Dipartimento di Scienze Chirurgiche e Gastroenterologiche, Sezione di Gastroenterologia, Università di Padova,
1 Istituto Oncologico, Università di Bologna and
2 Istituto di Anatomia Patologica, I° Cattedra, Università di Padova, Italy
Received 30 August 2000; in revised form 8 May 2001; accepted 9 July 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alcohol intake is also described as being one of the dietary factors epidemiologically linked to a greater risk of cancer, and numerous studies investigating the correlation between ethanol consumption and the risk of hepatocellular carcinoma (HCC) have been published (Farinati et al., 1992; French, 1996
; Seitz et al., 1998
). Several plausible mechanisms have been suggested by various investigators for the link between excessive consumption of alcoholic beverages and the increased risk of liver cancer, including: (1) the induction of cirrhosis; (2) ethanol's solvent effect; (3) exposure to carcinogens in alcoholic beverages; (4) dietary deficiencies and decreased immunological responsiveness, commonly associated with heavy drinking; (5) ethanol itself acting as a co-carcinogen at one or more stages in the multiphase process of carcinogenesis (Horie et al., 1965
; Walker et al., 1979
; Lieber et al., 1986
; Farinati et al., 1991
; Seitz et al., 1998
). We have also focused our attention in the past on this latter aspect of the problem, reporting that a co-carcinogenic activity of ethanol is also supported by ethanol's ability to increase the organism's capacity to activate environmental carcinogens, by inducing the cytochrome P450-dependent mixed function oxidase systems, and to interfere with DNA repair mechanisms (Farinati et al., 1989
).
Alterations in cell turnover, as expressed by changes in the cytoproliferation rate and/or in the process of apoptosis (programmed cell death) are also highly relevant in the process of carcinogenesis (Svegliati Baroni et al., 1994). Several papers suggest an increase in apoptosis (Higuchi et al., 1996
; Kurose et al., 1997
; Nanji, 1998
; Pianko et al., 2000
) or changes in cell growth rate in the liver of patients exposed to alcohol abuse (Hillan et al., 1996
; Zhang and Farrell, 1999
), but none has compared what happens in chronic alcohol-related liver disease with findings in patients with other types of liver disease, nor have they fully explored the link between free radical-mediated damage, iron metabolism and changes in cell growth rate. This paper reports the data we obtained from studying patients with alcohol-related liver damage in terms of the lipid peroxidation process, the levels of antioxidant defence and iron metabolism in relation to cytoproliferation and apoptosis, comparing them with the situation observed in chronic hepatitis C or hepatitis B virus-mediated damage.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
On the basis of their clinical history, biochemical and virological data, and histological examination, the patients were grouped as follows: (1) 10 patients (seven males and three females) with chronic alcoholic liver disease (CALD) (defined as chronic liver damage in patients who were both HBsAg- and HCV-negative, with a history of ethanol intake in excess of 80 g/day for males or 40 g/day for females for more than 10 years and lasting up until hospital admission, with no signs of autoimmunity (absence of any organ- and non-organ-specific autoantibody); (2) 24 patients (14 males and 10 females) were HCV-positive [HCV-related chronic hepatitis (HCV)]; (3) 11 patients (eight males and three females) were HBsAg-positive and anti-HCV-negative [HBV-related chronic hepatitis (HBV)]; (4) 10 control subjects (six males and four females).
The mean ethanol intake over the previous 10 years was 8 g/day in HCV-positive patients, 7 g/day in HBV-positive patients, 88 in CALD patients and 5 g/day in control subjects.
All the following studies were performed prior to any treatment.
