Use of rat liver slices for the study of oxidative DNA damage in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells

Abstract

Tissue slices are a useful biological system for Lipid peroxidation studies but their use for DNA damage studies is not well characterized. Hence, the present study investigates DNA damage in rat li,er slices, in comparison with isolated rat Liver nuclei and HepG2 human hepatoma cells, incubated with ferric nitrilotriacetate (Fe(III)-NTA), bromotrichloromethane (BrCCl3), bromobenzene (BrB) or 2-nitropropane (2-NP) at 37 degrees C for 2 hr. DNA damage was measured in slices, cells or nuclei after centrifugation as formation of as 8-hydroxy-2'-deoxyguanosine (8-OH-dGu) and loss of double-stranded (dsDNA) due to strand breakage using a fluorometric analysis of DNA unwinding (FADU). Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) released into the medium. The results show that in Liver slices and isolated nuclei, Fe/NTA (1 mM/4 mM) induced high levels of TEARS but low levels of 8-OH-dGu, whereas the oxidant induced low levels of TEARS and no formation of 8-OH-dGu in HepG2 cells. In all three systems, inclusion of ascorbate caused dose-dependent formation of 8-OH-dGu, and the levels were similar between liver slices and HepG2 cells but were far higher in isolated nuclei. In liver slices the FADU assay was not applicable due to limited solubilization of DNA from the slice, whereas the assay detected significant loss of dsDNA in HepG2 cells and slight loss in isolated nuclei induced by Fe/NTA with or without ascorbate, Liver slices incubated with 1 mm BrCCl3, BrB or 2-NP had elevated TBARS but had Little or no formation of 8-OH-dGu; none of these oxidants induced lipid peroxidation or DNA damage in HepG2 cells. When liver slices obtained from rats injected with diethylmaleate (to deplete GSH) were incubated with BrCCl3. BrB or 2-NP, le,els of TEARS and 8-OH-dGu increased markedly. Similarly, HepG2 cells with decreased GSH shelved marked elevation of TEARS and loss of dsDNA induced by these oxidants, although no formation of 8-OH-dGu was detected. The present study demonstrates the usefulness and limitations of liver slices for DNA damage studies and the importance of cellular GSH in the protection of DNA against environmental toxicants. (C) 2000 Elsevier Science Ltd. All rights reserved.