MEASUREMENT OF HEAT- AND PIPERIDINE-LABILE SITES IN PLASMID DNA EXPOSED TO DEPLETED URANIUM

picture of Janice Wilson presenting his/her poster: MEASUREMENT OF HEAT- AND PIPERIDINE-LABILE SITES IN PLASMID DNA EXPOSED TO DEPLETED URANIUM

Janice Wilson , Diane M. Stearns, PhD

MEASUREMENT OF HEAT- AND PIPERIDINE-LABILE SITES IN PLASMID DNA EXPOSED TO DEPLETED URANIUM

Uranium is an important emerging toxicant whose use is fast outpacing the rate at which we are learning about its health effects.  Previous work in our lab has shown that uranyl acetate (UA) could be activated by either ascorbate or UV radiation to form DNA strand breaks in pBR322 plasmid DNA.  The purpose of the current project was to develop a protocol to detect the presence of U-DNA adducts in these reactions by comparing the sensitivity of generated DNA lesions to heat or dilute piperidine.  Plasmid DNA was exposed to depleted uranium as uranyl acetate (UA) in the presence and absence of ascorbate, UVA light (368 nm), or UVB light (302 nm), and DNA damage was visualized by gel electrophoresis.  Post-treatment incubations of plasmid DNA at RT, 60 °C or with 30 μM piperidine at 60 °C were carried out to measure the propensity of any adduct-type lesions to be further converted to strand breaks.  We hypothesized that if these adduct-type lesions were present, than heat or piperidine treatment would degrade more supercoiled DNA compared to the same reaction conditions in the absence of heat or piperidine.  Results supported our hypothesis in that DNA lesions produced by reactions of UA with ascorbate were heat sensitive, lesions from reactions of UA and UVA light were piperidine-sensitive, and lesions produced by reactions of UA and UVB light were neither heat- nor piperidine-sensitive.  Results were interpreted to suggest that combined exposures to uranyl ion and either ascorbate or UV light are synergistically damaging, but by different mechanisms.  Furthermore, U-DNA adducts may serve as a “parent lesion” for other types of secondary DNA damage such as abasic sites and single strand breaks.  This work is significant because it establishes a new method to explore U-DNA adducts, which if present in humans exposed to uranium could be another source of mutations that may lead to cancer.  This work was supported by NIH grants ES019703 and U54CA143925, and Ms. Wilson was supported by the NAU Ottens Native American Undergraduate Research Program. 

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