Increased 4-Hydroxynonenal Levels in Experimental Alcoholic Liver Disease: Association of LiDid Peroxidation with Liver Fibrogenesis A
KARL GAAL,l* ROBERT s. BRITTON,3 BRUCER.BACON,3 GEORGE TRIADAFTLOPOULOS~. AND HIDEKAZU TSUKAMOTO',
SEIICHIRO KAMIMURA,',
'Division of Gastroenterology, Department of Medicine, Veterans Affairs Medical Center, Martinez 94553; 2University of California, Davis, California 95616; and 3Division of Gastroenterology and Hepatology, St. Louis University School of Medicine, St. Louis, Missouri 63110-0250
The precise role of lipid peroxidation in the pathogenesis of alcoholic liver disease is still being debated. To explore the issue, this study was undertaken to investigate the status of lipid peroxidation, antioxidants and prooxidants at two discrete stages of experimental alcoholic liver disease. Male Wistar rats were intragastrically fed a high-fat diet plus ethanol for 5 or 16 wk (the duration that resulted in initiation of centrilobular liver necrosis or liver fibrosis, respectively). Lipid peroxidation was assessed in isolated microsomes and mitochondria with three parameters: malondialdehyde equivalents as determined by thiobarbituric acid assay, conjugated diene formation and 4-hydroxynonenal as a 2,4dinitrophenylhydrazone derivative. To assess antioxidant systems, hepatic concentrations of glutathione, methionine and a-tocopherol were determined. The concentration of nonheme iron, a known prooxidant, was also measured. At wk 5, centrilobular liver necrosis was already evident in the ethanol-fedanimals, with two- or threefold increases in plasma AST and ALT levels. At this stage, neither malondialdehyde equivalentsnor conjugated diene values were elevated, and the 4-hydroxynonemallevel was below 0.2 nmol/mg protein. Hepatic concentrations of methionine and a-tocopherol in these animals were increased twoand threefold, respectively, whereas the reduced glutathione level remained unchanged. When alcoholic liver disease had progressed to perivenular or bridging fibrosis at wk 16, all three parameters of lipid peroxidation showed consistent increases that were accompanied by significant reductions in the hepatic glutathione and methionine levels. Interestingly, the control animals pair-fedwith the high-fat diet also had Received December 26, 1991; accepted March 23, 1992. This study was presented during Digestive Disease Week,May 19-22,1991,in New Orleans, Louisiana and published as an abstract (Gastroenterology 1991;100:A758). This study was supported by US. Public Health Service grant AA06603 and by the Department of Veterans Affairs. Address reprint requests to:Hidekazu Tsukamoto, D.V.M., Ph.D., Division of Gastroenterology and Hepatology, MetroHealth Medical Center, Case Western Reserve University, 2500 MetroHealth Dr., Cleveland, OH 44109. 31/1/38332
significantlyelevated 4-hydroxynonenallevels at wk 16 compared to the wk 5 level. The liver concentration of nonheme *an was not increased at wk 5 or wk 16. These results demonstrate (a) dissociation between the initiation of alcoholic liver necrosis and enhanced lipid peroxidation, (b) association of enhanced lipid peroxidation with liver fibrogenesis and depressed antioxidant system, ( c ) the first demonstration of increased 4-hydroxynonenallevel in experimental alcoholic liver disease and (d)possible implication of a high-fat diet in hepatic 4-hydroxynonenal generation. (HEPATOLOGY 1992;16:448-453.)
Peroxidative destruction of biological membranes is believed to be an underlying mechanism of a variety of hepatotoxic diseases (1, 2). However, the role of lipid peroxidation in alcoholic liver disease (ALD)is still a subject of controversy (3-8).Several studies have shown enhanced lipid peroxidation in animals rendered acutely or chronically alcoholic (3-5)and in patients with ALD (6); however, others could not confirm this result (7,8). The hypothesis that incriminates lipid peroxidation as an initiating event for alcohol-induced liver necrosis is based on (a) enhanced levels of conjugated dienes, thiobarbituric acid-reactive substances or chemiluminescence in liver homogenates or subcellular fractions (3-6); (b) decreased levels of antioxidants such as glutathione (4-6); and (c) increased generation of free radicals by liver microsomes of animals fed ethanol for long periods (9-11). However, these studies failed to provide critical information on a correlation between hepatocellular necrosis and the parameters used to put forth the hypothesis. In fact, the animal models used in some of these studies did not produce hepatocellular necrosis (3, 51, and this made it impossible to interpret the data in terms of the pathogenesis of alcoholic liver necrosis. Furthermore, even if the enhanced lipid peroxidation was evidenced in the animal model (4) or patients (6) with advanced ALD, careful longitudinal approaches were not taken to conclude whether this phenomenon was a cause or a consequence of hepatocellular necrosis. Increased hepatic lipid peroxidation
448
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4-JXYDROXYNONENAL IN EXPERIMENTAL ALCOHOLIC LrVER DISEASE
449
FIG.1.Alcoholic centrilobular liver necrosis and inflammation of the liver from a 5-wk ethanol-fed rat iH&E; original magnification x 100).
