Pro-oxidant and lifespan extension effects of caffeine and related methylxanthines in Caenorhabditis elegans

Abstract

Caffeine and related purine alkaloids are common ingredients of many stimulating drinks. Studies have shown that lower concentrations of caffeine have a protective role in aging-related disorders. However, the associated mode of action of caffeine and its related methylxanthines is still not clear. In this study, we demonstrated that caffeine and theophylline promote longevity in Caenorhabditis elegans. Lifespan studies with the wild type, DAF-16 and SKN-1 mutant strains indicated that the methylxanthines-mediated lifespan extension in C. elegans was independent of DAF-16/FOXO and SKN-1. All the tested methylxanthines could protect C. elegans against acute oxidative stress. At early stages of life, an increase of ROS (reactive oxygen species) induced the translocation of DAF-16 and SKN-1, resulting in upregulation of several antioxidant genes, for example, sod-3p::GFP, gst-4p::GFP, gcs-1p::GFP; and downregulation of hsp-16.2p::GFP. RT-PCR corroborates the upregulation of gst-4 and skn-1 genes. The expression of DAF-16 decreased although its nuclear translocation was induced.

Keywords: Caenorhabditis elegans; Caffeine; DAF-16/FOXO; Pro-oxidant effect; SKN-1; Theobromine; Theophylline.

Fig. 1

Chemical structures of methylxanthines.

Fig. 2

Effects of methyxanthines on the lifespan of wild-type (N2), daf-16 mutant (CF1038), and skn-1 mutant (EU1) worms. Lifespan experiments were performed at least three times. Additional information is shown in Supplementary Table 1.

Fig. 3

Antioxidant effects of methylxanthines. They protected against oxidative stress in wild type nematodes. A, in vitro evaluation of antioxidant activity using DPPH assay. B, in vivo anti-oxidant activity was detected by the quantification of intracellular ROS levels in N2 worms. C, the effect of methylxanthines on stress resistance (% survival) under a lethal dose of juglone. The experiments were carried out at least three times, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, and compared to the untreated control by one-way ANOVA following Dunnett.

Fig. 4

Methylxanthines induced DAF-16/FOXO and SKN-1 translocation to the nucleus and regulated target genes. A, three levels to determine the pattern of the DAF-16::GFP subcellular translocation. B, methylxanthines induced significant translocation of DAF-16::GFP. C. Two patterns of the SKN-1::GFP subcellular translocation. D, methylxanthine treatment induced a significant translocation of SKN-1::GFP in LD1. Data are presented as mean ± SEM, experiments were repeated at least three times, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, and compared to the untreated control by one-way ANOVA following Dunnett.

Fig. 5

Effect of methyxanthines on stress resistant genes. A, higher concentrations of methylxanthines increased sod-3 expression in CF1553. B, hsp:GFP was suppressed by methylxanthines. C, methylxanthine treatment increased gst-4 expression in mutant CL2166. D, methylxanthine treatment increased gcs-1 expression in LD1171. Data are presented as mean ± SEM. Experiments were carried out at least three times, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, and compared to the untreated control by one-way ANOVA following Dunnett. E, expression of candidate genes. *p < 0.05, **p < 0.01, and a two-tailed Student’s test were used to analyze the results. Data are presented as mean ± SD.

Fig. 6

Effect of methylxanthines on markers of toxicity in C. elegans. A, higher concentration of methylxanthines reduced the brood size. Data are presented as mean ± SEM. B, 5 mM caffeine treatment decreased the body length of worms. Data are presented as mean (μM) ± SEM. Experiments were carried out at least three times, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, and compared to the untreated control by one-way ANOVA following Dunnett.