Pollutant source or sink? Adsorption and mobilization of PFOS and PFOA from sediments in a large shallow lake with extended reed belt.

In this study, we i) assessed the occurrence of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in sediments, pore water, and bulk water from three different areas in Lake Neusiedl, Austria, and ii) investigated mechanisms regulating adsorption and remobilization of these substances under different conditions via multiple lab-scale experiments. The adsorption capacity was mainly influenced by sediments' organic matter content, oxide composition, and pre-loading. Results suggest that a further increase of PFAS-concentrations in the open lake can be partly buffered by sediment transport to the littoral zone and adsorption to sediments in the extended reed belt. But, under current conditions, the conducted experiments revealed a real risk for mobilization of PFOS and PFOA from reed belt sediments that may lead to their transport back into the lake. The amount of desorbed PFAS is primarily dependent on water/sediment- or pore water/water-ratios and the concentration gradient. In contrast, water matrix characteristics and oxygen levels played a minor role in partitioning. The highest risk for remobilizing PFOS and PFOA was observed in experiments with sediments taken near the only major tributary to the lake (river Wulka), which had the highest pre-loading. The following management advice for water transport between high and low polluted areas can be derived based on the results. First, to reduce emissions into Lake waters from polluted tributaries like the Wulka river, we recommend diffuse pathways through the reed belt in the lake's littoral to reduce pollutant transport into the Lake and avoid high local sediment loadings. Second, water exchange with dried-up areas with probable higher loadings should be carefully handled and monitored to avoid critical back transport in the open lake. And third, general work in the reed belt or generally in the reed should be accompanied by monitoring to prevent uncontrolled remobilization in the future.

Advanced wastewater treatment with ozonation and granular activated carbon filtration: Inactivation of antibiotic resistance targets in a long-term pilot study.

The inactivation of antibiotic resistant bacteria (ARB) and genes (ARGs) in an advanced plant combining ozonation and granular activated carbon (GAC) filtration applied for effluent after conventional activated sludge treatment at a full-scale urban wastewater treatment plant was investigated for over 13 consecutive months. The nitrite compensated specific ozone dose ranged between 0.4 and 0.7 g O3/g DOC with short-time sampling campaigns (0.2-0.9 g O3/g DOC). Samples were analysed with culture-dependent methods for bacterial targets and with qPCR for genes. The log removal values were correlated with a decrease of the matrix UV absorption at 254 nm (ΔUV254) and indicated a range of ΔUV254 that corresponds to a sufficient membrane damage to affect DNA. For trimethoprim/sulfamethoxazole resistant E. coli, sul1, ermB and tetW, this phase was observed at ΔUV254 of ~30 % (~0.5 g O3/g DOC). For ampicillin resistant E. coli and blaTEM-1, it was observed around 35-40 % (~0.7 g O3/g DOC), which can be linked to mechanisms related to oxidative damages in bacteria resistant to bactericidal antibiotics. GAC treatment resulted in a further abatement for trimethoprim/sulfamethoxazole E. coli, sul1 and tetW, and in increase in absolute and relative abundance of ermB and blaTEM-1.

Integrated analysis of genetic and proteomic data identifies biomarkers associated with adverse events following smallpox vaccination.

