Association of Cu,Zn-type superoxide dismutase with mitochondria and peroxisomes

The subcellular localization of Cu,Zn-type superoxide dismutase (Cu,Zn-SOD) was investigated in rat tissues and cultured human fibroblasts. Subcellular fractionation, Nycodenz gradient centrifugation, and immunoblot analysis using specific antibodies showed that Cu,Zn-SOD was localized in cytosol, mitochondria, and peroxisomes of rat liver and brain. Treatment of highly purified mitochondria from rat liver with either Chaps or Triton X-100 released the bound Cu,Zn-SOD into supernatant fraction. Depolarization of mitochondria by inorganic phosphate and Ca(2+) released both Cu,Zn-SOD and cytochrome c from mitochondria. Digitonin also released Cu,Zn-SOD but not cytochrome c from mitochondria. Confocal immunofluorescence microscopy revealed that anti-Cu,Zn-SOD antibody in cultured human fibroblasts was found to colocalize with antibodies to Mn-SOD and PMP-70, markers of mitochondria and peroxisomes, respectively. Incubation of human Cu,Zn-SOD with purified mitochondria resulted in their association. These results indicate that Cu,Zn-SOD associates with mitochondria and peroxisomes in various cell types such as those in brain, liver, and skin.     

Changes in Superoxide Dismutase Activity in Liver and Lung of Old Rats

Superoxide dismutase activity was measured in liver and lung from 3 and 24 month-old rats. Both total SOD and Mn-SOD activity decreased significantly in the liver of old rats. Recent results from our laboratory have indicated that during aging, the activity of Cu/Zn-SOD decreases in rat liver and that there is an accumulation of altered protein. It was also shown that the old Cu/Zn-SOD had one histidine fewer than the young one. In the present study, the immunoprecipitation experiments showed that the amount of immunoprecipitable Mn-SOD from liver of old rats was greater than from young ones, but when amino acid residues were measured in purified young and old Mn-SOD from liver, no change was observed. In lung, no significant age-related differences in total SOD, Cu/Zn-SOD and Mn-SOD activity were found, nor was there accumulation of altered protein during aging.     

Changes in Superoxide Dismutase Activity in Liver and Lung of Old Rats

Superoxide dismutase activity was measured in liver and lung from 3 and 24 month-old rats. Both total SOD and Mn-SOD activity decreased significantly in the liver of old rats. Recent results from our laboratory have indicated that during aging, the activity of Cu/Zn-SOD decreases in rat liver and that there is an accumulation of altered protein. It was also shown that the old Cu/Zn-SOD had one histidine fewer than the young one. In the present study, the immunoprecipitation experiments showed that the amount of immunoprecipitable Mn-SOD from liver of old rats was greater than from young ones, but when amino acid residues were measured in purified young and old Mn-SOD from liver, no change was observed. In lung, no significant age-related differences in total SOD, Cu/Zn-SOD and Mn-SOD activity were found, nor was there accumulation of altered protein during aging.     

Capn10, a candidate gene responsible for type 2 diabetes mellitus in the OLETF rat

The rat strain Otsuka Long-Evans Tokushima Fatty (OLETF) is an animal model for type 2 diabetes mellitus. Nidd8/of has been identified as one of 14 quantitative trait loci (QTLs) involved in the diabetes by a whole genome search in 160 F2 progenies obtained by mating the OLETF and F344 rats. Comparative mapping between human and rat indicated that the Nidd8/of genomic region, near D9rat21 on rat chromosome 9, contains the calpain10 (Capn10) gene, which is putative type 2 diabetes-susceptibility gene in humans. In this study, we found no difference in Capn10 mRNA expression in the heart, liver, skeletal muscle and pancreas between OLETF and F344 rats at 5 and 10 weeks of age. However, we found a single nucleotide polymorphism (SNP) (A/A genotype in OLETF and G/G genotype in F344 and LETO rats) at the base 583 downstream from the translation start site in the rat Capn10 cDNA sequence. This SNP was deduced to substitute serine (OLETF) for glycine (F344 and LETO) at the 195 amino acid residue within the protease domain of rat Capn10. Because serine is generally not interchangeable with glycine in respect of the protein structure and function, it was deduced that the A/A genotype in OLETF is not a 'safe' mutation. This non-conservative amino acid substitution might be associated with susceptibility to type 2 diabetes in OLETF rats.     

