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RESEARCH PROFILES A discriminating assay for endocrine disrupters Endocrine disrupters are synthetic chemicals and natural plant compounds that have been linked to developmental and reproductive defects in animals. Whether humans are at risk of health problems upon exposure to endocrine-disrupting chemicals (EDCs) is a hotly debated issue that many scientists and governmental organizations are currently investigating. Perhaps the most widely studied EDCs are those that affect estrogen regulation. To promote reproductive system development, estrogen binds to an estrogen receptor (ER), which causes a conformational change in the ER. In its new conformation, ER– estrogen can then bind to a coactivator, and this interaction stimulates transcription. A major hurdle in trying to predict which chemicals will interfere with estrogen’s effects is that many EDCs do not resemble an estrogen molecule. Thus, suspected estrogen EDCs must be screened using functional assays. In the April 15 issue of Analytical Chemistry (pp 2181–2186), Yoshio Umezawa and colleagues at the University of Tokyo and the Japan Science and Technology Agency describe their new in vivo assay for estrogen EDCs. According to Umezawa, current protocols have many shortcomings. Competitive receptor binding assays, in which a chemical is tested for its ability to disrupt an estrogen–ER complex, cannot distinguish between an agonist and an antagonist. Although both agonists and antagonists bind the ER, agonists activate estrogen effects, whereas antagonists do not. In reporter gene assays, cells are exposed to a chemical, and the transcription of a particular gene is monitored. Although these tests can classify EDCs as agonists or antagonists, Umezawa says that the assays can take >30 h. The researchers, already skilled at designing fluorescent and bioluminescent probes for biological systems, decided to adapt their methods to EDC screening. “Our simple assay with the SCCoR (single cell-coactivator recruitment) indicator has 154 A
A N A LY T I C A L C H E M I S T R Y / M A Y 1 , 2 0 0 4
CFP Flexible linker
CFP
Coactivator
Coactivator
+ Agonist (EDCs) ER
ER
Coactivator
ER
– Agonist
YFP CFP
YFP
YFP FRET CFP Coactivator
+ Antagonist (EDCs) ER No FRET – Antagonist YFP
A schematic of the SCCoR assay.
overcome the limitations of conventional methods and has enabled us to distinguish between agonists and antagonists within only several minutes,” says Umezawa. In the SCCoR (pronounced “score”) assay, mammalian cells are transfected with a piece of DNA, which encodes one large hybrid protein consisting of cyan fluorescent protein (CFP), a domain of ER, a domain of the coactivator, and yellow fluorescent protein (YFP). Umezawa’s group reasoned that when an agonist, such as an estrogenic compound, binds to the ER of the SCCoR indicator, the ER domain will change shape and bind to the coactivator domain. This event will bring CFP and YFP close to each other, which will result in an increase in fluorescence resonance energy transfer (FRET) from CFP to YFP. Antagonist binding to ER, however, will not cause a change in the ER domain, and an increase in FRET will not be observed. Umezawa’s group initially tested the SCCoR indicator with the estrogenic compound estradiol. An increase in FRET was observed almost immediately
after estradiol was added, and a plateau was reached after ~16 min. A SCCoR indicator that was mutated in the coactivator region showed no change in FRET upon estradiol addition. Genistein, diethylstilbestrol, bisphenol A, and nonylphenol, which are known estrogen EDCs, were also tested in the SCCoR assay. All of these chemicals resulted in increased FRET in a dosedependent manner, although some activated ER–coactivator binding to different extents. These results are consistent with those obtained by in vitro methods. The assay accurately pinpointed agonists, but could it distinguish those types of chemicals from antagonists? No change in FRET was observed when the known antagonists ICI 182,780 (ICI) and 4-hydroxytamoxifen (OHT) were tested using the SCCoR method. To see if estradiol could displace the antagonists, the researchers added estradiol after the cells were incubated with a 1.0-µM solution of either ICI or OHT for 15 min. Solutions of 1.0- and 10-µM estradiol had no effect when added to ICI-treated cells, but exposure to 100-µM estradiol caused an
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increase in FRET signal that was ~40% of that observed with 1.0-µM estradiol alone. Similar results were obtained with OHT. Therefore, the effects of these antagonists appear to dominate those of estradiol. According to Umezawa, the SCCoR assay has many potential applications.
hormone-disrupting activities. In fact, he says, “The assay can also be used to screen chemicals for pharmaceutical or medical purposes.” Such applications include screening drugs for antiestrogenic effects to find new drugs for breast cancer treatment. a —Katie Cottingham
“We have already used the androgen receptor for the same purpose,” he says. “The approach can likewise be applied to develop SCCoR indicators for other hormone receptors, such as progesterone, thyroid, and glucocorticoid receptors.” But SCCoR indicators are not only suited to screening chemicals for
alyte. A second UV photon is then subsequently absorbed, which raises the internal energy above the ionization energy, forming a molecular ion. The process is enhanced when the first step is in resonance with an electronic excited state. “You benefit from changing the wavelength of the laser. That provides you with a fingerprint spectrum of the compound of interest. Every molecule has its own distinct fingerprint,” explains Oudejans. “The technique is also selective in that you record the individual masses of the compounds. So you get a two-dimensional detection array that you can measure your concentrations with,” he adds. One advantage of using a real-time technique like REMPI-TOFMS is the ability to measure transient events, such as the start-up of a cold diesel engine. Because cold start-ups include a period of incomplete combustion, one would expect emissions to fluctuate during that time. And that is exactly what the researchers found. During the cold start-up of a diesel generator, they observed a sharp peak in benzene emissions lasting
