Samuel W. French
Department of Pathology
Harbor-UCLA Medical Center
It would be presumptuous to claim that the pathogenesis of alcoholic liver disease is understood. Most
studies done so far have focused on the early changes in the liver observed over one or two months of
alcohol feeding in rats or mice. More chronic studies utilized primates or pigs. Cirrhosis is rarely
observed experimentally except in primates1 and this result has not been confirmed in other laboratories.
Studies in man using repeated liver biopsies do show positive correlation between the progression of the
disease and the dose of alcohol consumed daily and the duration of alcohol consumption. Women required a
lower dose of ethanol to develop cirrhosis. The type of alcoholic beverage and the type of meat consumed
also influences the development of cirrhosis.2 Follow-up studied done on German veterans clearly showed
a progression from the initial fatty liver stage to the stage of alcoholic hepatitis and to the final stage
of cirrhosis3 depending on the dose of alcohol consumed. Of course, all three stages may co-exist. In
one large VA study of 281 patients with alcoholic hepatitis 26 had fatty liver, 106 had acute alcoholic
hepatitis, 39 had cirrhosis and 111 had both alcoholic hepatitis and cirrhosis.4 The histologic changes
included 96.8% showed fatty change, 3.8% showed cholestasis, 85.3% showed limiting plate erosion, 76.2%
showed Mallory body formation and 9.6% showed central hyaline sclerosis. At the alcoholic hepatitis stage
97% showed fatty change, 57% showed apoptosis, 78% showed Mallory bodies, 32% showed megamitochondria, 58%
showed inflammation. The inflammation was predominantly PMN's in the regions of necrosis and Mallory body
formation.5 Although many of these changes have been observed in experimental animal models of alcoholic
hepatitis, progression to cirrhosis has been seen only rarely.6
Investigations where animals are fed ethanol for 1 to several months have shown that the mechanisms of liver
injury caused by ethanol ingestion are numerous and this indicates that the pathogenesis of alcoholic liver
disease (ALD) is complex and, multifactorial. Liver injury is minimal if ethanol is fed for less than 15
days probably because it takes this long to induce cytochrome P450 2E1 (CYP2E1). Because of this fact it is
likely that the induction of CYP2E1 is one key player in the pathogenesis of the initial liver injury (see
1. CYP2E1 induction
CYP2E1 is induced by ethanol primarily by stabilizing the CYP2E1 protein. CYP2E1 is turned over by the
ubiquitin-proteasome pathway which is inhibited by ethanol. CYP2E1 knockout mice do not show this
inhibition of the proteasome in chronic ethanol fed animals indicating that CYP2E1 is responsible for the
inhibition of the proteasome.7
CYP2E1 induction by ethanol plays an important role in ALD pathogenesis because of the free radicals
generated when it oxidizes ethanol to acetaldehyde. Free radicals generated include superoxide and
hydroxyethyl radical. These free radicals generate reactive intermediates such as hydroxyethyl radical (HE)
and products of lipid peroxidation which form adducts with proteins such as malondialdehyde (MDA) and
4-hydroxylenoneal (4HNE).8,9,10,11 The protein adducts such as HE-CYP2E1 act as neoantigens which stimulate
antibody formation and immunocyte dependent liver cell damage.8 Circulating antibodies to adducts of
CYP2E1 are present in alcoholics with liver disease as well as rats fed ethanol12 (Figure 1).
2. Fatty Acid Metabolism
CYP2E1 induction alters fatty acid metabolism, which leads mainly to a decrease in arachidonic acid through
several mechanisms (Figure 1). Some arachidonic acid is lost through lipid peroxidation by free radicals
generated by CYP2E1. Some is used by cyclooxygenase-2 (COX-2) in prostaglandin synthesis. COX-2 expression
is increased by free radicals generated by CYP2E1. COX-2 gene expression is increased at low blood ethanol
levels (BAL) in the rat intragastric fed rat model of ALD13 (Figure 2). Alcohol ingestion also reduces
arachidonic acid synthesis by inhibiting delta 5 and 6 desaturase activity. Lastly, arachidonic acid and
other fatty acids are diminished through hydroxylation by CYP2E1 and 4A.14,15 The products of
hydroxylation (hydroxides and epoxides) are biologically active agents which modulate blood flow.
3. Oxidative Damage
Chronic ethanol feeding of rats increases oxidized proteins in the liver.16 This does not occur in
CYP2E1 knockout mice fed ethanol.7 This indicates that the increased oxidation of cytoplasmic proteins
in the liver results from free radicals generated by CYP2E1 oxidation of ethanol (Figure 1) which may overwhelm
the damaged protein disposal by the 20S proteasome system which is inhibited by ethanol.17,18
4. Kupffer cell-Stellate cell activation
Endotoxin is increased in the blood after chronic ethanol ingestion. The concentration achieved is
dependent on the BAL.19 Endotoxenemia activates the Kupffer cells which then express numerous cytokines,
chemokines and free radicals which initiate a local inflammatory response in the liver sinusoid20 (Figure 3).