Complex clinical outcomes, such as adverse reaction to vaccination, arise from the concerted interactions among the myriad components of a biological system. Therefore, comprehensive etiological models can be developed only through the integrated study of multiple types of experimental data. In this study, we apply this paradigm to high-dimensional genetic and proteomic data collected to elucidate the mechanisms underlying the development of adverse events (AEs) in patients after smallpox vaccination. As vaccination was successful in all of the patients under study, the AE outcomes reported likely represent the result of interactions among immune system components that result in excessive or prolonged immune stimulation. In this study, we examined 1442 genetic variables (single nucleotide polymorphisms) and 108 proteomic variables (serum cytokine concentrations) to model AE risk. To accomplish this daunting analytical task, we employed the Random Forests (RF) method to filter the most important attributes, then we used the selected attributes to build a final decision tree model. This strategy is well suited to integrated analysis, as relevant attributes may be selected from categorical or continuous data. Importantly, RF is a natural approach for studying the type of gene-gene, gene-protein and protein-protein interactions we hypothesize to be involved in the development of clinical AEs. RF importance scores for particular attributes take interactions into account, and there may be interactions across data types. Combining information from previous studies on AEs related to smallpox vaccination with the genetic and proteomic attributes identified by RF, we built a comprehensive model of AE development that includes the cytokines intercellular adhesion molecule-1 (ICAM-1 or CD54), interleukin-10 (IL-10), and colony stimulating factor-3 (CSF-3 or G-CSF) and a genetic polymorphism in the cytokine gene interleukin-4 (IL4). The biological factors included in the model support our hypothesized mechanism for the development of AEs involving prolonged stimulation of inflammatory pathways and an imbalance of normal tissue damage repair pathways. This study shows the utility of RF for such analytical tasks, while both enhancing and reinforcing our working model of AE development after smallpox vaccination.

Evaporative cooling feature selection for genotypic data involving interactions.

MOTIVATION: The development of genome-wide capabilities for genotyping has led to the practical problem of identifying the minimum subset of genetic variants relevant to the classification of a phenotype. This challenge is especially difficult in the presence of attribute interactions, noise and small sample size. METHODS: Analogous to the physical mechanism of evaporation, we introduce an evaporative cooling (EC) feature selection algorithm that seeks to obtain a subset of attributes with the optimum information temperature (i.e. the least noise). EC uses an attribute quality measure analogous to thermodynamic free energy that combines Relief-F and mutual information to evaporate (i.e. remove) noise features, leaving behind a subset of attributes that contain DNA sequence variations associated with a given phenotype. RESULTS: EC is able to identify functional sequence variations that involve interactions (epistasis) between other sequence variations that influence their association with the phenotype. This ability is demonstrated on simulated genotypic data with attribute interactions and on real genotypic data from individuals who experienced adverse events following smallpox vaccination. The EC formalism allows us to combine information entropy, energy and temperature into a single information free energy attribute quality measure that balances interaction and main effects. AVAILABILITY: Open source software, written in Java, is freely available upon request.

ARL 17477, a potent and selective neuronal NOS inhibitor decreases infarct volume after transient middle cerebral artery occlusion in rats.

We tested the effects of administration of a selective neuronal nitric oxide synthase (nNOS) inhibitor, ARL 17477, on ischemic cell damage and regional cerebral blood flow (rCBF), in rats subjected to transient (2 h) middle cerebral artery (MCA) occlusion and 166 h of reperfusion (n = 48) and in rats without MCA occlusion (n = 25), respectively. Animals were administered ARL 17477 (i.v.): 10 mg/kg; 1 mg/kg; 3mg/kg; N-nitro-L-arginine (L-NA) 10 mg/kg L-NA 1 mg/kg; and Vehicle. Administration of ARL 17477 1 mg/kg, 3 mg/kg and 10 mg/kg reduced ischemic infarct volume by 53 (p < 0.05), 23, and 6.5%, respectively. L-NA 1 mg/kg and 10 mg/kg increased infarct volume by 2 and 15%, respectively (p > 0.05). Administration of ARL 17477 (10 mg/kg) significantly (p < 0.05) decreased rCBF by 27 +/- 5.3 and 24 +/- 14.08% and cortical NOS activity by 86 +/- 14.9 and 91 +/- 8.9% at 10 min or 3 h, respectively, and did not alter mean arterial blood pressure (MABP). L-NA (10 mg/kg) significantly reduced rCBF by 23 +/- 9.8% and NOS activity by 81 +/- 7% and significantly (p < 0.05) increased MABP. Treatment with 3 mg/kg and 1 mg/kg ARL 17477 reduced rCBF by only 2.4 +/- 4.5 and 0%, respectively, even when NOS activity was reduced by 63 +/- 13.4 and 45 +/- 15.7% at 3 h, respectively, (p < 0.05). The data demonstrate that ARL 17477 inhibits nNOS in the rat brain and causes a dose-dependent reduction in infarct volume after transient MCA occlusion.

