DNA Damage

Project Outline (project on hold)

DNA damage is the underlying cause for mutations leading to cancer. It can be caused by a number of different agents ranging from chemical compounds to radiation sources. Detection of these different types of damages is important for understanding the mechanisms by which DNA damage is caused and propagated, particularly under real-world conditions of exposure to multiple sources. 

There are several methods available for detecting DNA damage; however, they suffer from high detection limits, low sensitivity and the use of radioactive materials. In addition, there are very few studies that have attempted to detect DNA damage from multiple sources simultaneously. Recently, capillary electrophoresis with laser-induced fluorescence detection has been developed to provide ultrasensitive detection of DNA damage using fluorescently labelled antibodies specific to the damaged site. The aim of this research is to develop a method using antibodies that have been fluorescently labelled with either yellow- and red-fluorescing quantum dots to specifically detect both ultraviolet radiation damage and chemical damage simultaneously. This method will be beneficial for studying the overall genotoxic effects within cells that occur upon exposure to multiple sources of DNA damaging agents. 

It has been estimated that the average number of DNA damages occurring in human cells under normal conditions per hour is about 800, and the number per day is about 19,200 [1]. This number is likely higher in cells that have been exposed to genotoxic agents such as UVR or cigarette smoke. These DNA damages can be removed naturally by DNA repair systems present within our cells; however, these processes are not 100% efficient.

When cells divide, unrepaired DNA damages can cause errors during DNA synthesis, which may lead to mutations that can give rise to cancer. Most of thee mutations result in deleterious or neutral effects on the cells, but some will result in uncontrolled growth. The growth advantage may arise from an increased rate of cell division or a decreased rate of cell death. These cells will tend to proliferate at the expense of neighbouring cells, resulting in a population of mutant cells [2]. Further mutations within these cells may result in additional growth advantages, which, through repetition of this process, may ultimately result in cancer.

Cellular DNA damage can occur from a number of different stresses and proliferate by several different mechanisms, including oxidative stress, DNA strand breaks, synergistic toxicity and DNA adduct formation. This research focuses on the detection of DNA adducts (covalent bonds to DNA) formed by exposure to benzo[a]pyrene (B[a]P) and ultraviolet radiation (UVR). 

B[a]P is a polycyclic aromatic hydrocarbon that is formed from the incomplete combustion of organic material. Common sources of B[a]P include cigarette smoke and automobile exhaust. Figure 1 shows the likely mechanism of DNA damage through the formation of a reactive metabolite trans-benzo[a]pyrene-7,8-diol-9,10-(BPDE), which covalently bonds to the guanine residue on DNA.  

UVR comes in three forms: UV-A (315 - 400 nm), UV-B (280 - 315 nm) and UV-C (< 280 nm). UV-C is largely absorbed by the ozone layer as is much of UV-B. UV-A is not absorbed by Ozone, but generally too weak to be much of a concern. The small amount of UV-B that does reach the Earth’s surface can have significant effects on the biota. DNA damage from UVR is caused through the formation reactive oxygen species and free radicals that result in the formation of  cyclubutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and their Dewer isomers (Figure 2)

Several methods of detection of DNA damage have been used including 32P post-labelling assays and High Performance Liquid Chromatography [3,4]. These methods may require the use of radioactive phosphorus, laborious sample preparation, and have relatively high limits of detection. CE-LIF enables detection of very low levels of DNA damage, which are especially important when dealing with 6-4PP’s since they are not formed in as high an abundance as CPDs, and the are short lived, readily converting to Dewer isomers in the presence of UVR [5]. 

References

[1] M.M Vilenchik, et al, Proc Natl Acad Sci USA, (2000) 97 5381-5386.

[2] C. Bernstein et al, Cancer Lett, (2008) 260 1-10.

[3] Shengwen Shen et al, Molecular Carcinogenesis (2008) 47 508–518

[4] R.P. Rastogi et al, J. Nucleic Acids, (2010) 1-32

[5] R.P. Sinha et al, Photochem. Photobiol. Sci., (2002) 1 225–236