Quantitation of Free Radicals in B6C3F1 Mouse Liver Slices on Exposure to Four Chemical Carcinogens: An EPR-Spin Trapping Study

摘要

Free radicals generated in mouse liver slices following exposure to four chemical carcinogens: tert-butyl hydroperoxide (TBOOH), bromotrichloromethane (BrCCl3), carbon tetrachloride (CCl4) and trichloroethylene (TCE), were quantitated using electron paramagnetic resonance spectroscopy (EPR)/spin trapping techniques. Precision cut liver slices (n= 128) were prepared from B6C3F1 mice and incubated in Waymouth's media supplemented with 10% fetal bovine serum and 10 mM N-tert-butyl-a-phenyl nitrone (PBN). Liver slices were exposed to control media or media with 1 mM TBOOH, BrCCl3, CCl4, or TCE. After 5 or 60 min. the slices and media were homogenized, frozen in liquid nitrogen and lyophilized. Radicals trapped by PBN were quantitated using an EMS 104 EPR analyzer (Bruker Instruments, MA). Standards were prepared by homogenizing the liver slices with known quantities of the spin label 2,2,5,5,- tetramethyl-1-pyrolidinyl oxy-3-carboxamide (3-CAR). EPR spectra were compared quantitatively and qualitatively. Two factorial analysis and analysis of variance showed there was significant differences in the radicals detected with time and chemical treatment (P <0.002). The EPR spectra of the lyophilized liver homogenate suggest there is a difference in the radical species trapped if slices are incubated in media containing 1 mM TBOOH, BrCCl3, CCl4, or TCE when compared to control media. The interaction of both time and concentration should be addressed in any studies designed to quantitate free radical-induced liver damage.