Harper: Environmental Pollutants and the Cytotoxic Stress Response in Vertebrate Cell Lines

Funding provided by the College of Sciences Dean’s Office

The use of cell culture to evaluate the effects of environmental pollutants is new to me and my lab. Prior to initiating this line of work, I worked exclusively with primary cell lines from a variety of species to ask questions about the comparative biology of aging among vertebrates. Consequently, anything accomplished during the course of the EURECA FAST project would be a significant step forward.

The specific project conducted during the summer focused on the effect of plasticizing agents on DNA damage in a neuron‐like cell line from rats known as PC12 cells. There is a long history of using these cells to address fundamental question about neurophysiology in multiple model systems, but to the best of our knowledge no one had attempted to assess whether the industrial plasticizer bisphenol‐A (BPA) rendered PC12 cells more susceptible to UV‐induced DNA damage. Since this work was new to the lab, a portion of the summer was devoted to simple protocol development. That is, how many cells should we irradiate, what doses of UV radiation and so on should be used. Once we established a non‐lethal working dose of UV radiation, the student working on this project set out to learn how to conduct a COMET assay which is routinely used to assess DNA damage under a variety of conditions in cells, tissues and intact animals. However, since this method was also new to the lab, the protocol had to be fine‐tuned for our own use prior to asking specific questions.

In the final month of the summer, the student had worked out all of the necessary details and was able to ask whether prior exposure of PC12 cells to BPA would render them more sensitive to UV radiation. In particular, BPA‐treated versus untreated cells were exposed to either a “low” or “high” dose of UV‐C radiation using a commercially available UV‐C source (Stratalinker) then the amount of damage assessed via the COMET assay. Control cells received no UV radiation. Since previous reports had suggested that BPA could cause DNA damage directly in other model systems, we expected to find some damage in BPA‐treated cells even in the absence of UV exposure and higher levels of damage in BPA‐treated cells after UV irradiation relative to untreated cells. Surprisingly, the results of four independent experiments indicated that there was no appreciable effect of BPA on DNA damage in PC12 cells before or after UV‐C irradiation. That is, none of our expectations were met. Although this finding was unexpected, that is the nature of experimental science.

Just because the degree of DNA damage does not differ among treatment groups, this does not rule out the possibility that BPA is altering the response to DNA damage in these cells. In fact, prior work conducted in my lab had indicated that BPA exposure rendered a mouse cell line more susceptible to UV‐induced death in a dose‐dependent manner. Consequently, we are beginning a new set of experiments to determine whether BPA exposure can alter the dynamics of the DNA damage repair (DDR) response. In brief, cell lines will be exposed to UV‐C radiation as before, but instead of using a general measure of DNA damage, i.e. a COMET assay, we will use an enzyme immunoassay (EIA) to quantify a specific form of UV‐induced DNA damage, namely cyclobutane pyrimidine dimers (CPD), among treatment group immediately after exposure and at multiple time points afterward. This will allow us to assess whether there are differences among groups with regard to this specific form of DNA damage, as well as monitoring the clearance of this specific lesion over time. A specific form of the DDR response is responsible for removing CPDs from DNA with time. Hence, it’s plausible that even if there is no appreciable difference in the amount of damage rendered to PC12 immediately after UV irradiation with prior BPA exposure, there may be a difference in the kinetics of the damage response. These experiments are currently underway and we hope to have some results soon.