Fast evaluation of oxidative DNA damage by liquid chromatography-electrospray tandem mass spectrometry coupled with precision-cut rat liver slices.
OBJECTIVE
To establish a fast and sensitive method for the detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in precision-cut rat liver slices by HPLC-MS/MS and to investigate isoniazid (INH) -induced oxidative DNA damage.


METHODS
Precision-cut liver slices (300 microm) were prepared from male rats, and incubated with INH (0.018 mol/L) for 2 h after 1 h preincubation. DNA in the slices was extracted and digested into free nucleosides at 37 degrees C. The samples were injected into HPLC-MS/MS after the proteins were removed. The level of oxidative DNA damage was estimated using the ratio of 8-OHdG to deoxyguanosine (dG).


RESULTS
The limit of detection of 8-OHdG was 1 ng/mL (S/N=3) and the intra-assay relative standard variation was 3.38% when one transition 284.3/168.4 was used as a quantifier and another two transitions 284.3/140.2, 306.1/190.2 as qualifiers. 8-OHdG and dG were well separated, as indicated by elution at 10.02 and 7.37 min, respectively. INH significantly increased the ratio of 8-OHdG to dG in rat liver slices (P<0.05).


CONCLUSIONS
8-OHdG in precision-cut liver slices could be sensitively determined by HPLC-MS/MS. HPLC-MS/MS coupled with precision-cut tissue slices is a fast and reliable analytical technique to evaluate oxidative DNA damage of target tissues caused by procarcinogens and cytotoxins.
Yue
Jiang
J
Department of Pharmacology, College of Basic Medicine, Wuhan University, Wuhan 430071, Hubei, China.
Wang
Peng
P
Liu
Ying-Hui
YH
Wu
Jun-Yu
JY
Chen
Jie
J
Peng
Ren-Xiu
RX
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002853:Chromatography, Liquid; D004249:DNA Damage / Q000187:drug effects*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine; D007538:Isoniazid