Antioxidant activity of Vitamin-C in
iron-overloaded human plasma
Berger T.M.; Polidori M.C.; Dabbagh A.; Evans
P.J.; Halliwell B.; Morrow J.D.; Roberts II L.J.;
Frei B.
B. Frei, Whitaker Cardiovascular Inst., Boston
University School of Medicine, 80 East Concord
St., Boston, MA 02118 USA
Journal of Biological Chemistry (USA), 1997,
272/25 (15656-15660)
Vitamin-C (ascorbic acid, AA) can act as an
antioxidant or a pro- oxidant in vitro, depending
on the absence or the presence, respectively, of
redox-active metal ions. Some adults with
iron-overload and some premature infants have
potentially redox-active, bleomycin-detectable
iron (BDI) in their plasma. Thus, it has been
hypothesized that the combination of AA and BDI
causes oxidative damage in vivo. We found that
plasma of preterm infants contains high levels of
AA and F2-isoprostanes, stable lipid peroxidation
end products. However, F2-isoprostane levels were
not different between those infants with BDI (138
plus or minus 51 pg/ml, n = 19) and those without
(126 plus or minus 41 pg/ml, n = 10), and the same
was true for protein carbonyls, a marker of
protein oxidation (0.77 plus or minus 0.31 and
0.68 plus or minus 0.13 nmol/mg protein,
respectively). Incubation of BDI-containing plasma
from preterm infants did not result in detectable
lipid hydroperoxide formation (less than or equal
to10 nM cholesteryl ester hydroperoxides) as long
as AA concentrations remained high. Furthermore,
when excess iron was added to adult plasma, BDI
became detectable, and endogenous AA was rapidly
oxidized. Despite this apparent interaction
between excess iron and endogenous AA, there was
no detectable lipid peroxidation as long as AA was
present at >10% of its initial concentration.
Finally, when iron was added to plasma devoid of
AA, lipid hydroperoxides were formed immediately,
whereas endogenous and exogenous AA delayed the
onset of iron-induced lipid peroxidation in a
dose-dependent manner. These findings demonstrate
that in iron-overloaded plasma, AA acts an
antioxidant toward lipids. Furthermore, our data
do not support the hypothesis that the combination
of high plasma concentrations of AA and BDI, or
BDI alone, causes oxidative damage to lipids and
proteins in vivo.
Effect of
vitamin E supplementation on hepatic fibrogenesis
in chronic dietary iron overload
Brown K.E.; Poulos J.E.; Li L.; Soweid A.M.;
Ramm G.A.; O'Neill R.; Britton R.S.; Bacon B.R.
B.R. Bacon, Div. of Gastroenterology/Hepatology,
Dept. of Internal Medicine, Saint Louis Univ.
Hlth. Sci. Center, 3635 Vista Ave., St. Louis, MO
63110-0250 USA
American Journal of Physiology - Gastrointestinal
and Liver Physiology (USA), 1997, 272/1 35-1
(G116-G123)
It has been suggested that lipid peroxidation
plays an important role in hepatic fibrogenesis
resulting from chronic iron overload. Vitamin E is
an important lipid-soluble antioxidant that has
been shown to be decreased in patients with
hereditary hemochromatosis and in experimental
iron overload. The aim of this study was to
determine the effects of vitamin E supplementation
on hepatic lipid peroxidation and fibrogenesis in
an animal model of chronic iron overload. Rats
were fed the following diets for 4, 8, or 14 mo:
standard laboratory diet (control), diet with
supplemental vitamin E (200 IU/kg, control + E),
diet with carbonyl iron (Fe), and diet with
carbonyl iron supplemented with vitamin E (200
IU/kg, Fe + E). Iron loading resulted in
significant decreases in hepatic and plasma
vitamin E levels at all time points, which were
overcome by vitamin E supplementation.
Thiobarbituric acid-reactive substances (an index
of lipid peroxidation) were increased three- to
fivefold in the iron-loaded livers;
supplementation with vitamin E reduced these
levels by at least 50% at all time points. Hepatic
hydroxyproline levels were increased twofold by
iron loading. Vitamin E did not affect
hydroxyproline content at 4 or 8 mo but caused an
18% reduction at 14 mo in iron-loaded livers. At 8
and 14 mo, vitamin E decreased the number of
alpha-smooth muscle actin-positive stellate cells
in iron-loaded livers. These results demonstrate a
dissociation between lipid peroxidation and
collagen production and suggest that the
profibrogenic action of iron in this model is
mediated through effects which cannot be
completely suppressed by vitamin E.
