Department of Pathology (S.M.H.S., A.A.N.), Beth Israel Deaconess
Medical Center and Harvard Medical, Boston, Massachusetts and
Biomedical Frontiers Inc. (P.E.H.) Minneapolis, Minnesota
We studied the effect of the long-acting parenteral iron chelator,
hydroxyethyl starch deferoxamine (HES-DFO) on liver nonheme iron, lipid
peroxidation and pathologic changes in the liver in the intragastric
feeding rat model for alcoholic liver disease. Male Wistar rats
(225-250 g) were fed liquid diet and ethanol for 2 months. In control
pair-fed animals, ethanol was isocalorically replaced by dextrose. Two
additional groups of animals (dextrose and ethanol fed) received
HES-DFO (25 mg deferoxamine equivalents/kg, three times a week). The
blood ethanol level in the ethanol-fed animals was maintained between
150 and 350 mg/dl. For each animal, the levels of hepatic nonheme iron,
lipid peroxidation and pathologic changes were evaluated. Ethanol
administration caused fatty liver, necrosis and inflammation. Addition
of HES-DFO to the ethanol diet increased the severity of pathologic
changes, particularly necrosis and inflammation. The nonheme iron in
alcohol-fed animals was significantly higher (18.3 ± 4.3 µg
liver) than in pair-fed dextrose controls (12.5 ± 1.5 µg,
P < .05). Addition of HES-DFO significantly increased nonheme
iron levels in the dextrose-fed rats (17.1 ± 2.0 µg/g, P < .02) but not in ethanol-fed rats (20.0 ± 2.0). Ethanol
increased levels of conjugated dienes; these levels were not altered by
HES-DFO. The most significant observations in this study were: 1) the
higher hepatic nonheme iron content in ethanol-fed rats compared with
pair-fed dextrose controls; 2) the absence of changes in hepatic
nonheme iron levels or lipid peroxidation in ethanol-fed groups treated
with HES-DFO; and 3) the worsening of liver injury in ethanol-fed rats
by HES-DFO.
 |
Introduction |
One of the mechanisms invoked to
explain alcoholic liver injury is an increase in the formation of
oxygen free radicals and lipid peroxidation (Cederbaum, 1989
; Nanji
et al., 1994; Reinke et al., 1987). Although the
mechanisms involved in lipid peroxidation are not completely
understood, iron, and especially catalytic iron, has been implicated as
an initiator of lipid peroxidation (Bacon and Britton, 1990; Minotti,
1992; Minotti and Aust, 1989). Although alcoholism and ALD per
se are not associated etiologically with marked hepatic iron
overload, there is growing evidence that only mild degrees of iron
overload are sufficient to enhance alcoholic-induced liver injury
(Bonkovsky et al., 1996). Recently, Tsukamoto et al. (1995), with the intragastric feeding rat model for ALD,
showed that when iron was supplemented in a high fat-ethanol diet to produce only a slight increase in liver iron concentrations, there was
a synergistic increase in levels of hepatic malondialdehyde and
4-hydroxynonenal in the liver. The increase in lipid peroxide levels
was accompanied by enhanced severity of liver injury.
We have previously shown that in ethanol-fed rats, therapy with an oral
iron-chelator, 1,2-dimethyl-3-hydroxypyridin-4-one, led to a reduction
in hepatic-free iron, lipid peroxidation and fat accumulation
(Sadrzadeh et al., 1994). To follow up on these observations, the present study was designed to investigate the efficacy of a more powerful iron chelator, DFO, in preventing alcoholic
liver injury. DFO is an extremely potent iron chelator which has been
shown to be useful in the management of patients with iron overload
(Hershko and Weatherall, 1988). DFO, however, has a very short
half-life which severely limits it utility. Hallaway et al.
(1989) have developed a technique of covalently attaching DFO by its
amino group to various polymers such as HES rendering a high molecular
weight compound (HES-DFO) with iron-binding properties virtually
identical with free DFO. The half-life of HES-DFO is about 22 h.
We tested the effectiveness of HES-DFO in preventing liver injury in
the intragastric feeding rat model for ALD (French et al.,
1986; Tsukamoto et al., 1990). This model allows for the evaluation of therapeutic interventions on ethanol-induced pathologic changes in the liver. When rats are fed polyunsaturated fatty acids
with ethanol, the animals develop pathologic injury which includes
fatty liver, necrosis and inflammation (Nanji et al., 1989;
Nanji and French, 1989).
| |
Materials and Methods |
Animal model.
The experimental animals were male Wistar rats
weighing between 225 and 250 g (Harlan-Sprague Dawley,
Indianapolis, IN). All animals were fed for 2 months by continuous
infusion of a liquid diet through permanently implanted gastric
cannulas, as described previously (Tsukamoto et al., 1990).
