Antioxidant properties of blirubin in the model organism, Caenorhabditis elegans

Abstract

We evaluated the effects of bilirubin on the nematode, C. elegans, specifically addressing the ability of bilirubin to induce oxidative stress and alter glutathione (GSH) content as measures of injury. Bilirubin exposure caused a doubling of the spectrophotometric absorption at 440 nm in wild-type C. elegans irrespective of the bilirubin concentration used, suggesting that bilirubin is readily taken up by the worms. No changes were noted in growth, phenotype or reproductive cycle at any bilirubin concentration at 24, 48, and 72 hrs. The oxidative stress inducible green fluorescent protein (GFP) expression in the gst-4::GFP strain was decreased upon exposure to bilirubin at a concentration of 0.5 mM at 24, 48 and 72 hrs. This trend was not statistically significant at 24 or 72 hrs, but did reach statistical significance at 48 hours (p < 0.05). Glutathione (GSH) content in the gst-4::GFP strain showed a significant increase as early as 20 hours post treatment with 0.5 mM bilirubin (p < 0.05). Microarray analysis showed that in bilirubin-exposed worms, 27 genes were up-regulated, and 90 genes were down-regulated (by >1.3 fold vs. controls). The transcription factor asc-1 was induced, whereas genes involved in transcription, trafficking and mitochondrial function were down-regulated. Our findings corroborate earlier findings of bilirubin's ability to act as an antioxidant, most likely by reducing the metabolic requirement in C. elegans.

Keywords: Antioxidant; Bilirubin; C. elegans.

Figure 1.

Structure of bilirubin

Figure 2.

The synthesis pathway of bilirubin

Figure 3.

GFP expression of gst-4::GFP upon biliverdin and bilirubin treatment for 24 hrs. Wildtype and gst-4::GFP strains were treated with 0.5mM biliverdin or bilirubin for 24 hrs, and GFP expression was measured. Wildtype GFP expression was used to correct for background autofluoresence in all treatment groups. All data are represented as mean percent control ± SEM, replicated in three separate experiments. No statistical difference was observed between treated and control groups.

Figure 4.

GFP Expression of gst-4::GFP upon biliverdin and bilirubin treatment for 48 hrs. Wildtype and gst-4::GFP strains were treated with 0.5 mM biliverdin or bilirubin for 48 hrs, and GFP expression was measured. Wildtype GFP expression was used as a baseline to correct for background autofluoresence in all treatment groups. All data are represented as mean percent control ± SEM, replicated in 3 separate experiments. Compared with untreated control gst-4::GFP worms, gst-4::GFP exposed to bilirubin for 48 hours had a significant reduction in GFP fluorescence (*p < 0.05).

Figure 5.

GFP Expression of gst-4::GFP upon biliverdin and bilirubin treatment for 72 hrs. Wildtype and gst-4::GFP strains were treated with 0.5mM biliverdin or bilirubin for 72 hrs, and GFP expression was measured. Wildtype GFP expression was used as a baseline to correct for background autofluoresence in all treatment groups. All data are represented as mean percent control ± SEM, replicated in 3 separate experiments. No statistical difference was observed between treated and control groups.

Figure 6.

Intracellular GSH measurement in gst-4::gfp strain treated with blank or 0.5 mM bilirubin for 20 hrs. Data are presented as mean GSH level ± SEM, replicated in 3 experiments. gst-4::GFP worms treated with bilirubin had a statistically higher GSH level (*p < 0.05) compared to control.