Iron in
liver diseases other than
hemochromatosis
Bonkovsky H.L.; Banner B.F.; Lambrecht R.W.;
Rubin R.B.
Div. of Digestive Disease/Nutrition, Univ. of
Massachusetts Med. Center, 55 Lake Avenue, North,
Worcester, MA 01655 USA
Seminars in Liver Disease (USA), 1996, 16/1
(65-82)
There is growing evidence that normal or only
mildly increased amounts of iron in the liver can
be damaging, particularly when they are combined
with other hepatotoxic factors such as alcohol,
porphyrogenic drugs, or chronic viral hepatitis.
Iron enhances the pathogenicity of microorganisms,
adversely affects the function of macrophages and
lymphocytes, and enhances fibrogenic pathways, all
of which may increase hepatic injury due to iron
itself or to iron and other factors. Iron may also
be a co-carcinogen or promoter of hepatocellular
carcinoma, even in patients without HC or
cirrhosis. Based on this and other evidence, we
hope that the era of indiscriminate iron
supplementation will come to an end. Bloodletting,
a therapy much in vogue 2 centuries ago, is
deservedly enjoying a renaissance, based on our
current understanding of the toxic effects of iron
and the benefits of its depletion.
Metal-induced
hepatotoxicity
Britton R.S.
Div. of Gastroenterology/Hepatology, Department
of Internal Medicine, Saint Louis Univ. School of
Medicine, 3635 Vista Ave., St. Louis, MO
63110-0250 USA
Seminars in Liver Disease (USA), 1996, 16/1
(3-12)
Figure 3 summarizes several proposed mechanisms
of iron- or copper- induced hepatotoxicity. It has
long been suspected that free radicals may play a
role in iron- and copper-induced cell toxicity
because of the powerful prooxidant action of iron
and copper salts in vitro. In the presence of
available cellular reductants, iron or copper in
low molecular weight forms may play a catalytic
role in the initiation of free radical reactions.
The resulting oxyradicals have the potential to
damage cellular lipids, nucleic acids, proteins,
and carbohydrates, resulting in wide-ranging
impairment in cellular function and integrity.
However, cells are endowed with cytoprotective
mechanisms (antioxidants, scavenging enzymes,
repair processes) that act to counteract the
effects of free radical production. Thus, the net
effect of metal-induced free radicals on cellular
function will depend on the balance between
radical production and the cytoprotective systems.
As a result, there may be a rate of free radical
production that must be exceeded before cellular
injury occurs. Evidence has now accumulated that
iron or copper overload in experimental animals
can result in oxidative damage to lipids in vivo,
once the concentration of the metal exceeds a
threshold level. In the liver, this lipid
peroxidation is associated with impairment of
membrane dependent functions of mitochondria
(oxidative metabolism) and lysosomes (membrane
integrity, fluidity, pH). Although these findings
do not prove causality, it seems likely that lipid
peroxidation is involved, since similar functional
defects are produced by metal-induced lipid
peroxidation in these organelles in vitro. Both
iron and copper overload impair hepatic
mitochondrial respiration, primarily through a
decrease in cytochrome c oxidase activity. In iron
overload, hepatocellular calcium homeostasis may
be impaired through damage to mitochondrial and
microsomal calcium sequestration. DNA has also
been reported to he a target of metal-induced
damage in the liver; this may have consequences as
regards malignant transformation. The levels of
some antioxidants in the liver are decreased in
rats with iron or copper overload, which is also
suggestive of ongoing oxidative stress. Reduced
cellular ATP levels, lysosomal fragility, impaired
cellular calcium homeostasis, and damage to DNA
may all contribute to hepatocellular injury in
iron and copper overload. There are few data
addressing the key issue of whether tree radical
production is increased in patients with iron or
copper overload. Patients with hereditary
hemochromatosis have elevated plasma levels of
TBA-reactants and increased hepatic levels of
MDA-protein and HNE-protein adducts, indicative of
lipid peroxidation. Mitochondria isolated from the
livers of Wilson disease patients have evidence of
lipid peroxidation, and some patients with Wilson
disease have decreased hepatic and plasma levels
of vitamin E. Additional investigation will be
required to fully assess oxidant stress and its
potential pathophysiologic role in patients with
iron or copper overload.
