Near-infrared chemiluminescence imaging of superoxide anion production in kidneys with iron3+-nitrilotriacetate-induced acute renal oxidative stress in rats.

Iron-catalyzed oxidative stress generates reactive oxygen species in the kidney and induces oxidative damage including lipid, protein, and DNA modifications which induces renal injury and may lead to cancer. An analysis of oxidative stress dynamics by reactive oxygen species has not been performed non-invasively in real time in intact kidneys and is a significant challenge in biology and medicine. Here, I report that MCLA-800 is a near-infrared chemiluminescent probe that visualizes the dynamics of superoxide anion (O2•-) production and the upstream generation of reactive oxygen species in living rat kidneys suffering acute renal oxidative stress induced by intraperitoneal administration of iron3+-nitrilotriacetate (Fe3+-NTA) as a representative Fe3+ chelate. MCLA-800 was intravenously injected at 250 nmol/kg body weight and immediately transported to the kidneys with the emitting light dependent on O2•- production. The magnitude of O2•- production correlated with the Fe3+-NTA dose. O2•- was continuously produced in the blood stream following Fe3+-NTA injection at 0.15 mmol/kg body weight, while peak production in the renal cortex occurred at 24 h, then decreased to the background level at 72 h. This study clearly revealed the dynamics of Fe3+-NTA-mediated O2•- production in the living kidney by chemiluminescent imaging of O2•- production using MCLA-800.

Effect of nitrilotriacetic acid and tea saponin on the phytoremediation of Ni by Sudan grass (Sorghum sudanense (Piper) Stapf.) in Ni-pyrene contaminated soil.

Phytoremediation is commonly used in the remediation of soils co-contaminated by heavy metals and polycyclic aromatic hydrocarbons (PAHs) because of its economy and effectiveness. Sudan grass (Sorghum sudanense (Piper) Stapf.) has well-developed roots and strong tolerance to heavy metals, so it has been widely concerned. In this study, nitrilotriacetic acid (NTA) and tea saponin (TS) were used as enhancers and combined with Sudan grass for improving the remediation efficiency of Ni-pyrene co-contaminated soil. The results of the pot experiment in soils showed that enhancers promoted the enrichment of Ni in plants. With the function of enhancers, more inorganic and water-soluble Ni were converted into low-toxic phosphate-bonded and residual Ni, so as to reinforce the tolerance of Sudan grass to Ni. In the pot experiment based on vermiculite, it was found that enhancers increased the accumulation of Ni in cell wall by 49.71-102.73%. Enhancers also had the positive effect on the relative abundance of Proteobacteria, Patescibacteria and Bacteroidetes that could tolerate heavy metals at phylum level. Simultaneously, the study found that pyrene reduced the exchangeable Ni in soils. More Ni entered the organelles and transfer to more high-toxic forms in Sudan grass when pynere coexisted. The study manifested that enhancers improved the phytoremediation effect of Ni significantly, yet the co-existence of pyrene weakened the process. Our results provided meaningful references for remediating actual co-contaminated soil of heavy metals and PAHs.

Bioinspired Metal-Free Fluorescent Carbon Nanozyme with Dual Catalytic Activity to Confront Cellular Oxidative Damage.

Development of metal-free, recyclable enzyme mimics is challenging and requires key chemical modifications at the molecular level. Here, nitrilotriacetic acid-functionalized carbon nanospheres (LC-CNS@NTA) were prepared from the nitrogen-rich weed Lantana camara (LC) using a simple hydrothermal reaction condition. Transmission electron microscopy (TEM) studies revealed size of ∼160 ± 20 nm for LC-CNS@NTA whereas, the same showed fluorescence emission at ∼520 nm with a ∼63% quantum yield. Furthermore, LC-CNS@NTA showed strong peroxidase (Pxrd) activity toward a wide range of substrate viz., H2O2, 3,3',5,5'-tetramethylbenzidine, and o-phenylenediamine with Km and Vmax values of ∼257 µM and 1.06 µM/s, 282 µM and 1.47 µM/s, and 270.8 µM and 1.647 µM/s, respectively. Interestingly, this also showed catalase (CAT) activity against H2O2 with Km and Vmax values of ∼0.374 µM and 1.87 µM/s, respectively. It was observed that LC-CNS@NTA could effectively reduce the oxidative stress-induced cytotoxicity of HEK293 cells via retention of mitochondrial membrane potential, prevention of lipid peroxidation and DNA damage. It was further found that LC-CNS@NTA-treated cells showed reduced level of intracellular protein carbonylation and protein aggregation. The finding of the present study is expected to pave the path for designing engineered metal-free carbon nanozyme with dual enzyme mimic activity.

