The covalent binding of genotoxins to DNA to form DNA adducts is considered to be a first step in chemically induced carcinogenesis. Measurement of DNA adducts in cells and tissues provides direct evidence of genetic damage and a means for assessing human health risk from exposure. However, the low levels at which adducts occur in affected organs and the limited amounts of DNA frequently available make their accurate measurement a highly challenging task. Methodology based on microcapillary liquid chromatography in combination with nano-electrospray mass spectrometry developed in this program has enabled us to achieve detection and quantification capabilities of adducts at levels similar to those frequently encountered in human exposure using low microgram quantities of DNA. Aromatic amines, compounds found in cigarette smoke, hair dyes and other environmental sources have been implicated in a variety of cancers including bladder cancer. 4-Aminobiphenyl (4-ABP), is a major carcinogen in this class of compounds and recent evidence has identified a potential relationship between mutational hot spots of the p53 gene in human bladder cancer and adduction sites of 4-ABP. A highly sensitive analytical protocol will be developed to quantify the DNA adducts of 4-ABP in urothelial cells from human volunteers involved in a smoke / cessation clinical study in order to assess the link between cigarette smoking and the risk for carcinogenesis. Use of urine as the physiological medium provides a non-invasive procedure which is more amenable for incorporation into human studies to assess the risk for bladder cancer from environmental chemicals. Agents which inhibit the formation of aromatic amine-DNA adducts will be tested in a human bladder cell line, thereby providing a broader perspective of their biological significance in their induction of bladder cancer. Development of methodology to characterize oligonucleotide adducts will expand the breadth of these clinical studies and will allow us to gain a better understanding of adduct formation in the context of base sequence and potentially identify the adduction in terms of specific domains of DNA and selected gene sequences. Additional novel technologies proposed here based on differential ion mobility spectrometry - mass spectrometry (DMS - MS) and the coupling of HPLC with MS and microNMR, will introduce new powerful tools to facilitate and improve the state of the art in the analysis of DNA adducts. We expect that the results of this work will help establish more definitively the degree of involvement of DNA adducts in the etiology of human cancer and define the relationship between bladder cancer and cigarette smoke.

Public Health Relevance

Damage of DNA from chemicals present in the environment is potentially the first step to carcinogenesis. This proposal addresses the development of sensitive and novel analytical methodology to detect and quantify this event at the molecular level. The study investigates carcinogens present in cigarette smoke, their role in the initiation of bladder cancer and approaches to prevent the infliction of DNA damage.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Johnson, Ronald L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Northeastern University
Schools of Arts and Sciences
United States
Zip Code
Flarakos, Caroline Ceailles; Weiskopf, Andrew; Robinson, Matthew et al. (2017) Metabolism of selective 20-epi-vitamin D3 analogs in rat osteosarcoma UMR-106 cells: Isolation and identification of four novel C-1 fatty acid esters of 1?,25-dihydroxy-16-ene-20-epi-vitamin D3. Steroids 119:18-30
Klaene, Joshua J; Flarakos, Caroline; Glick, James et al. (2016) Tracking matrix effects in the analysis of DNA adducts of polycyclic aromatic hydrocarbons. J Chromatogr A 1439:112-23
Bhattacharya, Arup; Klaene, Joshua J; Li, Yun et al. (2015) The inverse relationship between bladder and liver in 4-aminobiphenyl-induced DNA damage. Oncotarget 6:836-45
Sharma, Vaneet K; Xiong, Wennan; Glick, James et al. (2014) Determination of site selectivity of different carcinogens for preferential mutational hot spots in oligonucleotide fragments by ion-pair reversed-phase nano liquid chromatography tandem mass spectrometry. Eur J Mass Spectrom (Chichester) 20:63-72
Kafle, Amol; Coy, Stephen L; Wong, Bryan M et al. (2014) Understanding gas phase modifier interactions in rapid analysis by differential mobility-tandem mass spectrometry. J Am Soc Mass Spectrom 25:1098-113
Gathungu, Rose M; Bird, Susan S; Sheldon, Diane P et al. (2014) Identification of metabolites from liquid chromatography-coulometric array detection profiling: gas chromatography-mass spectrometry and refractionation provide essential information orthogonal to LC-MS/microNMR. Anal Biochem 454:23-32
Kafle, Amol; Klaene, Joshua; Hall, Adam B et al. (2013) A differential mobility spectrometry/mass spectrometry platform for the rapid detection and quantitation of DNA adduct dG-ABP. Rapid Commun Mass Spectrom 27:1473-80
Hall, Adam B; Coy, Stephen L; Kafle, Amol et al. (2013) Extending the dynamic range of the ion trap by differential mobility filtration. J Am Soc Mass Spectrom 24:1428-36
Rhieu, Steve Y; Annalora, Andrew J; Wang, Guochun et al. (2013) Metabolic stability of 3-epi-1?,25-dihydroxyvitamin D3 over 1 ? 25-dihydroxyvitamin D3: metabolism and molecular docking studies using rat CYP24A1. J Cell Biochem 114:2293-305
Gathungu, Rose M; Flarakos, Caroline C; Reddy, G Satyanarayana et al. (2013) The role of mass spectrometry in the analysis of vitamin D compounds. Mass Spectrom Rev 32:72-86

Showing the most recent 10 out of 54 publications