Hepatocyte proliferative activity in
chronic liver damage as assessed by the monoclonal
antibody MIB1 Ki67 in archival material: The role
of etiology, disease activity, iron, and lipid
peroxidation
Farinati F.; Cardin R.; D'Errico A.; De Maria
N.; Naccarato R.; Cecchetto A.; Grigioni W.
CMAD, Istituto di Medicina Interna, Policlinico
Universitario, Via Giustiniani 2, 35128 Padova
Italy
Hepatology (USA), 1996, 23/6 (1468-1475)
Hepatitis B virus (HBV)- and hepatitis C virus
(HCV)related liver damage is linked to an
increased risk of hepatocellular carcinoma, but
the mechanisms underlying hepatitis C viral
activity are not known. We therefore compared
hepatocellular proliferative activity in chronic C
virus-related hepatitis and in liver damage of
other etiology. Hepatocyte proliferation rate was
investigated in 56 patients with chronic hepatitis
using the Ki67 MIB1 monoclonal antibody in
archival material. According to etiology, the
patients were subgrouped as follows: HCV (34), HBV
(11), Alcohol (4), HCV + Alcohol (4), and
Hemochromatosis (3). Proliferation rate was
correlated with age, sex, etiology, disease
activity, liver iron storage, free-radical
production, and glutathione levels by
regression and discriminant analysis.
HCV-positive patients had significantly more
MIB1-positive hepatocytes in the periportal area
(P < .011) and in the low-proliferating
perivenular area (zones 2 and 3) (P < .05). The
number of MIB1-positive cells correlated directly
with alanine transaminase (ALT) levels, Knodell
index (KI), and, inversely, with iron saturation.
By stepwise discriminant analysis, ALT levels and
etiology were identified as single independent
variables. These data suggest that HCV infection
induces increased and abnormal hepatocyte
proliferation, which might be related to the
increased risk of hepatocellular carcinoma in
patients with HCV-related liver damage.
Hepatic
iron deposition in human disease and animal
models
Halliday J.W.; Searle J.
Liver Unit, Queensland Institute Med Research,
Royal Brisbane Hospital, 300 Herston Road,
Herston, Brisbane, QLD 4029 Australia
BioMetals (United Kingdom), 1996, 9/2
(205-209)
Iron deposition occurs in parenchymal cells of
the liver in two major defects in human subjects
(i) in primary iron overload (genetic
haemochromatosis) and (ii) secondary to anaemias
in which erythropolesis is increased
(thalassaemia). Transfusional iron overload
results in excessive storage primarily in cells of
the reticule endothelial system. The storage
patterns in these situations are quite
characteristic. Excessive iron storage,
particularly in parenchymal cells eventually
results in fibrosis and cirrhosis. There is no
animal model or iron overload which completely
mimics genetics haemochromatosis but dietary iron
loading with carbonyl iron or ferrocene does
produce excessive parenchymal iron stores in the
rat. Such models have been used to study iron
toxicity and the action of iron chelators in the
effective removal of excessive iron stores.
Long-term
intraperitoneal deferoxamine for
hemochromatosis
Swartz R.D.; Legault D.J.
Michigan University Medical Center, 3914 TC-Box
0364, Ann Arbor, MI 48109-0364 USA
American Journal of Medicine (USA), 1996, 100/3
(308-312)
Intraperitoneal deferoxamine is a
well-established treatment for aluminum
accumulation syndrome in patients with end-stage
renal disease receiving peritoneal dialysis, but
the use of intraperitoneal deferoxamine has not
been described outside of the setting of chronic
renal failure. We present here a case of secondary
hemochromatosis, complicated by cirrhosis and
cardiomyopathy, in which a chronic peritoneal
dialysis catheter was used both to treat ascites
and to deliver parenteral deferoxamine for iron
overload. Daily urinary iron excretion was similar
to that achieved when using standard routes of
deferoxamine administration. Over a 2-year period,
reversal of both the biochemical indicators and
the clinical manifestations of iron overload was
accomplished.
