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Iron toxicity and antioxidant nutrients
Toxicology 180 (2002) 23 /32 www.elsevier.com/locate/toxicol
Iron toxicity and antioxidant nutrients Cesar G. Fraga a,*, Patricia I. Oteiza b a
b
Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, 1113 Buenos Aires, Argentina Biological Chemistry-IQUIFIB (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, 1113 Buenos Aires, Argentina
Abstract Iron is an essential nutrient for the growth, development, and long-term survival of most organisms. High tissue iron concentrations have been associated with the development and progression of several pathological conditions, including certain cancers, liver and heart disease, diabetes, hormonal abnormalities, and immune system dysfunctions. In this review we discuss the relevance of iron toxicity on free radical-mediated tissue damage, and how iron interactions with nutrient antioxidants and other metals can affect the extent of oxidative damage to different biomolecules. It can be concluded that the ingestion of antioxidant rich foods may prevent or delay primary and secondary effects associated with iron overload-related diseases. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Free radicals; Oxidation; Flavonoids; Vitamin; Zinc
1. Iron metabolism and toxicity The average human male contains approximately 4.5 g of iron. About 65% of the iron is bound to haemoglobin, 10% is a constituent of myoglobin, cytochromes, and iron-containing enzymes, and 20 /30% is bound to the iron storage proteins, ferritin and hemosiderin. Ferritin, a lowaffinity, high-capacity storage protein, can store up to 4500 atoms of iron per molecule. Another protein relevant in iron homeostasis is transferrin, a high-affinity, low-capacity protein (two atoms of iron per molecule) that transports iron in the plasma. It is thought that only trace amounts of
* Corresponding author. Tel.: /54-11-4964-8244; fax: /5411-4508-3646 E-mail address: [email protected] (C.G. Fraga).
the metal remain free as non-chelated or loosely chelated iron. To prevent the potential health disturbances produced by both iron deficiency and iron overload, mammals have evolved with numerous, integrated mechanisms to regulate iron metabolism. Iron deficiency, is a widely spread condition that affects approximately 500 million people around the world. The consequences of iron deficiency can range from anemia to mental retardation in growing children. Iron overload is a less frequent condition, but high contents of tissue iron has been associated with several pathological conditions, including liver and heart disease (Rasmussen et al., 2001; Milman et al., 2001; Yang et al., 1998), cancer (Beckman et al., 1999; Parkkila et al., 2001), neurodegenerative disorders (Sayre et al., 2000; Berg et al., 2001), diabetes (Ellervik et al., 2001;
0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 3 7 9 - 7
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Parkkila et al., 2001; Perez de Nanclares et al., 2000), hormonal abnormalities (Wilkinson, 1996), and immune system abnormalities (Li et al., 2000; Walker and Walker, 2000). Table 1 shows a comprehensive list of pathological conditions associated with iron overload. In terms of toxicity, chronic iron toxicity is a condition that can be associated with: (a) primary hemochromatosis, a genetic disorder related to increased intestinal absorption of iron; (b) high dietary iron intake; and (c) frequent blood transfusions (often required for the treatment of certain refractory anemias). Cases of acute iron toxicity are rare and mostly related to hepatoxicity (Tenenbein, 2001).
2. Iron and free radicals Excessive tissue free radical-mediated damage is accepted as a major mechanism underlying the occurrence of certain chronic diseases, and undesired intoxication harm. The alterations in cell Table 1 Pathological situations associated with iron overload (a) Defects in iron absorption Hereditary hemochromatosis HFE3-type hemochromatosis Juvenile hemochromatosis African dietary iron overload (b) Defects in iron transport Porphyria cutanea tarda Erythropoietic protoporphyria Aceruloplasminemia Wilson’s disease Menkes Syndrome Friedreich’s ataxia (c) Secondary iron disorders Alcohol-induced iron abnormalities Non-alcoholic liver steatohepatitis Chronic hepatitis C Parkinson’s disease Alzheimer’s disease Hallervorden-Spatz syndrome Huntington‘s disease Diabetes, Arthritis Cardiomyopathies Liver cancer Leukemia
structure and function caused by iron overload seem to be fundamentally related to free radicalmediated damage of cell components. The chemical structure of iron and its capacity to drive one-electron reactions makes iron a major player in the production and metabolism of free radicals in biological systems. Ferrous iron has the capacity to reduce oxygen to superoxide radical, a reaction that, in aerobic organisms, is accomplished at cellular and extracellular levels (Eq. (1)). Both non-chelated ferrous iron and different forms of chelated ferrous iron can catalyse this reaction. Several reductants (ascorbate, thiol compounds, superoxide anion (O2 ), etc.) can restore ferrous iron from ferric iron (Eq. (2)), resulting in the production of superoxide anion from molecular oxygen. Two molecules of superoxide can dismutate, spontaneously or enzymatically, yielding molecular oxygen and hydrogen peroxide (Eq. (3)). Fe2 O2 0 Fe3 O 2
(1)
2 Fe A Aox red 0 Fe O 2H 0 O2 H2 O2 O 2 2
(2) (3)
3
Ferrous iron can catalyse the decomposition of peroxides, yielding hydroxyl radical from hydrogen peroxide (Eq. (4)) or alkoxyl radicals, if the substrate of the reaction is an organic peroxide (LOOH) (Eq. (5)). H2 O2 Fe2 0 Fe3 OH OH+
(4)
LOOHFe2 0 Fe3 LO OH+
(5)
Thus, the participation of iron is essential in: (a) the production of hydroxyl radical that can subsequently initiate lipid oxidation or oxidise almost every molecule present in biological systems; and (b) the propagation of free radical reactions by decomposing peroxides. The relevance of iron-catalysed reactions in vivo is restricted to the negligible availability of ‘free’ catalytic iron. Nevertheless, increased generation of superoxide can favour iron release from ferritin and from proteins, and hydrogen peroxide can degrade haem from haem-proteins to release iron (Halliwell and Gutteridge, 1999). For example, 5aminolevulinic acid (ALA) autoxidation generates superoxide anion that releases iron from ferritin,
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which could lead to a higher availability of free iron in patients with acute intermittent porphyria and lead poisoning (Oteiza et al., 1995a).
3. Antioxidants and iron toxicity An antioxidant is, as a general definition, any substance capable of preventing oxidation. Deleterious free radical-mediated oxidations occur in aerobic organism as a result of normal oxygen metabolism. As described above (Section 2), iron, especially ferrous iron, is able to trigger oxidations by reducing as well as by decomposing previously formed peroxides. Thus, an antioxidant that protects from iron toxicity is a substance that can: (a) chelate ferrous iron and prevent the reaction with oxygen or peroxides; (b) chelate iron and maintain it in a redox state that makes iron unable to reduce molecular oxygen; (c) trap already formed radicals, which is a putative action of any substance that can scavenge free radicals in biological systems, regardless if they are originated from iron-dependent reactions or not. A large number of substances can chelate iron, and many of them are present in biological systems. The proteins that participate in iron metabolism can sequester iron thus preventing it from participating in free radical reactions. Ferritin, transferrin, and several iron-containing enzymes such as catalase, maintain iron at a ferric or higher reduced state, making it less reactive to initiate and/or propagate free radical reactions. Several other compounds, both natural and synthetic, can chelate iron in vivo thus limiting its participation in free radical reactions. For example, EDTA is a synthetic compound, normally present in foods, that can chelate iron. However, EDTA chelation is unable to prevent iron mediated oxygen reduction, therefore, its efficiency as an antioxidant is low. By contrast, desferrioxamine mesylate (DFO) is a powerful iron-chelating substance that almost completely suppresses iron-mediated oxidations in biological systems. DFO is often used as a therapeutic tool in the treatment of iron overload (Kicic et al., 2001). There are a number of substances that have been defined as antioxidants, because of their
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capacity to protect biomolecules from free radical-mediated damage. Many of these substances can prevent iron-mediated oxidation in different in vitro and in vivo experimental systems (Packer and Cadenas, 1997). However, the relevance for human health of most of these substances remains uncertain. Among the antioxidant substances that are synthesised by mammals, thiol compounds, especially glutathione, afford significant antioxidant protection. This protection is related to the ability of glutathione to trap radicals, reduce peroxides, and work to maintain the redox state of the cells (Milchak and Douglas Bricker, 2002; Meister, 1995). The interaction between iron overload and dietary antioxidants has been well characterised, especially with respect to vitamins E and C. Vitamin E, a mixture of various tocopherols and tocotrienols, is the most important lipid-soluble antioxidant (Packer et al., 2001; Ricciarelli et al., 2001). Vitamin E has been extensively studied with respect to its capacity to protect molecules from the in vitro and in vivo effects of iron toxicity. Results from our group, as well as from other laboratories, have shown that vitamin E can prevent the majority of iron-mediated damage both, in in vitro systems, and in iron-loaded animals (Tappel and Dillard, 1981; Lucesoli and Fraga, 1999; Gavino et al., 1984; Galleano and Puntarulo, 1997; Bartfay et al., 1998). In hemodialysis patients, vitamin E administration (1200 U/day) attenuated the extent of lipid oxidation (Roob et al., 2000). Oxidative stress is a general condition in hemodialysis patients (Salahudeen et al., 2001; de Cavanagh et al., 1999), the periodic intravenous iron injection being a factor contributing to oxidative stress. Vitamin E may protect patients on hemodialysis from degenerative diseases, occurring in the long term, by decreasing the rate of free radical-mediated damage. The interaction of another dietary antioxidant, vitamin C (ascorbate) and iron is less clear. Ascorbate can reduce ‘free’ iron (ferric) to ferrous iron (see Eq. (3)), promoting the initiation (via hydroxyl radical formation, Eq. (4)), and propagation (via lipid alkoxyl radical formation, Eq. (5)) of free radical reactions (Buettner and Jurkiewicz,
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1996; Herbert et al., 1996). Although, this Fenton chemistry occurs readily in vitro, its occurrence under physiological conditions is unlikely, given the negligible availability of ‘free’ catalytic iron. As mentioned above, the levels of free iron are thought to be very low due to its sequestration by the various metal-binding proteins (ferritin, transferrin, etc.). However, in people under risk of iron overload (hemochromatosis, b-thalassemia, hemodialysis) in which the elevated levels of iron could lead to higher ‘free iron’ concentrations, an excess of vitamin C could have deleterious effects. In these cases, vitamin C tissue levels should be maintained in the low range, and vitamin C supplementation should be avoided. Supporting a pro-oxidant effect of the iron-vitamin C interaction in vivo, a recent report showed that a daily combined supplementation of iron (100 mg as fumarate) and vitamin C (500 mg as ascorbate) during the third trimester of pregnancy caused a 20% increase in plasma lipid oxidation (Lachili et al., 2001). We consider that the results of this study should be taken carefully, since only the plasma content of substances reactive to 2-thiobarbituric acid (TBARS) was determined and it is well known that this determination is not always a unequivocal evaluation of in vivo oxidative stress. More important, in several other studies, in both animal models and in humans, investigators have reported that there is no evidence of increased free radical damage due to the combined ingestion of iron and ascorbate (Proteggente et al., 2001, 2000; Chen et al., 2000; Carr and Frei, 1999).