Young leaf protection from cadmium accumulation and regulation of nitrilotriacetic acid in tall fescue (Festuca arundinacea) and Kentucky bluegrass (Poa pratensis).

Phytoextraction efficiency of cadmium (Cd) contaminated soil mainly depended upon the mechanism of plants in absorption, translocation, distribution, and detoxification of Cd. A pot experiment was designed to investigate Cd distribution and accumulation among the different leaves of tall fescue (Festuca arundinacea) and Kentucky bluegrass (Poa pratensis) and its regulation by Nitrilotriacetic acid (NTA), a biodegradable chelating agent. The results showed that Cd concentrations in the senescent and dead leaves were 3.2 and 5.3 fold of that in the emerging leaves of tall fescue, and 19.3 and 25.1 fold of that in the emerging leaves of Kentucky bluegrass, respectively. The lower Cd concentrations were maintained in the emerging and mature leaves to avoid Cd toxicity. In the emerging and mature leaves, Cd was mainly accumulated in the vascular bundles and epidermis. No Cd dithizonate color was observed in the mesophyll tissues of Kentucky bluegrass and only minor Cd was observed in the mesophyll tissues of tall fescue. In the senescent leaves, Cd dithizonate complexes were located in the protoplasts and cell walls of all leaf tissues. NTA greatly promoted Cd translocation and distribution to the senescent and dead leaves of tall fescue, but no significant effect was observed in Kentucky bluegrass. Our results indicate that a young leaf protection mechanism might be involved in their Cd hypertolerance. The Cd preferential accumulation could lead a novel phytoextraction strategy by the continuously harvesting the senescent and dead leaves of tall fescue and Kentucky bluegrass.

Simple and efficient knockdown of His-tagged proteins by ternary molecules consisting of a His-tag ligand, a ubiquitin ligase ligand, and a cell-penetrating peptide.

We designed and synthesized hybrid molecules for a protein knockdown method based on the recognition of a His-tag fused to a protein of interest (POI). The synthesized target protein degradation inducers contained three functional moieties: a His-tag ligand (nickel nitrilotriacetic acid [Ni-NTA]), an E3 ligand (bestatin [BS] or MV1), and a carrier peptide (Tat or nonaarginine [R9]). The designed hybrid molecules, BS-Tat-Ni-NTA, MV1-Tat-Ni-NTA, BS-R9-Ni-NTA, and MV1-R9-Ni-NTA, efficiently degraded His-tagged cellular retinoic acid binding protein 2 via the ubiquitin-proteasome system (UPS). This system will become a useful tool for research into selective protein degradation inducers that act via the UPS.

Synthesis and anti-diabetic activity of new N,N-dimethylphenylenediamine-derivatized nitrilotriacetic acid vanadyl complexes.