Biological markers of oxidative
stress induced by ethanol and iron overload in
rat.
Wisniewska-Knypl JM; Wronska-Nofer T
Department of Toxicological Biochemistry, Nofer
Institute of Occupational Medicine, Lodz,
Poland.
Int J Occup Med Environ Health (Poland) 1994, 7
(4) p355-63
Studies on rats treated for 15 months with
ethanol (10%, w/v, solution in drinking water)
revealed that the stimulation of hepatic
cytochrome P-450 monooxygenases activity was
accompanied by enhanced microsomal malondialdehyde
formation, a lipid peroxidation index and a
decreased level of the antioxidant,
alpha-tocopherol. The other components of the
prooxidant/antioxidant system, diene conjugates
and catalase, glutathione peroxidase and
superoxide dismutase activities were unaffected.
Oxidative stress in blood was shown by a
significant decrease in the alpha-tocopherol level
whereas lipid peroxidation and antioxidant enzyme
activity remained unchanged. The prooxidative
effect of ethanol was catalytically promoted by an
iron overload (Fe-saccharate, 100 mg Fe3+/kg body
wt. intraperitoneally, 2, 5 and 7 day before test)
to simulate the effect of alcoholic
hemochromatosis. Thus, the level of
malondialdehyde and alpha-tocopherol in the serum
may be recommended as biological markers of
ethanol-provoked oxidative stress, which is
especially useful in the evaluation of the
combined effect of ethanol and other chemicals
that affect the redistribution of active iron
complexes.
Antioxidant status and lipid
peroxidation in hereditary
haemochromatosis.
Young IS; Trouton TG; Torney JJ; McMaster D;
Callender ME; Trimble ER
Department of Clinical Biochemistry, Queen's
University of Belfast, UK.
Free Radic Biol Med (United States) Mar 1994, 16
(3) p393-7
Hereditary haemochromatosis is characterised by
iron overload that may lead to tissue damage. Free
iron is a potent promoter of hydroxyl radical
formation that can cause increased lipid
peroxidation and depletion of chain-breaking
antioxidants. We have therefore assessed lipid
peroxidation and antioxidant status in 15 subjects
with hereditary haemochromatosis and age/sex
matched controls. Subjects with haemochromatosis
had increased serum iron (24.8 (19.1-30.5) vs.
17.8 (16.1-19.5) mumol/l, p = 0.021) and %
saturation (51.8 (42.0-61.6) vs. 38.1 (32.8-44.0),
p = 0.025). Thiobarbituric acid reactive
substances (TBARS), a marker of lipid
peroxidation, were increased in haemochromatosis
(0.59 (0.48-0.70) vs. 0.46 (0.21-0.71) mumol/l, p
= 0.045), and there were decreased levels of the
chain-breaking antioxidants alpha-tocopherol (5.91
(5.17-6.60) vs. 7.24 (6.49-7.80) mumol/mmol
cholesterol, p = 0.001), ascorbate (51.3
(33.7-69.0) vs. 89.1 (65.3-112.9), p = 0.013), and
retinol (1.78 (1.46-2.10) vs. 2.46 (2.22-2.70)
mumol/l, p = 0.001). Patients with hereditary
haemochromatosis have reduced levels of
antioxidant vitamins, and nutritional antioxidant
supplementation may represent a novel approach to
preventing tissue damage. However, the use of
Vitamin-C may be deleterious in this setting as
ascorbate can have prooxidant effects in the
presence of iron overload.
Iron
storage, lipid peroxidation and glutathione
turnover in chronic anti-HCV positive
hepatitis.
Farinati F, Cardin R, De Maria N, Della Libera
G, Marafin C, Lecis E, Burra P, Floreani A,
Cecchetto A, Naccarato R
Cattedra Malattie Apparato Digerente, Universita
di Padova, Italy.