they can participate in: (a) iron chelation; and (b) trapping radicals (van Acker et al., 1998; Sestili et al., 1998; Morel et al., 1993; Moran et al., 1997). Several reports indicate that certain flavonoids can protect lipids in liposomes from iron-mediated oxidation (Gorelik and Kanner, 2001; Lotito et al., 2000; Shirai et al., 2001; Zago and Oteiza, 2001). Interestingly, when oligomeric flavonoids (procyanidins) isolated from cocoa were assayed as inhibitors of iron-induced lipid oxidation, we observed that the degree of oligomerisation significantly influenced the antioxidant capacity of the flavonoids. The capacity of procyanidins to protect against ferrous-mediated liposome oxidation was inversely associated with their chain length (Fig. 1). In the presence of other oxidising insults, the degree of oligomerisation had the opposite effect (the longer procyanidins protected better than the shorter ones) or had no effect (Lotito et al., 2000). There is a recent study showing that flavonoids protect culture cells from iron-mediated damage (Kostyuk et al., 2001). In vivo, dietary flavonoids have been reported to inhibit carbonyl iron-induced lipid oxidation in rat liver (Pietrangelo et al., 1995). As an indirect effect, polyphenol-containing foods
4. Flavonoids and iron toxicity Flavonoids represent one class of bioactive compounds that may have multiple beneficial effects on several chronic diseases (Hollman, 2001; Kris-Etherton and Keen, 2002). Flavonoids are polyphenol compounds that are synthesised by plants, and that can be incorporated in animals through the diet. In humans, dietary sources of flavonoids include tea, red wine, purple grape juice, cocoa products, apples, onions and certain nuts. Interestingly, these compounds can play a double role in reducing the rate of oxidation, as
Fig. 1. Cocoa monomers and procyanidins in the prevention of ferrous iron-initiated TBARS production. Multillamelar liposomes were added with 0.73 mg/ml (5 mM monomer equivalent) of monomers or procyanidin fractions (M, monomer; D, dimer; T, trimer; Te, tetramer; P, pentamer; and H, hexamer), and oxidised by the addition of 25 mM ferrous sulfate/25 mM ascorbate and incubated for 60 min at 37 8C. Results represent % of inhibition of the values obtained for control samples incubated without the addition of monomer or procyanidin fractions. Values taken from Lotito et al. (2000).
C.G. Fraga, P.I. Oteiza / Toxicology 180 (2002) 23 /32
may inhibit non haem /iron absorption as determined by erythrocyte incorporation of radio-Fe after a bread meal in adult human subjects (Hurrell et al., 1999). However, in weanling rats, chronic tea ingestion did not alter iron incorporation (Record et al., 1996). From the described studies, it can be concluded that: (a) flavonoids and related procyanidins reduce oxidative damage promoted by iron in vitro and in vivo; and (b) flavonoids can modulate iron intake, a function that may be beneficial for populations at risk for iron overload.
5. Iron interaction with other metals Other metals can interact with iron in biological systems, and this interaction can have beneficial effects preventing undesirable iron mediated damage. We consider of particular relevance the interaction between iron and zinc. One of the several mechanisms that have been shown to be involved in the antioxidant action of zinc, a metal that does not have redox activity, is the capacity of zinc to compete with iron for multiple cellular binding sites. The replacement of iron with zinc can prevent the redox-cycling of iron, thus minimising the rate of oxidising chemical groups in the close vicinity of the metal binding site. In this context, zinc can reduce the iron-mediated oxidation of lipids, proteins, and DNA. An early work showed that zinc could prevent iron-mediated oxidation of lipids in red blood cells (Girotti et al., 1985). To explore the mechanism(s) involved in the protection by zinc against iron-initiated lipid oxidation, we used synthetic liposomes and observed that the antioxidant effect of zinc depended on the negative charge density of the membrane (Zago and Oteiza, 2001). In this work we showed that zinc acted in part by competing with ferrous iron for negative binding sites in the membrane, and that the observed effects were within physiological membrane zinc concentrations. The competition of zinc for iron binding sites is particularly relevant, taking into account that one consequence of zinc deficiency can be a marked increase in membrane and intracellular iron concentration (Rogers et al., 1985, 1987). Interestingly, the
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simultaneous addition of zinc, a-tocopherol, and ( /)-epicatechin caused an additive inhibitory effect on TBARS formation when liposomes were incubated in the presence of iron (Fig. 2; Zago and Oteiza, 2001). It can be speculated that these three compounds could act as an antioxidant network to protect lipids from oxidation: (a) zinc, by replacing iron at the membrane surface; (b) vitamin E, inhibiting lipid oxidation propagation; and (c) epicatechin, both chelating iron, and trapping radicals in the aqueous phase. With respect to the inactivation of enzymes by iron, a described mechanism is triggered by the binding of iron to divalent cation sites localised in the catalytic portion of enzymes (Stadtman, 1992; Stadtman and Levine, 2000). Bonded iron can promote the local generation of hydroxyl radical through a Fenton reaction, leading to the oxidation of specific amino acids and then to enzyme inactivation. We found that the addition of zinc to rat brain supernatants does not prevent the ferrous iron-mediated inactivation of both, glutamine synthase and glucose-6-phosphate dehydrogenase (Zago et al., 2000). Zinc can markedly reduce the in vitro iron- and copper-induced breakage of DNA. The capacity of
Fig. 2. Zinc (Z), a-tocopherol (A), and ( /)-epicatechin (E) in the prevention of ferrous iron-initiated TBARS production. Multillamelar liposomes were added with 15 mM zinc sulphate, and/or 0.01 lipid mol% a-tocopherol, and/or 0.5 mM ( /)epicatechin, and oxidised by the addition of 25 mM ferrous sulphate and incubation for 90 min at 37 8C. Results represent % of inhibition of the values obtained for control samples incubated without addition of zinc, a-tocopherol, or ( /)epicatechin. Values taken from Zago and Oteiza (2001).
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zinc to protect DNA from oxidative breakage is greater against iron than against copper (Har-El and Chevion, 1991). This protection against DNA damage is particularly important since oxidative damage to DNA, especially strand breaks, is highly dependent on the amount of iron bound to DNA. Iron bound to DNA can catalyse the sitespecific generation of hydroxyl radical. In vivo we have observed that the increased DNA damage occurring in the testes of zinc deficient rats was associated with iron accumulation (Oteiza et al., 1995b). Iron accumulation has been associated with copper deficiency in different animal experimental models (Cohen et al., 1983, 1985; Fields et al., 2001). Also genetic disorders in copper metabolism can lead to elevated tissue iron. For example, aceruloplasminemia is a rare disease, in which individuals have no ceruloplasmin in their serum (Miyajima et al., 1987). The pathogenesis of the disease has been linked to a slow iron accumulation in tissues, with no changes in tissue copper levels. Selenium is another metal that can interact with iron at physiologically relevant levels (Bartfay and Bartfay, 2001; Christensen et al., 2000). It has been suggested that iron overload can modulate Se-dependent glutathione peroxidase activity (Madra et al., 1996; Dawson et al., 2000) Iron can also interact with other metals that have no redox capacity (aluminum, gallium, indium, scandium, yttrium, beryllium, lanthanum), but that can facilitate ferrous-initiated lipid and protein oxidation through the promotion of alterations in the rheology of membranes (Verstraeten et al., 1997). Aluminum (Al3) induced the formation of clusters of negatively charged phospholipids in the bilayer where the motion of fatty acids is restricted. In these clusters, the closer proximity of the phospholipids acyl chains facilitates the propagation of lipid oxidation. Galactolipids favour this mechanism by segregating phospholipids in domains where this mechanism can be amplified (Verstraeten et al., 1998). We have shown that Al3-induced changes in membrane-phospholipid phase state favouring the displacement of the bilayer to a gel state, facilitates the propagation of ferrous-initiated lipid oxidation (Verstraeten and Oteiza, 2000).
