DNA adducts in mammalian cells exposed to N-acetoxy-2-acetylaminofluorene* (NA-AAF), an activated derivative of the potent carcinogen 2-acetylaminofluorene (2-AAF), play significant roles in cell killing, chromosome aberration, sister-chromatid exchange, gene mutation and neoplastic transformation (1,2). NA-AAF binds covalently to guanine in the DNA of mammalian cells and produces three different DNA adducts. The C-8 adducts, N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) and deacetylated N-(deoxyguanosin-8-yl)- 2-aminofluorene (dG-C8-AF), account for the major portion of the DNA-bound products, while the minor N2 adduct, 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF), accounts for the rest of them. The relative induction levels of the two major C-8 adducts vary among cell types. These adducts distort the DNA helix as do UV-induced cyclobutane pyrimidine dimers (CPD), and therefore they are repaired by nucleotide excision repair in human cells. Iwamoto et al (3) have established monoclonal antibodies against dG-C8-AAF in denatured DNA. These antibodies enable one to detect dG-C8-AAFs in DNA from cultured cells using an enzyme-linked immunosorbent assay (ELISA) and to visualize them in cultured cells or rodent tissues using an immunofluorescence (IF). This technology would contribute to understanding of molecular mechanisms in AAF-related research fields including cancer research, anticancer research or toxicology.
*NA-AAF is available from NCI Chemical Carcinogen Reference Standards Repository (Midwest Research Institute, Kansas City, MO)
The hybridoma was established by fusion of mouse myeloma cells with Balb/c mouse splenocytes immunized with NA-AAF-modified single-stranded DNA conjugated with methylated BSA. This hybridoma (clone AAF-1) culture supernatant was collected and precipitated with ammonium sulfate. After centrifugation, the pellet dissolved in small volume of double-distilled water was dialysed against PBS. The dialysate was then lyophilized.
AAF-1 primarily binds to dG-C8-AAF in denatured DNA, although dG-C8-AF in DNA is also recognized with slightly less efficiency.
Optimal dilutions should be determined by the end user
[The dose-dependent formation of NA-AAF-induced DNA adducts in human cells]
Cells were exposed to NA-AAF for 0.5 h and the formation of DNA adducts in denatured
DNA (500 ng/well) was determined using a sensitive-direct-binding ELISA with AAF-1 (1/100).
(Details are shown in Ref. 3.)
[The formation of NA-AAF-induced DNA adducts in human cells]
Cells were exposed to 200 M NA-AAF or solvent for 0.5 h. After permeabilization and fixation, DNA adducts were visualized by sequential treatment of AAF-1 (1/25) and Alexa Fluor 488 goat anti-mouse IgG conjugate. Nuclear DNA was counterstained with DAPI.
(Details are shown in Ref. 3.)
1) R.H. Heflich and R.E. Neft, Genetic toxicity of 2-acetylaminofluorene, 2-aminofluorene and some of their metabolites and model metabolites. Mutation Res. 318 (1994) 73-174.
2) E. Kriek, Fifty years of research on N-acetyl-2-aminofluorene, one of the most versatile compounds in experimental cancer research. J. Cancer Res. Clin. Oncol. 118 (1992) 481-489.T. Iwamoto et al., In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies. DNA Repair 3 (2004) 1475-1482.
Anti AAF DNA Adducts
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.