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Alcohol — A major risk factor for hepatocellular carcinoma?
Journal of Hepatology, 1986; 2:513-519
513
Elsevier HEP 0123
Review
Alcohol A Major Risk Factor for Hepatocellular Carcinoma?
M . F . Bassendine Department of Medicine, The Universityof Newcastleupon Tyne, Newcastleupon Tyne, (U. K.)
Introduction
Primary hepatocellular carcinoma (PHC) is a relatively rare tumour in Northern Europe and North America but is common in many densely populated areas of Africa and Asia. The age-standardised incidence rates for men vary from < 4 per 100 000 in the U.K., Canada and Australia to > 100 per 100 000 in Mozambique and Taiwan [1]. This striking geographical variation in the incidence of PHC suggests that environmental factors might be important in its aetiology. The variation in the clinicopathological features between high and low incidence areas supports this concept. In temperate areas PHC usually occurs in patients over 60 years old and an association with alcoholic cirrhosis has been found, mainly from retrospective autopsy studies [2]. In contrast, in Africa and Asia, PHC is a cancer of younger males and the underlying cirrhosis is of the post-necrotic macronodular type. In these areas the majority of cases are associated with hepatitis B virus (HBV) infection [3]. Many of the early studies which established the sequence alcohol abuse ~ alcoholic cirrhosis ~ PHC are now open to question as data were obtained prior to the discovery of HBV or when methods available for its detection were relatively insensitive. This article aims to re-examine the possible role of alcohol in the development of PHC in the light of more recent literature. Hepatocarcinogenesis is a multistep process [4], development of the tumour being classically viewed in terms of initiation and promotion events. In examining the relationship between alcohol and PHC the possible role of alcohol as a carcinogen per se and as a co-carcinogen or promoting agent must be considered. Alcohol as a Hepatocarcinogen
Alcohol consumption has been shown to be a risk factor for the development of 0168-8278/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
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M.F. BASSENDINE
PHC in low-incidence areas. In a recent epidemiological study from Sweden a 4fold increase in risk of PHC was found in alcoholics (> 1 bottle of spirits/week) relative to teetotallers. No association between the use of wine and PHC could however be demonstrated and the role of HBV could not be evaluated due to lack of sufficient data [5]. Another case control study from Los Angeles reported similar findings: individuals who drank 80 g or more of ethanol per day had a relative risk (RR) of PHC of 4.2 compared to those drinking less than 10 g/day. Alcohol consumption was not as significant a risk factor for PHC as a history of hepatitis (RR = 13) or a history of blood transfusions (RR = 7) [6]. An earlier study from Ontario compared the death rate from cancer and other causes in a cohort population of 9 889 male alcoholics with those of the general male population and found an observed-to-expected PHC mortality ratio of 2.03. This was less than half the observed-to-expected mortality ratio found in alcoholics for cancer of the mouth, pharynx and larynx [7]. Similar studies corroborate this data and show that the association between chronic alcohol intake and PHC is less strong than between alcoholism and cancer of the mouth, larynx and oesophagus [8]. Alcohol consumption is also a much less significant risk factor for PHC than HBV, in both low and high PHC incidence areas [9,10] (Table 1). There is thus little evidence that alcohol is a hepatocarcinogen per se, a view supported by the paucity of convincing experimental evidence that alcohol is mutagenic [8]. Alcohol as a C o - c a r c i n o g e n
Other risk factors for the development of PHC are generally recognised as being persistent HBV infection [3], exposure'to dietary mycotoxins and other chemicals [11], underlying cirrhosis and male sex [12]. The interrelationship of these factors with each other and with alcohol abuse is complex and difficult to disentangle but TABLE 1 RELATIVE RISK OF DEVELOPING PHC IN RELATION TO AETIOLOGIC FACTORS IN LOW AND HIGH PHC INCIDENCE AREAS Country
Aetiologic factor
Sweden
Alcohol (> 1 bottle spirits/week)
4.2
Hardell et al. [5]
U.S.A.
Alcohol (> 80 g/day) Alcohol + smoking (> 80 g/day + 20 cigs/day)
4.2
Yu et al. [6]
7.8
Yu et al. [6]
Philippines
Relative risk
Reference
Alcohol + aflatoxin Heavy alcohol/light aflatoxin Light alcohol/heavy aflatoxin Heavy alcohol/heavy aflatoxin
3.9 17.5 35.0
Bulatao-Jayme et al. [22] Bulatao-Jayme et al. [22] Bulatao-Jayme et al. [22]
United Kingdom
HBV (HBsAg-positive)
42 (67) ~
Hall et al. [10]
Taiwan
HBV (HBsAg-positive)
223 (104) a
a Updated relative risk 1985.
Beasley et al. [9]
A L C O H O L - - M A J O R RISK F A C T O R F O R H E P A T O C E L L U L A R C A R C I N O M A ?
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recent literature suggests synergism of risk factors operating through indirect mechanisms.
