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Reliable 8oxodG assays?


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#1 jaydfox

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Posted 07 March 2005 - 11:14 PM


I found this abstract while perusing for references to Barja's work:
http://nar.oupjourna...ract/29/10/2117

A major controversy in the area of DNA biochemistry concerns the actual in vivo levels of oxidative damage in DNA. We show here that 8-oxo-2-deoxyguanosine (oxo8dG) generation during DNA isolation is eliminated using the sodium iodide (NaI) isolation method and that the level of oxo8dG in nuclear DNA (nDNA) is almost one-hundredth of the level obtained using the classical phenol method. We found using NaI that the ratio of oxo8dG/10^5 deoxyguanosine (dG) in nDNA isolated from mouse tissues ranged from 0.032 ± 0.002 for liver to 0.015 ± 0.003 for brain.

Those amounts are significantly smaller than those quoted by Barja and others, who normally give numbers between 1 and 10 oxo8dG/10^5 deoxyguanosine (dG), much larger than 0.032 or 0.015.

If the numbers are off by such large amounts, then drawing any conclusions about the relative rates of mtDNA and nuDNA lesions seems extremely impractical. For example, another notable quote from the abstract:

The levels of oxo8dG in mitochondrial DNA isolated from liver, heart and brain were 6-, 16- and 23-fold higher than nDNA from these tissues.

Those ratios are much higher than anything Barja quoted, as far as I can recall.

So, where do we go from here? Do we disregard this study, or find fault with its methodology? Comments from those with lab experience? I haven't had a chance to check any papers that reference this study (to refute or support it), so I may be jumping the gun here.

#2 jaydfox

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Posted 07 March 2005 - 11:16 PM

Or am I getting my 8oxo's mixed up? Is 8-oxo-2-deoxyguanosine even remotely similar to 8-oxo-7,8-dihydro-2'-deoxyguanosine?

#3 jaydfox

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Posted 08 March 2005 - 06:26 AM

Another study referencing the first, in the footnotes of the page I first linked to:

J.-L. Ravanat, T. Douki, P. Duez, E. Gremaud, K. Herbert, T. Hofer, L. Lasserre, C. Saint-Pierre, A. Favier, and J. Cadet
Cellular background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up
Carcinogenesis, November 1, 2002; 23(11): 1911 - 1918.

It basically says the same thing, that 8oxodG levels are grossly overreported in other studies, as the rates are closer to "~0.5 lesions/10^6 DNA nucleosides", not >1 per 10^5.

However, the Barja studies list levels per dG, not per nucleoside, so I don't know if that might account for the factor of 20-100 difference. I doubt it, but I'm just guessing.

#4

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Posted 08 March 2005 - 11:27 AM

Or am I getting my 8oxo's mixed up? Is 8-oxo-2-deoxyguanosine even remotely similar to 8-oxo-7,8-dihydro-2'-deoxyguanosine?

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#5 jaydfox

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Posted 08 March 2005 - 04:01 PM

Hmm, that picture you posted seems to have slightly different taxonomy.

Okay, so does that mean that this study does or does not affect Barja's studies?

What's the correlation between 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-2-deoxyguanosine? Is it proportional, and by what factor?

For that matter, is 8-oxo-7,8-dihydro-2'-deoxyguanosine the same as 8-hydroxy-7,8-dihydro-2'-deoxyguanosyl radical? Is 8-oxo-2-deoxyguanosine the same as 8-oxodGuo?

#6

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Posted 09 March 2005 - 12:14 PM

Have a read of this.




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