Date of Award:


Document Type:


Degree Name:

Doctor of Philosophy (PhD)


Animal, Dairy, and Veterinary Sciences

Department name when degree awarded

Interdepartmental Program in Toxicology

Committee Chair(s)

Roger Coulombe Jr.


Roger Coulombe Jr.


Raghubir Sharma


William A. Brindley


Michael R. Franklin


Lawrence H. Piette


A mechanism common to many carcinogens and mutagens is the interaction with cellular DNA. Alkaline elution is a sensitive and commonly used technique to detect cellular DNA damage in the form of DNA single-strand breaks (SSB), DNA-protein cross-links (DPC), and DNA interstrand cross-links (ISC). In the present study, gravity-flow alkaline elution was developed and validated to rapidly detect DNA damage in the MDBK cell culture system by genotoxic agents.

The system was first validated by detecting a dose dependent induction of DNA SSB in cells exposed to 25-1500 rad of X-ray. The assay reliably detected the DNA damage of seven genotoxic carcinogens and showed no detectable DNA damage in six non-genotoxic compounds.

Gravity-flow alkaline elution was then used to detect the induction of DNA SSB and subsequent repair in cells exposed to ultraviolet (UV) radiation from sunlight, suntan booth, or germicidal lamp (254 nm). The induction of DNA SSB was dose-dependent for all three exposure systems. Repair of sunlight and suntan induced DNA SSB were similar, whereas cells exposed to 254 nm UV did not show any repair. Repair studies using the DNA repair inhibitors 1-B-D-arabinofuranosylcytosine and hydroxyurea indicated DNA excision repair was involved. The mechanisms underlying UV induced DNA damage and repair appear similar for both sunlight and suntan booth, but different than 254 nm UV.

The gravity-flow system was modified to rapidly detect DNA cross-links in MDBK cells exposed the cross-linking agents, nitrogen mustard, mitomycin C, and 254 nm UV radiation. Both drugs induced DPC and ISC whereas UV radiation induced protein-associated DNA strand breaks.

The gravity-flow system was used to characterize the DNA damage in cells exposed to seven pyrrolizidine alkaloids (PAs). Five of the PAs induced both DPC and ISC, while heliosupine induced only DPC, retronecine induced no cross-links, and none of the PAs induced DNA SSB. Since these PAs differ only in the extent of substitution of the diester side chain, these results indicate that these substituents are an important determinant in the induction of DNA damage by pyrrolizidine alkaloids.



Included in

Genomics Commons