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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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