6. Iron intoxication in testes Iron overload is deleterious to the male reproductive system. In patients with hereditary hemochromatosis the incidence of hypogonadism is high, and it has been demonstrated that iron depletion could reverse the pathology. Testes have been shown to be susceptible to iron overload in several experimental models. Table 2 shows the changes in oxidative damage to lipids, proteins and DNA determined in the testes of rats subjected to different conditions leading to iron overload. It can be observed that the oxidation of cell components was associated with increases in testes iron content, supporting a widespread tissue damage. Models of acute (dextran-iron i.p. injection) and chronic (6 weeks of feeding with diets containing carbonyl-iron) iron overload lead to increased damage to testes (Lucesoli and Fraga, 1995; Lucesoli et al., 1999; Lucesoli and Fraga, 1999). Vitamin E content in the tissues was associated with a decrease in oxidative damage, especially in the chronic model (Lucesoli and Fraga, 1999). Similar protection had been shown in tissue slices from iron-overloaded rats (Gavino et al., 1984). After acute intoxication with increasing amount of iron dextran (250 /1000 mg/kg), we observed a dose-dependent accumulation of iron in both testes and sperm cells, and a reduced spermatogenesis in the rats receiving the highest dose (Lucesoli et al., 1999). These results agree with those obtained in rats that received an i.p. injection of iron sulphate and showed extensive necrosis of the germ epithelium and altered morphology of the spermatids, damages resembling those observed in zinc deficiency (Merker et al., 1996). Iron overload secondary to zinc deficiency also caused alterations in testes physiology and oxidative stress (Oteiza et al., 1995b, 1996, 1999). A model of chronic zinc deficiency imposed for 14 days to developing male rats caused alterations in the oxidant defence system (Oteiza et al., 1996) and high levels of oxidative damage to lipids, proteins and DNA (Oteiza et al., 1995b; Table 2). Significantly, as it was earlier reported (Rogers et al., 1985, 1987), in this work it was shown that iron accumulated in the testes as a consequence of the
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Table 2 Oxidative damage to testes in iron overload Treatment
Testes iron (nmol/g tissue)
TBARS (nmol/g tissue)
Carbonyls (nmol/mg protein)
oxo8dG (/105dG)
Chronica Chronic/VEa Acuteb Acutec Zn deficiencyd
71 27 1300 3000 67
32 (37)* /10 ( /13) 30 (90)* 44 (111)* 14 (56)*
5.43 0.11 0.32 1.98 1.20
0.5 0.3 0.6 1.7 0.3
(33)* (13) (7 /)* (10 /)* (33)*
(205)* (4) (23) (96)* (50)*
(12) (7) (25)* (74)* (31)*
* Significant difference from controls (P B/0.05). Values indicate increases over controls (non-treated rats), and figures between parenthesis (%) or times of increase ( /) over control values. a Lucesoli and Fraga, 1999. b Lucesoli and Fraga, 1995. c Lucesoli et al., 1999. d Oteiza et al., 1995b.
zinc deficiency. The low activity in the zinc deficient animals of glutamine synthase, an enzyme that is sensitive to oxidative inactivation, was correlated with testes iron content, and with protein carbonyl concentration (a product of iron-mediated protein oxidation) (Oteiza et al., 1995b). These results support the concept that part of the testes oxidative damage associated with zinc deficiency is due to a secondary iron overload. Zinc deficiency was also found to increase the sensitivity of testes to cadmium-induced oxidation (Oteiza et al., 1999). Significantly, in the zinc deficient groups the extent of cadmium-related haemorrhages and iron overload was higher than in the zinc-supplemented controls. Similarly, it has been shown that cadmium treatment results in iron and copper accumulation, a decrease in tissue zinc and in high rates of lipid oxidation, which could be attenuated by co-treatment with the antioxidant metal selenium (Yiin et al., 1999). Unpublished results from our laboratory, have shown that acute iron treatment leads to iron overload in rat sperm cells, increasing their physiological levels of DNA, protein and lipid oxidation, and affecting several parameters of sperm function (Lucesoli et al., unpublished).
mediated tissue damage rests on the availability of ferrous iron to promote free radical reactions. Since most organisms have a number of proteins that maintain most of iron sequestered, only trace amounts of iron are ‘free’ or available for catalysing free radical reactions. Under certain pathological conditions, iron can be released from storage and/or ‘free iron’ pools can be increased, leading to an oxidative stress condition. The strategies to control iron-mediated free radical damage, could be to: (a) prevent conditions that can lead to iron overload; (b) provide chelating substances that sequester excess iron; (c) protect functional molecules (lipid, proteins, DNA) from oxidation with the use of dietary antioxidants (vitamins, flavonoids, zinc, etc.). The overall results presented here support a beneficial effect of antioxidants, obtained either from the diet or by supplementation, in conditions of iron overload. In this regard, flavonoids are substances with both chelating and free radical scavenging properties that deserve further research in iron overload situations.
Acknowledgements 7. Conclusion Iron overload toxicity has been generally associated with a condition of free radical-mediated tissue damage. The free radical mechanism of iron-
This work was supported by grants from the University of Buenos Aires, CONICET B042 to CGF and B054 to PO), and Ministry of Health (Carrillo/Onativia fellowship).
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References Bartfay, W.J., Bartfay, E., 2001. Selenium and glutathione peroxidase with beta-thalassemia major. Nurs. Res. 50, 178 /183. Bartfay, W.J., Hou, D., Brittenham, G.M., Bartfay, E., Sole, M.J., Lehotay, D., Liu, P.P., 1998. The synergistic effects of vitamin E and selenium in iron-overloaded mouse hearts. Can. J. Cardiol. 14, 937 /941. Beckman, L.E., Van Landeghem, G.F., Sikstrom, C., Wahlin, A., Markevarn, B., Hallmans, G., Lenner, P., Athlin, L., Stenling, R., Beckman, L., 1999. Interaction between haemochromatosis and transferrin receptor genes in different neoplastic disorders. Carcinogenesis 20, 1231 /1233. Berg, D., Gerlach, M., Youdim, M.B., Double, K.L., Zecca, L., Riederer, P., Becker, G., 2001. Brain iron pathways and their relevance to Parkinson’s disease. J. Neurochem. 79, 225 /236. Buettner, G.R., Jurkiewicz, B.A., 1996. Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat. Res. 145, 532 /541. Carr, A., Frei, B., 1999. Does vitamin C act as a pro-oxidant under physiological conditions. FASEB J. 13, 1007 /1024. Chen, K., Suh, J., Carr, A.C., Morrow, J.D., Zeind, J., Frei, B., 2000. Vitamin C suppresses oxidative lipid damage in vivo, even in the presence of iron overload. Am. J. Physiol. Endocrinol. Metab. 279, E1406 /E1412. Christensen, M.J., Olsen, C.A., Hansen, D.V., Ballif, B.C., 2000. Selenium regulates expression in rat liver of genes for proteins involved in iron metabolism. Biol. Trace Elem. Res. 74, 55 /70. Cohen, N.L., Keen, C.L., Lonnerdal, B., Hurley, L.S., 1983. The effect of copper chelating drugs on liver iron mobilisation in the adult rat. Biochem. Biophys. Res. Commun. 113, 127 /134. Cohen, N.L., Keen, C.L., Lonnerdal, B., Hurley, L.S., May, J.N., 1985. Effects of varying dietary iron on the expression of copper deficiency in the growing rat: anaemia, ferroxidase I and II, tissue trace elements, ascorbic acid, and xanthine dehydrogenase. J. Nutr. 115, 633 /649. Dawson, E.B., Albers, J.H., McGanity, W.J., 2000. The apparent effect of iron supplementation on serum selenium levels in teenage pregnancy. Biol. Trace Elem. Res. 77, 209 / 217. de Cavanagh, E.M., Ferder, L., Carrasquedo, F., Scrivo, D., Wassermann, A., Fraga, C.G., Inserra, F., 1999. Higher levels of antioxidant defences in enalapril-treated versus non-enalapril-treated hemodialysis patients. Am. J. Kidney Dis. 34, 445 /455. Ellervik, C., Mandrup-Poulsen, T., Nordestgaard, B.G., Larsen, L.E., Appleyard, M., Frandsen, M., Petersen, P., Schlichting, P., Saermark, T., Tybjaerg-Hansen, A., Birgens, H., 2001. Prevalence of hereditary haemochromatosis in late-onset type 1 diabetes mellitus: a retrospective study. Lancet 358, 1405 /1409.