The amount of ethanol was initially 8 g/kg/day and increased up to 16 g/kg/day as tolerance developed. Blood alcohol levels were maintained
between 150 mg/d and 350 mg/dl. At the time when the highest ethanol
levels were achieved, the average caloric distribution for each
nutrient was 25% of total calories as fat, 21% as protein, 12% as
carbohydrate and 42% as ethanol. In control animals, ethanol was
isocalorically replaced by dextrose. For rats treated with the iron
chelator (HES-DFO), the dose administered was 25 mg/kg (expressed as
DFO equivalents) given intraperitoneally three times a week for a 2-month period. HES-DFO was kindly donated by Biomedical Frontiers Inc.
(Minneapolis, MN). Development and characterization of this iron
chelator has been described previously (Hallaway et al., 1989). The content of bound DFO in the compound used was 15% (w/w) of
the conjugated form. Both dextrose and ethanol-fed rats were treated
with HES-DFO. All rats tolerated the three-times-weekly injections
without complication. At sacrifice, the animals were anesthetized, and
blood was drawn from the aorta for enzyme measurements. The liver was
perfused with ice-cold, iron-free saline, cut into small pieces and
frozen immediately in liquid nitrogen. All animals received humane care
in accordance with the guidelines issued by the National Institutes of
Health.
Liver pathology.
At the termination of each experiment, a
small piece of liver was removed and fixed in formalin. The samples
were stained with hematoxylin and eosin for light microscopy. The
pathologist who carried out the histologic analysis had no prior
knowledge of the different experimental groups. The liver pathologic
findings were scored as follows (French et al., 1986):
steatosis (the percentage of liver cells containing fat): 1+, less than
25% of cells containing fat; 2+, 26% to 50%; 3+, 51% to 75%; and
4+, more than 75%, inflammation and necrosis: one focus/lobule 1+; two
or more foci/lobule, 2+. The total liver pathology score was calculated
by adding the scores from each of the parameters. At least five fields
in three different sections of the liver were examined.
Biochemical analyses.
Ethanol was measured in the blood by
the alcohol dehydrogenase method (Sigma Chemical Co., St. Louis, MO).
Plasma ALT was measured by an automated method in routine use in our
clinical laboratories. Conjugated dienes were measured according to the method of Recknagel and Glende (1984). Butylated hydroxy toluene (90 µM) was added to the homogenate to prevent lipid peroxidation during
the procedure.
Nonheme iron was determined in liver homogenate, with ferene S, as an
indicator with the molar absorptivity of 35,500 M
1
cm1 at 594 nm (Artiss et al., 1982). The liver
was homogenized in NaCl solution (7 mM NaCl/100 mg tissue) and
centrifuged at 1000 × g for 10 min. The clear
supernatant (150 µl) was mixed with dH2O (150 µl) and
150 µl of thiourea/ascorbate solution (4.4% and 2.68%, in
dH2O). Trichloroacetic acid (150 µl of 40% solution) was
added to the mixture, vortexed and centrifuged for 30-60 s. The
supernatant (500 µl) was then mixed with 125 µl of fresh ferene S
solution (35 mg ferene S in 10 ml of 50% ammonium acetate solution). The mixture was incubated at room temperature for 5 to 10 min, and the
absorbance was read at 594 nm. Control experiments were carried out to
ensure that the measured nonheme iron was not from nonspecific iron
released from ferruginous compounds during the procedures.
DFO measurement in liver.
Quantitation of HES-DFO in liver
samples was accomplished spectrophotometrically by measuring the
concentration of the iron-saturated form of the drug in deproteinized
liver homogenates. A 10% homogenate of liver was prepared by weighing
out a piece of liver weighing several hundred milligrams and then
homogenizing the tissue in the appropriate volume of 76 mM sodium
chloride to yield a final tissue concentration of 10%. Conversion to
the iron-saturated (ferrioxamine) form was accomplished by addition of
100 µl of 100 mM ferrous sulfate solution to 500 µl of liver
homogenate and allowing the mixture to stand for 45 min. The solution
was then deproteinized by adding 30 µl of 100% (w/v) trichloroacetic acid, vortexing thoroughly and then centrifuging the sample for 5 min
at 10,000 × g. The pH of the solution was then
adjusted by adding 250 µl of 2.5 M sodium acetate to 400 µl of
clear supernatant. A blank sample was prepared by use of 500 µl of
water in place of the liver homogenate. The absorbances of the
solutions were determined at 429 nm and the concentration of DFO
calculated with a molar absorptivity of 2700 M1
cm1.
Statistical analysis.