The Kupffer cell is the origin of TGFb which activates the stellate cell to produce collagen21 (Figure 1).
This activation is presumably the causative interaction which leads to pericellular fibrosis in
alcoholic hepatitis (Figure 3). This process is initiated very late (many months) in the course of
experimental liver fibrosis in rats fed ethanol.22
5. Role of Liver Hypoxia
Centrilobular fibrosis is in part the result of hypoxia which occurs in zone 3 of the liver lobule when high
BAL is achieved.19 (Figure 1 and Figure 2). Centrilobular hypoxia has been documented by a variety of means
including the measurement of O2 tension and measurement of liver ATP by NMR in vivo in rats and by HPLC in
rats.23,24 The ischemic necrosis followed by healing leads to focal centrilobular fibrosis as in scar
formation.22 Centrilobular hypoxia initiates multiple other processes through the activation of the
transcription factor H1F-1a (Figure 2). These factors include increased expression of VEGF and iNOS. iNOS
generates NO which reacted with O2 generated by CYP2E1 to form peroxynitrate which forms adducts with
proteins at tyrosine residues. The level nitrotyrosine in the liver is increased as are liver nitrates.
Hypoxia also increases serum lipopolysaccharide (LPS) which stimulates Kupffer cells to release macrophage
chemoattractant protein (MCP-1) (Figure 2) and inflammatory cytokines and chemokines (Figure 2), most notably TNFa
(Figure 1). The latter may induce apoptosis of hepatocytes. IL-8 which is chemotaxic for PMNs is released by
neighboring hepatocytes in response to TNFa from activated Kupffer cells (Figure 3).
Other important consequences of hypoxia includes a shift in the redox state of the liver cells, which
increases NADH and decreases NAD+ catalyzed reactions. Lack of O2 inhibits fatty acid b oxidation by the
mitochondria which leads to fatty liver (Figure 1). When BAL falls reoxygenation injury occurs in the ischemia
damaged liver (Figure 2). Mitochondria damage caused by free radical generation is partially prevented by the
induction of MnSOD. However, glutathione (GSH) is reduced in the mitochondria as the result of ethanol
reduction of methyl donor metabolism (methyl transferase activity reduction) and inhibition of GSH transport
from the cytoplasm into the mitochondria. Cox-2 is induced which increases prostaglandin (PGE-2) synthesis
(Figure 1 and Figure 2). Growth factors are upregulated which stimulate liver cell regeneration.25,26
6. Role of Immunologic Synapse
"Piecemeal" necrosis is a common feature in alcoholic hepatitis.27 T cells home to and are sequestered
in the liver28,29 presumably as a consequence of activation of T cell receptors on endothelial cells in
the portal tracts30 and attachment to target liver cells through the formation of immunological synapses.31
CD4 cells predominate but CD8 cells are also sequestered.32 Expression of class II MHC molecules
is enhanced and this correlates with hepatocellular necrosis and Mallory body (MB) formation.33 Damage
to hepatocytes by the lymphocytes that form an immunological synapse is thought to be due to endocytosis of
the synapse which overtime reduces the hepatocyte to the point that it ultimately disappears.34
7. Mechanism of MB formation
MB formation is an important component of alcoholic liver disease even in the fatty liver stage. Ethanol
fed mice form MBs35 if the mice are first fed a porphyrinogenic drug which transforms groups of
hepatocytes. This suggests that gene expression changes must first occur before hepatocytes can form MBs.
This idea is supported by the fact that MBs can be formed by hepatocellular carcinomas. It now appears that
MBs form in the same way that aggresomes form in cells containing mutant or misfolded proteins.36 First,
CK18 and 8 become hyperphosphorylated (Figure 1). This step can be shown using phosphatase inhibitors such as
ethanol or okadaic acid. Hyperphosphorylation leads to conformational changes in the cytokeratins as shown
by infrared spectra deconvolution studies. Ubiquitin covalent binding occurs in response to the misfolding
of the CK proteins. A mutant ubiquitin forms as a consequence of molecular misreading at the time of
transcription so that both normal ubiquitin and mutant ubiquitin coexist in the same cell. Mutant ubiquitin
inhibits the deubiquitination step of proteolysis of the CK proteins by the proteasome. Consequently, the
altered CK proteins accumulate and form aggresomes which move to a perinuclear region of the cells next to
the centrisome. At this point the aggresome (MB) is visible and stains positive with antibodies to CK8,
CK18, ubiquitin, mutant ubiquitin, transglutaminase, protreasome subunits, tubulin and HSPs 70 and 90.
