A method to determine RNA and DNA oxidation simultaneously by HPLC-ECD: greater RNA than DNA oxidation in rat liver after doxorubicin administration.
We developed a novel method for the simultaneous extraction and analysis of total tissue RNA and DNA to quantify the RNA and DNA oxidation products 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine using HPLC coupled to electrochemical detection (HPLC-ECD). The protein denaturing agents guanidine thiocyanate and phenol/chloroform at neutral pH were found to be very efficient for the isolation of RNA and DNA from rat brain, liver and muscle. The method is very fast, allows extraction at 0 degrees C, gives high yields of pure RNA and DNA with low background oxidation levels, and also determines the RNA/DNA ratio. Experiments with isolated RNA and DNA exposed to the Fenton reagents H2O2/ascorbate/Fe3+ (or Cu2+) resulted in significantly greater RNA oxidation. The RNase inhibitor 2-mercaptoethanol, commonly used for RNA extraction, acted as a pro-oxidant during nucleic acid extraction, an effect attenuated by the inclusion of the metal chelator deferoxamine mesylate. In vivo, administration of doxorubicin (an oxidant generator) to Fisher-344 rats resulted in a significant increase in liver RNA oxidation, but no significantly increased DNA oxidation. This new method could be useful to assess oxidatively damaged RNA and DNA simultaneously, and our data show that RNA is more susceptible to oxidative stress than DNA in vivo and in vitro.
Department of Aging and Geriatric Research, College of Medicine, University of Florida, Genomics and Biomarkers Core of the Institute on Aging, Biochemistry of Aging Laboratory, 1329 SW 16th Street, Gainesville, FL 32608, USA.
Tim
T
Hofer
Arnold Y
AY
Seo
Mercedes
M
Prudencio
Christiaan
C
Leeuwenburgh
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002725:Chloroform / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis; D004317:Doxorubicin / Q000008:administration & dosage / Q000494:pharmacology*; D004563:Electrochemistry; D006151:Guanosine / Q000031:analogs & derivatives / Q000032:analysis; D008099:Liver / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D019800:Phenol / Q000737:chemistry; D012313:RNA / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D012974:Sodium Iodide / Q000737:chemistry; D012985:Software Design
D006151:Guanosine; D019800:Phenol; C046215:8-hydroxyguanosine; D012313:RNA; D002725:Chloroform; D004317:Doxorubicin; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D012974:Sodium Iodide; D003849:Deoxyguanosine