Fields, M., Lewis, C.G., Bureau, I., 2001. Aspirin reduces blood cholesterol in copper-deficient rats: a potential antioxidant agent. Metabolism 50, 558 /561. Galleano, M., Puntarulo, S., 1997. Dietary alpha-tocopherol supplementation on antioxidant defences after in vivo iron overload in rats. Toxicology 124, 73 /81. Gavino, V.C., Dillard, C.J., Tappel, A.L., 1984. Release of ethane and pentane from rat tissue slices: effect of vitamin E, halogenated hydrocarbons, and iron overload. Arch. Biochem. Biophys. 233, 741 /747. Girotti, A.W., Thomas, J.P., Jordan, J.E., 1985. Inhibitory effect of Zn (II) on free radical lipid peroxidation in erythrocyte membranes. Free Radic. Biol. Med. 1, 395 /401. Gorelik, S., Kanner, J., 2001. Oxymyoglobin oxidation and membrane lipid peroxidation initiated by iron redox cycle: prevention of oxidation by enzymic and nonenzymic antioxidants. J. Agric. Food Chem. 49, 5945 /5950. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford University Press, Oxford, pp. 1 /936. Har-El, R., Chevion, M., 1991. Zinc(II) protects against metalmediated free radical induced damage: studies on single and double-strand DNA breakage. Free Radic. Res. Commun. 12, 509 /515. Herbert, V., Shaw, S., Jayatilleke, E., 1996. Vitamin C-driven free radical generation from iron. J. Nutr. 126, 1213S / 1220S. Hollman, P.C.H., 2001. Evidence for health benefits of plant phenols: local or systemic effects. J. Sci. Food Agric. 81, 842 /852. Hurrell, R.F., Reddy, M., Cook, J.D., 1999. Inhibition of nonhaem iron absorption in man by polyphenolic-containing beverages. Br. J. Nutr. 81, 289 /295. Kicic, A., Chua, A.C., Baker, E., 2001. Effect of iron chelators on proliferation and iron uptake in hepatoma cells. Cancer 92, 3093 /3110. Kostyuk, V.A., Potapovich, A.I., Vladykovskaya, E.N., Korkina, L.G., Afanas’ev, I.B., 2001. Influence of metal ions on flavonoid protection against asbestos-induced cell injury. Arch. Biochem. Biophys. 385, 129 /137. Kris-Etherton, P., Keen, C.L., 2002. Evidence that the antioxidant flavonoids in tea and cocoa are beneficial for cardiovascular health. Curr. Opin. Lipidol. 13, 41 /49. Lachili, B., Hininger, I., Faure, H., Arnaud, J., Richard, M.J., Favier, A., Roussel, A.M., 2001. Increased lipid peroxidation in pregnant women after iron and vitamin C supplementation. Biol. Trace Elem. Res. 83, 103 /110. Li, J., Zhu, Y., Singal, D.P., 2000. HFE gene mutations in patients with rheumatoid arthritis. J. Rheumatol. 27, 2074 / 2077. Lotito, S.B., Actis-Goretta, L., Renart, L., Caligiuri, M., Rein, D., Schmitz, H.H., Steinberg, F.M., Keen, C.L., Fraga, C.G., 2000. Influence of oligomer chain length on the antioxidant activity of procyanidins. Biochem. Biophys. Res. Commun. 276, 945 /951. Lucesoli, F., Fraga, C.G., 1995. Oxidative damage to lipids and DNA concurrent with decrease of antioxidants in rat testes
C.G. Fraga, P.I. Oteiza / Toxicology 180 (2002) 23 /32 after acute iron intoxication. Arch. Biochem. Biophys. 316, 567 /571. Lucesoli, F., Fraga, C.G., 1999. Oxidative stress in testes of rats subjected to chronic iron intoxication and alpha-tocopherol supplementation. Toxicology 132, 179 /186. Lucesoli, F., Caligiuri, M., Roberti, M.F., Perazzo, J.C., Fraga, C.G., 1999. Dose-dependent increase of oxidative damage in the testes of rats subjected to acute iron overload. Arch. Biochem. Biophys. 372, 37 /43. Madra, S., Mann, F., Francis, J.E., Manson, M.M., Smith, A.G., 1996. Modulation by iron of hepatic microsomal and nuclear cytochrome P450, and cytosolic glutathione Stransferase and peroxidase in C57BL/10ScSn mice induced with polychlorinated biphenyls (Aroclor 1254). Toxicol. Appl. Pharmacol. 136, 79 /86. Meister, A., 1995. Glutathione metabolism. Methods Enzymol. 251, 3 /7. Merker, H.J., Vormann, J., Gunther, T., 1996. Iron-induced injury of rat testis. Andrologia 28, 267 /273. Milchak, L.M., Douglas Bricker, J., 2002. The effects of glutathione and vitamin E on iron toxicity in isolated rat hepatocytes. Toxicol. Lett. 126, 169 /177. Milman, N., Pedersen, P., Steig, T., Byg, K.E., Graudal, N., Fenger, K., 2001. Clinically overt hereditary hemochromatosis in Denmark 1948 /1985: epidemiology, factors of significance for long-term survival, and causes of death in 179 patients. Ann. Hematol. 80, 737 /744. Miyajima, H., Nishimura, Y., Mizoguchi, K., Sakamoto, M., Shimizu, T., Honda, N., 1987. Familial apoceruloplasmin deficiency associated with blepharospasm and retinal degeneration. Neurology 37, 761 /767. Moran, J.F., Klucas, R.V., Grayer, R.J., Abian, J., Becana, M., 1997. Complexes of iron with phenolic compounds from soybean nodules and other legume tissues: prooxidant and antioxidant properties. Free Radic. Biol. Med. 22, 861 /870. Morel, I., Lescoat, G., Cogrel, P., Sergent, O., Pasdeloup, N., Brissot, P., Cillard, P., Cillard, J., 1993. Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures. Biochem. Pharmacol. 45, 13 /19. Oteiza, P.I., Kleinman, C.G., Demasi, M., Bechara, E.J., 1995a. 5-Aminolevulinic acid induces iron release from ferritin. Arch. Biochem. Biophys. 316, 607 /611. Oteiza, P.I., Olin, K.L., Fraga, C.G., Keen, C.L., 1995b. Zinc deficiency causes oxidative damage to proteins, lipids and DNA in rat testes. J. Nutr. 125, 823 /829. Oteiza, P.L., Olin, K.L., Fraga, C.G., Keen, C.L., 1996. Oxidant defence systems in testes from zinc-deficient rats. Proc. Soc. Exp. Biol. Med. 213, 85 /91. Oteiza, P.I., Adonaylo, V.N., Keen, C.L., 1999. Cadmiuminduced testes oxidative damage in rats can be influenced by dietary zinc intake. Toxicology 137, 13 /22. Packer, L., Cadenas, E., 1997. Handbook of Synthetic Antioxidants. Marcel-Dekker, New York, pp. 1 /442. Packer, L., Weber, S.U., Rimbach, G., 2001. Molecular aspects of alpha-tocotrienol antioxidant action and cell signalling. J. Nutr. 131, 369S /373S.
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Parkkila, S., Niemela, O., Savolainen, E.R., Koistinen, P., 2001. HFE mutations do not account for transfusional iron overload in patients with acute myeloid leukemia. Transfusion 41, 828 /831. Perez de Nanclares, G., Castano, L., Gaztambide, S., Bilbao, J.R., Pi, J., Gonzalez, M.L., Vazquez, J.A., 2000. Excess iron storage in patients with type 2 diabetes unrelated to primary hemochromatosis. New Engl. J. Med. 343, 890 / 891. Pietrangelo, A., Borella, F., Casalgrandi, G., Montosi, G., Ceccarelli, D., Gallesi, D., Giovannini, F., Gasparetto, A., Masini, A., 1995. Antioxidant activity of silybin in vivo during long-term iron overload in rats. Gastroenterology 109, 1941 /1949. Proteggente, A.R., Rehman, A., Halliwell, B., Rice-Evans, C.A., 2000. Potential problems of ascorbate and iron supplementation: pro-oxidant effect in vivo. Biochem. Biophys. Res. Commun. 227, 535 /540. Proteggente, A.R., England, T.G., Rice-Evans, C.A., Halliwell, B., 2001. Iron supplementation and oxidative damage to DNA in healthy individuals with high plasma ascorbate. Biochem. Biophys. Res. Commun. 288, 245 /251. Rasmussen, M., Folsom, A.R., Catellier, D.J., Tsai, M.Y., Garg, U., Eckfeldt, J.H., 2001. A prospective study of coronary heart disease and the hemochromatosis gene (HFE) C282Y mutation: the atherosclerosis risk in communities (ARIC) study. Atherosclerosis 154, 739 /746. Record, I.R., McInerney, J.K., Dreosti, I.E., 1996. Black tea, green tea, and tea polyphenols. Effects on trace element status in weanling rats. Biol. Trace Elem. Res. 53, 27 /43. Ricciarelli, R., Zingg, J.M., Azzi, A., 2001. Vitamin E: protective role of a Janus molecule. FASEB J. 15, 2314 / 2325. Rogers, J.M., Keen, C.L., Hurley, L.S., 1985. Zinc deficiency in pregnant Long /Evans hooded rats: teratogenicity and tissue trace elements. Teratology 31, 89 /100. Rogers, J.M., Lonnerdal, B., Hurley, L.S., Keen, C.L., 1987. Iron and zinc concentrations and 59Fe retention in developing foetuses of zinc-deficient rats. J. Nutr. 117, 1875 / 1882. Roob, J.M., Khoschsorur, G., Tiran, A., Horina, J.H., Holzer, H., Winklhofer-Roob, B.M., 2000. Vitamin E attenuates oxidative stress induced by intravenous iron in patients on hemodialysis. J. Am. Soc. Nephrol. 11, 539 /549. Salahudeen, A.K., Oliver, B., Bower, J.D., Roberts, L.J., 2001. Increase in plasma esterified F2-isoprostanes following intravenous iron infusion in patients on hemodialysis. Kidney Int. 60, 1525 /1531. Sayre, L.M., Perry, G., Atwood, C.S., Smith, M.A., 2000. The role of metals in neurodegenerative diseases. Cell. Mol. Biol. (Noisy-le-grand) 46, 731 /741. Sestili, P., Guidarelli, A., Dacha, M., Cantoni, O., 1998. Quercetin prevents DNA single strand breakage and cytotoxicity caused by tert -butylhydroperoxide: free radical scavenging versus iron chelating mechanism. Free Radic. Biol. Med. 25, 196 /200.
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Shirai, M., Moon, J.H., Tsushida, T., Terao, J., 2001. Inhibitory effect of a quercetin metabolite, quercetin 3-O -beta-D-glucuronide, on lipid peroxidation in liposomal membranes. J. Agric. Food Chem. 49, 5602 / 5608. Stadtman, E.R., 1992. Protein oxidation and ageing. Science 257, 1220 /1224. Stadtman, E.R., Levine, R.L., 2000. Protein oxidation. Ann. New York Acad. Sci. 899, 191 /208. Tappel, A.L., Dillard, C.J., 1981. In vivo lipid peroxidation: measurement via exhaled pentane and protection by vitamin E. Fed. Proc. 40, 174 /178. Tenenbein, M., 2001. Hepatotoxicity in acute iron poisoning. J. Toxicol. Clin. Toxicol. 39, 721 /726. van Acker, S.A., van Balen, G.P., van den Berg, D.J., Bast, A., van der Vijgh, W.J., 1998. Influence of iron chelation on the antioxidant activity of flavonoids. Biochem. Pharmacol. 56, 935 /943. Verstraeten, S.V., Oteiza, P.I., 2000. Effects of Al(3/) and related metals on membrane phase state and hydration: correlation with lipid oxidation. Arch. Biochem. Biophys. 375, 340 /346. Verstraeten, S.V., Nogueira, L.V., Schreier, S., Oteiza, P.I., 1997. Effect of trivalent metal ions on phase separation and
membrane lipid packing: role in lipid peroxidation. Arch. Biochem. Biophys. 338, 121 /127. Verstraeten, S.V., Keen, C.L., Golub, M.S., Oteiza, P.I., 1998. Membrane composition can influence the rate of Al3 mediated lipid oxidation: effect of galactolipids. Biochem. J. 333, 833 /838. Walker, E.M.J., Walker, S.M., 2000. Effects of iron overload on the immune system. Ann. Clin. Lab. Sci. 30, 354 /365. Wilkinson, H., 1996. Haemochromatosis associated with arthritis and hypopituitarism. Ann. Clin. Biochem. 333, 171 / 173. Yang, Q., McDonnell, S.M., Khoury, M.J., Cono, J., Parrish, R.G., 1998. Hemochromatosis-associated mortality in the United States from 1979 to 1992: an analysis of MultipleCause Mortality Data. Ann. Intern. Med. 129, 946 /953. Yiin, S.J., Chern, C.L., Sheu, J.Y., Lin, T.H., 1999. Cadmium indiced lipid peroxidation in rat testes and protection by selenium. Biometals 12, 353 /359. Zago, M.P., Oteiza, P.I., 2001. The antioxidant properties of zinc: interactions with iron and antioxidants. Free Radic. Biol. Med. 31, 266 /274. Zago, M.P., Verstraeten, S.V., Oteiza, P.I., 2000. Zinc in the prevention of Fe2 -initiated lipid and protein oxidation. Biol. Res. 33, 143 /150.
