Detection and evaluation of estrogen DNA-adducts and their carcinogenic effects in cultured human cells using biotinylated estradiol.
The normal female reproductive hormone estrogen has been linked with increased risk of breast and many other forms of cancer. This is largely due to metabolic conversion of estrogens into highly reactive catechol estrogen quinones which can interact with DNA and cause a variety of DNA adducts and lesions. Detection and analysis of these adducts and their associated cellular responses involve complex chemical, enzymatic, and LC-MS based methods, which are both laborious and require specialized expertise and instrumentation. Herein, we show that using a biotin-labeled estradiol allows immunodetection of estrogen-induced DNA adducts by slot blot and single-cell molecular combing and proximity ligation assays. The biotinylated and unlabeled estradiols induced similar levels of DNA single and double strand breaks as measured by comet assays. Using biotinylated estrogen, we further show that estrogens are able to activate the Fanconi anemia-BRCA tumor suppressor pathway and cause DNA strand breaks and oxidatively modified DNA bases as well as gross chromosomal aberrations. Utilization of biotin-labeled estrogens could be a powerful tool to detect estrogen adducts and associated DNA damage, and to track estrogen adduct-induced cellular responses and carcinogenic mechanisms in cultured cells. The techniques presented here allow simple and rapid detection and quantitation of estrogen adducts by slot blot as well as direct visualization on the DNA strand and could pave the way for developing new treatments to protect the genome from the effects of reactive estrogen metabolites. © 2016 Wiley Periodicals, Inc.
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama.
Kaushlendra
K
Tripathi
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama.
Chinnadurai
C
Mani
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama.
Ranganatha R
RR
Somasagara
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama.
David W
DW
Clark
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama.|Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad, Telangana, India.
Venkateshwari
V
Ananthapur
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama.|Department of Chemistry, Government First Grade College, Kadur, Karnataka, India.
Kambappa
K
Vinaya
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama.
Komaraiah
K
Palle
D019921:Biotinylation; D002273:Carcinogens / Q000378:metabolism*; D002478:Cells, Cultured; D002869:Chromosome Aberrations; D018736:DNA Adducts / Q000378:metabolism*; D004958:Estradiol / Q000737:chemistry*; D004967:Estrogens / Q000737:chemistry / Q000633:toxicity*; D051856:Fanconi Anemia Complementation Group Proteins / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D061986:MCF-7 Cells; D059010:Single-Cell Analysis
D002273:Carcinogens; D018736:DNA Adducts; D004967:Estrogens; D051856:Fanconi Anemia Complementation Group Proteins; D004958:Estradiol
DNA damage; genotoxic stress; hormones; mutagenesis