Vanadium compounds are promising anti-diabetic agents. However, reducing the metal toxicity while keeping/improving the hypoglycemic effect is still a big challenge towards the success of anti-diabetic vanadium drugs. To improve the therapeutic potency using the anti-oxidative strategy, we synthesized new N,N-dimethylphenylenediamine (DMPD)-derivatized nitrilotriacetic acid vanadyl complexes ([VO(dmada)]). The in vitro biological evaluations revealed that the DMPD-derivatized complexes showed improved antioxidant capacity and lowered cytotoxicity on HK-2 cells than bis(maltolato)oxidovanadium (IV) (BMOV). In type II diabetic mice, [VO(p-dmada)] (0.15mmolkg-1/day) exhibited better hypoglycemic effects than BMOV especially on improving glucose tolerance and alleviating the hyperglycemia-induced liver damage. These insulin enhancement effects were associated with increased expression of peroxisome proliferator-activated receptor α and γ (PPARα/γ) in fat, activation of Akt (v-Akt murine thymoma viral oncogene)/PKB (protein kinase-B) in fat and liver, and inactivation of c-Jun NH2-terminal protein kinases (JNK) in liver. Moreover, [VO(p-dmada)] showed no tissue toxicity at the therapeutic dose in diabetic mice and the oral acute toxicity (LD50) was determined to be 1640mgkg-1. Overall, the experimental results indicated that [VO(p-dmada)] can be a potent insulin enhancement agent with improved efficacy-over- toxicity index for further drug development. In addition, the results on brain Tau phosphorylation suggested necessary investigation on the effects of vanadyl complexes on the pathology of the Alzheimer's disease in the future.

Characterization and cytotoxic effect of aqua-(2,2',2''-nitrilotriacetato)-oxo-vanadium salts on human osteosarcoma cells.

The use of protonated N-heterocyclic compound, i.e. 2,2'-bipyridinium cation, [bpyH+], enabled to obtain the new nitrilotriacetate oxidovanadium(IV) salt of the stoichiometry [bpyH][VO(nta)(H2O)]H2O. The X-ray measurements have revealed that the compound comprises the discrete mononuclear [VO(nta)(H2O)]- coordination ion that can be rarely found among other known compounds containing nitrilotriacetate oxidovanadium(IV) moieties. The antitumor activity of [bpyH][VO(nta)(H2O)]H2O and its phenanthroline analogue, [phenH][VO(nta)(H2O)](H2O)0.5, towards human osteosarcoma cell lines (MG-63 and HOS) has been assessed (the LDH and BrdU tests) and referred to cis-Pt(NH3)2Cl2 (used as a positive control). The compounds exert a stronger cytotoxic effect on MG-63 and HOS cells than in untransformed human osteoblast cell line. Thus, the [VO(nta)(H2O)]- containing coordination compounds can be considered as possible antitumor agents in the osteosarcoma model of bone-related cells in culture.

Effect of alkyl polyglucoside and nitrilotriacetic acid combined application on lead/pyrene bioavailability and dehydrogenase activity in co-contaminated soils.

At present, few research focus on the phytoremediation for organic pollutants and heavy metals enhanced by surfactants and chelate agents in the combined contaminated soils or sediments. In this study, the effect of a novel combined addition of alkyl polyglucoside (APG) and nitrilotriacetic acid (NTA) into pyrene and lead (Pb) co-contaminated soils on bioaccessiblity of pyrene/Pb and dehydrogenase activities (DHA) was studied. Through the comparison of the results with the alone and combined application, synergistic effect on bioaccessiblity of pyrene and Pb was found while APG and NTA was applied together. Results also indicated a significant promotion on the DHA in mixed addition of APG and NTA. In addition, correlation and principal component analysis were performed to better understand the relationship among APG/NTA, bioaccessiblity of pyrene/Pb and the DHA. Results showed that APG and NTA can affect DHA directly by themselves but also can affect DHA indirectly by changing bioaccessible pyrene and exchangeable Pb.

Nephroprotective effect of ß-sitosterol on N-diethylnitrosamine initiated and ferric nitrilotriacetate promoted acute nephrotoxicity in Wistar rats.