J Hepatol 1995 Apr;22(4):449-56
BACKGROUND/AIMS: Little is known about the
pathogenesis of liver damage related to hepatitis
C virus. The presence of steatosis or increased
ferritin levels, and preliminary data on the
relevance of iron as a prognostic factor prompted
us to ascertain whether hepatitis C virus-related
liver damage might be mediated by iron
accumulation.
METHODS: We evaluated the degree of hepatic
inflammation and steatosis, serum ferritin,
transferrin saturation and iron levels, tissue
iron concentrations and iron index, liver
glutathione and malondialdehyde in 33 males and 20
females with chronic hepatitis C virus- or
hepatitis B virus-related hepatitis (42 + 11). We
also considered six patients with both alcohol
abuse and hepatitis C virus, four males with
chronic alcoholic liver disease and four males
with genetic hemochromatosis, giving a total of
67. All diagnoses were histologically confirmed.
Patients with cirrhosis were excluded.
RESULTS: Our data show that: 1. Steatosis is
more frequent in hepatitis C virus and hepatitis C
virus+alcohol abuse patients; 2. In males, serum
ferritin and tissue iron are significantly higher
in hepatitis C virus- than in hepatitis B
virus-positive patients (p < 0.01 and 0.05);
transferrin saturation is higher (p < 0.05) in
hepatitis C virus-positive than in hepatitis B
virus-positive patients only when males and
females are considered together; 3. Serum ferritin
and transferrin saturation only correlate with
liver iron (r = 0.833 and r = 0.695, respectively,
p = 0.00001); tissue iron is significantly higher
in hepatitis C virus- than in hepatitis B
virus-positive patients (p < 0.05); 4. In
patients with chronic hepatitis, serum ferritin is
a better marker of liver iron storage than
transferrin saturation, both in males and in
females; 5. Hepatitis C virus-positive patients
have higher malondialdehyde levels and activation
of turnover of glutathione, probably in response
to free-radical-mediated liver damage. Females
have lower liver iron levels but similar
trends.
CONCLUSIONS: These findings suggest that
hepatitis C virus-related liver damage is
characterized by increased iron storage (possibly
induced by the virus) which elicits a
free-radical-mediated peroxidation, with
consequent steatosis and activation of glutathione
turnover.
Induction of oxidative single- and
double-strand breaks in DNA by ferric
citrate.
Toyokuni S; Sagripanti JL
Molecular Biology Branch, Center for Devices and
Radiological Health, Food and Drug Administration,
Rockville, MD 20857.
Free Radic Biol Med (United States) Aug 1993, 15
(2) p117-23
The relative risk of primary hepatocellular
carcinoma in genetic hemochromatosis (GH) is
estimated at over 200 times as that of control
populations. Recently, ferric ion chelated to
citrate (Fe-citrate) was identified as the major
non-transferrin-bound iron in the serum of GH
patients. We investigated whether low
concentration of Fe-citrate plus reductant could
damage supercoiled plasmid DNA under physiological
pH and ionic strength. Incubation of Fe-citrate
with either H2O2, L-ascorbate, or L-cysteine
induced single- and double-strand breaks in
supercoiled plasmid pZ189 in a concentration- and
time-dependent fashion. DNA strand breaks produced
by Fe-citrate plus H2O2 increased at reduced pH
(< or = 6.9). Catalase and free radical
scavengers inhibited the DNA breakage produced by
Fe-citrate in combination with each reductant,
suggesting that H2O2 and finally .OH are
responsible DNA damaging species. The catalytic
ability of Fe-citrate to induce DNA strand breaks,
particularly double-strand breaks (DSBs), may
contribute to the carcinogenic processes observed
in GH.
A
unique rodent model for both the cardiotoxic and
hepatotoxic effects of prolonged iron
overload.
Carthew P, Dorman BM, Edwards RE, Francis JE,
Smith AG
MRC Toxicology Unit, University of Leicester,
United Kingdom.
Lab Invest 1993 Aug;69(2):217-22
BACKGROUND: Hemochromatosis is a disease of
excessive iron storage leading to tissue damage
and fibrosis. Both genetic hemochromatosis, which
can affect 1 in 500 of some populations, and the
form of this disease which occurs as a secondary
consequence of the hemoglobinopathy, homozygous
beta-thalassemia, with 40 million carriers
worldwide, have a common pathology. The
cardiotoxicity and hepatotoxicity, which occurs
with this disease, have never been produced
experimentally in other species.