Ferritin as a source of iron for oxidative damage.

The generation of deleterious activated oxygen species capable of damaging DNA, lipids, and proteins requires a catalyst such as iron. Once released, ferritin iron is capable of catalyzing these reactions. Thus, agents that promote iron release may lead to increased oxidative damage. The superoxide anion formed enzymatically, radiolytically, via metal-catalyzed oxidations, or by redox cycling xenobiotics reductively mobilizes ferritin iron and promotes oxidative damage. In addition, a growing list of compounds capable of undergoing single electron oxidation/reduction reactions exemplified by paraquat, adriamycin, and alloxan have been reported to release iron from ferritin. Because the rapid removal of iron from ferritin requires reduction of the iron core, it is not surprising that the reduction potential of a compound is a primary factor that determines whether a compound will mobilize ferritin iron. The reduction potential does not, however, predict the rate of iron release. Therefore, ferritin-dependent oxidative damage may be involved in the pathogenesis of diseases where increased superoxide formation occurs and the toxicity of chemicals that increase superoxide production or have an adequate reduction potential to mobilize ferritin iron.

Discovery and development of neuronal nitric oxide synthase inhibitors.

The role of neuronally derived nitric oxide (NO) in neurotransmission and neural injury remains an area of active investigation. NO generation has been postulated to be involved in the deleterious events surrounding ischemia/reperfusion injury either directly or via the production of more reactive oxidants such as peroxynitrite. In our search for novel therapeutics for the treatment of a variety of neurological diseases including stroke, we have discovered novel, potent, and selective inhibitors of the neuronal nitric oxide synthase (nNOS) isoform. These compounds have proven to be effective in models of ischemia/reperfusion supporting the role of nNOS in these processes. The effects of these compounds as well as additional aspects critical to their development will be presented.

Lack of involvement of neuronal nitric oxide synthase in the pathogenesis of a transgenic mouse model of familial amyotrophic lateral sclerosis.

A subset of familial cases of amyotrophic lateral sclerosis are linked to missense mutations in copper/zinc superoxide dismutase type 1. Patients with missense mutations in copper/zinc superoxide dismutase type 1 develop a paralytic disease indistinguishable from sporadic amyotrophic lateral sclerosis through an unknown toxic gain of function. Nitric oxide reacts with the superoxide anion to form the strong oxidant, peroxynitrite, which participates in neuronal injury in a variety of model systems. Peroxynitrite is an alternate substrate for copper/zinc superoxide dismutase type 1, causing catalytic nitration of tyrosine residues in other proteins. Mutations in copper/zinc superoxide dismutase type 1 may disrupt the active site of the enzyme and permit greater access of peroxynitrite to copper, leading to increased nitration by peroxynitrite of critical cellular targets. To investigate whether neuronal-derived nitric oxide plays a role in the pathogenesis of familial amyotrophic lateral sclerosis, we examined the effects of three different nitric oxide synthase inhibitors: a non-selective nitric oxide synthase inhibitor, nitro-L-arginine methyl ester; a relatively selective inhibitor of neuronal nitric oxide synthase, 7-nitroindazole; and a novel highly selective neuronal nitric oxide synthase inhibitor, AR-R 17,477, in transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 (Gly-->Ala at position 93; G93A) containing a high transgene copy number and a low transgene copy number. AR-R 17,477, but not nitro-L-arginine methyl ester or 7-nitroindazole, significantly prolonged survival in both the high and low transgene transgenic mice. To determine whether neuronal nitric oxide synthase is involved in the pathogenesis resulting from the familial amyotrophic lateral sclerosis copper/zinc superoxide dismutase type 1 mutation, we produced mice with the copper/zinc superoxide dismutase type 1 mutation which lack the neuronal nitric oxide synthase gene. The transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background do not live significantly longer than transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1. Western blot analysis indicates the presence of two neuronal nitric oxide synthase-like immunoreactive bands in spinal cord homogenates of the neuronal nitric oxide synthase null mice, and residual neuronal nitric oxide synthase catalytic activity ( > 7%) is detected in the spinal cord of the transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background. This amount of residual activity probably does not account for lack of protection afforded by the disrupted neuronal nitric oxide synthase gene in the familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 mice. Immunological nitric oxide synthase is not detected in the copper/zinc superoxide dismutase type 1 mutant mice at several different ages, thus excluding immunological nitric oxide synthase as a contributor to the pathogenesis of familial amyotrophic lateral sclerosis. Levels of neuronal nitric oxide synthase as well as Ca2+-dependent nitric oxide synthase catalytic activity in the copper/zinc superoxide dismutase type 1 mutant mice do not differ from wild type mice. Endothelial nitric oxide synthase levels may be decreased in the copper/zinc superoxide dismutase type 1 mutant mice. Together, these results do not support a significant role for neuronal-derived nitric oxide in the pathogenesis of familial amyotrophic lateral sclerosis transgenic mice.