Morphological evaluation
Biopsies (one per patient) were taken using a 1617 gauge modified Menghini needle under ultrasound guidance and local anaesthesia. Only patients whose biopsy material was adequate (i.e. 4 cm in length) were included in the study, to avoid taking a second biopsy. At least 2 cm of biopsy material were cut, fixed in 10% buffered formaldehyde and handed over to the pathologists. The tissue was embedded in paraffin, cut and routinely stained with haematoxylin and eosin (Fig. 1
) and periodic acidSchiff for routine evaluation, and Perl's stain for siderosis. Histological findings were classified as mild or moderatesevere chronic active hepatitis, with or without cirrhosis.
|
Biochemical tests
The tissue for biochemical determination was 15 mg in wet weight. Samples were either processed immediately or stored at -80°C for up to 2 weeks.
Serum ferritin. Serum ferritin (µg/l) levels were determined by the standard technique used at our clinical chemistry laboratory, the normal range of which is 31290 (for males) or 4233 (for females).
Tissue iron determination.
Liver iron concentrations were measured by atomic absorption spectrophotometry (Bassetet al., 1986), in a 4 mg wet weight piece of a needle biopsy specimen. The liver tissue was placed in a clean tube and dried at 37°C for 24 h. After cooling, the sample was transferred to volumetric flasks, adding 3 ml of concentrated nitric acid and incubating at 37°C for 24 h. The samples were then made up with de-ionized water. Iron standard solutions were prepared by dilution of a concentrated iron stock solution (Titrisol; Merck, Darmstadt, Germany) with de-ionized water. A Perkin Elmer atomic absorption spectrophotometer with a simultaneous background corrector was used with an acetylene/air mixture. A lean blue (oxidizing) flame was used with a cathode lamp current of 5 mA, a monochromator wavelength of 248.3 nm and a slit width of 0.2 mm. Under these conditions, the detection limit for iron was 0.005 µg/ml. The results were expressed as µmol/g tissue (dry weight), the normal range being <30 µmol/g.
Malondialdehyde (MDA) determination.
Liver lipoperoxide determination was performed by the thiobarbituric acid reaction using a modified version of Masugi's method (Masugi and Nakamura, 1977). The method identifies TBARS (thiobarbituric acid-reactive substances), i.e. aldehydes, in particular MDA. Liver biopsy samples weighing
5 mg were homogenized with a Teflon pestle in 0.5 ml of cold 50 mM phosphate buffer (pH 7.4). This homogenate was made up with 7% sodium dodecyl sulphate (SDS), 0.1 M HCl, phosphotungstic acid and 0.67% thiobarbituric acid aqueous solutions. The mixture was heated for 60 min in a boiling water bath. After cooling, 5 ml of n-butanol were added and the mixture was stirred vigorously, then spun at 3000 r.p.m. for 10 min. The separated n-butanol layer was assayed fluorometrically with excitation at 515 nm and emission at 553 nm. MDA tissue levels were expressed in nmol/g of wet tissue.
Liver glutathione and cysteine determination.
The liver tissue was homogenized in ice-cold 0.15 M KCl and the homogenate was treated with 5% (v/v) perchloric acid for protein precipitation. The samples were spun at 3000 r.p.m. for 10 min and the supernatant was retained. Liver glutathione in its reduced (GSH) and oxidized (GSSG) forms and cysteine (CYSH) were determined according to Reed's method (Reed et al., 1980). This method of analysis is based on the reaction of iodoacetic acid (80 mM) with thiols, in a solution containing excess sodium bicarbonate, to form S-carboxymethyl derivatives, followed by chromophore derivation of the amino groups with Sanger's reagent (1-fluoro-2,4-dinitrobenzene) 1.5%. These derivatives were rapidly separated by high-performance liquid chromatography, using a Shimadzu gradient liquid chromatograph equipped with an aminopropyl-NH2 column, to permit nanomol level analysis of GSH, GSSG and CYSH. Results were calculated in relation to peak areas of freshly prepared standards. GSH, GSSG and CYSH levels were expressed as nmol/mg of wet tissue.