Results are presented as mean ± S.D. Differences between groups were evaluated by analysis of variance,
and multiple comparisons were conducted with the Student-Newman-Keuls
method.
| |
Results |
The weight gain and blood ethanol levels (range, 150-350 mg/dl)
were not significantly different among the various experimental groups.
Animals treated with ethanol had a significantly greater (P < .01) degree of pathologic injury (pathology score, 4.4 ± 0.8; n = 5) than dextrose-fed controls (0.4 ± 0.2, n = 5). Administration of HES-DFO with ethanol resulted
in more severe injury (pathology score, 6.6 ± 0.5;
n = 5) (P < .02 compared with the ethanol-treated group). In particular, the degree of necrosis and inflammation was
greater in the HES-DFO-treated rats (table 1, fig.
1, B and C). The increased severity of liver injury was
confirmed by measurements of ALT in plasma (fig. 2).
HES-DFO did not enhance liver injury in dextrose-fed rats (pathology
score, 0.9 ± 0.4).
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Fig. 1.
(A) The section of the liver from a rat fed
dextrose and corn oil for 2 months shows normal histology with no
evidence of pathologic change (hematoxylin and eosin, magnification
×155). (B) Section of the liver from a rat fed corn oil and ethanol
for a 2-month period. The pathologic changes present include fatty liver (arrow head) and focal necrosis and inflammation (small arrow)
(hematoxylin and eosin, magnification ×155). (C) Liver from a rat fed
corn oil and ethanol with HES-DFO for 2 months. The severity of
necrosis and inflammatory change is increased compared with the corn
oil-ethanol-fed rat (small arrows) (hematoxylin and eosin,
magnification ×155).
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Fig. 2.
Measurements of plasma ALT in the various
experimental groups (n = 5 rats/group).
Administration of ethanol led to a significant increase (P < .01)
in ALT levels (45 ± 5 U/l) compared with dextrose-fed controls
(19 ± 2 U/l). Treatment with HES-DFO led to a significant increase (P < .01) in ALT levels (100 ± 301 U/l) compared
with the ethanol-treated rats and dextrose-fed rats treated with
HES-DFO (28 ± 2 U/l). *P < .01 vs. Dex;
**P < .01 vs. ETOH and H-DFO. Dex, dextrose; ETOH,
ethanol; H-DFO, HES-DFO.
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The concentrations of liver nonheme iron (µg/g wet weight) in the
different treatment groups are shown in figure 3.
Ethanol feeding resulted in a significant increase (P < .05) in
liver nonheme iron (18.3 ± 4.3 µg/g wet weight) compared with
the dextrose-fed group (12.5 ± 1.5 µg/g wet weight). Treatment
with HES-DFO significantly increased (P < .02) liver nonheme iron
in dextrose-fed rats (17.1 ± 2.0 µg/g liver). Administration of
HES-DFO to ethanol-fed animals did not result in any further increase
in nonheme iron levels (20.0 ± 2.0 µg/g liver).
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Fig. 3.
Changes in hepatic nonheme iron (micrograms per
gram) in the different experimental groups. Ethanol (ETOH)
significantly increased nonheme iron (18.3 ± 4.3) compared with
the dextrose (DEX) control (12.5 ± 1.5). Dextrose-fed rats
treated with HES-DFO (H-DFO) had significantly higher levels of nonheme
iron (17.1 ± 2.0) than the dextrose-fed rats. HES-DFO treatment
of ethanol-fed rats did not significantly alter the liver iron
concentration (20.0 ± 2.0) compared with ethanol-fed rats
(n = 5 rats/group).
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The extent of liver lipid peroxidation, expressed as conjugated dienes,
in the various experimental groups is shown in figure 4.
Ethanol feeding resulted in 2-fold increase in conjugated diene formation compared with dextrose-fed controls. Treatment with HES-DFO
did not increase conjugated diene levels in either dextrose-fed or
ethanol-fed rats. Measurement of DFO levels in liver confirmed that DFO
did indeed accumulate in the liver. The levels of DFO were not
significantly different in the dextrose-HES-DFO group (92 ± 27 µg/g) than in the respective ethanol group (112 ± 15 µg/g).
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Fig. 4.
Ethanol (ETOH) feeding increased the level of
conjugated dienes (0.24 ± 0.05) compared with dextrose (DEX)-fed
controls (0.12 ± 0.03, P < .01). Addition of HES-DFO
(H-DFO) to the dextrose-fed group did not increase levels of conjugated
dienes (0.14 ± 0.06). Ethanol-fed rats given HES-DFO also did not
show a significant increase in conjugated diene levels (0.29 ± 0.12) compared with ethanol-fed rats (n = 5 rats/group).
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Discussion |
Iron within the liver is found in several biochemical forms such
as ferritin, hemosiderin, heme and the putative "intracellular low
molecular weight" chelate pool (Voogd et al., 1992).