Radiolabeled neuronal nitric oxide synthase inhibitors: synthesis, in vivo evaluation, and primate PET studies.

UNLABELLED: The objectives of this study were to synthesize neuronal nitric oxide synthase (NOS-I)-selective imaging agents based on the 2 potent, selective inhibitors AR-R 17443 [N-(4-((2-((phenylmethyl) (methyl)-amino)ethyl)phenyl)-2-thiophenecarboximidamide)] and AR-R 18512 [(N(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-yl)-2-thiophenecarboxim idamide)] in positron-emitting form and to evaluate regional brain uptake in rodents and primates. METHODS: [11C]AR-R 17443 and [11C]AR-R 18512 were produced by N-alkylation of the corresponding desmethyl precursors using [11C]iodomethane. Regional brain uptake of [11C]AR-R 17443 and [11C]AR-R 18512 was assayed in rats and NOS-I knockout mice, and PET was performed in baboons. Tracer kinetic modeling used a 2-compartment plasma and brain tissue model. RESULTS: Yields of [11C]AR-R 17443 and [11C]AR-R 18512 ranged from 8% to 16% at the end of synthesis, with specific activities of 50-178 GBq/micromol (1,350-4,800 Ci/mmol) at the end of synthesis. In rat cerebellum and cortex at 30 min after injection, [11C]AR-R 17443 showed 1.01 +/- 0.01 and 1.63 +/- 0.12 percentage injected dose per gram (%ID/g) uptake, respectively, whereas [11C]AR-R 18512 showed 0.88 +/- 0.01 and 1.30 +/- 0.07 %ID/g uptake, respectively. Attempts to block tracer uptake by pretreatment with the NOS-I-selective inhibitor 7-nitroindazole or the corresponding unlabeled inhibitor (or desmethyl precursor to AR-R 17443 of similar potency) were unsuccessful. A small but significant (20%) decrease in cerebellar uptake of [11C]AR-R 18512 was present in NOS-I knockout mice compared with control mice. PET of [11C]AR-R 18512 in baboons with concurrent regional cerebral blood flow (rCBF) determination before and after administration of blocker showed dose-related decreases in cerebellar uptake that were greater than or equal to decreases in rCBF. Plasma metabolites accounted for 27% of total activity at 30 min after injection. Kinetic modeling of binding potentials revealed a distribution volume of 334 in cerebral blood that dropped 51% after blocker administration. CONCLUSION: Rodent studies for [11C]AR-R 17443 and [11C]AR-R 18512 showed little evidence of specific NOS-I binding. In baboons, we detected a higher uptake of [11C]AR-R 18512 in the cerebellum than in the cortex (approximately 5%, accounting for decreased rCBF because of blockade), indicating minimal specific binding. Analogs of higher affinity are likely required if this class of agents is to prove viable for PET.

Delayed production of nitric oxide contributes to NMDA-mediated neuronal damage.