Transglutaminase further covalently cross-links the aggregated proteins which form the MB making the MB
insoluble and resistant to proteolysis.
8. Mechanism of periportal fibrosis
Although fibrosis usually starts centrilobularly in alcoholic liver disease, sooner or later periportal
fibrosis appears as a result of bile duct metaplasia at the limiting plate of hepatocytes. The mechanism of
periportal fibrosis is quite different from the mechanism of centrilobular and pericellular fibrosis in this
way. Since bile duct metaplasia recapitulates the embryologic mechanism of bile duct formation from the
limiting plate of hepatocytes37 it's not surprising to find stellate cells surrounding and supporting the
metaplastic bile ducts. The transformed liver cells involved in forming bile ductules were originally
flanked by stellate cells. The stellate cells, in turn, form a collar of collagen around the metaplastic
ducts (Figure 4). This provides the collagen which leads to the periportal scarring.38
Bode C, Bode JC, Erhardt JG, French BA, French SW. Effect of the type of beverage and meat consumed by
alcoholics with alcoholic liver disease. Alcoholism: Clin Exp Res 22:1803-1805, 1998.
Lelbach WK. Cirrhosis in the alcoholic and its relation to the volume of alcohol abuse. Ann NY Acad
Sci 252:88-105, 1975.
Mendenhall CL and the VA Cooperative Study Group on Alcoholic Hepatitis. Pathogenesis, diagnosis and
treatment of alcoholic hepatitis. Clin Gastroenterol 10: 417-441, 1981.
French SW, Nash J, Shitabata P, Kachi K, Hara C, Chedid A, Mendenhall CL, and the VA Cooperative Study
Group 119. Seminars Liver Dis 13:154-169, 1993.
Hall Pdl M, Lieber CS, DeCarli LM, French SW, Lindros KO, Jarvelainen H, Bode C, Parlesak A, Bode JC.
Models of alcoholic liver disease in rodents: A critical evaluation. Alcoholic: Clin Exp Res
Bardag-Gorce F, Yuan QX, Li J, French BA, Fang C, Ingelman-Sundberg M, French SW. The effect of
ethanol-induced cytochrome P450 2E1 in the inhibition of proteasome activity by alcohol BBRC 279:23-29,
Albano E, French SW, Ingelman-Sundberg M. Hydroxyethyl radicals in ethanol hepatotoxicity. Frontiers
Biosci 4:d 533-540, 1999.
French SW, Kim W, Jui L, Albano E, Hagbjork A-L, Ingelman-Sundberg M. Effect of ethanol on cytochrome
P450 2E1 (CYP2E1) lipid peroxidation and serum protein adduct formation in relation to liver pathology
pathogenesis. Exp Mol Pathol 58: 61-75, 1993.
Albano E, Clot P, Morimoto M, Tamasi A, Ingelman-Sundberg M, French SW. Role of cytochrome P450
2E1-dependent formation of hydroxyethyl free radical in the development of liver damage in rats
intragastrically fed with ethanol. Hepatology 23: 155-163, 1966.
Gouillon ZQ, Lucas D, Li J, French BA, Fu P, Ingelman-Sundberg M, Donohue Jr TM, French SW: Effect of
chlormethiazole treatment on ethanol-induced liver disease in the intragastric feeding rat model. Proc Soc
Exp Biol Med 224:302-308, 2000.
Lytton D, Helander A, Zhang-Gouillon ZQ, Stokklan K, Bordone R, Arico S, Albano E, French SW,
Ingelman-Sundberg M. Auto-antibodies against cytochrome P450 2E1 and 3A in alcoholics. Molec Pharmacol 55:
French S. Gene expression of the liver at the peaks and troughs of the blood alcohol cycle in rats fed
ethanol continuously intragastrically: A most ingenious paradox: in Summary Report of a Symposium: Genes
and Genes Delivery for Diseases of Alcoholism, Crews FT, Editor, Alcoholism: Clin Exp Res 25:1778-1800,
French SW, Morimoto M, Reitz R, Koop D, Klopfenstein B, Ester K, Clot P, Ingelman-Sundberg M, Albano E.
Lipid peroxidation, CYP2E1 and fatty acid metabolism in alcoholic liver disease. J Nutr 127:9075-9115,
Amet Y, Berthou F, French SW. Alcohol-inducible P450 in rat liver and kidney microsomes. Fatty acid
and metabolism. Alcoholism: Clin Exp Res 22:744-746, 1998.
Rouach H, Fataccioli V, Gentil M, French SW, Morimoto M, Nordmann R. Effect of chronic ethanol feeding
on lipid peroxidation and protein oxidation in relation to liver pathology. Hepatology 25:351-355, 1997.
Fataccioli V, Andraud E, Gentil M, French SW. Rouach H. Effects of chronic ethanol administration on
rat liver proteasome activities. Hepatology 29: 14-20, 1999.