Iron toxicity and antioxidant nutrients Cesar G. Fraga a,*, Patricia I. Oteiza b a
b
Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, 1113 Buenos Aires, Argentina Biological Chemistry-IQUIFIB (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, 1113 Buenos Aires, Argentina
Abstract Iron is an essential nutrient for the growth, development, and long-term survival of most organisms. High tissue iron concentrations have been associated with the development and progression of several pathological conditions, including certain cancers, liver and heart disease, diabetes, hormonal abnormalities, and immune system dysfunctions. In this review we discuss the relevance of iron toxicity on free radical-mediated tissue damage, and how iron interactions with nutrient antioxidants and other metals can affect the extent of oxidative damage to different biomolecules. It can be concluded that the ingestion of antioxidant rich foods may prevent or delay primary and secondary effects associated with iron overload-related diseases. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Free radicals; Oxidation; Flavonoids; Vitamin; Zinc
1. Iron metabolism and toxicity The average human male contains approximately 4.5 g of iron. About 65% of the iron is bound to haemoglobin, 10% is a constituent of myoglobin, cytochromes, and iron-containing enzymes, and 20 /30% is bound to the iron storage proteins, ferritin and hemosiderin. Ferritin, a lowaffinity, high-capacity storage protein, can store up to 4500 atoms of iron per molecule. Another protein relevant in iron homeostasis is transferrin, a high-affinity, low-capacity protein (two atoms of iron per molecule) that transports iron in the plasma. It is thought that only trace amounts of
* Corresponding author. Tel.: /54-11-4964-8244; fax: /5411-4508-3646 E-mail address: [email protected] (C.G. Fraga).
the metal remain free as non-chelated or loosely chelated iron. To prevent the potential health disturbances produced by both iron deficiency and iron overload, mammals have evolved with numerous, integrated mechanisms to regulate iron metabolism. Iron deficiency, is a widely spread condition that affects approximately 500 million people around the world. The consequences of iron deficiency can range from anemia to mental retardation in growing children. Iron overload is a less frequent condition, but high contents of tissue iron has been associated with several pathological conditions, including liver and heart disease (Rasmussen et al., 2001; Milman et al., 2001; Yang et al., 1998), cancer (Beckman et al., 1999; Parkkila et al., 2001), neurodegenerative disorders (Sayre et al., 2000; Berg et al., 2001), diabetes (Ellervik et al., 2001;
0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 3 7 9 - 7
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Parkkila et al., 2001; Perez de Nanclares et al., 2000), hormonal abnormalities (Wilkinson, 1996), and immune system abnormalities (Li et al., 2000; Walker and Walker, 2000). Table 1 shows a comprehensive list of pathological conditions associated with iron overload. In terms of toxicity, chronic iron toxicity is a condition that can be associated with: (a) primary hemochromatosis, a genetic disorder related to increased intestinal absorption of iron; (b) high dietary iron intake; and (c) frequent blood transfusions (often required for the treatment of certain refractory anemias). Cases of acute iron toxicity are rare and mostly related to hepatoxicity (Tenenbein, 2001).
2. Iron and free radicals Excessive tissue free radical-mediated damage is accepted as a major mechanism underlying the occurrence of certain chronic diseases, and undesired intoxication harm. The alterations in cell Table 1 Pathological situations associated with iron overload (a) Defects in iron absorption Hereditary hemochromatosis HFE3-type hemochromatosis Juvenile hemochromatosis African dietary iron overload (b) Defects in iron transport Porphyria cutanea tarda Erythropoietic protoporphyria Aceruloplasminemia Wilson’s disease Menkes Syndrome Friedreich’s ataxia (c) Secondary iron disorders Alcohol-induced iron abnormalities Non-alcoholic liver steatohepatitis Chronic hepatitis C Parkinson’s disease Alzheimer’s disease Hallervorden-Spatz syndrome Huntington‘s disease Diabetes, Arthritis Cardiomyopathies Liver cancer Leukemia
structure and function caused by iron overload seem to be fundamentally related to free radicalmediated damage of cell components. The chemical structure of iron and its capacity to drive one-electron reactions makes iron a major player in the production and metabolism of free radicals in biological systems. Ferrous iron has the capacity to reduce oxygen to superoxide radical, a reaction that, in aerobic organisms, is accomplished at cellular and extracellular levels (Eq. (1)). Both non-chelated ferrous iron and different forms of chelated ferrous iron can catalyse this reaction. Several reductants (ascorbate, thiol compounds, superoxide anion (O2 ), etc.) can restore ferrous iron from ferric iron (Eq. (2)), resulting in the production of superoxide anion from molecular oxygen. Two molecules of superoxide can dismutate, spontaneously or enzymatically, yielding molecular oxygen and hydrogen peroxide (Eq. (3)). Fe2 O2 0 Fe3 O 2
(1)
2 Fe A Aox red 0 Fe O 2H 0 O2 H2 O2 O 2 2
(2) (3)
3
Ferrous iron can catalyse the decomposition of peroxides, yielding hydroxyl radical from hydrogen peroxide (Eq. (4)) or alkoxyl radicals, if the substrate of the reaction is an organic peroxide (LOOH) (Eq. (5)). H2 O2 Fe2 0 Fe3 OH OH+
(4)
LOOHFe2 0 Fe3 LO OH+
(5)
Thus, the participation of iron is essential in: (a) the production of hydroxyl radical that can subsequently initiate lipid oxidation or oxidise almost every molecule present in biological systems; and (b) the propagation of free radical reactions by decomposing peroxides. The relevance of iron-catalysed reactions in vivo is restricted to the negligible availability of ‘free’ catalytic iron. Nevertheless, increased generation of superoxide can favour iron release from ferritin and from proteins, and hydrogen peroxide can degrade haem from haem-proteins to release iron (Halliwell and Gutteridge, 1999). For example, 5aminolevulinic acid (ALA) autoxidation generates superoxide anion that releases iron from ferritin,
C.G. Fraga, P.I. Oteiza / Toxicology 180 (2002) 23 /32
which could lead to a higher availability of free iron in patients with acute intermittent porphyria and lead poisoning (Oteiza et al., 1995a).
3. Antioxidants and iron toxicity An antioxidant is, as a general definition, any substance capable of preventing oxidation. Deleterious free radical-mediated oxidations occur in aerobic organism as a result of normal oxygen metabolism. As described above (Section 2), iron, especially ferrous iron, is able to trigger oxidations by reducing as well as by decomposing previously formed peroxides. Thus, an antioxidant that protects from iron toxicity is a substance that can: (a) chelate ferrous iron and prevent the reaction with oxygen or peroxides; (b) chelate iron and maintain it in a redox state that makes iron unable to reduce molecular oxygen; (c) trap already formed radicals, which is a putative action of any substance that can scavenge free radicals in biological systems, regardless if they are originated from iron-dependent reactions or not. A large number of substances can chelate iron, and many of them are present in biological systems. The proteins that participate in iron metabolism can sequester iron thus preventing it from participating in free radical reactions. Ferritin, transferrin, and several iron-containing enzymes such as catalase, maintain iron at a ferric or higher reduced state, making it less reactive to initiate and/or propagate free radical reactions. Several other compounds, both natural and synthetic, can chelate iron in vivo thus limiting its participation in free radical reactions. For example, EDTA is a synthetic compound, normally present in foods, that can chelate iron. However, EDTA chelation is unable to prevent iron mediated oxygen reduction, therefore, its efficiency as an antioxidant is low. By contrast, desferrioxamine mesylate (DFO) is a powerful iron-chelating substance that almost completely suppresses iron-mediated oxidations in biological systems. DFO is often used as a therapeutic tool in the treatment of iron overload (Kicic et al., 2001). There are a number of substances that have been defined as antioxidants, because of their
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capacity to protect biomolecules from free radical-mediated damage. Many of these substances can prevent iron-mediated oxidation in different in vitro and in vivo experimental systems (Packer and Cadenas, 1997). However, the relevance for human health of most of these substances remains uncertain. Among the antioxidant substances that are synthesised by mammals, thiol compounds, especially glutathione, afford significant antioxidant protection. This protection is related to the ability of glutathione to trap radicals, reduce peroxides, and work to maintain the redox state of the cells (Milchak and Douglas Bricker, 2002; Meister, 1995). The interaction between iron overload and dietary antioxidants has been well characterised, especially with respect to vitamins E and C. Vitamin E, a mixture of various tocopherols and tocotrienols, is the most important lipid-soluble antioxidant (Packer et al., 2001; Ricciarelli et al., 2001). Vitamin E has been extensively studied with respect to its capacity to protect molecules from the in vitro and in vivo effects of iron toxicity. Results from our group, as well as from other laboratories, have shown that vitamin E can prevent the majority of iron-mediated damage both, in in vitro systems, and in iron-loaded animals (Tappel and Dillard, 1981; Lucesoli and Fraga, 1999; Gavino et al., 1984; Galleano and Puntarulo, 1997; Bartfay et al., 1998). In hemodialysis patients, vitamin E administration (1200 U/day) attenuated the extent of lipid oxidation (Roob et al., 2000). Oxidative stress is a general condition in hemodialysis patients (Salahudeen et al., 2001; de Cavanagh et al., 1999), the periodic intravenous iron injection being a factor contributing to oxidative stress. Vitamin E may protect patients on hemodialysis from degenerative diseases, occurring in the long term, by decreasing the rate of free radical-mediated damage. The interaction of another dietary antioxidant, vitamin C (ascorbate) and iron is less clear. Ascorbate can reduce ‘free’ iron (ferric) to ferrous iron (see Eq. (3)), promoting the initiation (via hydroxyl radical formation, Eq. (4)), and propagation (via lipid alkoxyl radical formation, Eq. (5)) of free radical reactions (Buettner and Jurkiewicz,
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1996; Herbert et al., 1996). Although, this Fenton chemistry occurs readily in vitro, its occurrence under physiological conditions is unlikely, given the negligible availability of ‘free’ catalytic iron. As mentioned above, the levels of free iron are thought to be very low due to its sequestration by the various metal-binding proteins (ferritin, transferrin, etc.). However, in people under risk of iron overload (hemochromatosis, b-thalassemia, hemodialysis) in which the elevated levels of iron could lead to higher ‘free iron’ concentrations, an excess of vitamin C could have deleterious effects. In these cases, vitamin C tissue levels should be maintained in the low range, and vitamin C supplementation should be avoided. Supporting a pro-oxidant effect of the iron-vitamin C interaction in vivo, a recent report showed that a daily combined supplementation of iron (100 mg as fumarate) and vitamin C (500 mg as ascorbate) during the third trimester of pregnancy caused a 20% increase in plasma lipid oxidation (Lachili et al., 2001). We consider that the results of this study should be taken carefully, since only the plasma content of substances reactive to 2-thiobarbituric acid (TBARS) was determined and it is well known that this determination is not always a unequivocal evaluation of in vivo oxidative stress. More important, in several other studies, in both animal models and in humans, investigators have reported that there is no evidence of increased free radical damage due to the combined ingestion of iron and ascorbate (Proteggente et al., 2001, 2000; Chen et al., 2000; Carr and Frei, 1999).
