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, and
Laboratory of Hepatobiology and Toxicology, Department of Pharmacology; * Department of Radiation Oncology; and Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC; and Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, NC
ABSTRACT
INTRODUCTION
STUDIES COMPARING FEMALES AND MALES
ROLE OF HYPOXIA AND FREE RADICALS
HYPOXIA
FREE RADICALS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Alcohol ingestion results in increases in the release of endotoxin from gut bacteria or membrane permeability of the gut to endotoxin, or both. Female rats are more sensitive to these changes. Elevated levels of endotoxin activate Kupffer cells to release substances such as eicosanoids, tumor necrosis factor-
and free radicals. Prostaglandins increase oxygen uptake and most likely are responsible for the hypermetabolic state in the liver. The increase in oxygen demand leads to hypoxia in the liver, and on reperfusion, -hydroxyethyl free radicals are formed that lead to tissue damage in oxygen-poor pericentral regions of the liver lobule.
INTRODUCTION
Alcohol-induced liver injury causes serious medical, financial and social problems. The disease progresses from fatty infiltration and follows a pernicious course of inflammation leading to irreversible damage; liver transplantation, at a cost of about $100,000 each, is the only known cure. The fourth leading cause of death in urban American males is alcoholic liver disease, and the number of female alcohol abusers has increased over the last 30 y. Although the hepatotoxic effects of alcohol have been described previously (Lieber 1991
), factors responsible have only been partially characterized.
Significant changes occur in host defense mechanisms after consumption of alcohol, including modified reticuloendothelial function as well as altered immune, lymphocyte, granulocyte and platelet functions (McCuskey 1991). Interest in the effect of alcohol on the reticuloendothelial system has mainly been in the context of the known predisposition of alcoholics to infection (Adams and Jordan 1984). Recently, however, attention has been directed toward the role of endotoxin and Kupffer cells (Adachi et al. 1995). Chronic ethanol ingestion produces fatty liver, hepatomegaly, alcoholic hepatitis, fibrosis and cirrhosis. It is now clear that Kupffer cells are key in at least the early aspects of this pathology.
It has been proposed that the cascade of events leading to alcohol-induced toxicity is initiated by endotoxin. We hypothesize that endotoxin initially activates the Kupffer cell, which is critical for producing a hypermetabolic state (swift increase in alcohol metabolism or SIAM) in parenchymal cells. This leads to hypoxia in pericentral regions of the liver lobule where toxic free radicals are formed upon reintroduction of oxygen, causing cell death. This hypothesis was supported by the discovery that treatment with antibiotics and destruction of Kupffer cells with gadolinium chloride (GdCl3) blocked alcohol-induced liver injury in the Tsukamoto-French model of continuous enteral alcohol administration in vivo (Adachi et al. 1994 and 1995). Interestingly, injury is dependent on unsaturated fat in the diet in this model (Nanji et al. 1989). The ability of Kupffer cells to remove and detoxify various exogenous and endogenous substances, such as endotoxin, is an important physiological process. Recent work has shown that Kupffer cells are required for the alcohol-induced hypermetabolic state in experimental animals (Bradford et al. 1993).
Several observations suggest that Kupffer cells are involved in liver injury caused by alcohol. First, alcohol has been reported to alter Kupffer cell functions such as phagocytosis, bactericidal activity and cytokine production (Martinez et al. 1992). Second, the increase in serum tumor necrosis factor- concentration in alcoholics (Stahnke et al. 1991) is consistent with the hypothesis that Kupffer cells of patients with alcoholic liver disease are activated; tumor necrosis factor- is produced exclusively by the monocyte-macrophage lineage, and the major cell type of this lineage is the hepatic Kupffer cell (Decker et al. 1989). Third, Kupffer cells, which are activated by calcium and contain Ca2+ channels, are opened by prolonged exposure to ethanol (Goto et al. 1994). Reports that the calcium channel blocker nimodipine decreased alcohol-induced liver injury in the Tsukamoto-French model suggests that Kupffer cell calcium channels play an important role in the mechanisms of alcoholic liver disease (Iimuro et al. 1995). Collectively, these observations are consistent with the hypothesis that prolonged exposure to alcohol leads to activation of Kupffer cells.
