholics, and hepatic synthesis creased dietary intake of folate, ethanol itself enhances re of retinol-binding protein also was found to be impaired (349). quirements for folic acid (228). Indeed, chronic ethanol admin Acute ethanol administration results also in a lowered vitamin istration prevents the normal hematological response to folic A (2). Vitamin A has been implicated in the differentiation of acid (356). Folic acid deficiency has been shown to interfere epithelial tissue, and it has been shown that vitamin A deficien with humoral and cellular immunity(201 ). Furthermore, the cies affect the induction of pulmonary tumors (55, 73, 328), adverse effect of folic acid deficiency on the hemopoietic bladder tumors (53), colon tumors (274, 314), and cervical system as well as on the gastrointestinal tract is well known. tumors (52). Moreover, a recent epidemiological study in over Whether those morphological changes associated with folic 8000 men in Norway suggests that relatively low dietary intake acid deficiency alter the effect of chemicals or other carcino of vitamin A is correlated with a relatively high incidence of genic agents is not known. lung cancer (34). A study in India (384) showed that three Iron. Alcoholics also may have severe long-standingiron

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1979 American Association for Cancer Research. C. S. Lieber et a!. deficiency, and the role of iron in carcinogenesis may be ularly in later stages). The relationship between alcohol-in significant. Iron deficiency without anemia produces profound duced necrosis in various tissues and carcinogenesis remains defects in cell-mediated immunity (382). Chronic prolonged to be further documented experimentally, particularly the pos and severe iron deficiency produces gastric atrophia in rats tulated link between regeneration associated with cirrhosis and (381 ). Recent studies from Colombia and from the National hepatocellular carcinoma. The status of the immune system is Cancer Institute Epidemiological Section indicate that chronic important in carcinogenesis and appears to be also affected iron deficiency may have a possible role in the etiology of by alcohol, but the available information is still sketchy. gastric cancer (383). In Colombia, there is a gastric cancer In any event, the recent description of the induction by rate 4 times higher than in the United States. Furthermore, alcohol consumption of microsomal systems which activate chronic iron deficiency and/or chronic riboflavin deficiency secondary carcinogens in various tissues raises the possibility have an effect on the development of Plummer-Vinson syn that this cocarcinogenic action of alcohol contributes to the drome (404). Plummer-Vinson syndrome is known to increase enhanced incidence of cancer in the alcoholic. the risk of cancer of the upper alimentary tract among women (who are more prone to the syndrome than are men) (199, 410). Acknowledgments Minerals. Magnesium deficiency is common in alcoholic patients, probably because of its increased excretion in urine The authors thank N. Holloway for preparation of the manuscript. (105, 162, 355, 397). Magnesium-free diets have been re ported to lead to leukemia in certain strains of rats (9). Also, References zinc deficiency has been reported in alcoholics (106) and has 1. Alcohol and Health. Second Special Report to the U.S. Congress, National been associated with a high incidence of esophageal carci Institute of Mental Health, National Institute on Alcohol Abuse and Alco noma in Iran (183). Zinc serves as cofactor for a number of holism, Department of Health. Education, and Welfare Publication No. (ADM) 75-21 2. Washington,0. C.: United States GovernmentPrinting enzymes. Zinc is needed for RNA metabolism and DNA synthe Office, 1975. sis. It is of interest that zinc has a protective effect on the 2. Althausen, T. L., Uyeyama, K., and Loran, M. R. Effects of alcohol on teratogenicity of cadmium (28). On the other hand, the absorp absorption of vitamin A in normal and gastrectomized subjects. Gastroen terology, 38: 942-945, 1960. tion of cadmium is significantly increased at a low serum iron 3. Ames, B. N., McCann, J.. and Yamasaki, E. Methods for detecting carcin level, which is common in alcoholics (104). Cadmium serum ogens and mutagens with the Salmonella/mammalian-microsomes muta levels were found to be elevated in patients with bronchial genicity test. Mutat. Res., 31: 347-364, 1975. 