FIG.2. Bridging fibrosis of the liver from a 16-wk ethanol-fed rat (reticulin stain; original magnification x 40).
may simply result from the liver necrosis attributable to other underlying mechanisms, but this peroxidative change may still constitute an important chemical stimulus to induce liver fibrogenesis as postulated by an in uitro study with fibroblasts (7). To provide insights into these unanswered questions, we designed this study to examine the status of lipid peroxidation and antioxidant systems in rat livers at the two stages of experimental ALD: the early stage of centrilobular liver necrosis and the advanced stage of active liver fibrogenesis. For assessment of hepatic lipid peroxidation, the level of 4-hydroxynonenal (4-HNE1,
one of the most common and important aldehydic products of lipid peroxidation, was measured in addition to two other commonly employed parameters, namely, thiobarbituric acid-reactive substances and conjugated dienes. MATERIALS AND METHODS Animals. Male Wistar rats (initialbody weight = 350 to 400 gm) were aseptically fitted with long-term gastrostomy catheters and continuously infused with a high-fat diet plus ethanol or isocaloric glucose solution for 5 and 16 wk (8, 12).
These durations have been shown to result in the initiation of
450
KAMIMURA ET AL.
HEPATOLOGY
TABLE1. Body and liver weights, serum transaminase levels and hepatic hydroxyproline contents 5 wk Parameters
Body weight gain (gm)
Liver weight (gm) AST (UL)
ALT (UL) Hydroxyproline (pmolfliver)
"Data expressed as mean
16 w k
Control
Alcohol-fed
108.8 % 13.7" 15.2 ? 2.0 88.0 f 9.5 25.7 f 2.8 30.3 t 5.9
94.0 f 8.7 21.5 f 1.8' 202.6 f 43.2d 126.1 f 55.6' 63.1 f 10.4
Control
219.5 ? 17.4 2 137.6 ? 23.4 ? 36.8 ?
Alcohol-fed
19.4 1.0 22.8 2.5 2.4
217.8 ? 16.9 32.5 -f 1.4' 329.4 t 50.8d 155.8 t 44.1d 107.3 t 4.0'
+ S.E.M.
'p < 0.05. cp < 0.001. dp < 0.01. ep = 0.073.
TABLE 2. Hepatic concentration of antioxidants and prooxidants 5 wk
16 w k
Parameters
Control
Alcohol-fed
Control
Alcohol-fed
GSH (WmoUgm liver) Methionine (nmougm liver) a-Tocopherol ( ~ g / g m liver) Nonheme iron (pg/gm liver)
8.46 -f 0.71" 50.0 f 9.0 16.4 ? 4.1 124.3 f 10.0
8.18 f 0.49 99.0 ? 9.06 50.9 ? 9.2' 102.7 f 9.9
8.18 f 0.30 33.6 f 5.6 31.3 t 2.2 149.2 2 6.8
6.82 rf: 0.57' 17.9 f 0.9' 27.7 ? 4.2 120.0 f 4.8'
"Data expressed as mean
f
S.E.M.
'p < 0.05. 'p < 0.01.
centrilobular liver necrosis and active liver fibrogenesis, respectively, in the ethanol-fed rats (13). The dose of ethanol was progressively increased to 42% and 49% of the total calories at the end of these periods, respectively. At the termination points, the animals were anesthetized with sodium pentobarbital, a blood sample was taken from the inferior vena cava and the liver was quickly removed for subcellular fractionation. All animals received humane care. Use of animals in this study was approved by the Institutional Animal Care and Use Committee of the Veterans Af'f'airs Medical Center, Martinez, CA, and was in compliance with guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" (NIH publication no. 86-23, revised 1985). Assessment of Lipid Peroxidation. Mitochondria and microsomes were isolated by differential centrifugation with 0.25 m o m sucrose and 5 mmol/L EDTA homogenization solution (8, 14). Isolated fractions were resuspended in 1.15% KCl containing 0.2% butylated hydroxytoluene at a concentration of 10 mg protein/ml. A 0.5-ml aliquot was used for each of the following three assays. The measurement of malondialdehyde equivalents (MDAs) was performed according to the method described by Uchiyama and Mihara (15). Briefly, the mixture of 0.5 ml sample, 3 ml of 1%phosphoric acid and 1.0 ml of 0.6% thiobarbituric acid was incubated for 45 min at 100" C; this was followed by extraction with 4 ml n-butanol. The supernatant was analyzed in a spectrophotometer to determine the difference in the absorbance wavelengths of 520 nm and 535 nm. The results were expressed as MDAs. For the assessment of conjugated dienes, microsomal and mitochondrial lipids were extracted by the method of Bligh and Dyer (161, and the absorbance at 234 nm was determined as described previously (8). To determine the level of free 4-HNE, the samples were first incubated with 5 ml of 0.1% 2,4-dinitrophenylhydrazine in ethanol and sulfuric acid (9: 1) in the dark at room
temperature for 12 hr to form dinitrophenylhydrazone derivatives of alkenals (17, 18). These derivatives were extracted with dichloromethane and separated by thin-layer chromatography with dichloromethane and benzene as developers (17, 18). The area corresponding to the dinitrophenyhydrazone derivative of authentic 4-HNE (kindly provided by Professor Hermann Esterbauer of the University of Graz, Graz, Austria) was scraped, extracted with chloroform and methanol (9: 1)) dissolved in 100% methanol and injected into an HPLC reverse-phase column (Ultrasphere, 5 km octadecyl silica gel, 4.6 mm x 25 cm; Alltech Associates, Inc., Deerfield, IL) to separate the 4-HNE derivative (17, 18). A slight modification was incorporated to employ a mobile phase of methanol with a linear gradient of 60% to 100% during the first 15 min, followed by isocratic elution with 100% methanol. The authentic 4-HNE in the amount of 20 to 360 pmol was reacted with dinitrophenyhydrazine, extracted and separated by TLC and HPLC; the derivative was monitored with a detector at a wavelength of 370 nm to establish the standard curve. Analyses of Antioxidants, Prooxidants and Hydroxyproline.