In situ end-labelling (ISEL)
The In Situ Cell Death Detection Kit (Boehringer, Indianapolis, IN, USA) was used to recognize DNA strand breaks (Gavrieli et al., 1992). Briefly, residues of digoxigenin-dUTP were catalytically added to 3'-OH ends of double- or single-stranded DNA by terminal deoxynucleotidyl transferase (TdT). The incorporated nucleotides were detected by a peroxidase-conjugated anti-digoxigenin antibody and visualized with 3,3'-diaminobenzidine (DAB) (Sigma Chemicals, St Louis, MO, USA). For negative control purposes, TdT was excluded from the reaction buffer. The apoptosis (APO) index (number of ISEL-positive cells/1000) was determined by counting 2000 cells per section (Fig. 2
).
|
Statistics
The results obtained were analysed statistically using Student's t-test, one-way analysis of variance (ANOVA), the MannWhitney U-test and the KruskalWallis test; linear regression analysis was also used, together with the 2 and Fisher's exact tests.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Reduced/oxidized glutathione and cysteine
Liver GSH levels were significantly lower in CALD and HBV patients and higher in HCV and control subjects by one-way ANOVA (P < 0.025), with a significant difference between HCV and CALD (P < 0.01 by Student's t-test) and between HCV and HBV (P < 0.05 by Student's t-test), when these subgroups were analysed separately (Table 3). No significant differences were detected in tissue GSSG levels (Table 3
). The results in Table 3
also show that CYSH levels were significantly higher in CALD than in the other three groups (P < 0.05 by one-way ANOVA). In summary, all of the above parameters of iron overloading and oxidative damage were higher in CALD and HCV, while only in CALD were GSH levels significantly lower than in the other patient groups.
|
|
When the data on apoptosis and cytoproliferation were considered separately for the periportal (zone 1) and centrilobular areas (zones 2 and 3), we obtained the following results: (1) patients with CALD had apoptotic cells more frequently in the centrilobular area (60%). The opposite was true for patients with HCV- or HBV-related hepatitis in whom the percentage of apoptotic cells in this area was only of 32 and 36%, respectively; (2) patients with CALD had cytoproliferation less frequently in zones 2 and 3 (15% of cases) than HCV (50%), but as frequently as HBV (18%) patients.
Correlation studies
APO index and cytoproliferation correlated one with the other (r = 0.51 P < 0.001) and both with ALT levels (r = 0.47, P < 0.005 and r = 0.84, P < 0.001 respectively). Similarly, both APO and MIB1 correlated with grading (r = 0.495, P < 0.025 and r = 0.4, P < 0.02). Serum ferritin and iron were highly correlated (r = 0.848, P < 0.00001) and MIB1 correlated with tissue iron (r = 0.300, P < 0.05). MDA correlated with tissue iron and with ferritin (r = 0.597, P < 0.001 and r = 0.437, P < 0.004 respectively). GSH, GSSG and CYSH were not significantly correlated with other parameters.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The causal relationship between oxidative events and the onset of alcoholic liver damage is supported by immunohistochemical analysis showing the presence of aldehydes derived from lipid peroxidation in the areas of fatty infiltration, focal necrosis and fibrosis (Tsukamoto et al., 1995).
While several mechanisms involved in the pathogenesis of alcohol-mediated liver damage, such as oxidative stress, have been studied in detail, little is known about some other potential mechanisms, e.g. apoptosis and cytoproliferation in alcoholic liver disease. Cytoproliferation has been evaluated only rarely in humans chronically exposed to ethanol (Zhang and Farrell, 1999), while several studies have demonstrated that apoptosis occurs in both clinical and experimental alcoholic liver disease, though the mechanisms involved are not entirely clear (Amin, 1998
). For instance, it has been suggested that oxidative stress may be one of the mechanisms underlying ethanol-induced apoptosis (Kurose et al., 1997
).