(i) Alcohol and hepatitis B virus In Europe hepatitis B virus infection appears to be a common event in the course of chronic alcoholism [13,14]. Conversely a study from Italy has shown that chronic symptomless carriers of H B V have an increased susceptibility to alcohol-induced liver damage [15]. In Japan the effect of habitual alcohol intake on the latency period for the development of PHC in chronic HBV carriers has been studied, and results suggest that alcoholism hastens the development of PHC in HBsAg carriers [16]. On the other hand in France application of the sensitive research methods of recombinant D N A technology has suggested that the development of PHC in 'alcoholic cirrhosis' is nearly always associated with HBV, as shown by the presence of integrated H B V D N A in malignantly transformed hepatocytes [17]. In this study 56% of patients had serological markers of H B V infection, raising the question whether alcohol or HBV was the main factor in the development of their cirrhosis. Certainly, estimation of the frequency of 'alcoholic cirrhosis' may depend on whether historical or morphological criteria are used. This is exemplified by studies in London where alcoholic cirrhosis was diagnosed in 65% of cases on historical criteria [18] but 29% of cases on morphological criteria [19]. The incidence of 'cryptogenic cirrhosis' on the other hand was found to be 8% on historical versus 36% on morphological grounds. Other British studies have shown an increased frequency of serological markers of H B V infection in cryptogenic cirrhosis and PHC suggesting that HBV is an aetiological agent which may give rise to both conditions in the same population [20]. In a prospective study of unselected hepatic cirrhosis carried out in West Germany, the highest incidence of PHC was observed among patients with H B V infection and coincidental excess alcohol consumption [21]. In view of the vast difference in scale of the relative risk of PHC in relation to alcohol consumption and HBV (see Table 1) it seems reasonable to conclude that alcohol may have a co-carcinogenic role with HBV, rather than vice versa. This could be accounted for simply by alcohol consumption being a cause of cirrhosis (see below). (ii) Alcohol and chemical carcinogens It has long been suggested that alcohol may cooperate with sub-threshold doses of chemical carcinogens to cause PHC. A recent case study from the Philippines supports this concept in relation to exposure to mycotoxins. Individuals consuming more than both 24 g of alcohol and an estimated > 4/~g of Aflatoxin B~ in contaminated food had a 35-fold increased relative risk of PHC whereas the risk was halved by similar mycotoxin exposure but little alcohol consumption (see Table 1). The effect of alcohol intake and dietary mycotoxin were thus synergistic [22]. The importance of other chemical carcinogens in hepatocarcinogenesis in man is less well documented [11]. One hypothesis that has been advanced to explain the enhanced risk of cancer in the alcoholic is that ethanol induces enzyme systems which activate procarcinogens to carcinogens [23]. Many environmental carcinogens may be activated to highly reactive intermediates by hepatic cytochrome p450-dependent mi-
516
M.F. BASSENDINE
crosomal mono-oxygenases. Recent studies however have shown there is broad impairment, rather than induction, of hepatic microsomal mono-oxygenase activity in alcoholic cirrhosis [24,25]. The picture is complicated by the fact that some substrates for human liver microsomes appear to involve a specific cytochrome p450 group of isoenzymes which is highly inducible by smoking [26]. This could explain the finding that excessive use of tobacco is an additional risk factor for PHC in alcoholics [6]. There is some evidence on the other hand that inactivation of reactive intermediates may be impaired in alcoholic cirrhosis. The concentration of a natural antioxidant, reduced glutathione, has been shown to be significantly lower in alcoholic cirrhosis, compared to normals [24]. In addition this antioxidant may play a more complex role than detoxification in hepatocarcinogenesis. It has been shown experimentally that administration of reduced glutathione to rats bearing aflatoxin Bl-induced liver tumours improves survival and may cause regression of tumour growth [27]. These studies serve to illustrate the interplay of factors that may alter the metabolic balance between activation and deactivation of procarcinogens and other xenobiotics and contribute to hepatocarcinogenesis. (iii) Cirrhosis - - A premalignant lesion?
The association between alcohol abuse and cirrhosis has long been recognised. In almost all European countries there is a strong correlation between national alcohol consumption and mortality rates from cirrhosis of the liver [28]. This has recently been evaluated prospectively when the risk of long-term alcohol abusers acquiring cirrhosis was shown to be about 15% [29]. The great majority of PHC arises in cirrhotic livers and the fact that alcohol is a cause of cirrhosis could account for the iiacreased risk of PHC in alcoholics. One hypothesis is that cirrhosis per se is a premalignant lesion - - i.e. that hyperplasia leads to neoplasia in the absence of additional factors such as exposure to chemical or viral carcinogens [12]. If this mechanism were universally operative a correlation in the geographic distribution of cirrhosis and PHC would be anticipated as has been documented for HBV and PHC [3]. This does not apply for alcoholic cirrhosis which is common in Northern Europe where the incidence of PHC is low. Moreover the risk of PHC is not similar for all types of cirrhosis; it is generally agreed that macronodular cirrhosis has the greatest malignant potential and some recent clinicopathological studies show that PHC is significantly more frequent in HBsAg-positive than in alcoholic cirrhosis [30]. In addition numerous studies from Europe and North America in recent years have suggested an increase in PHC in the western world and in some of these this appears to be independent of cirrhosis [31]. This would suggest that the relationship between cirrhosis and PHC is more complex than cirrhosis acting as a simple premalignant lesion. An alternative hypothesis has been advanced that cirrhosis tends to act as a powerful promoting agent in the presence of a primary carcinogenic factor or factors, whether viral or chemical [31]. Thus the raised rate of DNA synthesis that inevitably accompanies the formation of hyperplastic nodules in cirrhosis could act as a promotor for cells already initiated by some (unknown) stimulus. The association of HBV with PHC in 'alcoholic cirrhosis' is certainly compatible with this hypothesis, and it is supported by some ex-
A L C O H O L - - M A J O R RISK F A C T O R F O R H E P A T O C E L L U L A R C A R C I N O M A ?