For determination of hepatic concentrations of reduced glutathione (GSH), methionine, a-tocopherol, nonheme iron and hydroxyproline, the portions of the livers were immediately snap-frozen in liquid nitrogen and stored at - 80" C until they were assayed. The GSH level was determined by the method of Griffith (19) as modified by Allen and Arthur (20) using the glutathione reductased, 5'-dithiobis-(2-nitrobenzoicacid) recycling assay. For determination of the liver methionine concentration, the liver was homogenized with 6% sulfosalicylic acid and the supernatant was used for amino acid analysis with a Beckman Model 6300 amino acid analyzer (Beckman Instruments, Inc., Palo Alto, CA). a-Tocopherol was measured by a reverse-phase HPLC method (21))and nonheme iron was measured by a bathophenanthroline sulfonatethioglycolic acid chromogen assay (22). To estimate collagen concentration
I
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4-HYDROXYNONENAL IN EXPERIMENTAL ALCOHOLIC LrVER DISEASE
Vol. 16, No. 2, 1992
Mitochondria
Microsomes
5wk
5 wk
16wk
16wk
Mitochondria 5 wk
16 wk
***
-
Microsomes 5 wk
16 wk
T ***
0Control Ethanol-fed
***
FIG.4. 4-HNE levels in liver mitochondria and microsomes of 5-wk and 16-wk ethanol-fed rats and pair-fed control animals. Note significantly elevated 4-HNE concentrations in the 16-wk ethanol-fed animals compared with the controls (***p < 0.001). Also note that 4-HNE levels in the controls are increased significantly at 16 wk compared with those at 5 wk ( t p < 0.05; Sp < 0.01).
summarized in Figure 3. At wk 5, despite the centrilobular liver necrosis observed in the ethanol-fed rats, FIG.3. MDAs and conjugated d e n e levels in liver mitochondria and these parameters failed to provide evidence of enhanced microsomes from 5-wk and 16-wk ethanol-fed rats and pair-fed control lipid peroxidation in mitochondria or microsomes. In animals. Note consistent increases in MDAs and conjugated diene contrast, both parameters were significantly increased levels in the 16-wk ethanol-fed animals. *p < 0.05; **p < 0.01. at wk 16, when active fibrogenesis ensued. Measurement ***p < 0.001; t p = 0.07 compared with corresponding controls. of 4-HNE in these samples showed a similar pattern of changes: no change at wk 5 and significant elevations in the ethanol-fed animals at wk 16 (Fig. 4). Interestingly, in the liver, the release of hydroxyproline in hydrolysate of the the levels of this aldehyde in the controls were also liver was determined with Ehrlich’s reagent (23). markedly increased at wk 16 compared with those Liver Histological Study and Blood Aminotmnsferase measured at wk 5. A similar trend of an increase in heoye. Liver tissues were fixed in a 10% formalin solution, processed and stained with hematoxylin and eosin and Wilder’s MDAs was also noted in the controls at wk 16 compared reticulin stains for light-microscopical examination. Plasma with the early time point, even though the differences AST and ALT levels were determined on a Technicon high- were not significant. Table 2 summarizes the hepatic concentrations of speed computer-controlled biochemical analyzer (Technicon Instruments Corp., Tarrytown, NY). antioxidants and prooxidants. At wk 5, significant and marked increases were seen in the hepatic concentraRESULTS tions of methionine and a-tocopherol, whereas no Histological study of liver tissues (Figs. 1 and 2), change in GSH was noted. At wk 16, on the other hand, plasma aminotransferase levels and liver hydroxy- both methionine and GSH concentrations were signifiproline content (Table 1) confirmed the pathological cantly decreased, and the increased a-tocopherol level evolution of experimental ALD previously characterized was no longer present. The hepatic concentration of in this model (13). At wk 5, initiation of centrilobular nonheme iron, a known prooxidant, was not increased liver necrosis with local inflammation was evident (Fig. by ethanol feeding but was, rather, decreased at wk 16. 1); it was accompanied by moderate hepatomegaly The results obtained indicated the association of lipid ( 40%) and two- to fivefold increases in plasma AST and peroxidation with liver fibrogenesis. To statistically ALT levels (Table 1).At wk 16, induction of liver fibrosis address this question, the correlation of the combined was demonstrated by the presence of reticulin fibers 4-HNE level in liver microsomes and mitochondria with around and extending from central veins (Fig. 2). The the hepatic concentration of hydroxyproline was anahepatomegaly became more pronounced, with an 87% lyzed. The result shows a significant, positive correlation increase in the average liver weight, and the aminotrans- between these parameters, indicating the link between ferase levels continued to be elevated two to five times lipid peroxidation and liver fibrogenesis (Fig. 5 ) . compared with levels in control animals. The liver DISCUSSION hydroxyproline content was increased threefold in the alcohol-fed animals (Table 11,providing the biochemical In our previous study with the same model of ALD, we confirmation of liver fibrogenesis. proposed that enhanced hepatic lipid peroxidation might Results of MDA and conjugated diene assays are not be required for induction of alcoholic centrilobular
+
452
KAMIMURA ET AL.