Hyperoxia elevates Cu,Zn-superoxide dismutase of endothelial cells as detected by a sensitive ELISA.

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of bovine Cu,Zn-SOD. Accuracy of the ELISA and specificity of the antibody for cell-free extracts was established by: (1) measurement of antigen levels of bovine endothelial cell extracts reconstituted with pure antigen, and (2) immunoblotting with affinity purified antibody. The ELISA was highly sensitive and 0.05-0.10 ng of pure antigen could be accurately detected, which allowed the measurement of Cu,Zn-SOD in as few as 250 endothelial cells. With utilization of the ELISA for detection, DEAE-cellulose chromatography patterns of endothelial cell Cu,Zn-SOD overlapped those of pure bovine erythrocyte Cu,Zn-SOD. Exposure of cells in culture to 80% O2 for 48 h increased the relative abundance of the Cu,Zn-SOD as measured by the ELISA by 1.8-fold. Thus, endothelial cells in culture respond to hyperoxia by enhanced production of Cu,Zn-SOD protein. The ELISA developed in this study may be useful for assessing other factors that regulate cellular production of Cu,Zn-SOD.     

Developmental regulation of rat lung Cu,Zn-superoxide dismutase.

In the present investigation we found that lung Cu,Zn-superoxide dismutase (SOD) activity (units/mg of DNA) increases steadily in the rat from birth to adulthood. The specific activity (units/micrograms of enzyme) of Cu,Zn-SOD was unchanged from birth to adulthood, excluding enzyme activation as a mechanism responsible for the increase in enzyme activity. Lung synthesis of Cu,Zn-SOD peaked at 1 day before birth and decreased thereafter to adult values. Calculations, based on rates of Cu,Zn-SOD synthesis and the tissue content of the enzyme, indicated that lung Cu,Zn-SOD activity increased during development owing to the rate of enzyme synthesis exceeding its rate of degradation by 5-10%. These calculations were supported by measurements of enzyme degradation in the neonatal (half-life, t1/2, = 12 h) and adult lung (t1/2 = greater than 100 h); the difference in half-life did not reflect the rates of overall protein degradation in the lung, since these rates were not different in lungs from neonatal and adult rats. We did not detect differences in the Mr or pI of Cu,Zn-SOD during development, but the susceptibility of the enzyme to inactivation by heat or copper chelation decreased with increasing age of the rats. We conclude that the progressive increase in activity of Cu,Zn-SOD is due to a rate of synthesis that exceeds degradation of the enzyme. The data also suggest that increased stabilization of enzyme conformation accounts for the greater half-life of the enzyme in lungs of adult compared with neonatal rats.     

The effect of natural dicarbonyls on activity of antioxidant enzymes in vitro and in vivo

Natural dicarbonyls, which may be accumulated during oxidative stress in atherosclerosis (e.g. malondialdehyde) or carbonyl stress in diabetes mellitus (glyoxal and methylglyoxal) effectively inhibited activities of commercial preparations of the antioxidant enzymes: Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and Se-contained glutathione peroxidase from human and bovine erythrocytes, and also rat liver glutathione-S-transferase. After incubation of human erythrocytes with 10 mM of each investigated dicarbonyls the decrease of intracellular Cu,Zn-SOD was observed. The decreased activity of erythrocyte Cu,Zn-SOD was also detected in patients with diabetes mellitus type 2 with carbohydrate metabolism impairments but effective sugar-lowered therapy was accompanied by the increase of this enzyme activity. The increase of erythrocytes Cu,Zn-SOD activity in diabetic patients treated with metformin (which may utilize methylgly-oxal) was higher than in erythrocytes of diabetic patients subjected to traditional therapy.     