517
perimental data. One study examined the role of cell proliferation in the initiation of liver carcinogenesis induced by chemicals in rats. It was found that loci of preneoplastic hepatocytes only appeared in rats that received the carcinogen coupled with the cell proliferation stimulus of partial hepatectomy, whereas no loci were seen in rats that received the same dose of carcinogen and a sham operation. It is suggested that replication of D N A with carcinogen-induced lesions prior to repair offers a mechanism whereby a mutation may be transcribed and fixed in a daughter'initiated' cell [32]. There is hope that a greater understanding of the molecular mechanisms involved in the link between cell proliferation and malignant transformation may emerge in the next decade following the discovery of cellular protooncogenes (c-onc). Increased expression of c-onc has been reported in both liver regeneration and hepatocarcinogenesis in rats [33,34]. The role of these genes in the sequential events leading to tumour formation is as yet unclear. It could be that repetitive mitosis + viral or chemical carcinogens could induce mutations in specific genes, leading to increased and/or inappropriate expression of c-onc and malignant transformation. (iv) Alcohol and male sex
The male predominance of PHC, at least in the West, is almost completely accounted for by the association with cirrhosis [12]. This could merely reflect the fact that alcohol consumption is greater among men than women, but again evidence suggests that the explanation for the strong association of PHC with male sex is more complex. One possibility is that the hyperestrogenaemia commonly seen in male alcoholic cirrhosis may play some role in hepatocarcinogenesis. A recent study has shown elevated estrone levels in cirrhosis and elevated estrone to testosterone ratios in PHC arising in cirrhosis [35]. There is experimental evidence that estrogens promote neoplasia in rats [30] and that internalization of the estrogen receptor correlates with increased D N A synthesis in liver regeneration [37]. Another possibility is that enzyme systems involved in activating and deactivating carcinogens are influenced by the hormonal milieu. For example, higher levels of the reactive intermediate derived from aflatoxin are found in male rats than female rats because of rapid inactivation in females [38]. Conclusions
In conclusion there is an association between alcohol and hepatocellular carcinoma but evidence suggests it is an indirect one. Further studies are indicated on the effect of alcohol, sex and smoking, on the handling of environmental carcinogens by the liver and on the factors regulating liver cell proliferation and their role in hepatocarcinogenesis.
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M.F. BASSENDINE
References 1 Cook-Mozafferi, P. and Van Rensburg, S., Cancer of the liver, Brit. Med. Bull., 1984;40: 342-345. 2 Lee, F.I., Cirrhosis and hepatoma in alcoholics, Gut, 1966; 7: 77-85. 3 Szmuness, W., Hepatocellular carcinoma and the hepatitis B virus: Evidence for a causal association, Progr. Med. Virol., 1978;24: 40-69. 4 Goldfarb, S., Biologic mechanisms of hepatocarcinogenesis, Sem. Liver Dis., 1984; 4: 89-97. 5 Hardell, L., Bengtsson, N.O., Jonsson, U., Eriksson, S. and Larsson, L.G., Aetiological aspects on primary liver cancer with special regard to alcohol, organic solvents and acute intermittent porphyria - - A n epidemiological investigation, Brit. J. Cancer, 1984; 50: 389-397. 6 Yu, M.C., Mack, T., Hanisch, R., Peters, R.L., Henderson, B.E. and Pike, M.C., Hepatitis, alcohol consumption, cigarette smoking and hepatocellular carcinoma in Los Angeles, Cancer Res., 1983; 43: 6077-6079. 7 Schmidt, W. and Popham, R.E., The role of drinking and smoking in mortality from cancer and other causes in male alcoholics, Cancer, 1981; 47: 1031-1041. 8 MacSween, R.N.M., Alcohol and cancer, Brit. Med. Bull., 1982; 38: 31-33. 9 Beasley, R.P., Hwang, L.Y., Lin, C.C. and Chien, C.S., Hepatocellular carcinoma and hepatitis B virus: A prospective study of 22,707 men in Taiwan, Lancet, 1981; ii: 1129-1133. 10 Hall, A.J., Winter, P.D. and Wright, R., Mortality of hepatitis B positive blood donors in England and Wales, Lancet, 1985; i: 91-93. 11 Newberne, P.M., Chemical carcinogenesis: Mycotoxins and other chemicals to which humans are exposed, Sem. Liver Dis., 1984; 4: 122-135. 12 Zaman, S.N., Melia, W.M., Johnson, R.D., Portmann, B.C., Johnson, P.J. and Williams, R., Risk factors in development of hepatoeellular carcinoma in cirrhosis: prospective study of 613 patients, Lancet, 1985; i: 1357-1360. 13 Goudeau, A., Maupas, P., Dubois, F., Coursaget, P. and Bougnoux, P., Hepatitis B infection in alcoholic liver disease and primary hepatocellular carcinoma in France, Progr. Med. Virol., 1981; 27: 26-34. 14 Gluud, C., Aldershville, T., Henriksen, J., K~ger, P. and Mathieson, L., Hepatitis B and A virus antibodies in alcoholic steatosis and cirrhosis, J. Clin. Path., 1982; 35: 693-697. 