.**
*/
r.0.61 (PC0.05) Y=l1.8X-25.415 (n.13)
1.7
2.1
2.5
2.9
3.3
3.7
4.1
Hepatic concentration of hydroxyproline (pmolelg liver)
FIG.5. Correlation of the combined 4-HNE concentrations in liver microsomes and mitochondria with the hepatic concentration of hydroxyproline in the ethanol-fed rats.
liver necrosis (8). This proposal was derived from the observation that initiation of this pathological lesion was not accompanied by biochemical evidence of increased lipid peroxidation, as assessed by the level of conjugated dienes, or depressed GSH levels. Such dissociation has been confirmed by this study, which employed two additional parameters of lipid peroxidation (MDAs and 4-HNE) and the measurement of a-tocopherol, another important antioxidant. All three parameters for lipid peroxidation consistently show negative results at a time when the livers are undergoing induction of centrilobular liver necrosis (Figs. 1-4). In fact, the level of mitochondrial conjugated dienes is lower in these animals than in the controls, indicating a lower level of mitochondrial lipid peroxidation (Fig. 3). Similar observations were previously made by us (8)and others (24); this may reflect the reduced vulnerability of membrane lipids toward peroxidation by ethanol-induced reductions in highly unsaturated fatty acids (24). Alternatively, it may be related to the increased protective effects by the elevated levels of antioxidants in the ethanol-fed animals at this stage of ALD (Table 2). In particular, the liver concentration of a-tocopherol is markedly increased, more than threefold. This change and the increased liver methionine level in the 5-wk ethanol-fed animals may represent their mobilization defense response to the increased oxidative stress in the liver, as it was previously shown in the lung (25). In contrast, induction of active alcoholic liver fibrogenesis is associated with enhanced lipid peroxidation (Figs. 3 and 4) and decreased antioxidant levels (GSH and methionine) (Table 2). This can be interpreted to indicate that progressive liver injury results in compromised antioxidant systems because of overconsumption by the prolonged oxidant stress or because of impaired
HEPATOLOGY
homeostasis of antioxidants caused by other mechanisms. In any event, at this advanced stage of ALD antioxidant systems are surpassed by the oxidant stress, resulting in the net increase in lipid peroxidation. It is intriguing to note that this peroxidative change becomes evident only at the advanced fibrotic stage of ALD and not at the onset of centrilobular liver necrosis. The source of oxidant stress in these animals may be microsomal cytochrome P-450 2E1 coupled with NADPH reductase (9, 11,26), xanthine oxidase (27) or mitochondrial respiratory chain (28), all of which are theoretically capable of generating oxygen-centered free radicals. In this respect, we recently showed that microsomes isolated from this ALD model have marked induction of cytochrome P-450 2E1 and that this cytochrome is responsible in large part for the enhanced basal rate of microsomal lipid peroxidation in uitro (29). The mechanistic relationship between lipid peroxidation and fibrogenesis was proposed by Chojkier and colleagues in a study in which increased collagen gene expression was demonstrated in uitro by fibroblasts exposed to ascorbic acid (30). It was concluded that this stimulatory effect was mediated by formation of aldehydic metabolic products of lipid peroxidation induced by ascorbic acid (30).Our study shows a significant and positive correlation between the liver concentration of 4-HNE (Fig. 5) or MDAs (data not shown) and that of hydroxyproline, the parameter used to assess the degree of liver fibrosis. This in uiuo result supports the aforementioned hypothesis. 4-HNE is the main aldehyde produced after peroxidation of 0-6 polyunsaturated fatty acids (31, 32) and detected as a free form or adducts in several lipid peroxidation models such as hepatotoxicity induced by bromobenzene (181, ally1 alcohol (33) or iron overloading (34) and in ADP-Fez induced microsomal peroxidation (17) and oxidized low-density lipoprotein in atheromas (35). As far as we know, this study is the first to report detection of increased 4-HNE in ALD. This aldehyde is highly reactive toward sulfkydryl compounds and inhibits many -SH enzymes at high concentrations (32). At low concentrations, on the other hand, 4-HNE stimulates key enzymes involved in intracellular signal transduction such as adenylate cyclase (36) and phosphotidylinositol-4,5-bisphosphate-phospholipaseC (37, 38). Furthermore, it induces gene expression of heat-shock proteins (39) which may be mediated through formation of a DNA binding protein (40). In light of these effects of biological importance, 4-HNE, along with other aldehydes, may have influence over gene expression, synthesis or secretion of extracellular matrix proteins and glycoproteins. +
Acknowledgments: This paper is dedicated to the late Professor Richard 0. Recknagel of Case Western Reserve University, who inspired many of us through his spirited teaching and research in the field of lipid peroxidation. We also wish to thank Professor Hermann Esterbauer of the University of Graz for his generous gift of authentic 4-HNE.
