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 Fall/Winter
1997 |
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Outfoxing Toxins in Cells "We are all exposed to dioxin in low concentrations because it is released when you burn almost anything," explains Gary Perdew, professor and interim head of veterinary science. "Municipal incinerators, burning wood, automobiles, and barbecues are just some of the sources of dioxin. Humans absorb it through the food chain, particularly through meat, fish, and dairy products. It's been shown to be extremely toxic to rodents, so we need to know what the long-term effects are if we're going to continue to release this stuff into the air."
Dioxin molecules are structurally akin to natural hormones. Many hormones act as chemical messengers within the body, reacting biochemically with genes to trigger changes in the body such as growth control, physical development, and reproduction. At the cellular level, hormones bond with receptor proteins present in the cell. These receptor proteins then bond to a specific site on DNA within the cell's nucleus. There the hormone receptor complex can turn on or off biochemical "switches" that control bodily functions. Like hormones, dioxin can bond with receptor proteins and fit into DNA docking sites. Once the dioxin docks onto DNA, the dioxin-receptor complex can cause a variety of problems in laboratory animals. To make matters even more complicated scientifically, dioxin compounds cause a wide range of effects from species to species in laboratory experiments. For example, minuscule amounts of dioxin will kill hamsters, yet it takes 40 times more of the compound to kill mice. While rats and mice develop liver tumors upon exposure to dioxin, guinea pigs do not. Similarly, humans, monkeys, and rabbits exposed to dioxin develop severe forms of acne, but other species do not. These contradictory effects make it crucial for scientists to understand exactly how dioxin causes toxicity in human cells. That question is the focus of Perdew's research, a project he is pursuing with graduate students Jo Tsai, Brian Meyer, and Mohan Kumar. They are examining the effects of the most toxic dioxin compound, tetrachloro-dibenzo-p-dioxin (TCDD), by concentrating on a single receptor protein within most cells in the human body. This protein, called the Ah-receptor, is the site where dioxin enters the cell structure, binding dioxin molecules within the cell. "The body has complicated machinery that regulates how genes function," Perdew explains. "Dioxin can alter specific gene expression, acting like a hammer thrown into the gears of a machine."
To understand how dioxin affects the Ah-receptor, the research team must discover not only how the protein reacts to dioxin, but also how it functions normally–a process called regulation. Each graduate student in Perdew's lab is looking at a small piece of the overall puzzle. "Each project is interrelated, so eventually we will have a complete picture of how this receptor is regulated," Perdue says. Mohan Kumar, a graduate student in molecular biology and biochemistry from Bangalore, India, is examining how cellular proteins behave. "We know that once dioxin binds with the Ah-receptor, it is transported to the nucleus, where it binds with another protein called the Arnt protein, which is usually found within the nucleus," he points out. "Once bound, the proteins can find the docking sites on strands of DNA and act as switches that turn on specific genes. In the process, these bound proteins may interact with other proteins in the nucleus." Kumar is trying to identify the proteins that interact with the dioxin receptor complex. If those proteins can be isolated, then Kumar can pinpoint events that occur between the time the complex binds to DNA and the activation of specific genes. This time sequence could reveal more about how proteins affect the way the dioxin compound activates or alters the function of a gene. However, before searching for proteins that interact with the receptor complex, Kumar must first do some cellular demolition. He isolates a cell's nucleus by centrifugal force and extracts the contents of the nucleus. He then uses a gel to separate all the proteins in the extracted nucleus. Any one of these extracted proteins could be a protein that can interact with the Ah-receptor complex. Kumar's method for identifying likely candidates involves separating the dioxin receptor complex from a cell, allowing the receptor to bind with DNA, then labeling the strand of DNA with a radioactive molecule. The DNA strand marked by the radioactive molecule serves as a probe to locate proteins that interact with the dioxin receptor. "Any proteins that interact with the DNA strand can be easily identified using X-ray film, because the radioactive molecule exposes the film at the site of the interacting protein," Kumar says. Jo Tsai, a graduate student in biochemistry from Beijing, China, is focusing on the biological events required to create dioxin toxicity by studying the Arnt protein, which binds with the Ah-receptor, enabling it to be incorporated into the nucleus. "This bonding triggers many biochemical switches, such as induced or altered gene expression," she explains. "Very little is known about how this process is regulated." Tsai is looking at how dioxin affects genes within the cell through a process called phosphorylation, in which cells remove or add phosphate molecules from the Ah-receptor's protein, in turn activating or silencing the protein and thus regulating its function. Tsai examines these functional changes in somewhat the same way as a detective follows a car by observing its taillights. The phosphate compounds she introduces into the cell culture are radioactive, so she can easily pick up the trail of the receptor complex by using X-ray film. She then can analyze the functional changes caused by the phosphates. "It is amazing how different types of stimuli can modulate the function of living cells," she says. "Once we understand how the process is regulated, we might be able to block the toxic effects of dioxin exposure. By studying certain aspects of a complex system, we hope to gain insight into the whole process." Brian Meyer, a graduate student in molecular biology and biochemistry from Key Largo, Florida, is trying to isolate a single protein called p43, which is part of the dioxin receptor complex that binds with the cell's DNA. He hopes to learn what specific role this single protein plays in regulating or influencing the activity of the dioxin receptor complex. Meyer likens his search to fishing, although he has to create the hook, line, and bait in the lab. To isolate protein 43, he must take the gene that responds to the Ah-receptor and add onto it a sequence of genetic information called a flag, a scientific step best described as creating bait that can easily be seen on X-ray film. Meyer then introduces the flagged DNA into human liver cells, which can be thought of as fishing line, in order to isolate a cell that triggers the gene incorporating the flagged DNA. When the gene is triggered, it connects to its own docking site on the DNA strand. It can then be recognized by an antibody used as a hook to separate the p43 protein complex away from the rest of the cell. "Once the protein complex is isolated, p43 can be studied in more detail," Meyer says. "It sounds complicated but it's really a series of simple steps, like removing your car's alternator by taking off the bolts and the bracket first." The step-by-step nature of cellular research is painstaking, as scientists make their way slowly through unknown territory. Perdew says it may take years of intensive research before the long-term effects of dioxin can be understood. "In a lot of ways the work is very tedious," he admits. "But there is something exciting about finding information no one else has found. It's also very much a team approach. Every week, one of us learns something new, and we all try to see how that new information fits into the larger scheme." Perdew's role in overseeing the research is equal parts Socrates and Joe Paterno. One of his functions is to pose questions that make his students think more deeply about their research. Another is to keep the work driving forward until a goal is reached. "I tell my students, 'OK, you found this out. What does it really mean?'" Perdew says.
Kumar credits Perdew
with allowing an atmosphere of creativity within the demanding structure
of scientific research and being open to new techniques. "Dr.
Perdew is a real bench scientist," Kumar says. "He's actively
involved on every level of the project, even the nitty-gritty details."
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Penn State | College of Agricultural Sciences | ICT Copyright - Alternative
Media - Affirmative
Action |
"We
are all exposed to dioxin in low concentrations because it is
released when you burn almost anything."