Photoreactivity and ROS production: Looking at TiO2 (2011)

n the image above, the heads and tails of developing zebrafish are shown. In the top row, the developing transgenic ARE-gfp embryos were exposed to TiO2 nanoparticles without illumination, while in the bottom row the fish were exposed to particles and intense illumination, simulating sunlight. The yolk sacs of the fish normally produce some fluorescence, which can be seen in both sets of images. However, the individual green-marked cells producing GFP are seen only with the combination of the nanoparticles and illumination. The GFP signals the production of ROS by the TiO2 nanoparticles.

The properties of nanoparticles that make them special can also make them potentially dangerous. One type of nanoparticle with special properties is titanium dioxide (TiO2). Found in different crystalline forms, TiO2 nanoparticles can collect light and use that energy to drive the production of different very reactive forms of oxygen, known as reactive oxygen species, or ROS. The use of antioxidants in the diet to combat the effects of ROS in aging and disease is well publicized. While the interactions between TiO2 nanoparticles and light can be extremely useful, TiO2 nanoparticles will inevitably enter the waste stream. We wondered if TiO2 nanoparticle production of ROS could pose an environmental risk. To test this we used developing zebrafish. Developing organisms go through complex cellular maneuvers that are very sensitive to disruption. Thus, a good place to look for toxic effects is in a developing vertebrate like the zebrafish embryo. All animals have defense mechanisms against excess ROS levels. Genes needed for this defense lie waiting to be turned on by proteins that spread the cellular alarm. This defense mechanism works through DNA sequences called AREs. To measure the activation of this defense mechanism, we took the gene Green Fluorescent Protein (GFP), originally isolated from a marine jellyfish, and tied it to ARE activation in a transgenic zebrafish. In this newly developed line of fish, the tissues produce GFP when ROS activates the ARE. This produces a green florescent glow under ultraviolet light in the affected cells. To end this story, we found that TiO2 nanoparticles produce toxicity in zebrafish embryos, but only when activated by light. As seen below, this is accompanied by ROS production and ARE activation: embryos exposed to TiO2 nanoparticles and light produce glowing GFP. This presages toxic responses also observed in these experiments.