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KARL GAAL,l* ROBERT s. BRITTON,3 BRUCER.BACON,3 GEORGE TRIADAFTLOPOULOS~. AND HIDEKAZU TSUKAMOTO',
SEIICHIRO KAMIMURA,',
'Division of Gastroenterology, Department of Medicine, Veterans Affairs Medical Center, Martinez 94553; 2University of California, Davis, California 95616; and 3Division of Gastroenterology and Hepatology, St. Louis University School of Medicine, St. Louis, Missouri 63110-0250
The precise role of lipid peroxidation in the pathogenesis of alcoholic liver disease is still being debated. To explore the issue, this study was undertaken to investigate the status of lipid peroxidation, antioxidants and prooxidants at two discrete stages of experimental alcoholic liver disease. Male Wistar rats were intragastrically fed a high-fat diet plus ethanol for 5 or 16 wk (the duration that resulted in initiation of centrilobular liver necrosis or liver fibrosis, respectively). Lipid peroxidation was assessed in isolated microsomes and mitochondria with three parameters: malondialdehyde equivalents as determined by thiobarbituric acid assay, conjugated diene formation and 4-hydroxynonenal as a 2,4dinitrophenylhydrazone derivative. To assess antioxidant systems, hepatic concentrations of glutathione, methionine and a-tocopherol were determined. The concentration of nonheme iron, a known prooxidant, was also measured. At wk 5, centrilobular liver necrosis was already evident in the ethanol-fedanimals, with two- or threefold increases in plasma AST and ALT levels. At this stage, neither malondialdehyde equivalentsnor conjugated diene values were elevated, and the 4-hydroxynonemallevel was below 0.2 nmol/mg protein. Hepatic concentrations of methionine and a-tocopherol in these animals were increased twoand threefold, respectively, whereas the reduced glutathione level remained unchanged. When alcoholic liver disease had progressed to perivenular or bridging fibrosis at wk 16, all three parameters of lipid peroxidation showed consistent increases that were accompanied by significant reductions in the hepatic glutathione and methionine levels. Interestingly, the control animals pair-fedwith the high-fat diet also had Received December 26, 1991; accepted March 23, 1992. This study was presented during Digestive Disease Week,May 19-22,1991,in New Orleans, Louisiana and published as an abstract (Gastroenterology 1991;100:A758). This study was supported by US. Public Health Service grant AA06603 and by the Department of Veterans Affairs. Address reprint requests to:Hidekazu Tsukamoto, D.V.M., Ph.D., Division of Gastroenterology and Hepatology, MetroHealth Medical Center, Case Western Reserve University, 2500 MetroHealth Dr., Cleveland, OH 44109. 31/1/38332
significantlyelevated 4-hydroxynonenallevels at wk 16 compared to the wk 5 level. The liver concentration of nonheme *an was not increased at wk 5 or wk 16. These results demonstrate (a) dissociation between the initiation of alcoholic liver necrosis and enhanced lipid peroxidation, (b) association of enhanced lipid peroxidation with liver fibrogenesis and depressed antioxidant system, ( c ) the first demonstration of increased 4-hydroxynonenallevel in experimental alcoholic liver disease and (d)possible implication of a high-fat diet in hepatic 4-hydroxynonenal generation. (HEPATOLOGY 1992;16:448-453.)
Peroxidative destruction of biological membranes is believed to be an underlying mechanism of a variety of hepatotoxic diseases (1, 2). However, the role of lipid peroxidation in alcoholic liver disease (ALD)is still a subject of controversy (3-8).Several studies have shown enhanced lipid peroxidation in animals rendered acutely or chronically alcoholic (3-5)and in patients with ALD (6); however, others could not confirm this result (7,8). The hypothesis that incriminates lipid peroxidation as an initiating event for alcohol-induced liver necrosis is based on (a) enhanced levels of conjugated dienes, thiobarbituric acid-reactive substances or chemiluminescence in liver homogenates or subcellular fractions (3-6); (b) decreased levels of antioxidants such as glutathione (4-6); and (c) increased generation of free radicals by liver microsomes of animals fed ethanol for long periods (9-11). However, these studies failed to provide critical information on a correlation between hepatocellular necrosis and the parameters used to put forth the hypothesis. In fact, the animal models used in some of these studies did not produce hepatocellular necrosis (3, 51, and this made it impossible to interpret the data in terms of the pathogenesis of alcoholic liver necrosis. Furthermore, even if the enhanced lipid peroxidation was evidenced in the animal model (4) or patients (6) with advanced ALD, careful longitudinal approaches were not taken to conclude whether this phenomenon was a cause or a consequence of hepatocellular necrosis. Increased hepatic lipid peroxidation
448
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4-JXYDROXYNONENAL IN EXPERIMENTAL ALCOHOLIC LrVER DISEASE
449
FIG.1.Alcoholic centrilobular liver necrosis and inflammation of the liver from a 5-wk ethanol-fed rat iH&E; original magnification x 100).
