|
Flow cytometric detection of tandem repeat mutations induced by various chemical classes [An article from: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis | 
 enlarge | Authors: C. Healy, M. Wade, A. Mcmahon, A. Williams, Johnso
Publisher: Elsevier
Buy New: $7.95
Format: Html
Media: Digital
Pages: 17
| |
| Editorial Reviews:
Product Description
This digital document is a journal article from Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, published by Elsevier in 2006. The article is delivered in HTML format and is available in your Amazon.com Media Library immediately after purchase. You can view it with any web browser.
Abstract:
To facilitate detection of genotoxicity from environmental mutagen exposure, we generated an in vitro enhanced green fluorescence protein (EGFP) reactivation assay that quickly and effectively detects frameshift mutations in tandem repeat sequences (TRS). Two murine cell lines, C3H10T1/2 and mismatch repair deficient MC2a, were stably transfected with EGFP reporter plasmids in which the EGFP constructs contain TRS that put the EGFP sequence out of frame. These included several 2, 3, 4, 5 and 6bp repeat sequences, a control non-repetitive sequence and a human gene sequence containing a 4bp repeat motif. Transfected cultures were exposed to five model mutagens and carcinogens: hydrogen peroxide (H"2O"2), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), benzo-a-pyrene-diol-epoxide (BPDE), ethyl nitrosourea (ENU), 9-aminoacridine (9AA) and two controls: acetone and ethanol. Frameshift mutations resulted in green fluorescent revertants, as determined by flow cytometry, and were confirmed, for 9AA treatments, by sequencing. All five treatments with model agents induced statistically significant sequence- and exposure-dependent responses in MC2a cells and a negative response with the two negative control treatments, acetone and ethanol. Similar responses were seen in a smaller panel of treatments and plasmids in C3H10T1/2 cells. The mutation frequencies were higher in cells transfected with the plasmids containing TRS than those harbouring the control construct lacking repeats. The highest mutation frequencies were observed with H"2O"2 and 9AA treatments, yielding up to a 50-fold difference between vehicle and highest concentration treatment. ENU, BPDE, and to a lesser extent TPA treatments, also showed a statistically significant exposure response. Results from these experiments reveal that the assay responds robustly to various classes of mutagenic substances, as well as to rodent carcinogens that are inactive in conventional mutation assays, and that responses are not linked to cytotoxicity. This assay is a promising approach for detecting chemically induced frameshifts within certain DNA sequences of interest, but further characterization and validation are required prior to general use in genotoxicity screening.
|
|
|
Powered by Associate-O-Matic
| |
top