15 Villa, E., Barchi, T., Grisendi, A., Bellentani, S., Rubbiani, L., Ferr&ti, I., De Palma, M. and Manenti, F., Susceptibility of chronic symptomless HBsAg carriers to ethanol induced hepatic damage, Lancet, 1982; ii: 1243-1244. 16 Ohnishi, K., Iida, S., Iwana, S., Gato, N., Nomura, F., Takashi, M., Mishima, A., Kono, K., Kimura, K., Musha, H., Kotota, K. and Okuda, K., The effect of chronic habitual alcohol intake on the development of liver cirrhosis and hepatocellular carcinoma, Cancer, 1982;49: 672-677. 17 Brechot, C., Nalpas, B., Courouce, A.M., Duhamel, G., Callard, P., Carnot, F., Tiollais, P. and Berthelot, P., Evidence that hepatitis B virus has a role in liver cell carcinoma in alcoholic liver disease, N. Engl. J. Med., 1982; 306: 1384-1387. 18 Hodgson, H.J.F. and Thompson, R.P.H., Cirrhosis in South London, Lancet, 1976; ii: 118-121. 19 Blenkinsopp, W.K. and Hoffenden, G.P., Aetiology of cirrhosis, hepatic fibrosis and hepatocellular carcinoma, J. Clin. Path., 1977; 30: 579-584. 20 Bassendine, M.F., Delia Seta, L., Salmeron, J., Thomas, H.C. and Sherlock, S., Incidence of hepatitis B virus infection in alcoholic liver disease, HBsAg negative chronic active liver disease and primary liver cell cancer in Britain, Liver, 1983; 3: 65-70. 21 Lehmann, F.G. and Martini, G.A., Incidence of primary liver cell carcinoma in liver cirrhosis: 5 year prospective study, DigestiOn, 1976; 14: 530-531. 22 Bulatao-Jayme, J., Almero, E.M., Castro, C.A., Jardeleza, T.H. and Salamat, L.A., A case-control dietary study of primary liver cancer risk from aflatoxin exposure, Int. J. Epidemiol., 1982; 11: 112-119. 23 Lieber, C.S., Seitz, H.K., Garro, A.J. and Worner, T.M., Alcohol-related diseases and carcinogenesis, Cancer Res., 1979; 39: 2863-2886. 24 Woodhouse, K.W., Williams, F.M., Mutch, E., Wright, P., James, O.F.W. and Rawlins, M.D., The effect of alcoholic cirrhosis on the activities of microsomal aldrin epoxidase, 7-ethoxycoumarin, O-
A L C O H O L - MAJOR RISK FACTOR FOR HEPATOCELLULAR CARCINOMA?
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de-ethylase and epoxide hydrolase, and on the concentrations of reduced glutathione in human liver, Brit. J. Clin. Pharmac., 1983; 15: 667-672. 25 Woodhouse, K.W., Williams, F.M., Mutch, E., Wright, P., James O.F.W. and Rawlins, M.D., The effect of alcoholic cirrhosis on the two kinetic components (high and low affinity) of the microsomal O-deethylation of 7-ethoxycoumarin in human liver, Europ. J. Clin. Pharmacol., 1984;26: 61-64. 26 Williams, F.M., Mutch, E., Woodhouse, K.W., Lambert, D. and Rawlins, M.D., Ethoxyresurufin O-deethylation by human liver microsomes, Brit. J. Clin. Pharmacol., in press. 27 Novi, A.M., Regression of aflatoxin Bl-induced hepatocellular carcinomas by reduced glutathione, Science, 1981;212: 541-542. 28 Walsh, D., Alcohol-related medicosocial problems and their prevention, Copenhagen, World Health Organization Publications, 1982. 29 Serensen, T.I.A., Orholm, M., Bentsen, K., Hoybye, G., Eghoje, K. and Christoffersen, P., Prospective evaluation of alcohol abuse and alcohol liver injury in men as predictors of development of cirrhosis, Lancet, 1984, ii: 214-244. 30 Bartok, I., Remenar, E., Toth, J., Duschanek, P. and Kanyar, B., Clinicopathological studies of liver cirrhosis and hepatocellular carcinoma in a general hospital, Human Path., 1981; 12: 794-803. 31 Burnett, R.A., Patrick, R.S., Spilg, W.G.S., Buchanan, W.M. and MacSween, R.N.M., Hepatocellular carcinoma and hepatic cirrhosis in the West of Scotland: a 25-year necropsy review, J. Clin. Path., 1978;31: 108-110. 32 Columbano, A., Rajalakshmi, S. and Sarma, D.S.R., Requirement of cell proliferation for the initiation of liver carcinogenesis as assayed by three different procedures, Cancer Res., 1981; 41: 2079-2083. 33 Fausto, N. and Shank, P.R., Oncogene expression in liver regeneration and hepatocarcinogenesis, Hepatol., 1983;3: 1016-1023. 34 Yaswen, P., Goyette, M., Shank, P.R. and Fausto, N., Expression of c-Ki-ras, c-Ha-ras and c-myc in specific cell types during hepatocarcinogenesis, Mol. Cell. Biol., 1985; 5: 780-786. 35 Nagasue, N., Ogawa, Y., Yukaya, H., Ohta, N. and Ito, A., Serum levels of estrogens and testosterone in cirrhotic men with and without hepatocellular carcinoma, Gastroenterol., 1985; 88: 768-772. 36 Wanless, I.R. and Medline, A., Role of estrogens as promoters of hepatic neoplasia, Lab. Invest., 1982; 46: 313-320. 37 Francavilla, A., Di Leo, A., Eagon, P.K., Wu, S.Q., Ove, P., Van Thiel, D.H. and Starzl, T.E., Regenerating rat liver: correlations between estrogen receptor localizations and deoxyribonucleic acid synthesis, Gastroenterol., 1984; 86: 552-557. 38 O'Brien, K., Moss, E., Judah, D. and Neal, G., Metabolic basis of the species difference to aflatoxin induced hepatoxicity, Biochem. Biophys. Res. Comm., 1983; 114: 812-821.