Influence of age on Cu/Zn-superoxide dismutase and indole 2,3-dioxygenase activities in rat tissues

The aim of this study was to investigate in vitro the variations with age of the activities of the two antioxidant enzymes Cu/Zn-superoxide dismutase (SOD) and indole 2,3-dioxygenase (IDO) in metabolically active tissues of rats of various ages. In rats aged one week and 2-3 months the highest Cu/Zn-SOD activity was found in the liver and the lowest in the small intestine. At 12 and 18 months of age, the activity was higher in the brain and kidneys, when compared to the small intestine, lungs and liver. Cu/Zn-SOD activity decreased significantly after 2-3 months of age with advancing age in all tissues examined. In newborn rats IDO activity was present only in the small intestine. In the group of rats aged 2-3 months, the highest specific activity was observed in the small intestine and the lowest in the lungs and kidneys, whereas at 12 months of age, the highest IDO activity was found in the brain, with kidneys presenting the lowest activity. At 18 months, IDO returned to be more elevated in the small intestine. At 12 months of age the values of IDO in the tissues varied slightly, while at 18 months similar activities were found between the lungs and brain and between the small intestine and kidneys. In relation to age, IDO specific activity declined in the small intestine, after 2-3 months of age. In the lungs, the activity remained unchanged; in the brain and in the kidneys activity decreased significantly from 2-3 to 18 months of age. In conclusion, this study demonstrates an age-related decline in Cu/Zn-SOD and IDO activities, the two enzymes responsible for scavenging O2*-.     

Multiple isoelectric variants of copper, zinc-superoxide dismutase from rat liver

Isoelectric variants of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) have been reported to exist in various organs including rat liver. To elucidate the biochemical characteristics of the variants, rat liver Cu,Zn-SOD was purified and isolated into eight variants, i.e., pI 5.15, 4.88, 4.80, 4.75, 4.70, 4.65, 4.60, and 4.50. The pI 4.88 variant had the highest specific activity (4245 U/mg protein) and the highest yield (45% of original activity). The descending order of specific activity for the other variants was pI 4.80, 4.75, 5.15, 4.70, 4.65, 4.60, and 4.50. The specific activity correlated well with metal content. The specific activity for most variants was 5-9 times greater when determined at pH 10.0 than at pH 7.8. However, three preparations of pI 4.80 and 4.70 variants had 13.9-16.3 times greater specific activity at pH 10.0 versus 7.8, while one of the pI 4.60 variants was only 3.5 times greater. The rate of Coomasie brilliant blue G-250 binding was lowest with pI 4.88 followed by pIs 4.80 and 4.75. To evaluate the mechanisms which might produce these variants, the pI 4.88 variant was incubated with xanthine-xanthine oxidase or a mixture of rat liver microsome, NADPH, and sodium azide, and a shift to variants pI 4.80 and pI 4.75 was found. The shift was greatly inhibited by the presence of mannitol or by the omitting of azide, respectively. The existence of these variants was also confirmed by other methods: (i) direct application of rat liver 105,000g supernatant to an isoelectric focusing, and (ii) extraction of SOD from acetone powder prepared from rat liver homogenate. Results indicate that several variants most likely arise in tissue as a result of activated oxygen radical modification of variant pI 4.88.     

Long distance charge redistribution upon Cu,Zn-superoxide dismutase reduction: significance for dismutase function

Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is a ubiquitous enzyme with an essential role in antioxidant defense. To better understand structural factors at the origin of the highly efficient superoxide dismutation mechanism, we analyzed the consequence of copper reduction on the electronic properties of the backbone and individual amino acids by using electrochemistry coupled to Fourier transform infrared spectroscopy. Comparison of data recorded with bovine erythrocyte and recombinant chloroplastic Cu,Zn-SOD from Lycopersicon esculentum, expressed as a functional tetramer in Escherichia coli and (14)N- or fully (15)N-labeled, demonstrated that the infrared changes were dominated by reorganizations of peptide bonds and histidine copper ligands. Two main infrared modes of histidine side chain, markers of metal coordination, were identified by using Cu- and Zn-methylimidazole models: the nu(C(4)C(5))at 1605-1594 cm(-1) or approximately 1586 cm(-1) for Ntau or Npi coordination, and the nu(C(5)Ntau) at approximately 1113-1088 cm(-1). These modes, also identified in Cu,Zn-SOD by using (15)N labeling, showed that the electronic properties of the histidine Ntau ligands of copper are mostly affected upon copper reduction. A striking conclusion of this work is that peptide groups from loops and beta-sheet largely participate in charge redistribution upon copper reduction, and in contrast, electronic properties of polar and charged amino acids of the superoxide access channel remain unaffected. This is notably shown for the strictly conserved Arg-143 by site-directed mutagenesis on chloroplastic Cu,Zn-SOD. Charge compensation by the peptide backbone and preserved electronic properties of the superoxide access channel and docking site upon copper reduction may be the determinant factors for the high reaction kinetics of superoxide with both reduced and oxidized Cu,Zn-SOD.     

Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas

Cu, Zn-superoxide dismutase (SOD) has been purified to homogeneity from Japanese flounder Paralichthys olivaceus hepato-pancreas. The purification of the enzyme was carried out by an ethanol/chloroform treatment and acetone precipitation, and then followed by column chromatographies on Q-Sepharose, S-Sepharose and Ultrogel AcA 54. On SDS-PAGE, the purified enzyme gave a single protein band with molecular mass of 17.8 kDa under reducing conditions, and showed approximately equal proportions of 17.8 and 36 kDa molecular mass under non-reducing conditions. Three bands were obtained when the purified enzyme was subjected to native-PAGE, both on protein and activity staining, but the electrophoretic mobility of the purified enzyme differed from that of bovine erythrocyte Cu, Zn-SOD. Isoelectric point values of 5.9, 6.0 and 6.2, respectively, were obtained for the three components. The N-terminal amino acid sequence of the purified enzyme was determined for 25 amino acid residues, and the sequence was compared with other Cu, Zn-SODs. The N-terminal alanine residue was unacetylated, as in the case of swordfish SOD. Above 60°C, the thermostability of the enzyme was much lower than that of bovine Cu, Zn-SOD.     

Purification and characterization of Cu,Zn-superoxide dismutase from common carp liver

1. Common carp (Cyprinus carpio L.) liver Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was purified and characterized. 2. Its molecular weight, isoelectric point, electrophoretic mobility, amino acid pattern and some other characteristics were determined.     

Role of cytosolic superoxide dismutase as a stimulator in anthranilamide hydroxylation by a microsomal monooxygenase system in rat liver

We have isolated a protein factor from rat liver which stimulates anthranilamide hydroxylation by the microsomes in the presence of NADPH and oxygen and showed this factor to contain Cu and Zn and to have superoxide dismutase activity [Biochim. Biophys. Acta 365, 148-157 (1974)]. In the present study, this protein factor was confirmed to be a superoxide dismutase (SOD) by comparison of the recovery of SOD activity with that of anthranilamide hydroxylation-stimulating activity at each step of its purification, by inhibition of SOD activity with NaCN and hydrogen peroxide (H2O2), and by recovery of the SOD activity of the protein factor after reconstitution with Cu2+ and/or Zn2+. At a given SOD activity level, there was no difference among the rat liver SOD, Cu,Zn-SOD from bovine erythrocytes, and Mn-SOD from Serratia marcescens in their ability to stimulate anthranilamide hydroxylation not only by rat liver microsomes, but also by the reconstituted cytochrome P-450-containing monooxygenase system. Rat liver SOD stimulated anthranilamide hydroxylation by the reconstituted system in proportion to its amount below a protein concentration of 1 microgram/ml. In anthranilamide hydroxylation by the reconstituted system without SOD, only a slight hydroxylase activity was found at the initial stage of the reaction and a marked increase in the amounts of NADPH oxidized and H2O2 formed was observed after a lag time. In the presence of rat liver SOD, however, the hydroxylase activity was markedly and continuously increased almost proportionally to reaction time with a concomitant decrease in the amounts of NADPH oxidized and H2O2 formed. In addition, a trace of 3-OH anthranilamide, one of the products, not only stimulated NADPH-dependent H2O2 formation in the reconstituted system, but also inhibited the apparent reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-OH anthranilamide were diminished by rat liver SOD. When a trace of 3-OH anthranilamide were added to a system composed of NADPH-cytochrome c (P-450) reductase and NADPH, H2O2 formation and NADPH oxidation were markedly stimulated. However, on addition of 3-OH anthranilamide to the system containing rat liver SOD, no stimulation on either H2O2 formation or NADPH oxidation was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

The ability of (-)deprenyl to increase superoxide dismutase activities in the rat is tissue and brain region selective.