BACKGROUND: The most abundant plant sterol ß-sitosterol is widely used for treating heart diseases and chronic inflammatory conditions. The objective of the current study was to evaluate the nephroprotective effect of ß-sitosterol against nephrotoxicants which were studied using renal function markers, antioxidant and lipid peroxidation status, and inflammatory markers. METHODS: Male albino Wistar rats were randomly grouped into four: group 1 was vehicle control rats (0.1% carboxymethyl cellulose [CMC]); group 2 was rats treated with N-diethylnitrosamine (DEN) (200 mg/kg body weight [bw] i.p. on the 15th day) and ferric nitrilotriacetate (Fe-NTA) (9 mg/kg bw i.p. on 30th and 32nd days); group 3 was rats that received ß-sitosterol (20 mg/kg bw in 0.1% CMC, p.o. for 32 days) 2 weeks prior to the exposure to the nephrotoxicant; and group 4 was rats that received ß-sitosterol alone. The experiment was terminated after the 24 h of last dosage of Fe-NTA, and all the animals were sacrificed. The blood, liver and kidney from each group were analyzed for biochemical, molecular and histological changes. RESULTS: All the parameters showed significant changes in DEN and Fe-NTA treated animals, whereas ß-sitosterol pretreated animals' altered biochemical parameters were restored to near normal. Histopathological and immunoexpression studies on tissues also corroborate the biochemical endpoints. CONCLUSIONS: Administration of ß-sitosterol to nephrotoxicity induced rats showed significant positive changes in biochemical parameters, histopathological and immunohistochemical observations, and up-regulation of Nrf2 gene expression. From this, it was clear that ß-sitosterol showed renal protective function.

Quantitative profiling of prostaglandins as oxidative stress biomarkers in vitro and in vivo by negative ion online solid phase extraction - Liquid chromatography-tandem mass spectrometry.

Free radical-mediated oxidation of arachidonic acid to prostanoids has been implicated in a variety of pathophysiological conditions such as oxidative stress. Here, we report on the development of a liquid chromatography-mass spectrometry method to measure several classes of prostaglandin derivatives based on regioisomer-specific mass transitions down to levels of 20 pg/ml applied to the measurement of prostaglandin biomarkers in primary hepatocytes. The quantitative profiling of prostaglandin derivatives in rat and human hepatocytes revealed the increase of several isomers on stress response. In addition to the well-established markers for oxidative stress such as 8-iso-prostaglandin F2α and the prostaglandin isomers PE2 and PD2, this method revealed a significant increase of 15R-prostaglandin D2 from 236.1 ± 138.0 pg/1E6 cells in untreated rat hepatocytes to 2001 ± 577.1 pg/1E6 cells on treatment with ferric NTA (an Fe(3+) chelate with nitrilotriacetic acid causing oxidative stress in vitro as well as in vivo). Like 15R-prostaglandin D2, an unassigned isomer that revealed a more significant increase than commonly analyzed prostaglandin derivatives was identified. Mass spectrometric detection on a high-resolution instrument enabled high-quality quantitative analysis of analytes in plasma levels from rat experiments, where increased concentrations up to 23-fold change treatment with Fe(III)NTA were observed.

Reconstituting functional microtubule-barrier interactions.

Local interactions between the tips of microtubules and the cell cortex, or other cellular components such as kinetochores, play an important role in essential cellular processes like establishing cell polarity, distribution of organelles, and microtubule aster and chromosome positioning. Here we present two in vitro assays that specifically mimic microtubule-cortex interactions by employing selectively functionalized microfabricated barriers that allow for the immobilization of proteins with a range of affinities. We describe the microfabrication process to create gold or glass barriers and the subsequent functionalization of these barriers using self-assembled thiol monolayers or polylysine-poly(ethylene glycol), respectively. Near-permanent attachment of proteins is obtained using biotinylated surfaces combined with streptavidin and biotinylated proteins. Lower affinity interactions, further tunable with the addition of imidazole, are obtained using nickel-nitrilotriacetic acid (Ni(II)-NTA) functionalization combined with his-tagged proteins. Both mono-NTA and tris-NTA compounds are used. We show an assay to reconstitute the "end-on" interaction between dynamic microtubule tips and barrier-attached dynein, mimicking the cellular situation at the cortex and at kinetochores. In a second assay, we reconstitute microtubule-based delivery of end-tracking proteins to functionalized barriers, mimicking the transport of cell-end markers to the cell poles in interphase fission yeast cells.

Iron overload signature in chrysotile-induced malignant mesothelioma.