EXPERIMENTAL DESIGN: Using a regimen of iron
dextran administered subcutaneously to gerbils on
a weekly basis for 7 weeks, we have produced
severe hemosiderosis, especially of the liver and
heart. By examining gerbils at 1, 2 and 3 months
after the final iron injections we followed the
subsequent development of hemochromatosis in the
hearts and livers of iron overloaded animals.
RESULTS: Hemochromatosis of the liver was
evident as a scarring fibrosis in all cases
between 1 and 3 months after iron dextran
administration to gerbils. The iron burden in the
cardiac myocytes of gerbils gradually increased
between 1 and 3 months, resulting in
hemochromatosis of the heart 2 and 3 months after
the final iron dextran injections.
CONCLUSIONS: Repeated parenteral injections of
iron dextran to gerbils resulted in
hemochromatosis affecting the liver and heart with
a pathology which is the same as occurs in the
end-stage disease in man. This model will allow
the detailed study of the mechanism of iron
induced, free radical tissue damage, which is
thought to be the cause of these lesions and will
also be useful in the evaluation of iron chelating
therapies to determine whether the hepatic and
cardiac pathology of iron overload can be
modulated over a long period.
Biochemical and biophysical
investigations of the ferrocene-iron-loaded rat.
An animal model of primary
haemochromatosis.
Ward RJ; Florence AL; Baldwin D; Abiaka C;
Roland F; Ramsey MH; Dickson DP; Peters TJ;
Crichton RR
Department of Clinical Biochemistry, King's
College School of Medicine and Dentistry, London,
England.
Eur J Biochem (Germany) Dec 5 1991, 202 (2)
p405-10
Male Wistar rats fed with ferrocene had high
hepatic iron loading (7.24 +/- 1.97 mg Fe/g
tissue) after 6 weeks, principally located in
lysosomes, which was comparable to the levels and
distribution determined in human haemochromatosis.
The two iron-storage proteins, ferritin and
haemosiderin were isolated from the livers of the
ferrocene-loaded rats and their iron cores were
investigated by Mossbauer spectroscopy and
inductively coupled plasma-emission spectrometry.
Ferrihydrite was the predominant form of iron
present in both ferritin and haemosiderin, while
haemosiderin contained higher amounts of
phosphorus, magnesium, calcium and barium, then
either normal or ferrocene-loaded ferritin.
Free-radical-mediated damage in the iron-loaded
livers was inferred by the significant depletion
of alpha-tocopherol in both the livers and
subcellular hepatic lysosomal fraction, which
inversely correlated with the increasing iron
content (r = -0.61; P less than 0.05) and was
associated with increased fragility of the
lysosomal membranes.
Antioxidant and iron-chelating
activities of the flavonoids catechin, quercetin
and diosmetin on iron-loaded rat hepatocyte
cultures
Morel I, Lescoat G, Cogrel P, Sergent O,
Pasdeloup N, Brissot P, Cillard P, Cillard J
Laboratoire de Biologie Cellulaire et Vegetale,
UFR des Sciences Pharmaceutiques, Rennes,
France.
Biochem Pharmacol 1993 Jan 7;45(1):13-9
The cytoprotective effect of three flavonoids,
catechin, quercetin and diosmetin, was
investigated on iron-loaded hepatocyte cultures,
considering two parameters: the prevention of
iron-increased lipid peroxidation and the
inhibition of intracellular enzyme release. These
two criteria of cytoprotection allowed the
calculation of mean inhibitory concentrations
(IC50) which revealed that the effectiveness of
these flavonoids could be classified as follows:
catechin>quercetin>diosmetin. These IC50
values have been related to structural
characteristics of the flavonoids tested.
Moreover, the investigation of the capacity of
these flavonoids to remove iron from iron-loaded
hepatocytes revealed a good relationship between
this iron-chelating ability and the cytoprotective
effect. The cytoprotective activity of catechin,
quercetin and diosmetin could thus be ascribed to
their widely known antiradical property but also
to their iron-chelating effectiveness. These
findings increase further the prospects for the
development and clinical application of these
potent antioxidants.
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