Formation of a Hydrogen Radical in Hydrogen Nanobubble Water and Its Effect on Copper Toxicity in Chlorella

Hydrogen has potential as an antioxidant in preventive and therapeutic applications. Hydrogen nanobubble (NB) water is an emerging technique for hydrogen delivery into living organisms. Although hydrogen NB water exhibits significantly higher scavenging activity of exogenous and endogenous reactive oxygen species (ROS) than hydrogen water without NBs, the mechanisms are unclear. We investigated the role of hydrogen NB water in copper-induced acute toxicity against algae (Chlorella). Hydrogen water without NBs alleviated the Cu toxicity against Chlorella, as the 72 h half maximal inhibitory concentration (IC50) was 0.220 mg/L Cu, whereas IC50 for Chlorella in control water was only 0.021 mg/L. In contrast, the alleviating ability of hydrogen NB water against Cu toxicity to Chlorella proved to be the highest, as IC50 was 0.372 mg/L Cu. The copper-induced endogenous ROS in Chlorella was significantly lower in hydrogen NB water than that in hydrogen water without NBs. Fluorescence spectroscopy and electron paramagnetic resonance measurement revealed that enhanced antioxidant capacity of hydrogen NB water as NBs could produce a sub-micromolar hydrogen radical in water. Hydrogen NB water significantly decreased copper bioaccumulation in Chlorella and affected copper uptake kinetics by reducing the affinity between copper and the copper transport protein and by reducing copper transport protein concentrations. Hence, it can be concluded that hydrogen NB water alleviates copper-induced acute toxicity in Chlorella via two mechanisms: first, the preventive mechanism involving decreased copper uptake rate due to the alteration of the activity and conformation of the copper transport protein; second, the repair mechanism involving reduced endogenous ROS due to hydrogen molecules penetrated in the cell.