Antioxidant Properties of Fullerene Derivatives Depend on Their Chemical Structure: A Study of Two Fullerene Derivatives on HELFs

Abstract

Oxidative stress is a major issue in a wide number of pathologies (neurodegenerative, cardiovascular, immune diseases, and cancer). Because of this, the search for new antioxidants is an important issue. One of the potential antioxidants that has been enthusiastically discussed in the past twenty years is fullerene and its derivatives. Although in aqueous solutions fullerene derivatives have shown to be antioxidants, their properties in this regard within the cells are controversially discussed. We have studied two different water-soluble fullerene C60 and C70 derivatives on human embryonic lung fibroblasts at a wide range of concentrations. Both of them cause a decrease in cellular ROS at short times of incubation (1 hour). Their prolonged action, however, is fundamentally different: derivative GI-761 causes secondary oxidative stress whereas derivative VI-419-P3K keeps ROS levels under control values. To gain a better understanding of this effect, we assessed factors that could play a role in the response of cells to fullerene derivatives. Increased ROS production occurred due to NOX4 upregulation by GI-761. Derivative VI-419-P3K activated the transcription of antioxidant master regulator NRF2 and caused its translocation to the nucleus. This data suggests that the antioxidant effect of fullerene derivatives depends on their chemical structure.

Figure 1

Molecular structures of the investigated water-soluble fullerene derivatives GI-761 and VI-419-P3K.

Figure 2

ROS levels in cells treated with fullerene derivatives. (a, b) Fluorescence plate reader: dependence of DCF signal intensity on GI-761 (a) and VI-419-P3K (b) concentration and exposure time. (c, d) Dependence of the k _i/k ₀ index on GI-761 (c) and VI-419-P3K (d) concentration and exposure time.

Figure 3

Dependence of ROS levels in HELFs treated with GI-761 (a) and VI-419-P3K (b) on time of incubation.

Figure 4

Colocalization of fullerene derivatives GI-761 and VI-419-P3K (red stains) and ROS stained with DCF, fluorescent microscopy.

Figure 5

NOX4 expression in HELFs incubated with GI-761 and VI-419-P3K. (a, b) Fluorescent microscopy: localization of NOX4 within fixed cells stained with DAPI and antibodies to NOX4 in HELFs treated with (a) GI-761 and (b) VI-419-P3K. (d, e) FCA: FL1-NOX4 levels for HELFs treated with GI-761 (d) and VI-419-P3K (e). Concentrations and times of exposure shown in graphs.

Figure 6

Dependence of NOX4 protein levels on time for HELFs treated with GI-761 (a) and VI-419-P3K (b). Concentrations and times of exposure shown in graphs. (c, d) Dependence of NOX4 protein levels and ROS levels shown in one graph. NOX4 mRNA levels in HELFs treated with GI-761 (e) and VI-419-P3K (f).

Figure 7

NRF2 levels in HELFs treated with investigated substances. (b) FCA: levels of FL1-NRF2 in cells treated with GI-761 and VI-419-P3K. (c) Real-time PCR: levels of NRF2 mRNA in cells treated with GI-761 and VI-419-P3K. (e) Fluorescent microscopy: localization of NRF2 protein in cells treated with GI-761 and VI-419-P3K, nuclei of the cells stained with DAPI.

Figure 8

DNA damage in cells incubated with GI-761 and VI-419-P3K. (a–c) Comet assay: (a) digital photography of nuclei with varying degree of DNA damage; (b) dependence of “% of tail DNA” on fullerene derivative concentration; (c) dependence of DNA comet tail moment on the time of incubation with fullerene derivatives. (d) Fluorescent microscopy: cells stained with antibodies for H2AX. (e) FCA: average signal intensity of FL1-γH2AX given for times and concentrations of investigated fullerene derivatives.