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Sensing The Unseen

UW Researcher Clem Furlong Is Developing New Strategies To Detect Environmental Toxins And Guard Against Exposure

If you can't see it, how do you know it's there? This is a problem that environmentalists, toxicologists, and physicians face in trying to evaluate the health and safety of our environment. They've been looking for better ways to detect the presence of potentially hazardous chemicals in the water we drink or the air we breathe. And there may be a new approach to the problem, courtesy of a research group from the University of Washington (UW).

UW scientist Clem Furlong and members of his laboratory have been designing a new tool in the mission to monitor toxic substances in the environment. The biosensor that they've developed can be used to detect shellfish toxins in the water or airborne release of biowarfare agents. In addition, their research has led to better understanding of how the human body responds to some of these toxins, and how we can detect if an individual has been exposed to a toxic agent.

What may seem like an odd assortment of research subjects has actually come about in a linear, if somewhat unpredictable, progression.

Furlong's early experiences included house construction, auto repair and wiring of the Polaris submarines. These activities may seem removed from the world of science and medicine, but fixing cars isn't too far off from the type of engineering and problem-solving that Furlong's research entails. When I ask what spurred his interest in research, Furlong comments that he likes to solve problems. "I'm sort of a reductionist at heart,” says Furlong.

His reductionist tendencies led him to an early interest in biochemistry. "The first thing that really stimulated my attention in biochemistry was seeing enzymes work in a test tube,” Furlong recalls. This first spark would prove a constant theme in his research and lead to other endeavors further afield from biochemistry. Purification and observation of enzymes in the test tube allowed for development of genetic tests, animal models, and biosensors, among other tools.

One of the first tools to emerge from Furlong's research came from a casual observation. As a young investigator, Furlong made a name for himself as an expert in bacterial nutrient transport. In studying the proteins bacteria used to transport various sugars and amino acids across their membranes, he realized that each protein interacted specifically with the nutrient that it transported. Such a protein, Furlong reasoned, could be used as a biosensor to detect the particular small molecule to which it bound. This idea led to development of a sensor that can be used to detect a range of analytes that vary in size from small molecular weight molecules to viruses and whole microbes.

The sensor employs cutting-edge surface plasmon resonance (SPR) technology. Furlong and his research group developed the sensor along with collaborators in the electrical engineering department and with funding from Washington Sea Grant Project at UW. SPR technology utilizes the refractive properties of metals such as gold to detect the presence of molecules of interest. In each SPR sensor, a gold surface is coated with a target recognition protein or receptor. The protein could be an antibody targeting a bacterial pathogen or a small molecule toxin. When the biosensor protein binds the target, the refractive index at the gold surface is changed, and the sensor records this interaction. According to Furlong, these SPR sensors are a rapid and sensitive method for detecting the presence of potentially toxic molecules in the environment.

One toxin that Furlong's group has detected in clams with SPR sensors is domoic acid. This small molecule is produced by the marine algae Pseudo-nitzschia. The domoic acid produced by these algae can accumulate in the food chain and cause toxic effects in higher predators. Large doses of domoic acid cause neurological damage and can even cause death among marine mammals or humans who have eaten contaminated shellfish. This work was carried out in collaboration with Vera Trainer's research team from NOAA>

The SPR sensor for domoic acid developed by the Furlong lab can ultimately be used to monitor seawater and determine if a toxin-producing bloom of Pseudo-nitzschia is present. This type of environmental surveillance can help health officials warn the public when eating local shellfish could be unsafe. In addition to detecting algal toxins, the sensor has been adapted for bacterial pathogens, potential biowarfare agents and many other potential toxins.

The road to understanding environmental toxins has been a long and winding one for Clem Furlong. His research has touched upon a variety of topics that at first seem unrelated. What could heart disease, migrant workers, and jet engines possibly have in common? Furlong could tell you. He's found that these disparate subjects all relate to one very powerful human enzyme. And how he began to study that enzyme is another intriguing story.

When Clem Furlong first came to the University of Washington, he tells me, he became interested in the interactions between genes and the environment. It was at the UW that he began collaborations with the renowned medical geneticist, Arno Motulsky. At the time, Motulsky was making strides in the field of pharmacogenetics, the study of genetic variation and its influence on response to drugs. Under his tutelage, Furlong began research on a gene that was responsible for detoxification of organophosphates, including insecticides.

The gene, known as paraoxonase 1, or PON1, had been found to be polymorphic. In other words, there was more than one version of the gene, and each version had a different ability to detoxify organophosphates. Since organophosphates can lead to neurological damage if they are not properly metabolized, these different versions of PON1 have a dramatic effect on ability to withstand exposure to pesticides and other organophosphates.