Our aim was to evaluate all the above parameters in the same group of CALD patients and to compare this scenario with findings obtained in other chronic liver diseases, such as HBV- or HCV-mediated liver damage and with data derived from control subjects. However, we will discuss our results focusing on the comparison with patients affected by other types of chronic liver damage, since we consider this as the more relevant point of our study. Our data confirm that, in comparison with HCV- and HBV-related hepatitis, chronic alcohol intake increases tissue MDA levels, a product of lipid peroxidation, and reduces GSH availability in the liver. This latter alteration indicates a progressive decline in the liver's capacity to provide an adequate scavenging response and is associated with a build-up of hepatic cysteine a finding that is more difficult to explain. The accumulation of this glutathione precursor/metabolite in the liver might stem from an excess GSH catabolism, also due to the induction of gamma-glutamyltranspeptidase (an enzyme involved in GSH catabolism), as demonstrated by studying cysteine erythrocyte levels (Loguercio et al., 1999).
Alcohol intake therefore correlates inversely with GSH levels and directly with lipid peroxidation products. In addition, liver iron levels were found to be higher in patients with alcohol misuse, due to iron accumulation in Kupffer cells, as reported elsewhere (Fletcher et al., 1999), and were found to correlate with lipid peroxidation. We have already reported that the increased hepatic oxidative disease accompanied by iron overloading in HCV-related liver damage is accompanied by a build-up of DNA oxidative damage, a fact that is also thought to be relevant to liver carcinogenesis and which may well be important in alcohol-related liver carcinogenesis (Farinati et al., 1995
). On the whole, the type of damage found in HCV-related liver disease in many ways resembles the situation in CALD, with the exception of liver GSH and CYSH levels, thus suggesting that oxidative damage is important in both, and that HBV-related liver damage definitely follows different pathways.
As for hepatocellular proliferation and the apoptosis rate (factors that may be quite important in carcinogenesis), we found that, in chronic alcohol-mediated liver damage, both MIB-1-positive, proliferating hepatocytes and apoptotic, in situ end-labelling-positive cells are less frequently detectable than in viral liver damage. While inducing cell proliferation in the digestive tract (Seitz et al., 1998), ethanol exposure is generally linked to an impaired cell proliferation in the liver (Zhang and Farrell, 1999
), though data on chronic exposure in humans are apparently very scarce (Lieber and Leo, 1992
). With respect to cell proliferation, therefore, our data are consistent with other reports. On the other hand, our data do not confirm previous reports with respect to apoptosis, or at least they show that any increase in apoptosis in CALD is definitely of a lower extent than in viral hepatitis. Finally, the distribution of proliferating and apoptotic cells in the lobule is very different in CALD from that observed in chronic viral liver damage. In particular, apoptotic cells are found more frequently in the perivenular area in CALD, where alcohol exerts its damaging effect (Lieber, 1994
), whereas proliferating hepatocytes are observed in the periportal tract, i.e. at a more physiological site, which is not the case in HCV-related damage. The two observations are not significantly different due to the relatively small sample size.
Since oxidative damage is more relevant in patients with CALD and changes in cell turnover are less distinct than in patients with HBV- or HCV-mediated damage, the above findings suggest that the role of oxidative damage in modulating cell turnover in alcoholic liver disease is more limited than was previously reported (Higuchi et al., 1996), and that the turnover is lower and involves different functional areas of the liver. Whether the above aspects are also relevant in relation to the different prognosis and lower neoplastic risk for patients with alcoholic liver damage is open to debate.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altomare, E., Vendemiale, G. and Albano, O. (1988) Hepatic glutathione content in patients with alcoholic and non alcoholic liver disease. Life Sciences 43, 991998.[ISI][Medline]
Amin, A. N. (1998) Apoptosis and alcoholic liver disease. Seminars in Liver Disease 18, 187190.[ISI][Medline]
Basset, M. L., Halliday, J. W. and Powell, L. W. (1986) Value of hepatic measurement in early hemochromatosis and determination of the critical iron level associated with fibrosis. Hepatology 61, 2429.