513
Elsevier HEP 0123
Review
Alcohol A Major Risk Factor for Hepatocellular Carcinoma?
M . F . Bassendine Department of Medicine, The Universityof Newcastleupon Tyne, Newcastleupon Tyne, (U. K.)
Introduction
Primary hepatocellular carcinoma (PHC) is a relatively rare tumour in Northern Europe and North America but is common in many densely populated areas of Africa and Asia. The age-standardised incidence rates for men vary from < 4 per 100 000 in the U.K., Canada and Australia to > 100 per 100 000 in Mozambique and Taiwan [1]. This striking geographical variation in the incidence of PHC suggests that environmental factors might be important in its aetiology. The variation in the clinicopathological features between high and low incidence areas supports this concept. In temperate areas PHC usually occurs in patients over 60 years old and an association with alcoholic cirrhosis has been found, mainly from retrospective autopsy studies [2]. In contrast, in Africa and Asia, PHC is a cancer of younger males and the underlying cirrhosis is of the post-necrotic macronodular type. In these areas the majority of cases are associated with hepatitis B virus (HBV) infection [3]. Many of the early studies which established the sequence alcohol abuse ~ alcoholic cirrhosis ~ PHC are now open to question as data were obtained prior to the discovery of HBV or when methods available for its detection were relatively insensitive. This article aims to re-examine the possible role of alcohol in the development of PHC in the light of more recent literature. Hepatocarcinogenesis is a multistep process [4], development of the tumour being classically viewed in terms of initiation and promotion events. In examining the relationship between alcohol and PHC the possible role of alcohol as a carcinogen per se and as a co-carcinogen or promoting agent must be considered. Alcohol as a Hepatocarcinogen
Alcohol consumption has been shown to be a risk factor for the development of 0168-8278/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
514
M.F. BASSENDINE
PHC in low-incidence areas. In a recent epidemiological study from Sweden a 4fold increase in risk of PHC was found in alcoholics (> 1 bottle of spirits/week) relative to teetotallers. No association between the use of wine and PHC could however be demonstrated and the role of HBV could not be evaluated due to lack of sufficient data [5]. Another case control study from Los Angeles reported similar findings: individuals who drank 80 g or more of ethanol per day had a relative risk (RR) of PHC of 4.2 compared to those drinking less than 10 g/day. Alcohol consumption was not as significant a risk factor for PHC as a history of hepatitis (RR = 13) or a history of blood transfusions (RR = 7) [6]. An earlier study from Ontario compared the death rate from cancer and other causes in a cohort population of 9 889 male alcoholics with those of the general male population and found an observed-to-expected PHC mortality ratio of 2.03. This was less than half the observed-to-expected mortality ratio found in alcoholics for cancer of the mouth, pharynx and larynx [7]. Similar studies corroborate this data and show that the association between chronic alcohol intake and PHC is less strong than between alcoholism and cancer of the mouth, larynx and oesophagus [8]. Alcohol consumption is also a much less significant risk factor for PHC than HBV, in both low and high PHC incidence areas [9,10] (Table 1). There is thus little evidence that alcohol is a hepatocarcinogen per se, a view supported by the paucity of convincing experimental evidence that alcohol is mutagenic [8]. Alcohol as a C o - c a r c i n o g e n
Other risk factors for the development of PHC are generally recognised as being persistent HBV infection [3], exposure'to dietary mycotoxins and other chemicals [11], underlying cirrhosis and male sex [12]. The interrelationship of these factors with each other and with alcohol abuse is complex and difficult to disentangle but TABLE 1 RELATIVE RISK OF DEVELOPING PHC IN RELATION TO AETIOLOGIC FACTORS IN LOW AND HIGH PHC INCIDENCE AREAS Country
Aetiologic factor
Sweden
Alcohol (> 1 bottle spirits/week)
4.2
Hardell et al. [5]
U.S.A.
Alcohol (> 80 g/day) Alcohol + smoking (> 80 g/day + 20 cigs/day)
4.2
Yu et al. [6]
7.8
Yu et al. [6]
Philippines
Relative risk
Reference
Alcohol + aflatoxin Heavy alcohol/light aflatoxin Light alcohol/heavy aflatoxin Heavy alcohol/heavy aflatoxin
3.9 17.5 35.0
Bulatao-Jayme et al. [22] Bulatao-Jayme et al. [22] Bulatao-Jayme et al. [22]
United Kingdom
HBV (HBsAg-positive)
42 (67) ~
Hall et al. [10]
Taiwan
HBV (HBsAg-positive)
223 (104) a
a Updated relative risk 1985.
Beasley et al. [9]
A L C O H O L - - M A J O R RISK F A C T O R F O R H E P A T O C E L L U L A R C A R C I N O M A ?
515
recent literature suggests synergism of risk factors operating through indirect mechanisms.