In a previous study we have shown that chronic administration of (-)deprenyl increases activities of superoxide dismutase (SOD) and catalase (CAT) in rat striatum (1). The present study attempted to clarify how specific the effect of deprenyl is to certain tissues and brain regions in the rat. Two mg/kg/day of deprenyl was continuously infused s.c. in young male Fischer-344 rats. On the 22nd day, rats were sacrificed and enzyme activities of SOD and CAT were determined in several different brain regions and the liver. Activities of both SOD and CAT were significantly increased in striatum and substantia nigra but not in hippocampus, cerebellum or liver. Both types of SOD (i.e. Cu Zn-SOD and Mn-SOD) were significantly increased in striatum, substantia nigra. Interestingly, in cerebral cortices of three different regions, activities also tended to increase (especially those of Mn-SOD), although the increase was not so striking as in substantia nigra and striatum. The results confirm the previous observation that (-)deprenyl can increase free radical scavenger enzyme activities in striatum and provide further evidence that this effect is selective to certain brain regions and tissue types.     

Vascular aging in the longest-living rodent, the naked mole rat

The naked mole rat (NMR; Heterocephalus glaber) is the longest-living rodent known [maximum lifespan potential (MLSP): >28 yr] and is a unique model of successful aging showing attenuated declines in most physiological function. This study addresses age-related changes in endothelial function and production of reactive oxygen species in NMR arteries and vessels of shorter-living Fischer 344 rats (MLSP: approximately 3 yr). Rats exhibit a significant age-dependent decline in acetylcholine-induced responses in carotid arteries over a 2-yr age range. In contrast, over a 10-yr age range nitric oxide (NO)-mediated relaxation responses to acetylcholine and to the NO donor S-nitrosopencillamine (SNAP) were unaltered in NMRs. Cellular superoxide anion (O(2)(*-)) and H(2)O(2) production significantly increased with age in rat arteries, whereas they did not change substantially with age in NMR vessels. Indicators of apoptotic cell death (DNA fragmentation rate, caspase 3/7 activity) were significantly enhanced ( approximately 250-300%) in arteries of 2-yr-old rats. In contrast, vessels from 12-yr-old NMRs exhibited only a approximately 50% increase in apoptotic cell death. In the hearts of NMRs (2 to 26 yr old), expression of endothelial NO synthase, antioxidant enzymes (Cu,Zn-SOD, Mn-SOD, catalase, and glutathione peroxidase), the NAD(P)H oxidase subunit gp91(phox), and mitochondrial proteins (COX-IV, ATP synthase, and porin, an indicator of mitochondrial mass) did not change significantly with age. Thus long-living NMRs can maintain a youthful vascular function and cellular oxidant-antioxidant phenotype relatively longer and are better protected against aging-induced oxidative stress than shorter-living rats.     

Modification of Cu,Zn-superoxide dismutase by oxidized catecholamines

Oxidation of catecholamines may contribute to the pathogenesis of Parkinson's disease (PD). The effect of the oxidized products of catecholamines on the modification of Cu,Zn-superoxide dismutase (SOD) was investigated. When Cu,Zn-SOD was incubated with the oxidized 3,4-dihydroxyphenylalanine (DOPA) or dopamine, the protein was induced to be aggregated. The deoxyribose assay showed that hydroxyl radicals were generated during the oxidation of catecholamines in the presence of copper ion. Radical scavengers, azide, N-acetylcysteine, and catalase inhibited the oxidized catecholamine-mediated Cu,Zn-SOD aggregation. Therefore, the results indicate that free radicals may play a role in the aggregation of Cu,Zn-SOD. When Cu,Zn-SOD that had been exposed to catecholamines was subsequently analyzed by an amino acid analysis, the glycine and histidine residues were particularly sensitive. These results suggest that the modification of Cu,Zn-SOD by oxidized catecholamines might induce the perturbation of cellular antioxidant systems and led to a deleterious cell condition.     

