A number of metals are known to produce reactive oxygen species (ROS) as at least one mechanism of their toxicity, especially Hg(II) and Cr(VI). The evidence for production of ROS by Ni(II) or Cr(VI) compounds has been less convincing, partly because extremely toxic levels of these metals were required to produce oxidative damage. There is, however, evidence of the production of ROS by in vivo routes of exposure for nickel in rodent studies. This latter result is consistent with recent findings that both Ni(II) and Cd(II) stimulate polymorphonuclear leucocytes (PMNs) in vitro to release an oxidative burst. HeLa cells stimulated with TPA produce oxidative bursts of much lower magnitude than PMNs but oxidative damage to DNA is still detectable. In this proposal we are designing an assay that will couple the digestion of DNA from lymphocytes by enzymes that recognize oxidized nucleoside damage and cleave at these sites with the detection of the induced DNA single strand breaks (SSB) by the alkaline unwinding assay. The two E. coli enzymes to be used recognize oxidized damage and produce endonuclease cleavage at the site of damage. Endonuclease III (endo III) recognizes several types of oxidized damage to pyrimidines while the E. coli fpg protein is a formamido-pyrimidine-DNA glycosylase that recognizes imadazole ring-opened purines. In the coupled assay cells will be lysed, subjected to enzyme digestion, and treated with alkaline unwinding solution for detection of SSB. Initially, the characterization of the coupled assay will be done with cultured HeLa cells labeled with 3H-thymidine. When human lymphocytes are used, the quantitation of DNA will be performed by slot blot hybridization with a human specific Alu probe, Blur 8. We will focus on Cr(VI) and Cd(II) for our studies in coordination with the epidemiology core for the project. These two metals will provide a good comparison between two apparently different mechanisms for the production of ROS. We will quantitate the levels of oxidative DNA damage by the enzyme assay relative to a known marker of oxidative DNA damage, 8-hydroxy- 2'-deoxyguanosine (8-HG). The sister chromatid exchange assay will be used as a conventional assay capable of detecting chromate exposure to evaluate the sensitivity of our assay for oxidative damage and to determine how well levels of oxidized nucleoside damage in circulating lymphocytes correlates with the production of SCEs. In summary, we will measure levels of DNA damage due to oxidative stress, relate these levels to a known marker for this type of damage, and evaluate the effect of the DNA damage in a conventional assay.
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