Fast evaluation of oxidative DNA damage by liquid chromatography-electrospray tandem mass spectrometry coupled with precision-cut rat liver slices.

Abstract

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).

Conclusion: 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.