Although the exact identity of this low molecular weight iron is not
known, it is apparently bound to weak chelators such as AMP and ATP. In
this form, iron is able to catalyze free radical formation and lipid
peroxidation. Despite convincing clinical and experimental evidence for
liver injury as a consequence of excess iron, the specific
pathophysiologic mechanisms leading to hepatocellular damage are poorly
understood (Bonkovsky, 1991; Pietrangelo et al., 1995). One
of the suggested mechanisms leading to iron-mediated cell injury is
membrane lipid peroxidation. The reaction of nonheme iron with
molecular oxygen and/or hydrogen peroxide is currently envisioned as
the most likely source of reactive oxygen species (Minotti, 1992).
Relevant to the role of iron in ALD is the observation by Hulcrantz
et al. (1991) who showed that incubation of hepatocytes with
ethanol led to the generation of a lipid chemoattractant, the
production of which was blocked by DFO. The investigators suggested a
therapeutic role for iron chelation in ALD.
We have recently shown that treating ethanol-fed rats with an oral iron
chelator resulted in lower levels of nonheme iron and fat deposition in
the liver (Sadrzadeh et al., 1994). The iron chelator used
was 1,2-dimethyl-3-hydroxyprid-4-one (L1) which is a member
of bidentate orally effective iron chelators. These compounds have been
shown to be effective iron chelators both in vitro and in
various animal models (Brittenham, 1992). To follow up on this
observation, we used a potentially more potent iron chelator DFO. The
efficacy of DFO is limited because of its short half-life in plasma
(Hershko and Weatherall, 1988). In addition, chronic use of DFO is
usually associated with toxicity. To increase its half-life, DFO has
been covalently bound to biocompatible high molecular weight polymers
such as HES. HES-DFO retains the equivalent iron-chelating properties
of free DFO and exhibits lower toxicity (Hallaway et al.,
1989). To our surprise, HES-DFO was ineffective in lowering hepatic
levels of nonheme iron or reducing lipid peroxidation. Additionally,
the severity of pathologic changes in the liver of rats fed HES-DFO and
ethanol chronically was increased. Although the absence of change in
lipid peroxidation in HES-DFO-ethanol-fed rats was not anticipated,
this may not be surprising because of the lack of decrease in hepatic
iron.
One possible explanation for the absence of decreased free iron and
lipid peroxidation could be that the high molecular weight and size of
the chelator may prevent its diffusion from the extracellular space
into the intracellular compartment which is where most of the
iron-chelating activity takes place. Although we were able to show that
DFO was present in liver in HES-DFO-treated animals, the exact
intracellular site where DFO accumulates is unknown. Another
possibility is that, under certain conditions, DFO may independently
stimulate lipid peroxidation. One of these conditions, relevant to
alcohol-induced liver injury, is the presence of increased amounts of
lipid in the liver. The amount of DFO required to inhibit lipid
peroxidation is much higher when the lipid concentrations are increased
(Braughler et al., 1988). Finally, the HES-DFO complex with
iron may be more reactive than iron itself. Support for this hypothesis
is provided by studies which show that DFO can react with superoxide
anion radicals, resulting in the formation of a relatively stable
nitroxide free radical (Davies et al., 1987; Morehouse
et al., 1987). Our results which show an absence of protection against liver injury by HES-DFO are in contrast to the
observations of other investigators who have used HES-DFO as an iron
chelator. For example, HES-DFO administered over a 8-week period has
been used successfully to delay the development of diabetes in BB rats
(Roza et al., 1994). In rats in which diabetes was delayed
there were also significantly fewer inflammatory cells in the
pancreatic islets. In a model of ischemia-reperfusion injury, HES-DFO
limited lipid peroxidation and severity of liver injury (Jacobs
et al., 1991). In the ischemia-reperfusion experiment, the
effectiveness of HES-DFO was assessed over a 24-h period. DFO has also
been shown to be protective against other types of toxic liver injury.
DFO given to rats 1 h before a toxic tore of acetaminophen
decreased the mortality rate and the degree of increase in liver
enzymes (Sakaida et al., 1995). The mechanism(s) by which
DFO protected against liver injury involved a reduction in lipid
peroxidation probably secondary to iron chelation.
In conclusion, we have shown that HES-DFO fails to protect against
alcohol-induced liver injury. This observation does not, however,
negate the possible use of iron chelators in ALD. HES-DFO may have been
relatively inactive because of the nonpolar environment in which
iron-mediated free radical injury was occurring. Furthermore, the large
size of the molecule may have prevented access of DFO to the
intracellular site where free radicals were generated.
ALD, alcoholic liver disease;
ALT, alanine
aminotransferase, DFO, deferoxamine;
HES, hydroxyethyl starch.
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Abstract
Introduction