Farinati, F., Lieber, C. S. and Garro, A. J. (1989) Effects of chronic ethanol consumption on carcinogen activating and detoxifying systems in rat upper alimentary tract tissue. Alcoholism: Clinical and Experimental Research 13, 357360.[ISI][Medline]
Farinati, F., Fagiuoli, S., De Maria, N., Zotti, S., Chiaramonte, M., Salvagnini, M. and Naccarato, R. (1991) Risk of hepatocellular carcinoma in alcoholic cirrhosis. Liver 11, 190191.[ISI][Medline]
Farinati, F., Cardin, R., Zordan, M., Valiante, F., Garro, A. J., Burra, P., Venier, P., Nitti, D., Levis, A. G. and Naccarato, R. (1992) Alcohol metabolism in the upper digestive tract: its implications with respect to carcinogenesis. European Journal of Cancer Prevention 1 (Suppl. 3), 2532.[Medline]
Farinati, F., Cardin, R., De Maria, N., Della Libera, G., Marafin, C., Lecis, E., Burra, P., Floreani, A., Cecchetto, A. and Naccarato, R. (1995) Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis. Journal of Hepatology 22, 449456.[ISI][Medline]
Fletcher, L. M., Halliday, J. W. and Powell, L. W. (1999) Interrelationships of alcohol and iron in liver disease with particular reference to the iron-binding proteins, ferritin and transferrin. Journal of Gastroenterology and Hepatology 14, 202214.[ISI][Medline]
French, S. W. (1996) Ethanol and hepatocellular injury. Journal of Clinical and Laboratory Medicine 16, 289306.
Gavrieli, Y., Sherman, Y. and Ben-Sasson, S. A. (1992) Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. Journal of Cell Biology 119, 493501.[Abstract]
Grattagliano, I., Vendemiale, G., Sabbà, G., Buonamico, P. and Altomare, E. (1996) Oxidation of circulating proteins in alcoholics: role of acetaldehyde and xanthine oxidase. Journal of Hepatology 25, 3836.
Higuchi, H., Kurose, I., Kato, S., Miura, S. and Ishii, H. (1996) Ethanol-induced apoptosis and oxidative stress in hepatocytes. Alcoholism: Clinical and Experimental Research 20 (Suppl. to no. 9), 340A346A.[Medline]
Hillan, K. J., Logan, M. C., Ferrier, R. K., Bird, G. L., Bennett, G. L., McKay, I. C. and MacSween, R. N. (1996) Hepatocyte proliferation and serum hepatocyte growth factor levels in patients with alcoholic hepatitis. Journal of Hepatology 24, 385390.[ISI][Medline]
Horie, A., Hohchi, S. and Kuratsune, M. (1965) Carcinogenesis in the esophagus. II. Experimental production of esophageal cancer by administration of ethanolic solution of carcinogens. Gann 56, 429441.[ISI][Medline]
Ishak, K., Baptista, A., Bianchi, L., Callea, F., De Groote, J., Gudat, F., Denk, H., Desmet, V., Korb, G., MacSween, R. N. M., Phillips, M. J., Portmann, B. G., Poulsen, H., Scheuer, P. J., Schmid, M. and Thaler, H. (1995) Histological grading and staging of chronic hepatitis. Journal of Hepatology 22, 696699.[ISI][Medline]
Knodell, R. G., Ishak, K. G., Black, W. C., Chen, T. S., Craig, R., Kaplowittz, N., Kiernan, T. W. and Wollman, J. (1981) Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1, 431435.[ISI][Medline]
Kurose, I., Higuchi, H., Miura, S., Saito, H., Watanabe, N., Hokari, R., Hirokawa, M., Takaishi, M., Zeki, S., Nakamura, T., Ebinuma, H., Kato, S. and Ishii, H. (1997) Oxidative stress-mediated apoptosis of hepatocytes exposed to acute ethanol intoxication. Hepatology 25, 368378.[ISI][Medline]
Lieber, C. S. (1992) Metabolism of ethanol. In Medical and Nutritional Complications of Alcoholism. Mechanisms and Management, Lieber, C. S. ed, pp. 185225. Plenum Medical, New York.
