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.</p

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.</p

Radical-promoting "free" iron level in the serum of rats treated with ferric nitrilotriacetate: comparison with other iron chelate complexes.

&lt;p&gt;Iron plays a critical role in the production of activated oxygen species and the activity of chelated iron in the biological system depends on the chemical forms of the chelators. In the present study, we used ferric nitrolotriacetate (Fe-NTA, molar ratio of iron to chelators = 1:3), ferric ethylenediaminetetraacetate (Fe-EDTA, 1:3 complex) and ferric Desferal (Fe-Des, 1:1.1 complex) to see their &quot;free&quot; iron content in aqueous solutions in vitro and in the serum obtained after a single intraperitoneal injection of the chelates to rats (7.5 mg of iron/kg). &quot;Free&quot; iron was measured by the bleomycin-assay system. When Fe-NTA was dissolved in water, &quot;free&quot; iron increased linearly with total iron concentration up to 10 microM, whereas Fe-EDTA and Fe-Des showed no &quot;free&quot; iron with corresponding iron concentrations. When these three ferric chelates were dissolved in normal rat serum, &quot;free&quot; iron in Fe-NTA increased abruptly between 40 microM and 60 microM iron concentrations, then increased slowly up to 100 microM. Fe-Des did not show any &quot;free&quot; iron at comparable iron concentrations. Fe-EDTA had an intermediate &quot;free&quot; iron level in the serum. Among the ferric chelate complexes, Fe-NTA showed a much faster increase of and a higher content of &quot;free&quot; iron in the serum than the other two complexes after a single injection of the chelates into rats.(ABSTRACT TRUNCATED AT 250 WORDS)&lt;/p&gt;</p

Generation of active oxygen species by iron nitrilotriacetate (Fe-NTA).

Ferric nitrilotriacetate (Fe3+-NTA) solution showed maximum absorbance at pH 7.5. The iron was in ferric high-spin state and coordinated octahedrally with a relatively symmetric structure and also probably pentagonally. A spin trapping technique employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) yielded a DMPO spin adduct of unknown radical with three doublets (DMPO-Z) and a simple nitroxide radical (Y-NO.) in serum from rats injected intraperitoneally with Fe3+-NTA. When the Fe3+-NTA solution was diluted 500-fold with 50 mM NTA solution, DMPO-Z, Y-NO. and an additional signal, DMPO-OH were observed. The DMPO-Z signal was suppressed by a decrease in oxygen tension, alpha-tocopherol and 3-tert-butyl-4-hydroxy-anisole (BHA). The DMPO-OH signal was suppressed in the presence of ethanol and catalase. Fe2+-NTA solution hardly produced DMPO spin adducts. The Fe3+-NTA solution produced a strong DMPO-OH signal in the presence of H2O2. Rose Bengal solution, a singlet oxygen generating system, produced the same DMPO adducts. Fe3+-NTA reacted with oxygen in solution. The oxygen was activated and might be similar to singlet molecular oxygen. In the presence of H2O2, the Fe3+-NTA solution generated a hydroxyl radical. Fe3+-NTA itself generated free radicals, but Fe2+-NTA did not.</p

In vitro transformation of rat renal cells by treatment with ferric nitrilotriacetate.

Administration of ferric nitrilotriacetate (Fe-NTA) in vivo causes acute renal tubular injury and finally induces renal cell carcinoma. There is accumulating evidence that these processes involve free radicals generated by Fe-NTA. To study the mechanism of renal carcinogenesis by Fe-NTA, we attempted to induce malignant transformation of primary cultured renal cells by treatment with Fe-NTA. When primary cultured renal cells (PRC) were treated continuously with Fe-NTA, all of the PRC died without transformation. On the other hand, when PRC were treated intermittently with Fe-NTA, transformed epithelial colonies were observed at 3 weeks after the first treatment. The established transformed cell line (RK523) showed drastic morphological transformation, grew in soft agar, and formed tumors when transplanted into athymic nude mice. These results indicate that the balance between cytotoxicity and mutagenecity is important for Fe-NTA induced transformation. The RK523 cell line may be a useful model for studying renal carcinogenesis in vitro.</p

Non-mutagenicity of Fe3+-NTA and NTA in the Ames Salmonella Test

The mutagenic effects of ferric nitrilotriacetate (Fe3+-NTA) and nitrilotriacetate (NTA) were evaluated on 8 Salmonella typhimurium strains (TA97, TA98, TA102, TA1535, TA1537, TA100 1,8-DNP6 and TA100NR). Neither Fe3+-NTA nor NTA significantly increased the frequency of revertant colonies in any of the different experimental conditions adopted

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