(i) Alcohol and hepatitis B virus In Europe hepatitis B virus infection appears to be a common event in the course of chronic alcoholism [13,14]. Conversely a study from Italy has shown that chronic symptomless carriers of H B V have an increased susceptibility to alcohol-induced liver damage [15]. In Japan the effect of habitual alcohol intake on the latency period for the development of PHC in chronic HBV carriers has been studied, and results suggest that alcoholism hastens the development of PHC in HBsAg carriers [16]. On the other hand in France application of the sensitive research methods of recombinant D N A technology has suggested that the development of PHC in 'alcoholic cirrhosis' is nearly always associated with HBV, as shown by the presence of integrated H B V D N A in malignantly transformed hepatocytes [17]. In this study 56% of patients had serological markers of H B V infection, raising the question whether alcohol or HBV was the main factor in the development of their cirrhosis. Certainly, estimation of the frequency of 'alcoholic cirrhosis' may depend on whether historical or morphological criteria are used. This is exemplified by studies in London where alcoholic cirrhosis was diagnosed in 65% of cases on historical criteria [18] but 29% of cases on morphological criteria [19]. The incidence of 'cryptogenic cirrhosis' on the other hand was found to be 8% on historical versus 36% on morphological grounds. Other British studies have shown an increased frequency of serological markers of H B V infection in cryptogenic cirrhosis and PHC suggesting that HBV is an aetiological agent which may give rise to both conditions in the same population [20]. In a prospective study of unselected hepatic cirrhosis carried out in West Germany, the highest incidence of PHC was observed among patients with H B V infection and coincidental excess alcohol consumption [21]. In view of the vast difference in scale of the relative risk of PHC in relation to alcohol consumption and HBV (see Table 1) it seems reasonable to conclude that alcohol may have a co-carcinogenic role with HBV, rather than vice versa. This could be accounted for simply by alcohol consumption being a cause of cirrhosis (see below). (ii) Alcohol and chemical carcinogens It has long been suggested that alcohol may cooperate with sub-threshold doses of chemical carcinogens to cause PHC. A recent case study from the Philippines supports this concept in relation to exposure to mycotoxins. Individuals consuming more than both 24 g of alcohol and an estimated > 4/~g of Aflatoxin B~ in contaminated food had a 35-fold increased relative risk of PHC whereas the risk was halved by similar mycotoxin exposure but little alcohol consumption (see Table 1). The effect of alcohol intake and dietary mycotoxin were thus synergistic [22]. The importance of other chemical carcinogens in hepatocarcinogenesis in man is less well documented [11]. One hypothesis that has been advanced to explain the enhanced risk of cancer in the alcoholic is that ethanol induces enzyme systems which activate procarcinogens to carcinogens [23]. Many environmental carcinogens may be activated to highly reactive intermediates by hepatic cytochrome p450-dependent mi-
516
M.F. BASSENDINE
crosomal mono-oxygenases. Recent studies however have shown there is broad impairment, rather than induction, of hepatic microsomal mono-oxygenase activity in alcoholic cirrhosis [24,25]. The picture is complicated by the fact that some substrates for human liver microsomes appear to involve a specific cytochrome p450 group of isoenzymes which is highly inducible by smoking [26]. This could explain the finding that excessive use of tobacco is an additional risk factor for PHC in alcoholics [6]. There is some evidence on the other hand that inactivation of reactive intermediates may be impaired in alcoholic cirrhosis. The concentration of a natural antioxidant, reduced glutathione, has been shown to be significantly lower in alcoholic cirrhosis, compared to normals [24]. In addition this antioxidant may play a more complex role than detoxification in hepatocarcinogenesis. It has been shown experimentally that administration of reduced glutathione to rats bearing aflatoxin Bl-induced liver tumours improves survival and may cause regression of tumour growth [27]. These studies serve to illustrate the interplay of factors that may alter the metabolic balance between activation and deactivation of procarcinogens and other xenobiotics and contribute to hepatocarcinogenesis. (iii) Cirrhosis - - A premalignant lesion?
The association between alcohol abuse and cirrhosis has long been recognised. In almost all European countries there is a strong correlation between national alcohol consumption and mortality rates from cirrhosis of the liver [28]. This has recently been evaluated prospectively when the risk of long-term alcohol abusers acquiring cirrhosis was shown to be about 15% [29]. The great majority of PHC arises in cirrhotic livers and the fact that alcohol is a cause of cirrhosis could account for the iiacreased risk of PHC in alcoholics. One hypothesis is that cirrhosis per se is a premalignant lesion - - i.e. that hyperplasia leads to neoplasia in the absence of additional factors such as exposure to chemical or viral carcinogens [12]. If this mechanism were universally operative a correlation in the geographic distribution of cirrhosis and PHC would be anticipated as has been documented for HBV and PHC [3]. This does not apply for alcoholic cirrhosis