Exposure of primary murine cortical neuron cultures to N-methyl-D-aspartate (NMDA) resulted in neuronal death as evidenced by release of lactate dehydrogenase (LDH) into the media. The addition of N-nitro-L-arginine (N-Arg) protected the neurons from death in a concentration-dependent manner when added after the NMDA, but not when the N-Arg was present with the NMDA. Protection by N-Arg was lost if L-arginine containing media was added to the cultures prior to the addition of the N-Arg. Treatment of the neurons with kainate prior to NMDA reduced subsequent NMDA-induced damage which was not prevented with N-Arg. These results suggest that delayed production of nitric oxide (NO) contributes to NMDA-induced neuronal damage in culture.

Inhibition of neuronal nitric oxide synthase protects against MPTP toxicity.

Previous work showed that several relatively specific inhibitors of neuronal nitric oxide synthase (nNOS) produce protection against MPTP induced dopaminergic toxicity. We examined whether a highly specific novel inhibitor of nNOS, ARRI 7338, could also protect against MPTP toxicity. ARR17338 produced dose-dependent significant protection against MPTP induced depletion of dopamine and protected against MPTP induced depletions of tyrosine hydroxylase immunostained neurons in the substantia nigra. These results provide further evidence that inhibitors of nNOS may be useful for the treatment of Parkinson's disease.

A staff-managed ICU.

After hospital-wide efforts at empowerment, an ICU staff developed and implemented a self-governance model. Difficulties included lack of involvement by some staff and lack of recognition of the elected Professional Practice Council as "director" of ICU. After one year, these problems have been overcome in part.

Zebrafish developmental screening of the ToxCast™ Phase I chemical library.

Zebrafish (Danio rerio) is an emerging toxicity screening model for both human health and ecology. As part of the Computational Toxicology Research Program of the U.S. EPA, the toxicity of the 309 ToxCast™ Phase I chemicals was assessed using a zebrafish screen for developmental toxicity. All exposures were by immersion from 6-8 h post fertilization (hpf) to 5 days post fertilization (dpf); nominal concentration range of 1 nM-80 µM. On 6 dpf larvae were assessed for death and overt structural defects. Results revealed that the majority (62%) of chemicals were toxic to the developing zebrafish; both toxicity incidence and potency was correlated with chemical class and hydrophobicity (logP); and inter-and intra-plate replicates showed good agreement. The zebrafish embryo screen, by providing an integrated model of the developing vertebrate, compliments the ToxCast assay portfolio and has the potential to provide information relative to overt and organismal toxicity.

N-nitro-L-arginine and N-monomethyl-L-arginine exhibit a different pattern of inactivation toward the three nitric oxide synthases.

The ability of NG-nitro-L-arginine (NNA) and NG-methyl-L-arginine (NMMA) to inactivate native neuronal, endothelial cell, and macrophage nitric oxide synthases (nNOS, eNOS, and iNOS, respectively) was investigated. Each NOS isozyme (plus cofactors) was preincubated with either NNA or NMMA and then assayed for remaining activity by measuring the conversion of labeled L-arginine to labeled L-citrulline. Consistent with previous reports (Olken, N. M., et al., Biochem. Biophys. Res. Commun. 177, 828-833, 1991), NMMA was a mechanism-based irreversible inhibitor of iNOS, exhibiting time- and concentration-dependent inactivation of iNOS with a KI equal to 2.6 microM and a kinact equal to 0.042 min-1. When assayed without a preincubation period, NMMA exhibited typical reversible inhibition of iNOS (Ki = 3.9 microM). NMMA also reversibly inhibited nNOS and the eNOS with Ki equal to 0.65 and 0.7 microM, respectively. However, NMMA did not inactivate eNOS at concentrations up to 10 microM. In the presence, but not the absence, of 4 microM tetrahydrobiopterin, NMMA inactivated nNOS with a kinact equal to 0.022 min-1 and a KI equal to 2.0 microM. Since NNA did not inactivate iNOS at concentrations up to 25 microM, NNA is strictly a reversible inhibitor of iNOS (Ki = 8.1 microM). Neuronal NOS and eNOS, however, were rapidly inactivated by NNA with kintact equal to 0.083 and 0.047 min-1 and KI equal to 0.09 and 0.02 microM, respectively, when preincubated with NNA. Tetrahydrobiopterin did not affect the rate of inactivation of nNOS by NNA. In all cases, L-arginine protected against inactivation, suggesting that inactivation occurs at or near the active site. Thus, inactivation of the three NOS isozymes with NMMA and NNA reveals active-site differences between the isoforms.