Lieber, C. S. (1993) Biochemical factors in alcoholic liver disease. Seminars in Liver Disease 13, 136153.[ISI][Medline]
Lieber, C. S. (1994) Alcohol and the liver: 1994 update. Gastroenterology 106, 10851105.[ISI][Medline]
Lieber, C. S. (1997) Role of oxidative stress and antioxidant therapy in alcoholic and non-alcoholic liver diseases. Advances in Pharmacology 38, 601628.[Medline]
Lieber, C. S. and Leo, M. A. (1992) Alcohol and the liver. In Medical and Nutritional Complications of Alcoholism. Mechanisms and Management, Lieber, C. S. ed, pp. 185225. Plenum Medical, New York.
Lieber, C. S., Garro, A. J., Leo, M. A., Mark, K. M. and Worner, T. (1986) Alcohol and cancer. Hepatology 6, 10051019.[ISI][Medline]
Loguercio, C., Blanco, F. D., De Girolamo, V., Disalvo, D., Nardi, G., Parente, A. and Blanco, C. D. (1999) Ethanol consumption, amino acid and glutathione blood levels in patients with and without chronic liver disease. Alcoholism: Clinical and Experimental Research 23, 17801784.[ISI][Medline]
Masugi, F. and Nakamura, T. (1977) Measurement of thiobarbituric acid value in liver homogenate solubilized with sodium dodecyl sulphate and variation of the values affected by vitamin E and drugs. Vitamins 52, 2129.
Nanji, A. A. (1998) Apoptosis and alcoholic liver disease. Seminars in Liver Disease 18, 187190.[ISI][Medline]
Niemela, O., Parkkila, S., Britton, R. S., Brunt, E., Janney, C. and Bacon, B. (1999) Hepatic lipid peroxidation in hereditary hemochromatosis and alcoholic liver injury. Journal of Clinical and Laboratory Medicine 133, 451460.
Nordmann, R. (1994) Alcohol and antioxidant systems. Alcohol and Alcoholism 29, 513522.[Abstract]
Nordmann, R., Ribiere, C. and Rouach, H. (1992) Implication of free radical mechanisms in ethanol induced cellular injury. Free Radicals Biology and Medicine 12, 219240.
Pianko, S., Patella, S. and Sievert, W. (2000) Alcohol consumption induces hepatocyte apoptosis in patients with chronic hepatitis C infection. Journal of Gastroenterology and Hepatology 15, 798805.[ISI][Medline]
Reed, D. J., Babson, J. R., Beatty, P. W., Brodie, A. E., Ellis, W. W. and Potter, D. W. (1980) High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulphide, and related thiols and disulphides. Analytical Biochemistry 106, 5562.[ISI][Medline]
Seitz, H. K., Poschl, G. and Simanowski, U. A. (1998) Alcohol and cancer. Recent Developments in Alcoholism 14, 6795.[Medline]
Sternberger, L. A. (1979) Immunocytochemistry, 2nd edn, pp. 90130. Wiley, New York.
Svegliati Baroni, G., Marucci, L., Benedetti, A., Mancini, R., Jezequel, A-M. and Orlandi, F. (1994) Chronic ethanol feeding increases apoptosis and cell proliferation in rat liver. Journal of Hepatology 20, 508513.[ISI][Medline]
Tsukamoto, H., Horne, W., Kamimura, S., Niemela, O., Parkkila, S., Yla-Herttuala, S. and Brittenham, G. M. (1995) Experimental liver cirrhosis induced by alcohol and iron. Journal of Clinical Investigation 96, 620630.[ISI][Medline]
Walker, E. A., Castegnaro, M., Garren, L., Toussaint, G. and Kowalski, B. (1979) Intake of volatile nitrosamines from consumption of alcohol. Journal of the National Cancer Institute 63, 947951.[ISI][Medline]
Zhang, B. H. and Farrell, G. C. (1999) Chronic ethanol consumption disrupts complexation between EGF receptor and phospholipase C-gamma1: relevance to impaired hepatocyte proliferation. Biochemical and Biophysical Research Communications 257, 8994.[ISI][Medline]