High-throughput, quantitative analysis of acrolein-derived DNA adducts in human oral cells by immunohistochemistry.
Acrolein (Acr) is a ubiquitous environmental pollutant as well as an endogenous compound. Acrolein-derived 1,N(2)-propanodeoxyguanosines (Acr-dG) are exocyclic DNA adducts formed following exposure to cigarette smoke or from lipid peroxidation. Acr-dG is mutagenic and potentially carcinogenic and may represent a useful biomarker for the early detection of cancers related to smoking or other oxidative conditions, such as chronic inflammation. In this study, we have developed a high-throughput, automated method using a HistoRx PM-2000 imaging system combined with MetaMorph software for quantifying Acr-dG adducts in human oral cells by immunohistochemical detection using a monoclonal antibody recently developed by our laboratory. This method was validated in a cell culture system using BEAS-2B human bronchial epithelial cells treated with known concentrations of Acr. The results were further verified by quantitative analysis of Acr-dG in DNA of BEAS-2B cells using a liquid chromatography/tandem mass spectrometry/multiple-reaction monitoring method. The automated method is a quicker, more accurate method than manual evaluation of counting cells expressing Acr-dG and quantifying fluorescence intensity. It may be applied to other antibodies that are used for immunohistochemical detection in tissues as well as cell lines, primary cultures, and other cell types.
Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA.
Emily J
EJ
Greenspan
Hanjoo
H
Lee
Marcin
M
Dyba
Jishen
J
Pan
Kepher
K
Mekambi
Tierra
T
Johnson
Jan
J
Blancato
Susette
S
Mueller
Deborah L
DL
Berry
Fung-Lung
FL
Chung
D000171:Acrolein / Q000032:analysis*; D000818:Animals; D001980:Bronchi / Q000166:cytology; D002460:Cell Line; D002478:Cells, Cultured; D018736:DNA Adducts / Q000032:analysis*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D009055:Mouth / Q000166:cytology*; D009153:Mutagens / Q000032:analysis*; D012984:Software
D018736:DNA Adducts; D009153:Mutagens; D000171:Acrolein