In vivo carcinogenicity testing is an expensive and time-consuming process, and as a result, only a relatively small fraction of new and existing chemicals has been tested in this manner. Therefore, the development and validation of alternative approaches is desirable. We previously developed a mammalian in vitro assay for genotoxicity based on the ability of cells to increase their level of the tumor-suppressor protein p53 in response to DNA damage. Cultured (11,16,17) or by initiating a cell's apoptotic pathway (18)(19)(20)(21).The assay itself is simple (9). Cultured mammalian cells (NCTC 929) are treated with various doses of the test agent, and at specified time points following treatment, the cells are harvested and lysed. The level of p53 in the lysates is measured by p53 enzymelinked immunosorbent assay (ELISA) and/or Western blot analysis, and compared to the level in untreated cells.Our previous work (9) indicated that both Western blot and ELISA analyses yielded similar results. Therefore, our current work focused on the more quantitative ELISA assay. We also found that the NCTC 929 cell line was a useful model system. NCTC 929 cells were initially selected because previously published results (10) indicated that their levels of p53 rise following genotoxin treatment. We verified this, and found in addition that p53 in these cells can be immunoprecipitated by a monoclonal antibody specific for wild-type p53. We also found that the level of p53 in these cells rises significantly following treatment with indirect genotoxins such as aflatoxin BI and 2-acetylaminofluorene, indicating that they possess significant amounts of the metabolic activities necessary for biotransformation (9.To determine the ability of this assay to predict carcinogenicity in rodents and to compare its performance with other proposed alternatives, we subjected 25
From a normal human brain phage display library screen we identified the gamma (A)-globin chain of fetal hemoglobin (Hb F) as a protein that bound strongly to Aβ1-42. We showed the oxidized form 1 These authors are co-corresponding authors for all stages of refereeing and publication and contributed equally to this work. Rodney T. Perry; Rm. 210H, 1665 University Blvd.; University of Alabama at Birmingham, Birmingham, USA; Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Disclosure Statement:I so state for myself and on behalf of the other authors on the manuscript, Hemoglobin binding to Aβ and HBG2 SNP association suggest a role in Alzheimer's disease, that we have no conflicts and sources of funding that influence the results of this paper, the data is not published or submitted elsewhere, and that appropriate approval and procedures were used concerning human subjects. I also verify that all authors on this manuscript have reviewed the contents of the manuscript, approve of its contents, and validate the accuracy of the data. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of adult Hb (metHb A) binds with greater affinity to Aβ1-42 than metHb F. MetHb is more toxic than oxyhemoglobin because it loses its heme group more readily. Free Hb and heme readily damage vascular endothelial cells similar to Alzheimer's disease (AD) vascular pathology. The XmnI polymorphism (C→T) at −158 of the gamma (G)-globin promoter region can contribute to increased Hb F expression. Using family-based association testing, we found a significant protective association of this polymorphism in the NIMH sibling dataset (n=489) in families, with at least two affected and one unaffected sibling (p=0.006), with an age of onset >50 years (p=0.010) and >65 years (p=0.013), and families not homozygous for the APOE4 allele (p=0.041). We hypothesize that Hb F may be less toxic than adult Hb in its interaction with Aβ and may protect against the development of AD.
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