1. Kinetics of DNA Damage and Repair in Fish using the Zebrafish (Danio rerio) as a model Chris A. McCabe 1, Chris W. Theodorakis 2, Theodore B. Henry 1 and Mark G. J. Hartl 1 Introduction Environmental stressors can damage the DNA of aquatic organisms In fish, DNA damage may negatively affect the cellular physiology and higher-level consequences including reproductive success and recruitment 1 Centre for Marine Biodiversity & Biotechnology, School of Life Sciences, Heriot-Watt University, UK m.hartl@hw.ac.uk 2 Department of Biological Sciences and Environmental Sciences Program, Southern Illinois University, Edwardsville, USA Exposure to ultra violet irradiation (UVR) can induce DNA damage directly Or may cause photoactivation of substances, such as PAHs and TiO 2 nanoparticles, generating reactive oxygen species which lead to DNA damage, especially in vulnreable, early life-stages Benzo[a]pyrene TiO 2 Our Aim is to investigate the the kinetics of DNA damage and repair in zebrafish (Danio rerio). The effects of UV irradiation with and without the presence of the nanoparticle TiO 2 on DNA damage and expression of genes involved in DNA repair DNA repair and damage is ongoing, however negative effects on physiology occur when damage exceeds repair. Therefore the kinetics of DNA damage and repair in fish are an important system to investigate Slide 1 of 3

2. Methods and Optimisations A Comet Assay method was developed to identify and assess DNA Damage and Repair Slide 2 of 3 Individual Larvae (72 hr post- fertilisation) were exposed to UVR with the aim to damage the DNA and assess repair. Comet Head Start Comet Nucleus Comet Tail End Spectromicroscopy and specialised software is used to assess the comet cells from the assay A comet cell with tail, indicating damage and subsequent loss of DNA fragments from the cell nucleus. DNA Damage was assessed by the intensity of Comet ‘tails’- a quantification of the loss of fragmented DNA from the cell post- damage Repair was quantified by the disappearance of damage, and a reduction in this measure of intensity The differences in DNA damage were compared between treatments in order to optimise the variables within the experiment. Electrophoresis Duration Optimisations have included: Ultraviolet Radiation Types Damage Specific Enzyme dilutions - Formamidopyrimadine glycosyase (FaPy): Makes single-strand nicks wherever there is an oxidized purine or an abasic site. - Endonuclease III(EndoIII): Makes single-strand nicks wherever there is an oxidized pyrimidine - T4 pyrimadine dimer glycosylase (PDG) (Shown) Makes single-strand nicks wherever there is a UV photoproduct UV Optimisation Larvae were exposed to 10 J/cm2 UVA or 0.333 J/cm2 UVC. Larvae were subjected to mixture of Fpg (1:400) and Endonuclease III (1:100) enzymes in buffer dilution to detect oxidized bases. The results show that both UVA and UVC induced oxidised base DNA damage. Although the amount of UVC damage was lower, the dose was also significantly lower. Therefore UVC may be more effective in causing base oxidative damage than UV-A. (n=2 in all treatments) Electrophoresis Duration Assessment of dimer-specific damage to different electrophoresis times. Graph depicts the difference between samples that were incubated with and without T4 PDG enzyme (1:80 dilution). Larvae were either exposed to 1 min UVC (0.333 J/cm2) (red) or unexposed (blue). The relative difference between control and exposed larvae was greatest at 35 min. (n= 2 for all treatments) Enzyme Dilutions In this optimisation larvae were exposed to 1 min UVC (0.333 J/cm2) and treated with various T4 PDG enzyme dilutions in buffer (5x PDG buffer). Slides were incubated with 50 uL of enzyme dilutions. From the results, the optimal appears at 1:100. At higher dilutions fewer dimers were detected in exposed fish; while at 1:50 dilution, there were more apparent “dimers” in the control. This may be due to non-specific cutting of the DNA by the enzyme if the concentration is too high. (n=2 in all treatments)

3. Conclusions A method to assess DNA Damage in larval fish was established, using Danio rerio as a model. Slide 3 of 3 Initial experimental results investigating a recovery or repair period, post- exposure, suggest the amount of DNA damage decreases in proportion to recovery time. Most damage was observed to be repaired after a 1- hour period. Future Studies Aim to analyse the kinetics of expression of genes involved in DNA repair using qRT- PCR in fish exposed to photoactivated TiO 2 nanoparticles to establish the potential effects of this substance in the environment. Thank You for watching Acknowledgements I would like to thank Heriot-watt Univeristy and for the use of the Centre for Marine Biodiversity & Biotechnology for experimentation, Mihalis Panagiotidis for the use of scientific equipment, and Jade Middlemiss for her tireless help in lab work and comet scoring. I would like to take this opportunity to thank my Co-Authors: Dr. Theodore Henry, Dr. Chris Theodorakis, and Dr. Mark Hartl for their continuous support and guidance. http://markhartl.sls.hw.ac.uk/