Exposure to asbestos is a risk for malignant mesothelioma (MM) in humans. Among the commercially used types of asbestos (chrysotile, crocidolite, and amosite), the carcinogenicity of chrysotile is not fully appreciated. Here, we show that all three asbestos types similarly induced MM in the rat peritoneal cavity and that chrysotile caused the earliest mesothelioma development with a high fraction of sarcomatoid histology. The pathogenesis of chrysotile-induced mesothelial carcinogenesis was closely associated with iron overload: repeated administration of an iron chelator, nitrilotriacetic acid, which promotes the Fenton reaction, significantly reduced the period required for carcinogenesis; massive iron deposition was found in the peritoneal organs with high serum ferritin; and homozygous deletion of the CDKN2A/2B/ARF tumour suppressor genes, the most frequent genomic alteration in human MM and in iron-induced rodent carcinogenesis, was observed in 92.6% of the cases studied with array-based comparative genomic hybridization. The induced rat MM cells revealed high expression of mesoderm-specific transcription factors, Dlx5 and Hand1, and showed an iron regulatory profile of active iron uptake and utilization. These data indicate that chrysotile is a strong carcinogen when exposed to mesothelia, acting through the induction of local iron overload. Therefore, an intervention to remove local excess iron might be a strategy to prevent MM after asbestos exposure.

Oxidation and turnover of renal metallothioneins after an injection of ferric nitrilotriacetate.

Metallothioneins (MTs) have demonstrated strong antioxidant properties, however the biological significance of their effect against hydroxyl radical toxicity remains unclear. We investigated the oxidation and turnover of renal MTs in MT-preinduced mice after an injection of ferric nitrilotriacetate (Fe-NTA). Incubation of MTs with Fe-NTA and H(2)O(2) resulted in a loss of their metal-binding properties and a decrease in their thiol concentration independent of binding potential and isoforms. Moreover, in vitro reduction of renal oxidized MT with dithiothreitol (DTT) reversed these oxidative changes. An injection of Fe-NTA oxidized renal preinduced MT in Zn- and Cd-pretreated mice. The metal-binding properties of renal MTs were lost when the Fe-NTA dose was increased. However, analysis of renal MTs using an immunoassay showed that its protein concentration did not decrease 4h after the injection with various Fe-NTA doses. Furthermore, in vitro reduction of renal oxidized MTs with DTT resulted in an increase in the concentration of metals in the MT fraction. These data indicate that radicals produced by Fe-NTA may oxidize MTs in vitro and in vivo. When we investigated the turnover of oxidized MTs in Fe-NTA-treated mice, effects on the concentration of renal (35)S-labeled MTs were opposite to those observed in Cd-pretreated mice. The concentration of preinduced (35)S-labeled MTs in the kidneys of Cd-pretreated mice showed a significant decrease (p<0.05), whereas that of newly synthesized (35)S-labeled MTs showed a considerable increase. These data suggest that degradation of oxidized MTs may be faster than intact MTs. Therefore, the radical scavenging system of MTs may include their induction and degradation during oxidative stress conditions.

Induction of apoptotic change in the rat hippocampus caused by ferric nitrilotriacetate.

Iron, a source of oxidative stress, plays a major role in the pathology of neurodegenerative disease. In Alzheimer's disease, the hippocampus is vulnerable to oxidative stress, leading to impairment in memory formation. In our previous study, a brain oxidative reaction was induced after intraperitoneal injection of ferric nitrilotriacetate (Fe-NTA). However, since only a small amount of iron reached the brain in the previous study, Fe-NTA was administered into the hippocampus using an osmotic pump in this study. After continuous injection of Fe-NTA for 2 weeks, a high level of apoptotic change was induced in the hippocampus, in accordance with the iron localization. After injection for 4 weeks, the hippocampus was totally destroyed. A small amount of iron infiltrated into the cerebral cortex and the striatum, and deposition was observed at the choroid plexus and ependymal cells. However, no apoptotic reaction or clear tissue injury was observed in these areas. In addition, muscarinic acetylcholine receptors (M1, M2, and M4) were decreased in both the cortex and hippocampus while it increased in the striatum. Thus, the hippocampus is likely vulnerable to oxidative stress from Fe-NTA, and the oxidative stress is considered to bring the disturbance in the muscarinic acetylcholine receptors.