STUDIES COMPARING FEMALES AND MALES
), we developed a new animal model to study female susceptibility to alcohol-induced liver injury. Female and male Wistar rats were given ethanol [11-12 g/(kg·d)] continuously for up to 4 wk via intragastric feeding using the Tsukamoto-French model. Control rats were fed an isoenergetic high fat diet without ethanol. There were no significant differences in body weight, mean (± SEM) ethanol concentrations (230 ± 11 and 228 ± 7 mg/100 mL in females and males, respectively) or the cyclic pattern of ethanol levels in urine (5.2 ± 0.2 d in females, 5.5 ± 0.2 d in males). Furthermore, rates of ethanol elimination were similar in males and females. Ethanol treatment elevated serum asparate aminotransferase concentrations (means ± SEM) in male rats to 122 ± 10 IU/L after 4 wk, whereas in female rats values increased more rapidly and reached higher levels (168 ± 18 IU/L, P < 0.05). Steatosis, inflammation and necrosis assessed histologically developed more rapidly and were more severe in females than in males. Indeed, steatosis due to ethanol exposure, which was localized in centrilobular areas in males, was panlobular in females. Plasma endotoxin concentrations were more than twofold higher in females than in males following prolonged ethanol exposure (P < 0.005), and intercellular adhesion molecule-1 expression in hepatic endothelial cells was also about twofold greater in females than in males. Thus, a new model to study female susceptibility to alcoholic hepatitis has been developed (Iimuro et al. 1996). Initial work with this model fits the idea that endotoxin and Kupffer cells are responsible for the increased susceptibility to alcohol of women compared with men.
ROLE OF HYPOXIA AND FREE RADICALS
), and Ji et al. (1982) reported that increased tissue respiration caused by prolonged ethanol treatment made the intralobular oxygen gradient steeper. This stimulation in oxygen uptake is due to an alcohol-induced hypermetabolic state (Yuki and Thurman 1980). Subsequent centrilobular hypoxia may be responsible for pericentral liver injury induced by ethanol. Destruction of Kupffer cells with GdCl3 treatment prevented the elevation in oxygen uptake due to ethanol (Bradford et al. 1993). In the Tsukamoto-French model, the rate of ethanol elimination, which is oxygen dependent, was elevated two- to threefold in rats exposed to ethanol for 2-4 wk; however, when Kupffer cells were destroyed by GdCl3, this phenomenon was blocked. Indeed, centrilobular pathological changes are compatible with a mechanism involving hypoxia, and injury was increased when O2 tension in the liver was decreased (French et al. 1984). Importantly, conditioned media from isolated Kupffer cells from ethanol-treated rats stimulated parenchymal cell oxygen consumption. Interestingly, this media contained elevated levels of prostaglandin E2, which has been shown to elevate O2 uptake (Qu et al. 1996). Kupffer cells likely participate in the alcohol-induced liver injury by stimulating oxygen uptake, thereby contributing to pericentral hypoxia.
HYPOXIA
) by producting endothelin-1 (Oshita et al. 1993), and we recently detected hypoxia directly in rats on the Tsukamoto-French protocol (Knecht et al. 1995) and in the perfused liver after acute exposure to ethanol in vivo (Arteel et al. 1996). These findings support the idea that hypoxia directly contributes to alcohol-induced liver injury. Because alcohol also causes a compensatory increase in hepatic blood flow, resulting in an elevated oxygen delivery, it has been argued that any effect of hypoxia due to hypermetabolism or microcirculatory disturbances would be blocked (Shaw et al. 1977). However, use of the hypoxia marker pimonidazole confirmed that downstream hypoxia occurs after acute ethanol treatment (Arteel et al. 1996). Indeed, when livers were perfused in the retrograde direction, hypoxia was switched from pericentral to periportal regions (Arteel et al. 1995).
). Oxygen tension on the surface of livers of rats on the Tsukamoto-French protocol was measured with miniature oxygen electrodes, utilizing the fact that the terminal portal venule stops about 200 µm from the liver surface. Lower values reflect relative hypoxia irrespective of mechanism (e.g., microcirculatory disturbances or hypermetabolism). Surface hepatic oxygen tension was decreased more than 30% by alcohol treatment (Adachi et al. 1994). Thus, direct evidence has now been obtained demonstrating that hypoxia begins after ethanol treatment in the Tsukamoto-French model and hypoxia was blocked when Kupffer cells were inactivated with GdCl3. This was the first suggestion that an oxygen-sensing system was present in the liver. The importance of oxygen is underscored by the fact that rapid metabolic functions of the liver, such as urea and glucose synthesis, are dependent on the oxygen concentration gradient, whereas very slow metabolic processes, such as cytochrome P-450-dependent metabolism of drugs, predominate in pericentral areas irrespective of oxygen (Thurman and Kauffman 1985).