Relationship between dietary copper concentration and acetylcholine-induced vasodilation in the microcirculation of rats

Dietary copper deficiency has been shown to significantly reduce acetylcholine (Ach)-induced vascular smooth muscle relaxation. The current study was designed to examine the relative relationship between dietary copper and the vasodilator response to Ach in the microcirculation of the rat. Male weanling rats were fed a purified basal diet supplemented with 6.0, 3.0, 1.5 or 0.0 microg Cu/g diet for 4 weeks to provide an adequate, two marginal, and deficient intakes of dietary copper. Arteriole dilation in response to increasing concentrations of acetylcholine (10(-7) to 10(-4) M) was measured in the in vivo cremaster muscle microcirculation for each dietary group. Liver copper and both aortic and erythrocyte Cu,Zn-SOD activity were used as indices of systemic copper status. Dilation to the increasing concentrations of Ach was only different in the 0 microg Cu supplemented group compared to the copper-adequate control values. However, the combined results showed an exponential increase in 10(-5) M Ach-induced vasodilation as liver copper concentration increases from 0 microg Cu/g dry wt. This relationship suggests that dilation is attenuated at liver Cu concentrations below 5 microg/g dry wt. The results indicate that Ach-induced vasodilation is copper-dependent but that the pathway is not very sensitive to short-term marginal restriction of copper intake.     

The lack of binding of methyl-n-amyl ketone (MAK) to rat liver DNA as demonstrated by direct binding measurements, and 32P-postlabeling techniques.

It has been reported that 14C-labeled methyl-n-amyl ketone (MAK, 2-heptanone) is able to bind spontaneously, in vitro, to isolated rat liver DNA to the extent of 400 pmol/mg DNA; and that 14C-MAK, when given by gavage to female Fischer 344 rats, resulted in HPLC chromatograms of isolated, hydrolyzed liver DNA in which some radiolabel was not associated with the four normal DNA bases dA, dT, dC, and dG. The present studies were undertaken to re-examine the hypothesis that MAK is able to bind to rat liver DNA. In the in vitro study, liver nuclear DNA was incubated with [2-14C]-labeled MAK (25 mCi/mmol) in the absence, or in the presence of rat liver microsomes, precipitated, washed free of unbound MAK, and counted by scintillation spectrometry. No binding to DNA by MAK was detectable. In the in vivo study, groups of five female F344 rats were exposed by inhalation to 0, 80, 400, or 1000 ppm MAK for 6 h/day for 10 days. DNA was purified from the liver nuclei of the 0 and 1000 ppm dosed animals, and 32P-postlabeling techniques were used to assay for adducts. No DNA adducts were detected using these techniques. It was concluded that MAK lacks the ability to bind to rat liver DNA in vitro and in vivo.     

Limited effects of combined dietary copper deficiency/iron overload on oxidative stress parameters in rat liver and plasma

Copper (Cu) deficiency decreases the activity of Cu-dependent antioxidant enzymes such as Cu,zinc-superoxide dismutase (Cu,Zn-SOD) and may be associated with increased susceptibility to oxidative stress. Iron (Fe) overload represents a dietary oxidative stress relevant to overuse of Fe-containing supplements and to hereditary hemochromatosis. In a study to investigate oxidative stress interactions of dietary Cu deficiency with Fe overload, weanling male Long–Evans rats were fed one of four sucrose-based modified AIN-93G diets formulated to differ in Cu (adequate 6 mg/kg diet vs. deficient 0.5 mg/kg) and Fe (adequate 35 mg/kg vs. overloaded 1500 mg/kg) in a 2×2 factorial design for 4 weeks prior to necropsy. Care was taken to minimize oxidation of the diets prior to feeding to the rats. Liver and plasma Cu content and liver Cu,Zn-SOD activity declined with Cu deficiency and liver Fe increased with Fe overload, confirming the experimental dietary model. Liver thiobarbituric acid reactive substances were significantly elevated with Fe overload (pooled across Cu treatments, 0.80±0.14 vs. 0.54±0.08 nmol/mg protein; P<.0001) and not affected by Cu deficiency. Liver cytosolic protein carbonyl content and the concentrations of several oxidized cholesterol species in liver tissue did not change with these dietary treatments. Plasma protein carbonyl content decreased in Cu-deficient rats and was not influenced by dietary Fe overload. The various substrates (lipid, protein and cholesterol) appeared to differ in their susceptibility to the in vivo oxidative stress induced by dietary Fe overload, but these differences were not exacerbated by Cu deficiency.