Allosteric effects of sulfonate anions on the rates of iron release from serum transferrin.

Serum transferrin is the protein that transports ferric ion through the bloodstream and is thus a potential target for iron chelation therapy. However, the release of iron from transferrin to low-molecular-weight chelating agents is usually quite slow. Thus a better understanding of the mechanism for iron release is important to assist in the design of more effective agents for iron removal. This paper describes the effect of sulfonate anions on the rates of iron removal from C-terminal monoferric transferrin by acetohydroxamic acid, deferiprone, nitrilotriacetic acid (NTA), and diethylenetriaminepentaacetic acid at 25°C in 0.1M N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (Hepes) buffer at pH 7.4. These ligands remove iron via a combination of pathways that show saturation and first order dependence on the ligand concentration. The kinetic effects of the anions methanesulfonate, methylenedisulfonate, and ethylenedisulfonate were evaluated. All these anions increase the overall rates of iron release, presumably by binding to an allosteric anion binding site on the protein. The two disulfonates produce a larger acceleration in iron release than the monosulfonate. More detailed studies using methylenedisulfonate show that this anion accelerates the rate of iron release via the saturation pathway. The addition of methylenedisulfonate results in the appearance of a large saturation pathway for iron release by NTA, which otherwise removes iron by a simple first-order process. The sulfonate group was selected for these studies because it represents an anionic functional group that can be covalently linked to a therapeutic ligand to accelerate iron release in vivo. The current studies indicate that the binding of the sulfonates to the allosteric site on the protein is quite weak, so that one would not expect a significant acceleration in iron release at clinically relevant ligand concentrations.

Characterization of the decaheme c-type cytochrome OmcA in solution and on hematite surfaces by small angle x-ray scattering and neutron reflectometry.

The outer membrane protein OmcA is an 85 kDa decaheme c-type cytochrome located on the surface of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. It is assumed to mediate shuttling of electrons to extracellular acceptors that include solid metal oxides such as hematite (alpha-Fe(2)O(3)). No information is yet available concerning OmcA structure in physiologically relevant conditions such as aqueous environments. We purified OmcA and characterized its solution structure by small angle x-ray scattering (SAXS), and its interaction at the hematite-water interface by neutron reflectometry. SAXS showed that OmcA is a monomer that adopts a flat ellipsoidal shape with an overall dimension of 34 x 90 x 65 A(3). To our knowledge, we obtained the first direct evidence that OmcA undergoes a redox state-dependent conformational change in solution whereby reduction decreases the overall length of OmcA by approximately 7 A (the maximum dimension was 96 A for oxidized OmcA, and 89 A for NADH and dithionite-reduced OmcA). OmcA was also found to physically interact with electron shuttle molecules such as flavin mononucleotide, resulting in the formation of high-molecular-weight assemblies. Neutron reflectometry showed that OmcA forms a well-defined monomolecular layer on hematite surfaces, where it assumes an orientation that maximizes its contact area with the mineral surface. These novel insights into the molecular structure of OmcA in solution, and its interaction with insoluble hematite and small organic ligands, demonstrate the fundamental structural bases underlying OmcA's role in mediating redox processes.

Lipid raft-dependent endocytosis: a new route for hepcidin-mediated regulation of ferroportin in macrophages.