they can participate in: (a) iron chelation; and (b) trapping radicals (van Acker et al., 1998; Sestili et al., 1998; Morel et al., 1993; Moran et al., 1997). Several reports indicate that certain flavonoids can protect lipids in liposomes from iron-mediated oxidation (Gorelik and Kanner, 2001; Lotito et al., 2000; Shirai et al., 2001; Zago and Oteiza, 2001). Interestingly, when oligomeric flavonoids (procyanidins) isolated from cocoa were assayed as inhibitors of iron-induced lipid oxidation, we observed that the degree of oligomerisation significantly influenced the antioxidant capacity of the flavonoids. The capacity of procyanidins to protect against ferrous-mediated liposome oxidation was inversely associated with their chain length (Fig. 1). In the presence of other oxidising insults, the degree of oligomerisation had the opposite effect (the longer procyanidins protected better than the shorter ones) or had no effect (Lotito et al., 2000). There is a recent study showing that flavonoids protect culture cells from iron-mediated damage (Kostyuk et al., 2001). In vivo, dietary flavonoids have been reported to inhibit carbonyl iron-induced lipid oxidation in rat liver (Pietrangelo et al., 1995). As an indirect effect, polyphenol-containing foods
4. Flavonoids and iron toxicity Flavonoids represent one class of bioactive compounds that may have multiple beneficial effects on several chronic diseases (Hollman, 2001; Kris-Etherton and Keen, 2002). Flavonoids are polyphenol compounds that are synthesised by plants, and that can be incorporated in animals through the diet. In humans, dietary sources of flavonoids include tea, red wine, purple grape juice, cocoa products, apples, onions and certain nuts. Interestingly, these compounds can play a double role in reducing the rate of oxidation, as
Fig. 1. Cocoa monomers and procyanidins in the prevention of ferrous iron-initiated TBARS production. Multillamelar liposomes were added with 0.73 mg/ml (5 mM monomer equivalent) of monomers or procyanidin fractions (M, monomer; D, dimer; T, trimer; Te, tetramer; P, pentamer; and H, hexamer), and oxidised by the addition of 25 mM ferrous sulfate/25 mM ascorbate and incubated for 60 min at 37 8C. Results represent % of inhibition of the values obtained for control samples incubated without the addition of monomer or procyanidin fractions. Values taken from Lotito et al. (2000).
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may inhibit non haem /iron absorption as determined by erythrocyte incorporation of radio-Fe after a bread meal in adult human subjects (Hurrell et al., 1999). However, in weanling rats, chronic tea ingestion did not alter iron incorporation (Record et al., 1996). From the described studies, it can be concluded that: (a) flavonoids and related procyanidins reduce oxidative damage promoted by iron in vitro and in vivo; and (b) flavonoids can modulate iron intake, a function that may be beneficial for populations at risk for iron overload.
5. Iron interaction with other metals Other metals can interact with iron in biological systems, and this interaction can have beneficial effects preventing undesirable iron mediated damage. We consider of particular relevance the interaction between iron and zinc. One of the several mechanisms that have been shown to be involved in the antioxidant action of zinc, a metal that does not have redox activity, is the capacity of zinc to compete with iron for multiple cellular binding sites. The replacement of iron with zinc can prevent the redox-cycling of iron, thus minimising the rate of oxidising chemical groups in the close vicinity of the metal binding site. In this context, zinc can reduce the iron-mediated oxidation of lipids, proteins, and DNA. An early work showed that zinc could prevent iron-mediated oxidation of lipids in red blood cells (Girotti et al., 1985). To explore the mechanism(s) involved in the protection by zinc against iron-initiated lipid oxidation, we used synthetic liposomes and observed that the antioxidant effect of zinc depended on the negative charge density of the membrane (Zago and Oteiza, 2001). In this work we showed that zinc acted in part by competing with ferrous iron for negative binding sites in the membrane, and that the observed effects were within physiological membrane zinc concentrations. The competition of zinc for iron binding sites is particularly relevant, taking into account that one consequence of zinc deficiency can be a marked increase in membrane and intracellular iron concentration (Rogers et al., 1985, 1987). Interestingly, the
27
simultaneous addition of zinc, a-tocopherol, and ( /)-epicatechin caused an additive inhibitory effect on TBARS formation when liposomes were incubated in the presence of iron (Fig. 2; Zago and Oteiza, 2001). It can be speculated that these three compounds could act as an antioxidant network to protect lipids from oxidation: (a) zinc, by replacing iron at the membrane surface; (b) vitamin E, inhibiting lipid oxidation propagation; and (c) epicatechin, both chelating iron, and trapping radicals in the aqueous phase. With respect to the inactivation of enzymes by iron, a described mechanism is triggered by the binding of iron to divalent cation sites localised in the catalytic portion of enzymes (Stadtman, 1992; Stadtman and Levine, 2000). Bonded iron can promote the local generation of hydroxyl radical through a Fenton reaction, leading to the oxidation of specific amino acids and then to enzyme inactivation. We found that the addition of zinc to rat brain supernatants does not prevent the ferrous iron-mediated inactivation of both, glutamine synthase and glucose-6-phosphate dehydrogenase (Zago et al., 2000). Zinc can markedly reduce the in vitro iron- and copper-induced breakage of DNA. The capacity of
Fig. 2. Zinc (Z), a-tocopherol (A), and ( /)-epicatechin (E) in the prevention of ferrous iron-initiated TBARS production. Multillamelar liposomes were added with 15 mM zinc sulphate, and/or 0.01 lipid mol% a-tocopherol, and/or 0.5 mM ( /)epicatechin, and oxidised by the addition of 25 mM ferrous sulphate and incubation for 90 min at 37 8C. Results represent % of inhibition of the values obtained for control samples incubated without addition of zinc, a-tocopherol, or ( /)epicatechin. Values taken from Zago and Oteiza (2001).
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zinc to protect DNA from oxidative breakage is greater against iron than against copper (Har-El and Chevion, 1991). This protection against DNA damage is particularly important since oxidative damage to DNA, especially strand breaks, is highly dependent on the amount of iron bound to DNA. Iron bound to DNA can catalyse the sitespecific generation of hydroxyl radical. In vivo we have observed that the increased DNA damage occurring in the testes of zinc deficient rats was associated with iron accumulation (Oteiza et al., 1995b). Iron accumulation has been associated with copper deficiency in different animal experimental models (Cohen et al., 1983, 1985; Fields et al., 2001). Also genetic disorders in copper metabolism can lead to elevated tissue iron. For example, aceruloplasminemia is a rare disease, in which individuals have no ceruloplasmin in their serum (Miyajima et al., 1987). The pathogenesis of the disease has been linked to a slow iron accumulation in tissues, with no changes in tissue copper levels. Selenium is another metal that can interact with iron at physiologically relevant levels (Bartfay and Bartfay, 2001; Christensen et al., 2000). It has been suggested that iron overload can modulate Se-dependent glutathione peroxidase activity (Madra et al., 1996; Dawson et al., 2000) Iron can also interact with other metals that have no redox capacity (aluminum, gallium, indium, scandium, yttrium, beryllium, lanthanum), but that can facilitate ferrous-initiated lipid and protein oxidation through the promotion of alterations in the rheology of membranes (Verstraeten et al., 1997). Aluminum (Al3) induced the formation of clusters of negatively charged phospholipids in the bilayer where the motion of fatty acids is restricted. In these clusters, the closer proximity of the phospholipids acyl chains facilitates the propagation of lipid oxidation. Galactolipids favour this mechanism by segregating phospholipids in domains where this mechanism can be amplified (Verstraeten et al., 1998). We have shown that Al3-induced changes in membrane-phospholipid phase state favouring the displacement of the bilayer to a gel state, facilitates the propagation of ferrous-initiated lipid oxidation (Verstraeten and Oteiza, 2000).
6. Iron intoxication in testes Iron overload is deleterious to the male reproductive system. In patients with hereditary hemochromatosis the incidence of hypogonadism is high, and it has been demonstrated that iron depletion could reverse the pathology. Testes have been shown to be susceptible to iron overload in several experimental models. Table 2 shows the changes in oxidative damage to lipids, proteins and DNA determined in the testes of rats subjected to different conditions leading to iron overload. It can be observed that the oxidation of cell components was associated with increases in testes iron content, supporting a widespread tissue damage. Models of acute (dextran-iron i.p. injection) and chronic (6 weeks of feeding with diets containing carbonyl-iron) iron overload lead to increased damage to testes (Lucesoli and Fraga, 1995; Lucesoli et al., 1999; Lucesoli and Fraga, 1999). Vitamin E content in the tissues was associated with a decrease in oxidative damage, especially in the chronic model (Lucesoli and Fraga, 1999). Similar protection had been shown in tissue slices from iron-overloaded rats (Gavino et al., 1984). After acute intoxication with increasing amount of iron dextran (250 /1000 mg/kg), we observed a dose-dependent accumulation of iron in both testes and sperm cells, and a reduced spermatogenesis in the rats receiving the highest dose (Lucesoli et al., 1999). These results agree with those obtained in rats that received an i.p. injection of iron sulphate and showed extensive necrosis of the germ epithelium and altered morphology of the spermatids, damages resembling those observed in zinc deficiency (Merker et al., 1996). Iron overload secondary to zinc deficiency also caused alterations in testes physiology and oxidative stress (Oteiza et al., 1995b, 1996, 1999). A model of chronic zinc deficiency imposed for 14 days to developing male rats caused alterations in the oxidant defence system (Oteiza et al., 1996) and high levels of oxidative damage to lipids, proteins and DNA (Oteiza et al., 1995b; Table 2). Significantly, as it was earlier reported (Rogers et al., 1985, 1987), in this work it was shown that iron accumulated in the testes as a consequence of the
C.G. Fraga, P.I. Oteiza / Toxicology 180 (2002) 23 /32
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Table 2 Oxidative damage to testes in iron overload Treatment
Testes iron (nmol/g tissue)
TBARS (nmol/g tissue)
Carbonyls (nmol/mg protein)
oxo8dG (/105dG)
Chronica Chronic/VEa Acuteb Acutec Zn deficiencyd
71 27 1300 3000 67
32 (37)* /10 ( /13) 30 (90)* 44 (111)* 14 (56)*
5.43 0.11 0.32 1.98 1.20
0.5 0.3 0.6 1.7 0.3
(33)* (13) (7 /)* (10 /)* (33)*
(205)* (4) (23) (96)* (50)*
(12) (7) (25)* (74)* (31)*
* Significant difference from controls (P B/0.05). Values indicate increases over controls (non-treated rats), and figures between parenthesis (%) or times of increase ( /) over control values. a Lucesoli and Fraga, 1999. b Lucesoli and Fraga, 1995. c Lucesoli et al., 1999. d Oteiza et al., 1995b.