FREE RADICALS
). By applying the electron spin resonance (ESR) technique of spin trapping to the study of ethanol-treated alcohol dehydrogenase-deficient deermice (Peromyscus maniculatus), we detected the -(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN)/-hydroxyethyl radical adduct in bile in vivo from animals given [1-13C]ethanol and the spin trap POBN for the first time. Spin trapping is a technique in which a diamagnetic molecule reacts with a free radical to produce a more stable species, called a radical adduct, which is readily detectable by ESR. Radical adducts are substituted nitroxide free radicals, which tend to be relatively long-lived compared with free radicals, which are very transient in biological systems. The POBN radical adducts characteristically exhibit a six-line ESR spectrum (Fig. 1B), whereas 13C substitution on the -carbon of a spin-trapped species causes six additional lines because of the magnetic interaction of the 13C with the unpaired electron. The subsequent production of a 12-line spectrum after administration of [13C]ethanol provides unequivocal physical evidence that the trapped radical arises from the labeled parent ethanol. Computer simulation of such an ESR spectrum gives spectral values called "hyperfine coupling constants" that can be compared with values of well-characterized radical adducts. The 13C spectral effect and the hyperfine coupling constants identify the radical adduct as the POBN/-hydroxyethyl radical adduct derived from 13C-labeled ethanol. The absorption of microwave energy at that particular magnetic field strength is rectified and detected by a microwave diode and recorded as the first derivative of the absorption peak.
-(4-pyridyl-1-oxide)-N-t-butylnitrone was administered intraperitoneally. Bile samples were collected into vials containing 5 mmol/L Desferal in order to prevent ex vivo free radical formation for 3-4 h, frozen on dry ice, and analyzed for free radical adducts with ESR spectroscopy.
). Importantly, we recently detected a free radical in the bile from rats exposed to ethanol on the Tsukamoto-French model (Fig. 1A). This free radical signal was reduced dramatically when Kupffer cells were eliminated with GdCl3. A six-line radical adduct spectrum was also detected from the bile of rats treated with an ethanol-containing high fat diet (Fig. 1A) but not in bile from rats fed a chow diet (data not shown). Bile from animals fed the control corn oil diet also contained low concentrations of radical adducts. The free radical adduct was identified as -hydroxyethyl by use of [13C]ethanol (Fig. 1B). Thus, ethanol-derived free radical formation can be detected in the bile of Tsukamoto-French rats treated intragastrically with a high fat, ethanol-containing diet. Superoxide-dismutase/catalase-insensitive free radicals have also been obtained from livers of alcohol-treated rats after transplantation (Gao et al. 1995). Exact pathways responsible for formation of free radicals in alcohol-treated rats remain unclear; however, because the ESR signal was reduced with GdCl3 treatment (Fig. 1C), a likely candidate is oxygen radical production by the NADPH oxidase system in Kupffer cells and neutrophils (Fig. 2). On the other hand, a reperfusion injury involving hypoxia and free radical formation via the xanthine-xanthine oxidase system cannot be ruled out, especially since radicals in bile would be expected to arise from parenchymal cells.
Fig. 2.
Scheme depicting working hypothesis for the involvement of endotoxin, Kupffer cells, hypoxia and free radicals in the mechanism of alcohol-induced liver injury. Alcohol consumption alters the membrane fluidity of the gut wall, making it more permeable to endotoxin. Blood endotoxin is elevated and enters the liver, where it is engulfed by Kupffer cells, which become activated and release tumor necrosis factor-, prostaglandin E2 and superoxide. Hepatocyte oxygen and ethanol metabolism is elevated, resulting in hypoxia in downstream regions of the liver lobule, which leads to generation of -hydroxyethyl radicals. Blocking this cascade of events by sterilization of the gut with antibiotics or destruction of Kupffer cells with GdCl3 prevents alcoholic liver injury.
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FOOTNOTES
LITERATURE CITED
and IL-6 in alcoholic liver disease. In: Cells of the Hepatic Sinusoid (Wisse, E., Knook, D. L. & McCuskey, R. S., eds.), 3rd ed., pp. 472-475. Kupffer Cell Foundation, Leiden, The Netherlands.
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