BACKGROUND: Expression of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and is decreased by hepcidin. We previously showed that ferroportin is present in specific cell surface domains suggestive of lipid rafts. Herein, we have clarified the localization of ferroportin in macrophage membranes and tested whether raft-mediated endocytosis plays a role in hepcidin activity. DESIGN AND METHODS: Raft/detergent-resistant membranes from murine bone marrow-derived macrophages and J774a1 cells were analyzed by Western blotting. The effect of lipid raft- or clathrin-dependent endocytosis inhibitors was studied on hepcidin activity. For this purpose, after treatment, ferroportin expression was analyzed by fluorescence microscopy, Western blotting of total protein extracts or plasma membrane protein samples, and by quantitative immunofluorescence assay (In-Cell-Western). RESULTS: Macrophage ferroportin was mostly detected in detergent-resistant membranes containing raft markers (caveolin 1, flotillin 1). Interestingly, iron overload strongly increased the presence of ferroportin in the lightest raft fraction. Moreover, lipid raft breakdown by cholesterol sequestration (filipin) or depletion (methyl-beta-cyclodextrin) decreased hepcidin activity on macrophage ferroportin. Cell surface biotinylation and immunofluorescence studies indicated that the process of both hepcidin mediated endocytosis and degradation of ferroportin were affected. By contrast, the inhibition of clathrin dependent endocytosis did not interfere with hepcidin effect. CONCLUSIONS: Macrophage ferroportin is present in lipid rafts which contribute to hepcidin activity. These observations reveal the existence of a new cellular pathway in hepcidin mediated degradation of ferroportin and open a new area of investigation in mammalian iron homeostasis.

Dietary supplementation of silymarin protects against chemically induced nephrotoxicity, inflammation and renal tumor promotion response.

Ferric nitrilotriacetate (Fe-NTA) is a potent nephrotoxicant and a renal carcinogen that induces its effect by causing oxidative stress. The present study was undertaken to explore protective effect of silymarin, a flavonolignan from milk thistle (Silybum marianum), against Fe-NTA mediated renal oxidative stress, inflammation and tumor promotion response along with elucidation of the implicated mechanism(s). Administration of Fe-NTA (10 mg/kg bd wt, i.p.) to Swiss albino mice induced marked oxidative stress in kidney, evident from augmentation in renal metallothionein (MT) expression, depletion of glutathione content and activities of antioxidant and phase II metabolizing enzymes, and enhancement in production of aldehyde products such as 4-hydroxy-2-nonenal. Fe-NTA also significantly activated nuclear factor kappa B (NFkappaB) and upregulated the expression of downstream genes: cyclooxygenase 2 and inducible nitric oxide synthase and enhancing the production of proinflammatory cytokines: tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). However, feeding of 0.5% and 1% silymarin diet conferred a significant protection against Fe-NTA induced oxidative stress and inflammation. It further augmented MT expression, restored the antioxidant armory, ameliorated NFkappaB activation and decreased the expression of proinflammatory mediators. Silymarin also suppressed Fe-NTA induced hyperproliferation in kidney, ameliorating renal ornithine decarboxylase activity and DNA synthesis. From these results, it could be concluded that silymarin markedly protects against chemically induced renal cancer and acts plausibly by virtue of its antioxidant, anti-inflammatory and antiproliferative activities.

Oxidative changes in the rat brain by intraperitoneal injection of ferric nitrilotriacetate.

Iron is known to be involved in neuronal diseases such as neurodegenerative diseases, brain ischemia and epilepsy. However, it is unclear if a high level of peripheral iron induces these pathological conditions. Since ferric nitrilotriacetate (Fe-NTA), a low molecule iron chelate, causes kidney carcinoma and diabetes in animals due to its strong and unique oxidative stress, it is also considered to cause pathological conditions in the brain. Therefore, we studied brain changes after intraperitoneal (i.p.) injection of Fe-NTA. We investigated iron distribution in the brain and evaluated heme oxygenase (HO)-1 mRNA, IL-6 mRNA and 4-hydroxy-2-nonenal (4-HNE) quantitatively. In addition, changes in muscarinic acetylcholine receptor mRNAs were measured. It was found that iron was localized in the cortex and the hypothalamus, but not in other areas of the brain. HO-1 was induced in both the cortex and hypothalamus, and the levels of IL-6 and 4-HNE were raised in the hypothalamus, but not in the cortex. In the cortex, expression in M1 and M2 mAChRs were suppressed. In conclusion, iron reached the brain parenchyma after i.p. injection of Fe-NTA, and Fe-NTA caused oxidative reactions and suppression of mAChRs in the brain.