which is common in Northern Europe where the incidence of PHC is low. Moreover the risk of PHC is not similar for all types of cirrhosis; it is generally agreed that macronodular cirrhosis has the greatest malignant potential and some recent clinicopathological studies show that PHC is significantly more frequent in HBsAg-positive than in alcoholic cirrhosis [30]. In addition numerous studies from Europe and North America in recent years have suggested an increase in PHC in the western world and in some of these this appears to be independent of cirrhosis [31]. This would suggest that the relationship between cirrhosis and PHC is more complex than cirrhosis acting as a simple premalignant lesion. An alternative hypothesis has been advanced that cirrhosis tends to act as a powerful promoting agent in the presence of a primary carcinogenic factor or factors, whether viral or chemical [31]. Thus the raised rate of DNA synthesis that inevitably accompanies the formation of hyperplastic nodules in cirrhosis could act as a promotor for cells already initiated by some (unknown) stimulus. The association of HBV with PHC in 'alcoholic cirrhosis' is certainly compatible with this hypothesis, and it is supported by some ex-
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perimental data. One study examined the role of cell proliferation in the initiation of liver carcinogenesis induced by chemicals in rats. It was found that loci of preneoplastic hepatocytes only appeared in rats that received the carcinogen coupled with the cell proliferation stimulus of partial hepatectomy, whereas no loci were seen in rats that received the same dose of carcinogen and a sham operation. It is suggested that replication of D N A with carcinogen-induced lesions prior to repair offers a mechanism whereby a mutation may be transcribed and fixed in a daughter'initiated' cell [32]. There is hope that a greater understanding of the molecular mechanisms involved in the link between cell proliferation and malignant transformation may emerge in the next decade following the discovery of cellular protooncogenes (c-onc). Increased expression of c-onc has been reported in both liver regeneration and hepatocarcinogenesis in rats [33,34]. The role of these genes in the sequential events leading to tumour formation is as yet unclear. It could be that repetitive mitosis + viral or chemical carcinogens could induce mutations in specific genes, leading to increased and/or inappropriate expression of c-onc and malignant transformation. (iv) Alcohol and male sex
The male predominance of PHC, at least in the West, is almost completely accounted for by the association with cirrhosis [12]. This could merely reflect the fact that alcohol consumption is greater among men than women, but again evidence suggests that the explanation for the strong association of PHC with male sex is more complex. One possibility is that the hyperestrogenaemia commonly seen in male alcoholic cirrhosis may play some role in hepatocarcinogenesis. A recent study has shown elevated estrone levels in cirrhosis and elevated estrone to testosterone ratios in PHC arising in cirrhosis [35]. There is experimental evidence that estrogens promote neoplasia in rats [30] and that internalization of the estrogen receptor correlates with increased D N A synthesis in liver regeneration [37]. Another possibility is that enzyme systems involved in activating and deactivating carcinogens are influenced by the hormonal milieu. For example, higher levels of the reactive intermediate derived from aflatoxin are found in male rats than female rats because of rapid inactivation in females [38]. Conclusions
In conclusion there is an association between alcohol and hepatocellular carcinoma but evidence suggests it is an indirect one. Further studies are indicated on the effect of alcohol, sex and smoking, on the handling of environmental carcinogens by the liver and on the factors regulating liver cell proliferation and their role in hepatocarcinogenesis.
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References 1 Cook-Mozafferi, P. and Van Rensburg, S., Cancer of the liver, Brit. Med. Bull., 1984;40: 342-345. 2 Lee, F.I., Cirrhosis and hepatoma in alcoholics, Gut, 1966; 7: 77-85. 3 Szmuness, W., Hepatocellular carcinoma and the hepatitis B virus: Evidence for a causal association, Progr. Med. Virol., 1978;24: 40-69. 4 Goldfarb, S., Biologic mechanisms of hepatocarcinogenesis, Sem. Liver Dis., 1984; 4: 89-97. 5 Hardell, L., Bengtsson, N.O., Jonsson, U., Eriksson, S. and Larsson, L.G., Aetiological aspects on primary liver cancer with special regard to alcohol, organic solvents and acute intermittent porphyria - - A n epidemiological investigation, Brit. J. Cancer, 1984; 50: 389-397. 6 Yu, M.C., Mack, T., Hanisch, R., Peters, R.L., Henderson, B.E. and Pike, M.C., Hepatitis, alcohol consumption, cigarette smoking and hepatocellular carcinoma in Los Angeles, Cancer Res., 1983; 43: 6077-6079. 7 Schmidt, W. and Popham, R.E., The role of drinking and smoking in mortality from cancer and other causes in male alcoholics, Cancer, 1981; 47: 1031-1041. 8 MacSween, R.N.M., Alcohol and cancer, Brit. Med. Bull., 1982; 38: 31-33. 9 Beasley, R.P., Hwang, L.Y., Lin, C.C. and Chien, C.S., Hepatocellular carcinoma and hepatitis B virus: A prospective study of 22,707 men in Taiwan, Lancet, 1981; ii: 1129-1133. 10 Hall, A.J., Winter, P.D. and Wright, R., Mortality of hepatitis B positive blood donors in England and Wales, Lancet, 1985; i: 91-93. 