zinc deficiency. The low activity in the zinc deficient animals of glutamine synthase, an enzyme that is sensitive to oxidative inactivation, was correlated with testes iron content, and with protein carbonyl concentration (a product of iron-mediated protein oxidation) (Oteiza et al., 1995b). These results support the concept that part of the testes oxidative damage associated with zinc deficiency is due to a secondary iron overload. Zinc deficiency was also found to increase the sensitivity of testes to cadmium-induced oxidation (Oteiza et al., 1999). Significantly, in the zinc deficient groups the extent of cadmium-related haemorrhages and iron overload was higher than in the zinc-supplemented controls. Similarly, it has been shown that cadmium treatment results in iron and copper accumulation, a decrease in tissue zinc and in high rates of lipid oxidation, which could be attenuated by co-treatment with the antioxidant metal selenium (Yiin et al., 1999). Unpublished results from our laboratory, have shown that acute iron treatment leads to iron overload in rat sperm cells, increasing their physiological levels of DNA, protein and lipid oxidation, and affecting several parameters of sperm function (Lucesoli et al., unpublished).
mediated tissue damage rests on the availability of ferrous iron to promote free radical reactions. Since most organisms have a number of proteins that maintain most of iron sequestered, only trace amounts of iron are ‘free’ or available for catalysing free radical reactions. Under certain pathological conditions, iron can be released from storage and/or ‘free iron’ pools can be increased, leading to an oxidative stress condition. The strategies to control iron-mediated free radical damage, could be to: (a) prevent conditions that can lead to iron overload; (b) provide chelating substances that sequester excess iron; (c) protect functional molecules (lipid, proteins, DNA) from oxidation with the use of dietary antioxidants (vitamins, flavonoids, zinc, etc.). The overall results presented here support a beneficial effect of antioxidants, obtained either from the diet or by supplementation, in conditions of iron overload. In this regard, flavonoids are substances with both chelating and free radical scavenging properties that deserve further research in iron overload situations.
Acknowledgements 7. Conclusion Iron overload toxicity has been generally associated with a condition of free radical-mediated tissue damage. The free radical mechanism of iron-
This work was supported by grants from the University of Buenos Aires, CONICET B042 to CGF and B054 to PO), and Ministry of Health (Carrillo/Onativia fellowship).
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References Bartfay, W.J., Bartfay, E., 2001. Selenium and glutathione peroxidase with beta-thalassemia major. Nurs. Res. 50, 178 /183. Bartfay, W.J., Hou, D., Brittenham, G.M., Bartfay, E., Sole, M.J., Lehotay, D., Liu, P.P., 1998. The synergistic effects of vitamin E and selenium in iron-overloaded mouse hearts. Can. J. Cardiol. 14, 937 /941. Beckman, L.E., Van Landeghem, G.F., Sikstrom, C., Wahlin, A., Markevarn, B., Hallmans, G., Lenner, P., Athlin, L., Stenling, R., Beckman, L., 1999. Interaction between haemochromatosis and transferrin receptor genes in different neoplastic disorders. Carcinogenesis 20, 1231 /1233. Berg, D., Gerlach, M., Youdim, M.B., Double, K.L., Zecca, L., Riederer, P., Becker, G., 2001. Brain iron pathways and their relevance to Parkinson’s disease. J. Neurochem. 79, 225 /236. Buettner, G.R., Jurkiewicz, B.A., 1996. Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat. Res. 145, 532 /541. Carr, A., Frei, B., 1999. Does vitamin C act as a pro-oxidant under physiological conditions. FASEB J. 13, 1007 /1024. Chen, K., Suh, J., Carr, A.C., Morrow, J.D., Zeind, J., Frei, B., 2000. Vitamin C suppresses oxidative lipid damage in vivo, even in the presence of iron overload. Am. J. Physiol. Endocrinol. Metab. 279, E1406 /E1412. Christensen, M.J., Olsen, C.A., Hansen, D.V., Ballif, B.C., 2000. Selenium regulates expression in rat liver of genes for proteins involved in iron metabolism. Biol. Trace Elem. Res. 74, 55 /70. Cohen, N.L., Keen, C.L., Lonnerdal, B., Hurley, L.S., 1983. The effect of copper chelating drugs on liver iron mobilisation in the adult rat. Biochem. Biophys. Res. Commun. 113, 127 /134. Cohen, N.L., Keen, C.L., Lonnerdal, B., Hurley, L.S., May, J.N., 1985. Effects of varying dietary iron on the expression of copper deficiency in the growing rat: anaemia, ferroxidase I and II, tissue trace elements, ascorbic acid, and xanthine dehydrogenase. J. Nutr. 115, 633 /649. Dawson, E.B., Albers, J.H., McGanity, W.J., 2000. The apparent effect of iron supplementation on serum selenium levels in teenage pregnancy. Biol. Trace Elem. Res. 77, 209 / 217. de Cavanagh, E.M., Ferder, L., Carrasquedo, F., Scrivo, D., Wassermann, A., Fraga, C.G., Inserra, F., 1999. Higher levels of antioxidant defences in enalapril-treated versus non-enalapril-treated hemodialysis patients. Am. J. Kidney Dis. 34, 445 /455. Ellervik, C., Mandrup-Poulsen, T., Nordestgaard, B.G., Larsen, L.E., Appleyard, M., Frandsen, M., Petersen, P., Schlichting, P., Saermark, T., Tybjaerg-Hansen, A., Birgens, H., 2001. Prevalence of hereditary haemochromatosis in late-onset type 1 diabetes mellitus: a retrospective study. Lancet 358, 1405 /1409.
Fields, M., Lewis, C.G., Bureau, I., 2001. Aspirin reduces blood cholesterol in copper-deficient rats: a potential antioxidant agent. Metabolism 50, 558 /561. Galleano, M., Puntarulo, S., 1997. Dietary alpha-tocopherol supplementation on antioxidant defences after in vivo iron overload in rats. Toxicology 124, 73 /81. Gavino, V.C., Dillard, C.J., Tappel, A.L., 1984. Release of ethane and pentane from rat tissue slices: effect of vitamin E, halogenated hydrocarbons, and iron overload. Arch. Biochem. Biophys. 233, 741 /747. Girotti, A.W., Thomas, J.P., Jordan, J.E., 1985. Inhibitory effect of Zn (II) on free radical lipid peroxidation in erythrocyte membranes. Free Radic. Biol. Med. 1, 395 /401. Gorelik, S., Kanner, J., 2001. Oxymyoglobin oxidation and membrane lipid peroxidation initiated by iron redox cycle: prevention of oxidation by enzymic and nonenzymic antioxidants. J. Agric. Food Chem. 49, 5945 /5950. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford University Press, Oxford, pp. 1 /936. Har-El, R., Chevion, M., 1991. Zinc(II) protects against metalmediated free radical induced damage: studies on single and double-strand DNA breakage. Free Radic. Res. Commun. 12, 509 /515. Herbert, V., Shaw, S., Jayatilleke, E., 1996. Vitamin C-driven free radical generation from iron. J. Nutr. 126, 1213S / 1220S. Hollman, P.C.H., 2001. Evidence for health benefits of plant phenols: local or systemic effects. J. Sci. Food Agric. 81, 842 /852. Hurrell, R.F., Reddy, M., Cook, J.D., 1999. Inhibition of nonhaem iron absorption in man by polyphenolic-containing beverages. Br. J. Nutr. 81, 289 /295. Kicic, A., Chua, A.C., Baker, E., 2001. Effect of iron chelators on proliferation and iron uptake in hepatoma cells. Cancer 92, 3093 /3110. Kostyuk, V.A., Potapovich, A.I., Vladykovskaya, E.N., Korkina, L.G., Afanas’ev, I.B., 2001. Influence of metal ions on flavonoid protection against asbestos-induced cell injury. Arch. Biochem. Biophys. 385, 129 /137. Kris-Etherton, P., Keen, C.L., 2002. Evidence that the antioxidant flavonoids in tea and cocoa are beneficial for cardiovascular health. Curr. Opin. Lipidol. 13, 41 /49. Lachili, B., Hininger, I., Faure, H., Arnaud, J., Richard, M.J., Favier, A., Roussel, A.M., 2001. Increased lipid peroxidation in pregnant women after iron and vitamin C supplementation. Biol. Trace Elem. Res. 83, 103 /110. Li, J., Zhu, Y., Singal, D.P., 2000. HFE gene mutations in patients with rheumatoid arthritis. J. Rheumatol. 27, 2074 / 2077. Lotito, S.B., Actis-Goretta, L., Renart, L., Caligiuri, M., Rein, D., Schmitz, H.H., Steinberg, F.M., Keen, C.L., Fraga, C.G., 2000. Influence of oligomer chain length on the antioxidant activity of procyanidins. Biochem. Biophys. Res. Commun. 276, 945 /951. Lucesoli, F., Fraga, C.G., 1995. Oxidative damage to lipids and DNA concurrent with decrease of antioxidants in rat testes
