By Saren Johnston
They surround us. There's no way we can completely escape them. But if we can learn more about the damage they do to our DNA, we might be able to combat them.
Cancer-producing pollutants, such as those found in cigarette smoke, automobile exhaust fumes and power plant emissions, invade our bodies every day. Over time, the onslaught can create internal havoc as these carcinogens mix with our cellular DNA, the macromolecule that carries our genetic code of life. The chemistry that takes place causes DNA damage and produces byproducts known as DNA adducts, which can lead to mutations. If scientists can identify these byproducts, they may be able to discover which ones lead to cancer.
Looking for specific DNA adducts may sound like an uncomplicated plan, but if you're the person doing the searching, you know just how frustrating it can be. Identifying structurally similar cancer-causing compounds and closely related DNA adducts can be a chemical analysis nightmare. A complex biomolecular mixture, such as urine, can contain almost a thousand different compounds. And it can become extremely exacting trying to identify the seven or eight metabolites of interest from the hundreds of others.
Hoping to ease this predicament, Ryszard Jankowiak, scientist, and Gerald Small, senior chemist, combined two well-established analytical methods to create an innovative, on-line technique called capillary electrophoresis-fluorescence line-narrowing spectroscopy (CE-FLNS).
"The marriage of capillary electrophoresis (CE) and fluorescence line-narrowing spectroscopy (FLNS) is an exciting addition to the rapidly evolving field concerned with selective detection methods that can provide the information necessary to distinguish between structurally similar molecular compounds," says Jankowiak.
The novel CE-FLNS combination can provide more detailed information on complex biomolecular samples than either CE or FLNS can do on its own. CE-FLNS first takes advantage of the ability of CE to separate minute amounts of closely related biological analytes, or compounds. The second half of the technology combination, FLNS, is a high-resolution, fluorescence-based detection method that was perfected at Ames Lab for analytical purposes. FLNS makes its contribution to resolving the "molecular identity crisis" by characterizing the CE-separated molecular samples. The process differentiates structurally related analytes by laser exciting them to fluoresce and emit fluorescence line-narrowed spectra, which can be collected and measured.
The power of CE-FLNS was demonstrated in work with the Eppley Institute for Research in Cancer and Allied Diseases at the University of Nebraska Medical Center. The research led to the discovery of a new pathway that chemical carcinogens take for their attack on DNA. "But CE-FLNS makes such research easier and misidentification of analytes far less likely," notes Jankowiak. "Its on-line capability is especially important when dealing with minute quantities of biological materials."
Michael Gross, professor of chemistry and medicine at Washington University in St. Louis, has collaborated with Jankowiak and Small on research to detect and identify trace amounts of pieces of DNA that have been modified by carcinogens. He expresses high regard for the capabilities of the CE-FLNS system. "I watched with interest the design, assembly and successful testing of the prototype CE-FLNS system, and I know first-hand that it has played a vital role in detecting modified DNA," he says. "The new CE-FLNS methodology for on-line structural characterization should significantly enhance the potential of high-resolution spectroscopy, which has already been shown to be the most powerful method to analyze DNA adducts derived from poly-cyclic aromatic hydrocarbons." (Polycyclic aromatic hydrocarbons make up a potent class of chemical carcinogens.)
Fundamental to the CE-FLNS technique is Jankowiak's and Small's design of a compact and reliable capillary cryostat that regulates and maintains the constant low temperature required for FLNS characterization. The uniquely designed cryostat consists of a double-walled quartz cell that encloses the capillary (a tube with a very small diameter) housing the CE-separated analytes. The capillary cryostat is the critical link that unites the CE and FLNS techniques. Gases and vapors are removed from the outer portion of the cryostat, and inlet and return lines introduce and circulate cryogenic liquids, either liquid nitrogen or liquid helium.
Liquid nitrogen is used to cool analytes to 77 K (-321 F) when low-resolution spectroscopy is sufficient for identification purposes. But when high-resolution spectroscopy is required, liquid helium is used to cool the capillary and CE-separated analytes to 4.2 K (-452 F) in less than one minute. The frozen and stationary analytes can then be sequentially characterized, or "fingerprinted," by FLNS as the capillary cryostat with the enclosed capillary moves automatically along a transmission stage and passes through the laser-excitation region.
Although Jankowiak and Small have used CE-FLNS primarily for research on DNA damage from chemical carcinogens, they'll tell you there's definitely more in store for the unique technology combination. Potential applications include other areas of biological research, as well as forensic science.
"Our technology has applicability well beyond just the problem of cancer," says Small. "It could be used for virtually any biological problem where one has to deal with and unravel complex biological mixtures. We haven't even begun to explore all of the possibilities yet."
The CE-FLNS technology is already being investigated by several companies, and Jankowiak and Small hope to have it licensed and on the market in three years. While all this activity is taking place, the two researchers are simultaneously looking at ways to enhance the capabilities of CE-FLNS.
"We'd like to do a second-generation cryostat and then improve the collection optics to give us more sensitivity," says Small. "At the present time, the capillary cryostat moves only in the direction of its axis. We'd like to put a three-dimensional, or x-y-z, transmission mode in there, too, so it makes it very easy to align the capillary."
But perhaps it is Jankowiak's and Small's fundamental idea and method of pairing two exceptional analytical techniques that will allow them to best improve upon and expand their efforts in on-line characterization of structurally similar compounds. "The versatility of our basic concept can be increased by coupling FLNS with as many different separation techniques as possible," says Jankowiak.
For more information:
Gerald Small, (515) 294-3859
gsmall@ameslab.gov
Current research funded by:
DOE Biological & Environmental Research Office
Last revision: 12/17/99 sd
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