11 Newberne, P.M., Chemical carcinogenesis: Mycotoxins and other chemicals to which humans are exposed, Sem. Liver Dis., 1984; 4: 122-135. 12 Zaman, S.N., Melia, W.M., Johnson, R.D., Portmann, B.C., Johnson, P.J. and Williams, R., Risk factors in development of hepatoeellular carcinoma in cirrhosis: prospective study of 613 patients, Lancet, 1985; i: 1357-1360. 13 Goudeau, A., Maupas, P., Dubois, F., Coursaget, P. and Bougnoux, P., Hepatitis B infection in alcoholic liver disease and primary hepatocellular carcinoma in France, Progr. Med. Virol., 1981; 27: 26-34. 14 Gluud, C., Aldershville, T., Henriksen, J., K~ger, P. and Mathieson, L., Hepatitis B and A virus antibodies in alcoholic steatosis and cirrhosis, J. Clin. Path., 1982; 35: 693-697. 15 Villa, E., Barchi, T., Grisendi, A., Bellentani, S., Rubbiani, L., Ferr&ti, I., De Palma, M. and Manenti, F., Susceptibility of chronic symptomless HBsAg carriers to ethanol induced hepatic damage, Lancet, 1982; ii: 1243-1244. 16 Ohnishi, K., Iida, S., Iwana, S., Gato, N., Nomura, F., Takashi, M., Mishima, A., Kono, K., Kimura, K., Musha, H., Kotota, K. and Okuda, K., The effect of chronic habitual alcohol intake on the development of liver cirrhosis and hepatocellular carcinoma, Cancer, 1982;49: 672-677. 17 Brechot, C., Nalpas, B., Courouce, A.M., Duhamel, G., Callard, P., Carnot, F., Tiollais, P. and Berthelot, P., Evidence that hepatitis B virus has a role in liver cell carcinoma in alcoholic liver disease, N. Engl. J. Med., 1982; 306: 1384-1387. 18 Hodgson, H.J.F. and Thompson, R.P.H., Cirrhosis in South London, Lancet, 1976; ii: 118-121. 19 Blenkinsopp, W.K. and Hoffenden, G.P., Aetiology of cirrhosis, hepatic fibrosis and hepatocellular carcinoma, J. Clin. Path., 1977; 30: 579-584. 20 Bassendine, M.F., Delia Seta, L., Salmeron, J., Thomas, H.C. and Sherlock, S., Incidence of hepatitis B virus infection in alcoholic liver disease, HBsAg negative chronic active liver disease and primary liver cell cancer in Britain, Liver, 1983; 3: 65-70. 21 Lehmann, F.G. and Martini, G.A., Incidence of primary liver cell carcinoma in liver cirrhosis: 5 year prospective study, DigestiOn, 1976; 14: 530-531. 22 Bulatao-Jayme, J., Almero, E.M., Castro, C.A., Jardeleza, T.H. and Salamat, L.A., A case-control dietary study of primary liver cancer risk from aflatoxin exposure, Int. J. Epidemiol., 1982; 11: 112-119. 23 Lieber, C.S., Seitz, H.K., Garro, A.J. and Worner, T.M., Alcohol-related diseases and carcinogenesis, Cancer Res., 1979; 39: 2863-2886. 24 Woodhouse, K.W., Williams, F.M., Mutch, E., Wright, P., James, O.F.W. and Rawlins, M.D., The effect of alcoholic cirrhosis on the activities of microsomal aldrin epoxidase, 7-ethoxycoumarin, O-
A L C O H O L - MAJOR RISK FACTOR FOR HEPATOCELLULAR CARCINOMA?
519
de-ethylase and epoxide hydrolase, and on the concentrations of reduced glutathione in human liver, Brit. J. Clin. Pharmac., 1983; 15: 667-672. 25 Woodhouse, K.W., Williams, F.M., Mutch, E., Wright, P., James O.F.W. and Rawlins, M.D., The effect of alcoholic cirrhosis on the two kinetic components (high and low affinity) of the microsomal O-deethylation of 7-ethoxycoumarin in human liver, Europ. J. Clin. Pharmacol., 1984;26: 61-64. 26 Williams, F.M., Mutch, E., Woodhouse, K.W., Lambert, D. and Rawlins, M.D., Ethoxyresurufin O-deethylation by human liver microsomes, Brit. J. Clin. Pharmacol., in press. 27 Novi, A.M., Regression of aflatoxin Bl-induced hepatocellular carcinomas by reduced glutathione, Science, 1981;212: 541-542. 28 Walsh, D., Alcohol-related medicosocial problems and their prevention, Copenhagen, World Health Organization Publications, 1982. 29 Serensen, T.I.A., Orholm, M., Bentsen, K., Hoybye, G., Eghoje, K. and Christoffersen, P., Prospective evaluation of alcohol abuse and alcohol liver injury in men as predictors of development of cirrhosis, Lancet, 1984, ii: 214-244. 30 Bartok, I., Remenar, E., Toth, J., Duschanek, P. and Kanyar, B., Clinicopathological studies of liver cirrhosis and hepatocellular carcinoma in a general hospital, Human Path., 1981; 12: 794-803. 31 Burnett, R.A., Patrick, R.S., Spilg, W.G.S., Buchanan, W.M. and MacSween, R.N.M., Hepatocellular carcinoma and hepatic cirrhosis in the West of Scotland: a 25-year necropsy review, J. Clin. Path., 1978;31: 108-110. 32 Columbano, A., Rajalakshmi, S. and Sarma, D.S.R., Requirement of cell proliferation for the initiation of liver carcinogenesis as assayed by three different procedures, Cancer Res., 1981; 41: 2079-2083. 33 Fausto, N. and Shank, P.R., Oncogene expression in liver regeneration and hepatocarcinogenesis, Hepatol., 1983;3: 1016-1023. 34 Yaswen, P., Goyette, M., Shank, P.R. and Fausto, N., Expression of c-Ki-ras, c-Ha-ras and c-myc in specific cell types during hepatocarcinogenesis, Mol. Cell. Biol., 1985; 5: 780-786. 35 Nagasue, N., Ogawa, Y., Yukaya, H., Ohta, N. and Ito, A., Serum levels of estrogens and testosterone in cirrhotic men with and without hepatocellular carcinoma, Gastroenterol., 1985; 88: 768-772. 36 Wanless, I.R. and Medline, A., Role of estrogens as promoters of hepatic neoplasia, Lab. Invest., 1982; 46: 313-320. 37 Francavilla, A., Di Leo, A., Eagon, P.K., Wu, S.Q., Ove, P., Van Thiel, D.H. and Starzl, T.E., Regenerating rat liver: correlations between estrogen receptor localizations and deoxyribonucleic acid synthesis, Gastroenterol., 1984; 86: 552-557. 38 O'Brien, K., Moss, E., Judah, D. and Neal, G., Metabolic basis of the species difference to aflatoxin induced hepatoxicity, Biochem. Biophys. Res. Comm., 1983; 114: 812-821.