Curcumin's Radioprotective Effects on Zebrafish Embryos

Abstract

Radiation modifiers are largely studied for their contribution to enlarging the treatment window. Curcumin is already known for its antioxidant properties; however, its role as a radioprotector in preclinical studies is affected by the well-known low absorption and bioavailability of curcumin. In this study, curcumin's radioprotection ability has been evaluated in zebrafish larvae, by taking advantage of quantifying curcumin absorption and evaluating its fluorescence in transparent embryos. A curcumin range of 1-10 μM was tested to select the non-toxic concentrations to be used for a pre-treatment of photon beam irradiation using a 2-15 Gy range of doses. The post-treatment analysis within 120 h post-fertilization (hpf) included an assessment of mortality and malformation rates and behavioral and gene expression analysis. A total of 2.5 and 5 μM of curcumin pre-treatment showed a radioprotective role, significantly reducing the frequency of embryo malformations and damaged entities. This sparing effect disappeared using 15 Gy, showing the radiation effect's prevalence. Gene expression analysis reconducted this radioprotective ability for antioxidant gene network activation. The curcumin-induced activation of the antioxidant gene network promoted radioprotection in zebrafish.

Keywords: curcumin; radiation; radiomodifier; radioprotection; zebrafish.

Figure 1

A synoptic diagram of the experimental plan. The evaluation of the mortality and morphological alteration rate was completed from 24 to 120 hpf, while the assessment of the hatching rate was completed at 48 and 72 hpf. The molecular analysis, heart rate evaluation, and behavioral assays were performed at 48, 72, and 119 hpf, respectively.

Figure 2

The evaluation of the curcumin auto-fluorescence signal (%) at 24 hpf in treated embryos with 1–10 μM curcumin (Cur), with respect to untreated controls.

Figure 3

Representative images of the main malformations observed after treatment with 5 μM curcumin (Cur), such as YM (from 24 hpf), SC (from 24 hpf), PE (from 72 hpf), and PIGM (from 48 hpf). Pictures were taken in a light field.

Figure 4

Normal (green bar), dead (black bar), and abnormal (red bar) embryo rates of developing zebrafish embryos exposed to a combination of curcumin (Cur) pre-treatment with concentrations of 2.5 or 5 μM, followed by irradiation with 0, 2, 4, 8, 10, or 15 Gy of X-rays. Data are presented as the mean of 3 experiments. Error bar = ±SD.

Figure 5

The distribution (%) of the main malformations observed in 96 and 120 hpf malformed zebrafish embryos exposed to a combination of curcumin (Cur) pre-treatment with concentrations of 2.5 or 5 μM, followed by irradiation with 0, 2, 4, 8, 10, or 15 Gy of X-rays: SC (blue bar), PE (orange bar), YM (gray bar), PIGM (yellow bar). Data are presented as the mean of 3 experiments.

Figure 6

Representative images of the main malformations (PE, SC, YM) observed, at 120 hpf, after treatment with 10 Gy or 15 Gy alone or combined with both 2.5 and 5 μM curcumin (Cur) concentration, vs. controls.

Figure 7

A 96 hpf measurement (mm) of morphological parameters (body length, yolk sac diameter, eye length, head length, and PE diameter) after 10 and 15 Gy of IR treatment, with or without 2.5 and 5 μM curcumin (Cur) pre-treatment. Error bar = ±SD.

Figure 8

Heart rate values (bpm) of 72 hpf embryos exposed to the experimental doses of 0, 2, 4, 8, and 15 Gy of X-rays in combination with 2.5 or 5 μM curcumin (Cur) pre-treatment. Data are presented as the mean of 3 experiments. Error bar = ±SD.

Figure 9

The gene expression variation in targets involved in oxidative stress regulation, in embryos treated with 5 μM curcumin (Cur) and 10 Gy, as single or combined treatments, analyzed by qPCR. Reference sample (Control) = 1. Error bar = ±SD.