FIG.2. Bridging fibrosis of the liver from a 16-wk ethanol-fed rat (reticulin stain; original magnification x 40).
may simply result from the liver necrosis attributable to other underlying mechanisms, but this peroxidative change may still constitute an important chemical stimulus to induce liver fibrogenesis as postulated by an in uitro study with fibroblasts (7). To provide insights into these unanswered questions, we designed this study to examine the status of lipid peroxidation and antioxidant systems in rat livers at the two stages of experimental ALD: the early stage of centrilobular liver necrosis and the advanced stage of active liver fibrogenesis. For assessment of hepatic lipid peroxidation, the level of 4-hydroxynonenal (4-HNE1,
one of the most common and important aldehydic products of lipid peroxidation, was measured in addition to two other commonly employed parameters, namely, thiobarbituric acid-reactive substances and conjugated dienes. MATERIALS AND METHODS Animals. Male Wistar rats (initialbody weight = 350 to 400 gm) were aseptically fitted with long-term gastrostomy catheters and continuously infused with a high-fat diet plus ethanol or isocaloric glucose solution for 5 and 16 wk (8, 12).
These durations have been shown to result in the initiation of
450
KAMIMURA ET AL.
HEPATOLOGY
TABLE1. Body and liver weights, serum transaminase levels and hepatic hydroxyproline contents 5 wk Parameters
Body weight gain (gm)
Liver weight (gm) AST (UL)
ALT (UL) Hydroxyproline (pmolfliver)
"Data expressed as mean
16 w k
Control
Alcohol-fed
108.8 % 13.7" 15.2 ? 2.0 88.0 f 9.5 25.7 f 2.8 30.3 t 5.9
94.0 f 8.7 21.5 f 1.8' 202.6 f 43.2d 126.1 f 55.6' 63.1 f 10.4
Control
219.5 ? 17.4 2 137.6 ? 23.4 ? 36.8 ?
Alcohol-fed
19.4 1.0 22.8 2.5 2.4
217.8 ? 16.9 32.5 -f 1.4' 329.4 t 50.8d 155.8 t 44.1d 107.3 t 4.0'
+ S.E.M.
'p < 0.05. cp < 0.001. dp < 0.01. ep = 0.073.
TABLE 2. Hepatic concentration of antioxidants and prooxidants 5 wk
16 w k
Parameters
Control
Alcohol-fed
Control
Alcohol-fed
GSH (WmoUgm liver) Methionine (nmougm liver) a-Tocopherol ( ~ g / g m liver) Nonheme iron (pg/gm liver)
8.46 -f 0.71" 50.0 f 9.0 16.4 ? 4.1 124.3 f 10.0
8.18 f 0.49 99.0 ? 9.06 50.9 ? 9.2' 102.7 f 9.9
8.18 f 0.30 33.6 f 5.6 31.3 t 2.2 149.2 2 6.8
6.82 rf: 0.57' 17.9 f 0.9' 27.7 ? 4.2 120.0 f 4.8'
"Data expressed as mean
f
S.E.M.
'p < 0.05. 'p < 0.01.
centrilobular liver necrosis and active liver fibrogenesis, respectively, in the ethanol-fed rats (13). The dose of ethanol was progressively increased to 42% and 49% of the total calories at the end of these periods, respectively. At the termination points, the animals were anesthetized with sodium pentobarbital, a blood sample was taken from the inferior vena cava and the liver was quickly removed for subcellular fractionation. All animals received humane care. Use of animals in this study was approved by the Institutional Animal Care and Use Committee of the Veterans Af'f'airs Medical Center, Martinez, CA, and was in compliance with guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" (NIH publication no. 86-23, revised 1985). Assessment of Lipid Peroxidation. Mitochondria and microsomes were isolated by differential centrifugation with 0.25 m o m sucrose and 5 mmol/L EDTA homogenization solution (8, 14). Isolated fractions were resuspended in 1.15% KCl containing 0.2% butylated hydroxytoluene at a concentration of 10 mg protein/ml. A 0.5-ml aliquot was used for each of the following three assays. The measurement of malondialdehyde equivalents (MDAs) was performed according to the method described by Uchiyama and Mihara (15). Briefly, the mixture of 0.5 ml sample, 3 ml of 1%phosphoric acid and 1.0 ml of 0.6% thiobarbituric acid was incubated for 45 min at 100" C; this was followed by extraction with 4 ml n-butanol. The supernatant was analyzed in a spectrophotometer to determine the difference in the absorbance wavelengths of 520 nm and 535 nm. The results were expressed as MDAs. For the assessment of conjugated dienes, microsomal and mitochondrial lipids were extracted by the method of Bligh and Dyer (161, and the absorbance at 234 nm was determined as described previously (8). To determine the level of free 4-HNE, the samples were first incubated with 5 ml of 0.1% 2,4-dinitrophenylhydrazine in ethanol and sulfuric acid (9: 1) in the dark at room
temperature for 12 hr to form dinitrophenylhydrazone derivatives of alkenals (17, 18). These derivatives were extracted with dichloromethane and separated by thin-layer chromatography with dichloromethane and benzene as developers (17, 18). The area corresponding to the dinitrophenyhydrazone derivative of authentic 4-HNE (kindly provided by Professor Hermann Esterbauer of the University of Graz, Graz, Austria) was scraped, extracted with chloroform and methanol (9: 1)) dissolved in 100% methanol and injected into an HPLC reverse-phase column (Ultrasphere, 5 km octadecyl silica gel, 4.6 mm x 25 cm; Alltech Associates, Inc., Deerfield, IL) to separate the 4-HNE derivative (17, 18). A slight modification was incorporated to employ a mobile phase of methanol with a linear gradient of 60% to 100% during the first 15 min, followed by isocratic elution with 100% methanol. The authentic 4-HNE in the amount of 20 to 360 pmol was reacted with dinitrophenyhydrazine, extracted and separated by TLC and HPLC; the derivative was monitored with a detector at a wavelength of 370 nm to establish the standard curve. Analyses of Antioxidants, Prooxidants and Hydroxyproline.