Donohue Jr TM, Zetterman RK, Zhang-Gouillon ZQ, French SW. Peptidase activities of the math catalytic
proteasome in rat liver after voluntary and intragastric ethanol administration. Hepatology 28: 486-491,
Li J, Nguyen V, French BA, Parlow AF, Su GL, Fu P, Yuan QX, French SW. Mechanisms of the cycle pattern
of urinary ethanol levels in rats fed ethanol. The role of the hypothalamic-pituitary thyroid axis. Am J
Physiol 279: G118-125, 2000.
French SW. Mechanisms of alcoholic liver injury. Canad J Gastroenterol 14: 327-332, 2000.
Tsukamoto H, Gaal K, French SW. In sight into the pathogenesis of alcoholic liver necrosis and
fibrosis: Use of Tsukamoto-French rat model of alcoholic liver disease. Hepatology 12:599-608, 1990.
Takahashi H, Wong K, Jui L, Nanji A, McKibbon D, Mendenhall CS, French SW. Effect of dietary fat on Ito
cell activation by chronic ethanol intake: A long term serial morphometric study on alcohol-fed and control
rats. Alcoholism: Clin Exp Res 15: 1060-1066, 1991.
Miyamoto K, French SW. Hepatic adenine nucleotide metabolism measured in vivo in rats fed ethanol and a
high fat-low protein diet. Relation to pathogenesis of alcohol-induced liver injury in the rat. Hepatology
8: 53-60, 1988.
Takahashi H, Geoffrion Y, Butler KW, French SW. In vivo hepatic energy metabolism during the
progression of alcoholic liver disease: A non-invasive 31P nuclear magnetic resonance study in rats.
Hepatology 11: 65-73, 1990.
Gouillon ZQ, Miyamoto K, Donohue TM, Wan YY, French BA, Nagao Y, Fu P, Reitz RC, Hagbjork A-L, Yap C,
Ingelman-Sundberg M, French SW. Role of CYP2E1 in the pathogenesis of alcoholic liver disease.
Modifications by cAMP and ubiquitin-proteasome pathway. Frontiers of Bioscience 4: 16-25, 1999.
French SW. Ethanol and hepatocellular injury. Clinics Lab Med 16: 289-306, 1996.
French SW. Burbige EJ. Alcohol hepatitis: Clinical morphologic pathogenic and therapeutic in H Popper
and F schaffner (eds). Progress in Liver Disease Vol IV, New York, Grune Stratton p 557-579, 1979.
French SW, Burbige EJ, Tarder G, Bourke E, Harkin CG, Denton T. Lymphocyte sequestration by the liver in
alcoholic hepatitis. Arch Pathol Lab Med 103: 146-152, 1979.
Lalor PF, Adams DH. Adhesion of lymphocytes to hepatic endothelium. Molec Pathol 52: 214-219, 1999.
Dustin MZ, Shaw AS. Co-stimulation building an immunological synapse. Science 283: 649-650, 1999.
Wang MX, Morgan T, Lungo W, Wang L, Sze GZ, French SW. "Piecemeal" necrosis: renamed troxis necrosis.
Exp Molec Pathol 71: 137-146, 2001.
Chedid A, Mendenhall CL, Moritz TE, French SW. Chen TS, Morgan TR, Roselle GA, Nemchausky BA, Tamburro
CH, Schiff ER, McClain CJ, Marsano LS, Allen J, Samanta A, Weesner RE, Henderson WG, and Veterans Affairs
Cooperative Study Group 275. Cell-mediated hepatic injury in alcoholic liver disease. Gastroenterology
105: 254-266, 1993.
Hwang JF, Yang Y, Sepulveda H, Shi W, Hwang I, Peterson PA, Jackson MR, Sprent J, Cai Z. TCR-mediated
internalization of peptide-MHC complexes acquired by T cells. Science 286:952-954, 1999.
Zhang-Gouillon ZQ, Yuan QX, Hu B, Gaal K, Marceau N, French BA, French SW. Alcohol induces Mallory body
formation in drug primed mice. Hepatology 27:116-122, 1998.
French BA, van Leeuwen F, Riley NE, Yuan QX, Bardag-Gorce F, Lue YH, Marceau N, French SW. Aggresome
formation in liver cells in response to different toxic mechanisms. Role of the ubiquitin-proteasome
pathway and the frameshift mutant of ubiquitin. Exp Molec Pathol 71: 241-246, 2001.
Cocjin J, Rosenthal P, Buslon V, Luk L, Barajas L, Geller SA, Ruebner B, French S. Bile ductule
formation in fetal, neonatal and infant livers compared with extrahepatic biliary atresia. Hepatology 24:
Ray MB, Mendenhall CL, French SW, Gartside PS. The nature of bile duct change in alcoholic liver
disease. Liver 13: 36-45, 1993.