Assessing Curcumin Uptake and Clearance and Their Influence on Superoxide Dismutase Activity in Drosophila melanogaster

Abstract

While normal levels of reactive oxygen and nitrogen species (RONS) are required for proper organismal function, increased levels result in oxidative stress. Oxidative stress may be managed via the scavenging activities of antioxidants (e.g., curcumin) and the action of enzymes, including superoxide dismutase (SOD). In this work, the uptake and clearance of dietary curcuminoids (consisting of curcumin, demethoxycurcumin, and bisdemethoxycurcumin) was assessed in Drosophila melanogaster larvae following chronic or acute exposure. High levels of curcuminoid uptake and loss were observed within a few hours and leveled off within eight hours post treatment onset. The addition or removal of curcuminoids from media resulted in corresponding changes in SOD activity, and the involvement of each of the three SOD genes was assessed for their contribution to total SOD activity. Taken together, these data provide insight into the uptake and clearance dynamics of curcuminoids and indicate that, while SOD activity generally increases following curcuminoid treatment, the individual SOD genes appear to contribute differently to this response.

Keywords: Drosophila; curcumin; superoxide dismutase.

Figure 1

Determination of the influence of time and concentration on curcuminoid uptake in wild type (WT) larvae. Larvae were reared for seven days and the level of curcuminoids in tissues was quantified from groups of 10 crawling third-instar larvae (n = 10 per treatment). Larvae were reared on varying curcuminoid concentrations (A) on 0 mM curcuminoids and transferred to 25 mM curcuminoids (B), or on 25 mM curcuminoids and transferred to 0 mM curcuminoids (C), which resulted in the following equations of best fit: $y=10.23+\frac{294.17}{1+{10}^{10.26-x}}$, (r² = 0.7382); $y=\frac{115.4\ast X^{1.608}}{2.151+X^{1.608}},$ (r² = 0.7650); and $y={359.38}^{-0.885\ast X}+43.42$, (r² = 0.7562), respectively. The mean of experiments conducted in n = 10 is presented, and error bars represent the standard error of the mean.

Figure 2

Parameters of the SOD activity assay used herein. A standard curve was prepared with purified SOD (A) with an equation of best fit: $y=2.156\ast X+1.274$, (r² = 0.9945). The addition of varying concentrations of curcuminoids to the SOD assay, conducted with a constant level of 3 units/mL SOD, is expressed as a percentage of the activity values recorded without the addition of curcuminoids (B). Each of three replicates was conducted in technical duplicate, averaged, and the mean of experiments conducted in triplicate is presented (A,B). Larvae from all strains were reared for seven days on 25 mM curcuminoids and the level of curcuminoids in tissues was quantified from groups of 10 crawling third-instar larvae (C) (n = 10 per treatment). The mean of experiments conducted in n = 10 is presented. Error bars in all panels represent the standard error of the mean. No significant differences were found in the data presented in panels (B,C).

Figure 3

The influence of chronic and acute exposure to curcuminoids on SOD activity in the wild type (WT) and three SOD mutants. Larvae from all strains were reared for seven days on 0 or 25 mM curcuminoids; total protein was extracted, and SOD activity was quantified from groups of 5 crawling third-instar larvae (A) (n = 5 per treatment). WT larvae were reared for seven days on 0 or 25 mM curcuminoids, transferred to 25 mM or 0 mM (the opposite condition) for 2–12 h, total protein was extracted, and SOD activity was quantified from groups of 5 crawling third-instar larvae (B) (n = 5 per treatment). Larvae from all strains were reared for seven days on 0 curcuminoids, transferred to 25 mM for 2–12 h, total protein extracted, and SOD activity was quantified from groups of 5 crawling third-instar larvae (C) (n = 5 per treatment). The mean of experiments conducted in n = 5 is presented, and error bars represent the standard error of the mean; * p < 0.05, ** p < 0.005.