For determination of hepatic concentrations of reduced glutathione (GSH), methionine, a-tocopherol, nonheme iron and hydroxyproline, the portions of the livers were immediately snap-frozen in liquid nitrogen and stored at - 80" C until they were assayed. The GSH level was determined by the method of Griffith (19) as modified by Allen and Arthur (20) using the glutathione reductased, 5'-dithiobis-(2-nitrobenzoicacid) recycling assay. For determination of the liver methionine concentration, the liver was homogenized with 6% sulfosalicylic acid and the supernatant was used for amino acid analysis with a Beckman Model 6300 amino acid analyzer (Beckman Instruments, Inc., Palo Alto, CA). a-Tocopherol was measured by a reverse-phase HPLC method (21))and nonheme iron was measured by a bathophenanthroline sulfonatethioglycolic acid chromogen assay (22). To estimate collagen concentration
I
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4-HYDROXYNONENAL IN EXPERIMENTAL ALCOHOLIC LrVER DISEASE
Vol. 16, No. 2, 1992
Mitochondria
Microsomes
5wk
5 wk
16wk
16wk
Mitochondria 5 wk
16 wk
***
-
Microsomes 5 wk
16 wk
T ***
0Control Ethanol-fed
***
FIG.4. 4-HNE levels in liver mitochondria and microsomes of 5-wk and 16-wk ethanol-fed rats and pair-fed control animals. Note significantly elevated 4-HNE concentrations in the 16-wk ethanol-fed animals compared with the controls (***p < 0.001). Also note that 4-HNE levels in the controls are increased significantly at 16 wk compared with those at 5 wk ( t p < 0.05; Sp < 0.01).
summarized in Figure 3. At wk 5, despite the centrilobular liver necrosis observed in the ethanol-fed rats, FIG.3. MDAs and conjugated d e n e levels in liver mitochondria and these parameters failed to provide evidence of enhanced microsomes from 5-wk and 16-wk ethanol-fed rats and pair-fed control lipid peroxidation in mitochondria or microsomes. In animals. Note consistent increases in MDAs and conjugated diene contrast, both parameters were significantly increased levels in the 16-wk ethanol-fed animals. *p < 0.05; **p < 0.01. at wk 16, when active fibrogenesis ensued. Measurement ***p < 0.001; t p = 0.07 compared with corresponding controls. of 4-HNE in these samples showed a similar pattern of changes: no change at wk 5 and significant elevations in the ethanol-fed animals at wk 16 (Fig. 4). Interestingly, in the liver, the release of hydroxyproline in hydrolysate of the the levels of this aldehyde in the controls were also liver was determined with Ehrlich’s reagent (23). markedly increased at wk 16 compared with those Liver Histological Study and Blood Aminotmnsferase measured at wk 5. A similar trend of an increase in heoye. Liver tissues were fixed in a 10% formalin solution, processed and stained with hematoxylin and eosin and Wilder’s MDAs was also noted in the controls at wk 16 compared reticulin stains for light-microscopical examination. Plasma with the early time point, even though the differences AST and ALT levels were determined on a Technicon high- were not significant. Table 2 summarizes the hepatic concentrations of speed computer-controlled biochemical analyzer (Technicon Instruments Corp., Tarrytown, NY). antioxidants and prooxidants. At wk 5, significant and marked increases were seen in the hepatic concentraRESULTS tions of methionine and a-tocopherol, whereas no Histological study of liver tissues (Figs. 1 and 2), change in GSH was noted. At wk 16, on the other hand, plasma aminotransferase levels and liver hydroxy- both methionine and GSH concentrations were signifiproline content (Table 1) confirmed the pathological cantly decreased, and the increased a-tocopherol level evolution of experimental ALD previously characterized was no longer present. The hepatic concentration of in this model (13). At wk 5, initiation of centrilobular nonheme iron, a known prooxidant, was not increased liver necrosis with local inflammation was evident (Fig. by ethanol feeding but was, rather, decreased at wk 16. 1); it was accompanied by moderate hepatomegaly The results obtained indicated the association of lipid ( 40%) and two- to fivefold increases in plasma AST and peroxidation with liver fibrogenesis. To statistically ALT levels (Table 1).At wk 16, induction of liver fibrosis address this question, the correlation of the combined was demonstrated by the presence of reticulin fibers 4-HNE level in liver microsomes and mitochondria with around and extending from central veins (Fig. 2). The the hepatic concentration of hydroxyproline was anahepatomegaly became more pronounced, with an 87% lyzed. The result shows a significant, positive correlation increase in the average liver weight, and the aminotrans- between these parameters, indicating the link between ferase levels continued to be elevated two to five times lipid peroxidation and liver fibrogenesis (Fig. 5 ) . compared with levels in control animals. The liver DISCUSSION hydroxyproline content was increased threefold in the alcohol-fed animals (Table 11,providing the biochemical In our previous study with the same model of ALD, we confirmation of liver fibrogenesis. proposed that enhanced hepatic lipid peroxidation might Results of MDA and conjugated diene assays are not be required for induction of alcoholic centrilobular
+
452
KAMIMURA ET AL.
