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Purdue Water-Monitoring System Has Potential for Use in ADME/Tox

Purdue University researchers have studied the real-time effects of five environmental contaminants with differing modes of action (atrazine, cadmium, chloride, pentachlorophenol, malathion, and potassium) on respiratory oxygen consumption in 2-day post-fertilization fathead minnow (Pimephales promelas) eggs. 
The investigators wanted to assess the sensitivity of fathead minnow eggs to brief exposures to large concentrations of contaminants using a micro-optrode that they developed to detect instantaneous changes in the eggs’ oxygen consumption.
Their work was the first step in developing a new technique for physiologically-coupled biomonitoring as a reliable environmental toxicology tool, with potential for use in drug discovery. The work was published online on August 6 in Environmental Science and Technology
Marisol Sepulveda, lead author and an assistant professor of forestry and natural resources at Purdue University, and Brian Sanchez, primary author on the paper and a doctoral student in the department of forestry and natural resources at Purdue, spoke with CBA News recently about their work, its potential for use in drug discovery, and how they plan to continue developing the technology.

Can you give me a little background on this work?
Sepulveda: In the big picture, [Purdue Associate Professor and co-author] Marshall Porterfield’s lab has the equipment to run all these assays, and had done similar work before with plant seeds. He has never used this with vertebrate or with animals before. So, it’s kind of a new adventure for him as well, and he thought it could be a good model for looking at water quality, basically.
Looking in the literature to see what other people had done, we felt that this work was very novel because most people had done similar work either with in vitro cell lines or single-celled organisms such as bacteria or algae. As far as we could tell, no one had done such work with vertebrate organisms.
How exactly does this assay work?
Sanchez: It actually measures the fluorescence lifetime of the fluorophore that is at the tip of the optical electrode, or optrode. The fluorescence lifetime varies predictably with the oxygen concentration. It is so sensitive that it can measure concentration differences in a microgradient.
Why did you select the fathead minnow for use in your work?  
MS: The fathead minnow is the model that has been used in ecotoxicology work for many years. It has actually been selected by the US Environmental Protection Agency as the model vertebrate or the aquatic model for ecotoxicology in the US, because we know so much about its biology and many aspects of its ecology.
The fathead minnow is a widely used model, and by using it, it is more likely that use of the system would be adopted in more areas of the country.
How would this compare to the use of zebrafish?
BS: That is a good question. We have not actually tested the system with zebrafish. Whether it would be a more sensitive model would be something that could be explored. They are both widely used ecotoxicological models.
MS: I would think that they would behave very similarly at the embryo level that we tested and with the chemicals that we tested. Both species have a very similar developmental biology.
How does the system that you developed compare to other systems that are currently in use? 
BS: Our system has proven to be more sensitive, in terms of detecting chemicals more quickly than the van der Schiale model mentioned in the paper. That system also uses adult bluegill fish, and we use fathead minnows at an embryonic stage. The literature suggests that earlier developmental stages are more sensitive to environmental contaminants, compared to adults.
That is another advantage of our system. It also detected lower concentrations of contaminants and was also quicker to detection time.
What is the next step in this project? How do you plan to continue to develop and use this technology? 
MS: More than testing other chemicals, we are interested in expanding the platform to measure other endpoints in which we are interested. For example, Dr. Porterfield’s lab has the ability to create probes that detect other types of oxygen-reactive species, such as hydrogen peroxide, that would be a measure of oxidative stress due to chemical exposure.
We are thinking about developing a more diverse platform that one can use to measure not just oxygen and hydrogen peroxide, but other things as well, so that the system itself will be more specific, and one can use it not only to detect the presence or absence of a particular chemical; but also pinpoint the particular class of chemicals that are present.
Do you feel that this system is applicable in some way to drug discovery?    
MS: Possibly. I guess one could potentially do toxicity studies. One of the things that we wanted to do was link these physiological responses in terms of metabolism and changes in oxygen consumption with molecular markers. Brian has been doing a lot of genomics and proteomics work, so our idea was to, after the animals have been exposed to the chemical and after we have gotten the signal with [the] optrode, saturate the organisms and look at the differences in gene and protein expression, and see if we could determine the specific modes of actions of different chemicals.
One can also consider taking the same approach with unknown types of compounds for drug discovery to unravel new mechanism of action for drugs or other types of compounds.
Is this assay something you plan to commercializes or to license? 
MS: We did apply for a patent last year through Purdue, because we do think that it has a lot of potential for being commercialized and applicable in the real world.
The way that I like to think about it is that water treatment facilities are required to do a lot of toxicity testing with things such as invertebrates and even fish. However, they are just doing mortality studies, they are just doing LC50 studies. So I would suspect that because these plants have advanced so much in technology over the last few years, that they probably do not see a lot of mortality anymore. 
That does not mean that chemicals are being removed from the water treated at these plants, because we do not even know what the chemicals are. We believe that our system could serve as an early warning system for things in the water, and then those plants would have to do a broad survey of samples on that water.
Right now, they are probably looking for certain things. They are looking for heavy metals, pesticides, the typical kinds of chemicals one looks for. Many new chemicals are coming out, however, such as pharmaceuticals, et cetera, that people are not looking for, but that are present in the water systems.

That is sort of the long-term potential of this platform — if we keep adding certain probes that will lead us to certain types or classes of chemicals, that would even increase its potential, because it will serve as an alarm and tell us what kinds of chemicals to look for in water samples.            

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