Endohedral Gd-Containing Fullerenol: Toxicity, Antioxidant Activity, and Regulation of Reactive Oxygen Species in Cellular and Enzymatic Systems

Abstract

The Gd-containing metallofullerene derivatives are perspective magnetic resonance imaging contrast agents. We studied the bioeffects of a water-soluble fullerene derivative, gadolinium-endohedral fullerenol, with 40−42 oxygen groups (Gd@Fln). Bioluminescent cellular and enzymatic assays were applied to monitor toxicity and antioxidant activity of Gd@Fln in model solutions; bioluminescence was applied as a signaling physiological parameter. The Gd@Fln inhibited bioluminescence at high concentrations (>2·10−1 gL−1), revealing lower toxicity as compared to the previously studied fullerenols. Efficient activation of bioluminescence (up to almost 100%) and consumption of reactive oxygen species (ROS) in bacterial suspension were observed under low-concentration exposure to Gd@Fln (10−3−2·10−1 gL−1). Antioxidant capability of Gd@Fln was studied under conditions of model oxidative stress (i.e., solutions of model organic and inorganic oxidizers); antioxidant coefficients of Gd@Fln were determined at different concentrations and times of exposure. Contents of ROS were evaluated and correlations with toxicity/antioxidant coefficients were determined. The bioeffects of Gd@Fln were explained by hydrophobic interactions, electron affinity, and disturbing of ROS balance in the bioluminescence systems. The results contribute to understanding the molecular mechanism of “hormetic” cellular responses. Advantages of the bioluminescence assays to compare bioeffects of fullerenols based on their structural characteristics were demonstrated.

Keywords: antioxidant activity; bioluminescence bioassay; endohedral fullerenol; gadolinium; hormesis; oxidative stress; reactive oxygen species; toxicity.

Figure 1

(A) Relative bioluminescence intensity, I^rel, at different concentrations of fullerenol Gd@Fln in bacterial suspension (1) and enzymatic system (2). The 5-min exposure. (B). Scheme of hormesis dose-effect model is presented according to [67]. Hormetic stages: I—stress recognition, II—physiological activation, III—inhibition of vital functions. “Control” corresponds to the absence of Gd@Fln in the experimental solutions.

Figure 2

Relative bioluminescence intensity, I^rel, (1) and relative ROS content, ROS^rel, (2) in bacterial suspension (A), enzymatic system (B) and distilled water (C) at different concentrations of fullerenol Gd@Fln. Time of exposure to Gd@Fln was 1 min. Concentration of ROS in the control bacterial suspension was ~4.5·10⁻⁶ M, in the control enzymatic sample—1.9·10⁻⁵ M, in distilled water—3·10⁻⁷ M. “Control” corresponds to the absence of Gd@Fln in the experimental solutions.

Figure 3

Antioxidant coefficients, I^rel_Ox, (1) and relative ROS content, ROS^rel, (2) in bacterial suspension (A) and enzymatic system (B) vs. concentration of fullerenol Gd@Fln. Time of exposure to Gd@Fln was 45 min. Concentrations of ROS in the control bacterial suspension (bacteria + 1,4-benzoquinone at EC₅₀ = 8·10⁻⁷ M) and control enzymatic system (enzymes + 1,4-benzoquinone at EC₅₀ = 10⁻⁵ M) were 5.8·10⁻⁶ M and 4.9·10⁻⁵ M, respectively. “Control” corresponds to the absence of Gd@Fln in the experimental solutions.

Figure 4

Relative ROS content, ROS^rel, in bacterial suspension (1), physiological 3%NaCl solution without bacteria (2) at different concentrations of 1,4-benzoquinone, 5 min exposure. Concentrations of ROS were 1.3·10⁻⁵ M and 4.8·10⁻⁶ M in the control physiological 3%NaCl solution and control bacterial suspension, respectively. Relative ROS content at EC₅₀ marked with green ellipse. “Control” corresponds to the absence of 1,4-benzoquinone in the experimental solutions.

Figure 5

Relative ROS content, ROS^rel, in distilled water (1), enzymatic system (2) at different concentrations of 1,4-benzoquinone, 5 min exposure. Concentrations of ROS were 4.5·10⁻⁷ M and 1.9·10⁻⁵ M in distilled water and enzymatic system, respectively. “Control” corresponds to the absence of 1,4-benzoquinone in the experimental solutions.

Figure 6

Bioluminescence kinetics in a solution of model oxidizer (Ox) and fullerenol (F): (a) cellular assay; (b) enzymatic assay.