Induction of oxidative DNA damage in human foreskin fibroblast Hs68 cells by oxidized β-carotene and lycopene

Abstract

Two recent clinical trials suggest that β-carotene may be harmful to smokers. In this study we examined the hypothesis that β-carotene may become toxic when degradation occurs. β-Carotene (BC) and lycopene (LP) with or without prior heat treatment (60°C for 1h in open air) were incubated at 20 and 40 μM with calf thymus DNA or human fibroblasts Hs68 cells. The heat treatment resulted in ca. 80% and 35% bleaching of BC and LP, respectively. When Hs68 cells were incubated with the oxidized β-carotene (OBC) or oxidized lycopene (OLP) at 37°C for 20h, cell viability was significantly and dose-dependently decreased whereas cell viability was not affected by BC or LP. Cell death, which was already evident at 4h after incubation with OBC or OLP, was possibly attributable to apoptosis, as shown by the increased histone-associated DNA fragmentation. However, cell lysis, measured as release of lactate dehydrogenase, also occurred at 4h after incubation with OBC and OLP, although the extent was relatively small and was greater for OLP than for OBC. When calf thymus DNA was incubated with OBC or OLP at 37°C for 20h, the 8-hydroxy-2-deoxyguanosine (8-OH-dG) level was significantly and dose-dependently increased by OLP whereas the increase by OBC was only significant at 40 μM. When Hs68 cells were incubated with OBC and OLP for 20h, both compounds increased the 8-OH-dG level, but the effect was only significant for 40 μM OLP. Comet (single-cell gel electrophoresis) assay of DNA damage in Hs68 cells was determined at 2h after incubation with OBC or OLP because of its high sensitivity. Both OBC and OLP significantly and dose-dependently increased DNA breakage while BC and LP had no effect. Inclusion of BHT during incubation of cells with 40 μM OBC or OLP partially inhibited (ca. 40%, p<.05) the extent of comet formation. Intriguingly, OBC and OLP neither induce lipid peroxidation in Hs68 cells (measured as thiobarbituric acid-reactive substances released into the medium) nor increased the intracellular level of reactive oxygen species. Although it is presently unclear about what degradation products are formed, this study has demonstrated that, when oxidized, BC and LP lead to oxidative damage to both purified DNA and cellular DNA. The results suggest that such damage may contribute to the adverse effects of β-carotene reported in recent clinical studies and caution that it is important to prevent oxidation of BC and LP for human uses such as in supplemental studies.

Keywords:

• β-carotene
• lycopene
• autoxidation
• DNA damage
• 8-OH-dG
• comet assay
• lipid peroxidation