Nitric oxide mediates iron release from ferritin.

Nitric oxide (NO) synthesis by cytotoxic activated macrophages has been postulated to result in a progressive loss of iron from tumor target cells as well as inhibition of mitochondrial respiration and DNA synthesis. In the present study, the addition of an NO-generating agent, sodium nitroprusside, to the iron storage protein ferritin resulted in the release of iron from ferritin and the released iron-catalyzed lipid peroxidation. Hemoglobin, which binds NO, and superoxide anion, which reacts with NO, inhibited nitroprusside-dependent iron release from ferritin, thereby providing evidence that NO can mobilize iron from ferritin. These results suggest that NO generation in vivo could lead to the mobilization of iron from ferritin disrupting intracellular iron homeostasis and increasing the level of reactive oxygen species.

Iron release from ferritin and lipid peroxidation by radiolytically generated reducing radicals.

Iron is involved in the formation of oxidants capable of damaging membranes, protein, and DNA. Using 137Cs gamma radiation, we investigated the release of iron from ferritin and concomitant lipid peroxidation by radiolytically generated reducing radicals, superoxide and the carbon dioxide anion radical. Both radicals released iron from ferritin with similar efficiencies and iron mobilization from ferritin required an iron chelator. Radiolytically generated superoxide anion resulted in peroxidation of phospholipid liposomes as measured by malondialdehyde formation only when ferritin was included as an iron source and the released iron was found to be chelated by the phospholipid liposomes.

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes.

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.

Effect of diquat on the distribution of iron in rat liver.

Diquat toxicity is proposed to be mediated through the generation of active oxygen species; however, the exact role of active oxygen in toxicity is not known. The generation of damaging oxygen radicals requires transition metals such as iron. In vitro studies have shown that redox cycling of diquat results in the release of iron from ferritin, thus, increasing the potential for active oxygen species generation. We sought to determine if diquat administration to male Sprague-Dawley rats would result in the release of iron from ferritin in vivo. Rats were treated with diquat dibromide (20 mg/kg body weight) and the effect on the iron distribution in liver was determined. The results show that diquat-treated animals had increased levels of hepatic low molecular weight chelatable iron (LMWC-Fe) and decreased levels of hepatic ferritin iron when compared to saline-treated animals. These results suggest that diquat toxicity may be associated with the release of iron from ferritin in vivo and that iron release from ferritin may be a process common to other free radical mediated toxicities.

Inhibition of superoxide and ferritin-dependent lipid peroxidation by ceruloplasmin.

Ceruloplasmin (CP) was found to inhibit xanthine oxidase and ferritin-dependent peroxidation of phospholipid liposomes, as evidenced by decreased malondialdehyde formation. Ceruloplasmin was also shown to inhibit superoxide-mediated mobilization of iron from ferritin, in a concentration-dependent manner, as measured spectrophotometrically using the iron(II) chelator bathophenanthroline sulfonate. Ceruloplasmin failed to function as a peroxyl radical-scavenging antioxidant as evidenced by its inability to inhibit free radical-initiated peroxidation of linoleic acid, suggesting that CP inhibited lipid peroxidation by affecting the availability of ferritin-derived iron. In addition, CP scavenged xanthine oxidase-derived superoxide as measured spectrophotometrically via its effect on cytochrome c reduction. However, the extent of the superoxide scavenging of CP did not quantitatively account for its effects on iron release, suggesting that CP inhibits superoxide-dependent mobilization of ferritin iron independently of its ability to scavenge superoxide. The effects of CP and apoferritin on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. In the absence of apoferritin, CP exhibited a concentration-dependent prooxidant effect. However, CP-dependent, iron-catalyzed lipid peroxidation was inhibited by the addition of apoferritin. Apoferritin did not function as a peroxyl radical-scavenging antioxidant but was shown to incorporate iron in the presence of CP. These data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation largely via its ability to reincorporate reductively mobilized iron back into ferritin.

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.