Furlong and his research group went on to link the polymorphic versions to an amino acid change in the protein produced by PON1. The difference between a glutamine and an arginine at a single position in the protein could affect the ability to detoxify organophosphates. This change in the protein, as well as other genetic variants that influence how much PON1 the body produces can affect how well a person tolerates exposure to these potential toxins. In the case of individuals frequently exposed to organophosphates, including migrant farm workers exposed to pesticides, the genetic variations described by Furlong can have a huge impact on their overall health. More recent work in Furlong's laboratory has addressed the question of why some airline workers are more susceptible to exposures of triaryl phosphates used in jet engine lubricants.

The PON1 genetic variants that Furlong has characterized have another effect on human health. In addition to its role in detoxification, PON1 also has another function. The protein circulates in the blood and binds to HDL ("the good cholesterol particles”), protecting them as well as LDL ("the bad cholesterol particles”)from oxidative damage. Not surprisingly, this protective effect is influenced by the amount of PON1 present. Because oxidation of lipids is among the first steps in atherosclerosis, low PON1 levels have been associated with vascular disease.

With so many factors, including heart disease, exposure to chemical toxins, and genetic variants, understanding PON1 has been a long-term project. According to Furlong, this type of challenge is what drives his desire to do research. "The puzzle part of science is what I find really motivating,” he tells me. His ability to work on many interrelating stories has led to a great diversity of research projects in the lab. Becky Richter, who has worked as a research scientist in the lab for 26 years, observes that Furlong "gets a lot of satisfaction out of making progress and helping people answer questions about why.”

In the case of PON1, Furlong himself notes that exploring such a complicated story required persistence over many years of research. Understanding each factor in turn, from the genetic variants, to developing a mouse model to understand how PON1 affects susceptibility to organophosphate toxins, allowed the research group to "narrow down until you get answers.”

Keeping such a long, complicated research project going has been a collaborative effort between Furlong and laboratory members. Richter notes that lab members "balance each other out with strengths and weaknesses.” She adds that Furlong's sense of humor creates a good working environment in the lab, where the daily ups and downs of research can be challenging. "There are some professors here that don't have a sense of humor. And I just don't want to work for them,” Richter says with a smile. Furlong's sense of humor seems matched by a curiosity and drive to create useful tools to help better understand toxin exposure.

One of the tools under current development in the Furlong lab is a biomarker that would help to pinpoint the time and extent of exposure to harmful organophosphates. Again, this tool emerged from prior research in the Furlong laboratory and was developed to serve a need in the community. And once more, there is an intriguing story in the genesis of the biomarker.

The story begins over eighty years ago, before any of the researchers involved were even born. During the Prohibition era, an odd medical ailment was described. Victims had neurologic damage that led to paralysis of the limbs and an odd, flapping gait. The illness came to be known as "jake leg” for its association with victims who drank ginger extract, or jake. These victims drank the extract for its high alcohol content and easy availability. The malady, it was eventually discovered, was caused by an adulterant added to the ginger extract, tricresyl phosphate (TCP). This organophosphate, a potent neurotoxin, is now added to jet engine lubricants as a fire retardant and to prevent wear on engine parts.

The use of TCP in airliners was what eventually drew Furlong into the picture. A number of airline workers had found themselves with unusual neurological symptoms including memory loss and uncontrollable shaking. TCP had been implicated as a potential cause, as most airplanes draw cabin air into the airplane through the jet engine. The air is then circulated through the cabin. If there are engine seal leaks, the air may be contaminated with jet engine lubricant components including TCP. Furlong was contacted by a pilot from the United Kingdom who, in searching for answers to this bizarre illness, came across reference to Furlong's expertise on PON1 and organophosphate toxicity.

Furlong was able to use his knowledge of organophosphate toxicity to find a biomarker of exposure to TCP and other organophosphates. These chemicals, once bioactivated in the body, can then poison key enzymes in the body by spontaneously forming harmful phosphate adducts. Furlong and collaborators Mike MacCoss and Dave Goodlett at UW were able to pinpoint one particular enzyme adduct that serves as a biomarker of exposure to TCP. Furlong hopes to discover other biomarkers specific to other types of organophosphate toxins. In addition, according to Furlong, the biomarker can be monitored in each patient over a period of time. This type of time course could be used to observe natural degradation of the biomarker by the body and extrapolate the original time of exposure.

Furlong and his collaborators are currently collecting patient samples and refining their biomarker assay. They hope that time course analysis in patients will be able to establish a concrete relationship between exposure to jet engine fumes and presence of the biomarker in people. "That's the missing link, as far as I'm concerned,” says Furlong. He goes on to add that this evidence, along with data from his lab demonstrating that chemicals with similar properties to TCP are less toxic to human enzymes, may help convince oil companies to switch to safer jet fuel additives.

Sara Selgrade recently completed a doctorate in genome sciences at the University of Washington.

Images:

Top: University of Washington researcher Clem Furlong studies a variety of subjects including organophosphate toxicity, sensor chips used to detect biowarfare agents, and risk factors for heart disease. Photo courtesy of Clem Furlong.

Bottom: Members of the Furlong lab. From left to right: Stephanie Suzuki, Becky Richter, Wanfen Li, Rick Stevens, and Clem Furlong. Photo courtesy of Becky Richter.

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