Artifactual formation of 8-oxo-2'-deoxyguanosine: role of fluorescent light and inhibitors

  • Authors:
    • Jamal M. Arif
    • Ramesh C. Gupta
  • View Affiliations

  • Published online on: November 1, 2003     https://doi.org/10.3892/or.10.6.2071
  • Pages: 2071-2074
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Abstract

The pro-mutagenic oxidative DNA lesion, 8-oxo-2'-deoxyguanosine (8-oxodG) has been a subject of numerous studies. However, the absolute 8-oxodG levels in tissue DNA reported by various methods have been debated due to its artifactual production during DNA isolation and/or the DNA processing. We have investigated factors that may result in such artifacts during isolation and analysis of DNA as well as means for its prevention. 8-OxodG content was measured by a recently described TLC enrichment-mediated 32P-postlabeling. Liver DNA from 3 month-old, female Sprague-Dawley rats was isolated by a standard solvent-extraction procedure (phenol, phenol:Sevag, and Sevag; 23°C), a modified solvent-extraction procedure (phenol:Sevag, and Sevag; 4°C; KCl-SDS-protein precipitation) or sodium iodide extraction procedure. 8-OxodG was analyzed in the DNA by the 32P-postlabeling assay using a fluorescent light box during the workup, as well as in its absence. The 8-oxodG levels, when the fluorescent light box was used, were in similar range irrespective of the DNA isolation procedure (16.4±1.6 to 28.7±6 8-oxodG/106 nucleotides). However, the values were significantly lower (3.1±0.4 to 3.4±0.2 8-oxodG/106 nucleotides) in the absence of fluorescence light box, room fluorescent light (suspended through the ceiling) and natural room light did not alter the 8-oxodG levels. Further, the addition of 0.3 mM of PBN (N-t-butyl-α-phenyl nitrone) or TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), or 6.8 mM 8-hydroxy-quinoline during the DNA isolation resulted in still lower values (0.8±0.1 to 1.8±0.5 8-oxodG/106 nucleotides) although this reduction was not consistently observed in different experiments. These results suggest that fluorescent light is the major ‘culprit’ in artifactual production and variability reported in the 8-oxodG levels.

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November-December 2003
Volume 10 Issue 6

Print ISSN: 1021-335X
Online ISSN:1791-2431

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Spandidos Publications style
Arif JM and Arif JM: Artifactual formation of 8-oxo-2'-deoxyguanosine: role of fluorescent light and inhibitors. Oncol Rep 10: 2071-2074, 2003
APA
Arif, J.M., & Arif, J.M. (2003). Artifactual formation of 8-oxo-2'-deoxyguanosine: role of fluorescent light and inhibitors. Oncology Reports, 10, 2071-2074. https://doi.org/10.3892/or.10.6.2071
MLA
Arif, J. M., Gupta, R. C."Artifactual formation of 8-oxo-2'-deoxyguanosine: role of fluorescent light and inhibitors". Oncology Reports 10.6 (2003): 2071-2074.
Chicago
Arif, J. M., Gupta, R. C."Artifactual formation of 8-oxo-2'-deoxyguanosine: role of fluorescent light and inhibitors". Oncology Reports 10, no. 6 (2003): 2071-2074. https://doi.org/10.3892/or.10.6.2071