Iron-Induced Oxidative Stress in Human Diseases.

Iron is responsible for the regulation of several cell functions. However, iron ions are catalytic and dangerous for cells, so the cells sequester such redox-active irons in the transport and storage proteins. In systemic iron overload and local pathological conditions, redox-active iron increases in the human body and induces oxidative stress through the formation of reactive oxygen species. Non-transferrin bound iron is a candidate for the redox-active iron in extracellular space. Cells take iron by the uptake machinery such as transferrin receptor and divalent metal transporter 1. These irons are delivered to places where they are needed by poly(rC)-binding proteins 1/2 and excess irons are stored in ferritin or released out of the cell by ferroportin 1. We can imagine transit iron pool in the cell from iron import to the export. Since the iron in the transit pool is another candidate for the redox-active iron, the size of the pool may be kept minimally. When a large amount of iron enters cells and overflows the capacity of iron binding proteins, the iron behaves as a redox-active iron in the cell. This review focuses on redox-active iron in extracellular and intracellular spaces through a biophysical and chemical point of view.

Oxidative renal tubular injuries induced by aminocarboxylate-type iron (III) coordination compounds as candidate renal carcinogens.

Oxidative renal tubular injuries and carcinogenesis induced by Fe(III)-nitrilotriacetate (NTA) and Fe(III)-ethylenediamine-N,N'-diacetate (EDDA) have been reported in rodent kidneys, but the identity of iron coordination structure essential for renal carcinogenesis, remains to be clarified. We compared renal tubular injuries caused by various low molecular weight aminocarboxylate type chelators with injuries due to NTA and EDDA. We found that Fe(III)-iminodiacetate (IDA), a novel iron-chelator, induced acute tubular injuries and lipid peroxidation to the same extent. We also prepared Fe(III)-IDA solutions at different pHs, and studied resultant oxidative injuries and physicochemical properties. The use of Fe(III)-IDA at pH 5.2, 6.2, and 7.2 resulted in renal tubular necrosis and apoptotic cell death, but neither tubular necrosis nor apoptosis was observed at pH 8.2. Spectrophotometric data suggested that Fe(III)-IDA had the same dimer structure from pH 6.2 to 7.2 as Fe(III)-NTA; but at a higher pH, iron polymerized and formed clusters. Fe(III)-IDA was crystallized, and this was confirmed by X-ray analysis and magnetic susceptibility measurements. These data indicated that Fe(III)-IDA possessed a linear mu-oxo bridged dinuclear iron (III) around neutral pH.

Role of recA/RAD51 family proteins in mammals.

DNA damage causes chromosomal instability leading to oncogenesis, apoptosis, and severe failure of cell functions. The DNA repair system includes base excision repair, nucleotide excision repair, mismatch repair, translesion replication, non-homologous end-joining, and recombinational repair. Homologous recombination performs the recombinational repair. The RAD51 gene is an ortholog of Esherichia coli recA, and the gene product Rad51 protein plays a central role in the homologous recombination. In mammals, 7 recA-like genes have been identified: RAD51, RAD51L1/B, RAD51L2/C, RAD51L3/D, XRCC2, XRCC3, and DMC1. These genes, with the exception of meiosis-specific DMC1, are essential for development in mammals. Disruption of the RAD51 gene leads to cell death, whereas RAD51L1/B, RAD51L2/C, RAD51L3/D, XRCC2, and XRCC3 genes (RAD51 paralogs) are not essential for viability of cells, but these gene-deficient cells exhibit a similar defective phenotype. Yeast two-hybrid analysis, co-immunoprecipitation, mutation analysis, and domain mapping of Rad51 and Rad51 paralogs have revealed protein-protein interactions among these gene products. Recent investigations have shown that Rad51 paralogs play a role not only in an early step, but also in a late step of homologous recombination. In addition, identification of alternative transcripts of some RAD51 paralogs may reflect the complexity of the homologous recombination system.

Genomic structure and multiple alternative transcripts of the mouse TRAD/RAD51L3/RAD51D gene, a member of the recA/RAD51 gene family.

The RecA/RAD51 family plays a central role in DNA recombinational repair. The targeted disruption of mouse RAD51L3/TRAD is lethal during embryogenesis, suggesting that this protein is essential for development. Recently, we reported multiple alternative splice variants of human RAD51L3/TRAD transcripts. In this study, we have identified multiple mouse transcript variants. Complete sequence analysis of the genomic and cDNA clones has confirmed that the exon-intron structures obey the GT/AG splicing rule, and that the multiplicity of the transcripts is due to alternative splicing. In addition, we have determined the transcription initiation site by rapid amplification of cDNA 5'-end (5'-RACE). These results show that the mouse gene structure is very similar to that of humans.

DNA rearrangement activity during retinoic acid-induced neural differentiation of P19 mouse embryonal carcinoma cells.

Because of the many superficial similarities between the immune system and the central nervous system, it has long been speculated that somatic DNA recombination is, like the immune system, involved in brain development and function. To examine whether or not the V(D)J recombination signals of the immune system work in an in vitro neural differentiation model, the P19 mouse embryonal carcinoma cell line was transfected with a reporter gene that is designed, when rearranged, to express bacterial beta-galactosidase, which was previously reported to exhibit somatic DNA recombination in the transgenic mouse brain. The cloned cells were then induced into neural cells by retinoic acid treatment. This neural induction treatment resulted in the cloning of a P19 cell line that showed a high incidence of beta-galactosidase-positive cells. Most of these beta-galactosidase-positive cells were immunocytochemically identified as either neurons, neuroepithelial cells, or astrocytes. The 5'-end sequences of the beta-galactosidase transcripts expressed in the induced cells were analyzed, and sequences were found that seemed to reflect DNA rearrangement through re-integration of the reporter gene into the host genome. However, the V(D)J recombination signals did not work in the in vitro model. These results suggested that DNA rearrangement activity though integration increased during neural differentiation of P19 cells.

Saccharated colloidal iron enhances lipopolysaccharide-induced nitric oxide production in vivo.

We investigated the effects of iron on the production of nitric oxide (NO), inducible NO synthase (iNOS), and plasma cytokines induced by lipopolysaccharide (LPS) in vivo. Male Wistar rats were preloaded with a single intravenous injection of saccharated colloidal iron (Fesin, 70 mg iron/kg body weight) or normal saline as a control, and then given an intraperitoneal injection of LPS (5.0 mg/kg body weight). Rats, preloaded with iron, had evidence of both iron deposition and strong iNOS induction in liver Kupffer cells upon injection of LPS; phagocytic cells in the spleen and lung had similar findings. LPS-induced NO production in iron-preloaded rats was significantly higher than control rats as accessed by NO-hemoglobin levels measured by ESR (electron spin resonance) and NOx (nitrate plus nitrite) levels. Western blot analysis showed that iron preloading significantly enhanced LPS-induced iNOS induction in the liver, but not in the spleen or lung. LPS-induced plasma levels of IL-6, IL-1beta, and TNF-alpha were also significantly higher in iron-preloaded rats as shown by ELISA, but IFN-gamma levels were unchanged. We conclude that colloidal-iron phagocytosed by liver Kupffer cells enhanced LPS-induced NO production in vivo, iNOS induction in the liver, and release of IL-6, IL-1beta, and TNF-alpha.