Spencer C. Davis and Dr. James Thorne, Chemistry
Photodynamic Therapy (PDT) is a new technique currently used to treat malignant cancer tumors in humans. This treatment uses the principles of light in a manner similar to types of radiation therapy. In PDT a photosensitizing dye is injected intraveneously diffusing to all cells. Tumor cell absorption of the dye is targeted by tailoring chemical properties of the photosensitizer, concentrating it in the tumor. The sensitizers absorb light energy, producing an excited singlet oxygen that readily reacts with double bonds and oxidizes adjacent molecules. PDT is very effective because the extremely reactive singlet oxygen travels only a short distance before it destroys neighboring molecules. If optimized, it can be lethal to a target tissue while not doing any permanent damage to surrounding normal tissue.
The goal of my project was to use the principles of PDT to cure a common fish disease called Ichthyophthirus Multifilis (ICK). This disease is caused by the parasitic single-celled protozoan ichthyophthirus multifilis, and occurs in wild fish populations, but more commonly in hatchery-raised fish. ICK rapidly penetrates the outer skin layer and lives between the skin and underlying tissue, feeding on the body fluids and skin cells. When mature, the parasites bore through the skin, fall to the bottom of the aquarium, and start to divide. Within 15 to 20 hours, up to 1000 free-swimming tomites (infant paramecia) are released into the water, starting the cycle all over again. The protozoan can be seen on the outside of the fish as bumpy whitish spots, giving the appearance of salt. Once the fish have become infected by the protozoans, there is no cure.
The first step towards my goal of finding a cure was to determine what dye would most effectively produce singlet oxygen, yet wouldn’t hurt fish. I selected several photosensitizing dyes on the basis of structure and spectroscopy and then measured dose-effect curves of these dyes on paramecium and tetrahymena. These protozoan were chosen because their body structure most closely resembles ICK. Of the dyes tested (including acridine orange, acridine yellow, erythrorine B, rose bengal, eosin Y, basic blue, methylene blue, gemisa stain, and phloxine B), the most promising photosensitizer was rose bengal (the chemical that was first shown to produce photodynamic effects).
Once I determined that rose bengal was the most effective photosensitizer available to me, my next task was to test the dye on fish. Dr. Richard Heckmann helped me catch some local sculpin (Cottus bairdi) infected with ICK and other gill parasites. Dr. Thorne then assisted me in getting the proper clearance to run experiments on the fish. These sculpin were treated using the same methods used in gathering dose curves on the protozoan.
The results of this testing indicated that the original levels of exposure (dye concentration in aquarium, length of exposure, delay period, and duration of light of a given intensity) caused severe damage to the parasites. Close examination of stained thin sections and direct microscopic examination also showed that there was some minimal collateral damage to the gills of the fish; minor damage that can quickly heal.
While this treatment did not cure the fish of ICK, the experiment has some exciting results. Because I now know that rose bengal is an effective photosensitizer for ICK, I plan on changing my test protocol by experimenting with heavier doses of dye and light intensity. In addition, adjusting the pH and partition coefficient might also be effective changes to the protocol that will enhance photosensitizer selectivity. By continuing this testing I hope to find a protocol that maximizes the therapeutic ratio for specific species of fish and parasites. In the long term, this research could be extended to other animals and other maladies where tissue needs to be specifically destroyed or inactivated.
As a result of this research, Dr. Thorne, Dr. Heckmann and I were invited and gave a fifteen minute presentation on our findings at the annual United States Parasitologists conference on May 16 in Boise, Idaho. At this conference many parasitologists expressed interest in our PDT protocol for treating other diseases. One parasitologist at the conference came to BYU to further examine our findings and treatments. He is currently using our protocol to test PDT on whirling disease in fish. Results and publications are pending.