Effects of Endohedral Gd-Containing Fullerenols with a Different Number of Oxygen Substituents on Bacterial Bioluminescence.

Gadolinium (Gd)-containing fullerenols are perspective agents for magnetic resonance imaging and cancer research. They combine the unique paramagnetic properties of Gd with solubility in water, low toxicity and antiradical activity of fullerenols. We compared the bioeffects of two Gd-containing fullerenols with a different number of oxygen groups-20 and 42: Gd@C82O20H14 and Gd@C82O42H32. The bioluminescent bacteria-based assay was applied to monitor the toxicity of fullerenols, bioluminescence was applied as a signal physiological parameter, and bacterial enzyme-based assay was used to evaluate the fullerenol effects on enzymatic intracellular processes. Chemiluminescence luminol assay was applied to monitor the content of reactive oxygen species (ROS) in bacterial and enzymatic media. It was shown that Gd@C82O42H32 and Gd@C82O20H14 inhibited bacterial bioluminescence at >10-1 and >10-2 gL-1, respectively, revealing a lower toxicity of Gd@C82O42H32. Low-concentration (10-3-10-1 gL-1) bacterial bioluminescence activation by Gd@C82O42H32 was observed, while this activation was not found under exposure to Gd@C82O20H14. Additional carboxyl groups in the structure of Gd@C82O42H32 were determined by infrared spectroscopy and confirmed by quantum chemical calculations. The groups were supposed to endow Gd@C82O42H32 with higher penetration ability through the cellular membrane, activation ability, lower toxicity, balancing of the ROS content in the bacterial suspensions, and lower aggregation in aqueous media.

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

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.

Antioxidant Activity and Toxicity of Fullerenols via Bioluminescence Signaling: Role of Oxygen Substituents.

Fullerenols are nanosized water-soluble polyhydroxylated derivatives of fullerenes, a specific allotropic form of carbon, bioactive compounds, and perspective basis for drug development. Our paper analyzes the antioxidant activity and toxicity of a series of fullerenols with different number of oxygen substituents. Two groups of fullerenols were under investigation: (1) C60Oy(OH)x, C60,70Oy(OH)x, where x + y = 24-28 and (2) C60,70Oy(OH)x, Fe0,5C60Oy(OH)x, Gd@C82Oy(OH)x, where x + y = 40-42. Bioluminescent cellular and enzymatic assays (luminous marine bacteria and their enzymatic reactions, respectively) were applied to monitor toxicity in the model fullerenol solutions and bioluminescence was applied as a signaling physiological parameter. The inhibiting concentrations of the fullerenols were determined, revealing the fullerenols' toxic effects. Antioxidant fullerenol' ability was studied in solutions of model oxidizer, 1,4-benzoquinone, and detoxification coefficients of general and oxidative types (DGT and DOxT) were calculated. All fullerenols produced toxic effect at high concentrations (>0.01 g L-1), while their antioxidant activity was demonstrated at low and ultralow concentrations (<0.001 g L-1). Quantitative toxic and antioxidant characteristics of the fullerenols (effective concentrations, concentration ranges, DGT, and DOxT) were found to depend on the number of oxygen substituents. Lower toxicity and higher antioxidant activity were determined in solutions of fullerenols with fewer oxygen substituents (x + y = 24-28). The differences in fullerenol properties were attributed to their catalytic activity due to reversible electron acceptance, radical trapping, and balance of reactive oxygen species in aqueous solutions. The results provide pharmaceutical sciences with a basis for selection of carbon nanoparticles with appropriate toxic and antioxidant characteristics. Based on the results, we recommend, to reduce the toxicity of prospective endohedral gadolinium-fullerenol preparations Gd@C82Oy(OH)x, decreasing the number of oxygen groups to x + y = 24-28. The potential of bioluminescence methods to compare toxic and antioxidant characteristics of carbon nanostructures were demonstrated.

Bioluminescent Enzymatic Assay as a Tool for Studying Antioxidant Activity and Toxicity of Bioactive Compounds.

A bioluminescent assay based on a system of coupled enzymatic reactions catalyzed by bacterial luciferase and NADH:FMN-oxidoreductase was developed to monitor toxicity and antioxidant activity of bioactive compounds. The assay enables studying toxic effects at the level of biomolecules and physicochemical processes, as well as determining the toxicity of general and oxidative types. Toxic and detoxifying effects of bioactive compounds were studied. Fullerenols, perspective pharmaceutical agents, nanosized particles, water-soluble polyhydroxylated fullerene-60 derivatives were chosen as bioactive compounds. Two homologous fullerenols with different number and type of substituents, C60 O2-4 (OH)20-24 and Fe0.5 C60 (OH) x Oy (x + y = 40-42), were used. They suppressed bioluminescent intensity at concentrations >0.01 g L-1 and >0.001 g L-1 for C60 O2-4 (OH)20-24 and Fe0.5 C60 (OH)x Oy , respectively; hence, a lower toxicity of C60 O2-4 (OH)20-24 was demonstrated. Antioxidant activity of fullerenols was studied in model solutions of organic and inorganic oxidizers; changes in toxicities of general and oxidative type were determined; detoxification coefficients were calculated. Fullerenol C60 O2-4 (OH)20-24 revealed higher antioxidant ability at concentrations 10-17 -10-5 g L-1 . The difference in the toxicity and antioxidant activity of fullerenols was explained through their electron donor/acceptor properties and different catalytic activity. Principles of bioluminescent enzyme assay application for evaluating the toxic effect and antioxidant activity of bioactive compounds were summarized and the procedure steps were described.