4. Anderson, D. L. lntraoral site distribution of malignancy and preinvasive carcinoma (146). The deficiency of another trace element, malignant cell transformation in dental patients and alcoholism. Acta Cytol., molybdenum, has been associated with an increased incidence 16: 322â€”326, 1972. 5. Anonymous. Heavy drinking, smoking linked with oral cancer. J. Am. Med. of esophageal cancer in certain parts of the world (373), but Assoc., 236: 435, 1976. its status in the alcoholic is unknown. Rogers and Gawienowsky 6. Anthony, P. P. Primary carcinoma of the liver. Ann. A. CoIl. Surg. Engi. 58: (31 3) reported that serum copper was elevated in alcoholics. 285-292, 1976. 7. Arakawa, M., Taketomi, S., Furuno, K., Matsuo, T., Iwatsuka, H., and There is a high incidence of lung cancer among coppersmiths Suzuoki, Z. Metabolic studies on the development of ethanol-induced fatty (28); however, the lack of a significant correlation in experi liver in [email protected]. Nutr., 105: 1500â€”1508,1975. mental animals has led to the conclusion that copper cannot 8. Arlyoshi, T., Takabatake, E.. and Remmer, H. Drug metabolism in ethanol induced fatty liver. Life Sci.. 9 (Part 2): 361 -369, 1970. be classified as a carcinogen. 9. Artizzu, M., and Messier, B. Histological changes in rat liver during chronic magnesium deficiency. Rev. Can. Biol., 31: 31â€”37,1972. Summary and Recommendations 10. Ashworth, C. T., Wrightsman, F.. and Buttram, V. Hepatic lipids. Compar ative study of effects of high-fat, choline-deficient, and high-cholesterol diets upon serum and hepatic lipids. Arch. Pathol., 72: 620â€”624,1961. In this paper, some medical complications of alcoholism were 11. Autrup, H., Harris, C. C., Fugaro, S., and Selkirk, J. K. Effect of various described, particularly their interrelationships with carcinogen chemicals on the metabolism of benzo(a)pyrene by cultured rat colon. esis. Emphasis was placed on mechanisms whereby alcohol Chem.-Biol. Interact., 18: 337-347, 1977. 12. Autrup, H., Harris, C. C., Stoner, G. D., Jesudason, M. L., and Trump, B. may contribute to carcinogenesis through (a) contact related F. Binding of chemical carcinogens to macromolecules in cultured human local effects, (b) the induction of microsomal enzymes which colon. J. NatI. Cancer Inst., 59: 351 â€”354,1977. 13. Avogaro, P., and Cazzolato, G. Changes in the composition and physico activate procarcinogens, (c) general mechanisms of tissue chemical characteristics of serum lipoproteins during ethanol-induced Ii injury and regeneration (particularly in the liver), or (d) asso paemia in alcoholic subjects. Metab. Cm. Exp., 24: 1231â€”1242,1975. ciated nutritional disturbances. Because of limitation of space 14. Axelrod, A. E., and Trakatellis, A. C. Relationship of pyridoxine to immu nologic phenomenon. Vitam. Horm., 22: 591-607, 1964. and/or lack of information, a number of significant questions 15. Ballard, H., Bernstein, M., and Farrar, J. T. Fatty liver presenting as were neglected. For instance, the potential impact of the al obstructive jaundice. Am. J. Med., 30: 196â€”201,1961. cohol-induced increase in microsomal metabolism and/or the 16. Barak, A. J., Tuma, D. J., and Sorrell, M. F. Relationship of ethanol to choline metabolism in the liver. Am. J. Clin. Nutr., 26: 1234-1241, 1973. effect of alcohol-related liver injury on chemotherapy and its 17. Baraona, E., Leo, M. A., Borowsky, S. A.. and Lieber, C. S. Alcoholic complications have not been considered. There is an obvious hepatomegaly: accumulation of protein in the liver. Science, 190: 794â€” need for additional information in this important area. In addi 795, 1975. 18. Baraona, E., Leo, M. A., Borowsky, S. A., and Lieber, C. S. Pathogenesis tion, more information is required concerning the possible of alcohol-induced accumulation of protein in the liver. J. Clin. Nutr., 60: impact of alcohol-induced disease status on carcinogen me 546-554, 1977. 19. Baraona, E., and Lieber, C. S. Effects of chronic ethanol feeding on serum tabolism. Several important opposing factors may interact: lipoprotein metabolism in the rat. J. Clin. Invest., 49: 769â€”778,1970. increased activation of secondary carcinogens and deactiva 20. Baraona, E., and Lindenbaum, J. 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Fig. 1. Cirrhosis in a baboon fed alcohol for 4 years. Fat is regularly distributed through nodules surrounded by connective tissue septa. Chromotrope-aniline blue, x 60 (216).

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Charles S. Lieber, Helmut K. Seitz, Anthony J. Garro, et al.

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