.**
*/
r.0.61 (PC0.05) Y=l1.8X-25.415 (n.13)
1.7
2.1
2.5
2.9
3.3
3.7
4.1
Hepatic concentration of hydroxyproline (pmolelg liver)
FIG.5. Correlation of the combined 4-HNE concentrations in liver microsomes and mitochondria with the hepatic concentration of hydroxyproline in the ethanol-fed rats.
liver necrosis (8). This proposal was derived from the observation that initiation of this pathological lesion was not accompanied by biochemical evidence of increased lipid peroxidation, as assessed by the level of conjugated dienes, or depressed GSH levels. Such dissociation has been confirmed by this study, which employed two additional parameters of lipid peroxidation (MDAs and 4-HNE) and the measurement of a-tocopherol, another important antioxidant. All three parameters for lipid peroxidation consistently show negative results at a time when the livers are undergoing induction of centrilobular liver necrosis (Figs. 1-4). In fact, the level of mitochondrial conjugated dienes is lower in these animals than in the controls, indicating a lower level of mitochondrial lipid peroxidation (Fig. 3). Similar observations were previously made by us (8)and others (24); this may reflect the reduced vulnerability of membrane lipids toward peroxidation by ethanol-induced reductions in highly unsaturated fatty acids (24). Alternatively, it may be related to the increased protective effects by the elevated levels of antioxidants in the ethanol-fed animals at this stage of ALD (Table 2). In particular, the liver concentration of a-tocopherol is markedly increased, more than threefold. This change and the increased liver methionine level in the 5-wk ethanol-fed animals may represent their mobilization defense response to the increased oxidative stress in the liver, as it was previously shown in the lung (25). In contrast, induction of active alcoholic liver fibrogenesis is associated with enhanced lipid peroxidation (Figs. 3 and 4) and decreased antioxidant levels (GSH and methionine) (Table 2). This can be interpreted to indicate that progressive liver injury results in compromised antioxidant systems because of overconsumption by the prolonged oxidant stress or because of impaired
HEPATOLOGY
homeostasis of antioxidants caused by other mechanisms. In any event, at this advanced stage of ALD antioxidant systems are surpassed by the oxidant stress, resulting in the net increase in lipid peroxidation. It is intriguing to note that this peroxidative change becomes evident only at the advanced fibrotic stage of ALD and not at the onset of centrilobular liver necrosis. The source of oxidant stress in these animals may be microsomal cytochrome P-450 2E1 coupled with NADPH reductase (9, 11,26), xanthine oxidase (27) or mitochondrial respiratory chain (28), all of which are theoretically capable of generating oxygen-centered free radicals. In this respect, we recently showed that microsomes isolated from this ALD model have marked induction of cytochrome P-450 2E1 and that this cytochrome is responsible in large part for the enhanced basal rate of microsomal lipid peroxidation in uitro (29). The mechanistic relationship between lipid peroxidation and fibrogenesis was proposed by Chojkier and colleagues in a study in which increased collagen gene expression was demonstrated in uitro by fibroblasts exposed to ascorbic acid (30). It was concluded that this stimulatory effect was mediated by formation of aldehydic metabolic products of lipid peroxidation induced by ascorbic acid (30).Our study shows a significant and positive correlation between the liver concentration of 4-HNE (Fig. 5) or MDAs (data not shown) and that of hydroxyproline, the parameter used to assess the degree of liver fibrosis. This in uiuo result supports the aforementioned hypothesis. 4-HNE is the main aldehyde produced after peroxidation of 0-6 polyunsaturated fatty acids (31, 32) and detected as a free form or adducts in several lipid peroxidation models such as hepatotoxicity induced by bromobenzene (181, ally1 alcohol (33) or iron overloading (34) and in ADP-Fez induced microsomal peroxidation (17) and oxidized low-density lipoprotein in atheromas (35). As far as we know, this study is the first to report detection of increased 4-HNE in ALD. This aldehyde is highly reactive toward sulfkydryl compounds and inhibits many -SH enzymes at high concentrations (32). At low concentrations, on the other hand, 4-HNE stimulates key enzymes involved in intracellular signal transduction such as adenylate cyclase (36) and phosphotidylinositol-4,5-bisphosphate-phospholipaseC (37, 38). Furthermore, it induces gene expression of heat-shock proteins (39) which may be mediated through formation of a DNA binding protein (40). In light of these effects of biological importance, 4-HNE, along with other aldehydes, may have influence over gene expression, synthesis or secretion of extracellular matrix proteins and glycoproteins. +
Acknowledgments: This paper is dedicated to the late Professor Richard 0. Recknagel of Case Western Reserve University, who inspired many of us through his spirited teaching and research in the field of lipid peroxidation. We also wish to thank Professor Hermann Esterbauer of the University of Graz for his generous gift of authentic 4-HNE.
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4-HYDROXYNONENALIN EXPERIMENTAL ALCOHOLIC LIVER DISEASE
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