C.G. Fraga, P.I. Oteiza / Toxicology 180 (2002) 23 /32 after acute iron intoxication. Arch. Biochem. Biophys. 316, 567 /571. Lucesoli, F., Fraga, C.G., 1999. Oxidative stress in testes of rats subjected to chronic iron intoxication and alpha-tocopherol supplementation. Toxicology 132, 179 /186. Lucesoli, F., Caligiuri, M., Roberti, M.F., Perazzo, J.C., Fraga, C.G., 1999. Dose-dependent increase of oxidative damage in the testes of rats subjected to acute iron overload. Arch. Biochem. Biophys. 372, 37 /43. Madra, S., Mann, F., Francis, J.E., Manson, M.M., Smith, A.G., 1996. Modulation by iron of hepatic microsomal and nuclear cytochrome P450, and cytosolic glutathione Stransferase and peroxidase in C57BL/10ScSn mice induced with polychlorinated biphenyls (Aroclor 1254). Toxicol. Appl. Pharmacol. 136, 79 /86. Meister, A., 1995. Glutathione metabolism. Methods Enzymol. 251, 3 /7. Merker, H.J., Vormann, J., Gunther, T., 1996. Iron-induced injury of rat testis. Andrologia 28, 267 /273. Milchak, L.M., Douglas Bricker, J., 2002. The effects of glutathione and vitamin E on iron toxicity in isolated rat hepatocytes. Toxicol. Lett. 126, 169 /177. Milman, N., Pedersen, P., Steig, T., Byg, K.E., Graudal, N., Fenger, K., 2001. Clinically overt hereditary hemochromatosis in Denmark 1948 /1985: epidemiology, factors of significance for long-term survival, and causes of death in 179 patients. Ann. Hematol. 80, 737 /744. Miyajima, H., Nishimura, Y., Mizoguchi, K., Sakamoto, M., Shimizu, T., Honda, N., 1987. Familial apoceruloplasmin deficiency associated with blepharospasm and retinal degeneration. Neurology 37, 761 /767. Moran, J.F., Klucas, R.V., Grayer, R.J., Abian, J., Becana, M., 1997. Complexes of iron with phenolic compounds from soybean nodules and other legume tissues: prooxidant and antioxidant properties. Free Radic. Biol. Med. 22, 861 /870. Morel, I., Lescoat, G., Cogrel, P., Sergent, O., Pasdeloup, N., Brissot, P., Cillard, P., Cillard, J., 1993. Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures. Biochem. Pharmacol. 45, 13 /19. Oteiza, P.I., Kleinman, C.G., Demasi, M., Bechara, E.J., 1995a. 5-Aminolevulinic acid induces iron release from ferritin. Arch. Biochem. Biophys. 316, 607 /611. Oteiza, P.I., Olin, K.L., Fraga, C.G., Keen, C.L., 1995b. Zinc deficiency causes oxidative damage to proteins, lipids and DNA in rat testes. J. Nutr. 125, 823 /829. Oteiza, P.L., Olin, K.L., Fraga, C.G., Keen, C.L., 1996. Oxidant defence systems in testes from zinc-deficient rats. Proc. Soc. Exp. Biol. Med. 213, 85 /91. Oteiza, P.I., Adonaylo, V.N., Keen, C.L., 1999. Cadmiuminduced testes oxidative damage in rats can be influenced by dietary zinc intake. Toxicology 137, 13 /22. Packer, L., Cadenas, E., 1997. Handbook of Synthetic Antioxidants. Marcel-Dekker, New York, pp. 1 /442. Packer, L., Weber, S.U., Rimbach, G., 2001. Molecular aspects of alpha-tocotrienol antioxidant action and cell signalling. J. Nutr. 131, 369S /373S.
31
Parkkila, S., Niemela, O., Savolainen, E.R., Koistinen, P., 2001. HFE mutations do not account for transfusional iron overload in patients with acute myeloid leukemia. Transfusion 41, 828 /831. Perez de Nanclares, G., Castano, L., Gaztambide, S., Bilbao, J.R., Pi, J., Gonzalez, M.L., Vazquez, J.A., 2000. Excess iron storage in patients with type 2 diabetes unrelated to primary hemochromatosis. New Engl. J. Med. 343, 890 / 891. Pietrangelo, A., Borella, F., Casalgrandi, G., Montosi, G., Ceccarelli, D., Gallesi, D., Giovannini, F., Gasparetto, A., Masini, A., 1995. Antioxidant activity of silybin in vivo during long-term iron overload in rats. Gastroenterology 109, 1941 /1949. Proteggente, A.R., Rehman, A., Halliwell, B., Rice-Evans, C.A., 2000. Potential problems of ascorbate and iron supplementation: pro-oxidant effect in vivo. Biochem. Biophys. Res. Commun. 227, 535 /540. Proteggente, A.R., England, T.G., Rice-Evans, C.A., Halliwell, B., 2001. Iron supplementation and oxidative damage to DNA in healthy individuals with high plasma ascorbate. Biochem. Biophys. Res. Commun. 288, 245 /251. Rasmussen, M., Folsom, A.R., Catellier, D.J., Tsai, M.Y., Garg, U., Eckfeldt, J.H., 2001. A prospective study of coronary heart disease and the hemochromatosis gene (HFE) C282Y mutation: the atherosclerosis risk in communities (ARIC) study. Atherosclerosis 154, 739 /746. Record, I.R., McInerney, J.K., Dreosti, I.E., 1996. Black tea, green tea, and tea polyphenols. Effects on trace element status in weanling rats. Biol. Trace Elem. Res. 53, 27 /43. Ricciarelli, R., Zingg, J.M., Azzi, A., 2001. Vitamin E: protective role of a Janus molecule. FASEB J. 15, 2314 / 2325. Rogers, J.M., Keen, C.L., Hurley, L.S., 1985. Zinc deficiency in pregnant Long /Evans hooded rats: teratogenicity and tissue trace elements. Teratology 31, 89 /100. Rogers, J.M., Lonnerdal, B., Hurley, L.S., Keen, C.L., 1987. Iron and zinc concentrations and 59Fe retention in developing foetuses of zinc-deficient rats. J. Nutr. 117, 1875 / 1882. Roob, J.M., Khoschsorur, G., Tiran, A., Horina, J.H., Holzer, H., Winklhofer-Roob, B.M., 2000. Vitamin E attenuates oxidative stress induced by intravenous iron in patients on hemodialysis. J. Am. Soc. Nephrol. 11, 539 /549. Salahudeen, A.K., Oliver, B., Bower, J.D., Roberts, L.J., 2001. Increase in plasma esterified F2-isoprostanes following intravenous iron infusion in patients on hemodialysis. Kidney Int. 60, 1525 /1531. Sayre, L.M., Perry, G., Atwood, C.S., Smith, M.A., 2000. The role of metals in neurodegenerative diseases. Cell. Mol. Biol. (Noisy-le-grand) 46, 731 /741. Sestili, P., Guidarelli, A., Dacha, M., Cantoni, O., 1998. Quercetin prevents DNA single strand breakage and cytotoxicity caused by tert -butylhydroperoxide: free radical scavenging versus iron chelating mechanism. Free Radic. Biol. Med. 25, 196 /200.
32
C.G. Fraga, P.I. Oteiza / Toxicology 180 (2002) 23 /32
Shirai, M., Moon, J.H., Tsushida, T., Terao, J., 2001. Inhibitory effect of a quercetin metabolite, quercetin 3-O -beta-D-glucuronide, on lipid peroxidation in liposomal membranes. J. Agric. Food Chem. 49, 5602 / 5608. Stadtman, E.R., 1992. Protein oxidation and ageing. Science 257, 1220 /1224. Stadtman, E.R., Levine, R.L., 2000. Protein oxidation. Ann. New York Acad. Sci. 899, 191 /208. Tappel, A.L., Dillard, C.J., 1981. In vivo lipid peroxidation: measurement via exhaled pentane and protection by vitamin E. Fed. Proc. 40, 174 /178. Tenenbein, M., 2001. Hepatotoxicity in acute iron poisoning. J. Toxicol. Clin. Toxicol. 39, 721 /726. van Acker, S.A., van Balen, G.P., van den Berg, D.J., Bast, A., van der Vijgh, W.J., 1998. Influence of iron chelation on the antioxidant activity of flavonoids. Biochem. Pharmacol. 56, 935 /943. Verstraeten, S.V., Oteiza, P.I., 2000. Effects of Al(3/) and related metals on membrane phase state and hydration: correlation with lipid oxidation. Arch. Biochem. Biophys. 375, 340 /346. Verstraeten, S.V., Nogueira, L.V., Schreier, S., Oteiza, P.I., 1997. Effect of trivalent metal ions on phase separation and
membrane lipid packing: role in lipid peroxidation. Arch. Biochem. Biophys. 338, 121 /127. Verstraeten, S.V., Keen, C.L., Golub, M.S., Oteiza, P.I., 1998. Membrane composition can influence the rate of Al3 mediated lipid oxidation: effect of galactolipids. Biochem. J. 333, 833 /838. Walker, E.M.J., Walker, S.M., 2000. Effects of iron overload on the immune system. Ann. Clin. Lab. Sci. 30, 354 /365. Wilkinson, H., 1996. Haemochromatosis associated with arthritis and hypopituitarism. Ann. Clin. Biochem. 333, 171 / 173. Yang, Q., McDonnell, S.M., Khoury, M.J., Cono, J., Parrish, R.G., 1998. Hemochromatosis-associated mortality in the United States from 1979 to 1992: an analysis of MultipleCause Mortality Data. Ann. Intern. Med. 129, 946 /953. Yiin, S.J., Chern, C.L., Sheu, J.Y., Lin, T.H., 1999. Cadmium indiced lipid peroxidation in rat testes and protection by selenium. Biometals 12, 353 /359. Zago, M.P., Oteiza, P.I., 2001. The antioxidant properties of zinc: interactions with iron and antioxidants. Free Radic. Biol. Med. 31, 266 /274. Zago, M.P., Verstraeten, S.V., Oteiza, P.I., 2000. Zinc in the prevention of Fe2 -initiated lipid and protein oxidation. Biol. Res. 33, 143 /150.