The dioxin they studied is one of the most potent of an important class of toxins, the polychlorinated aromatic hydrocarbons. Such toxins can cause cancer, birth defects, and altered hormone levels, among other things. In the body, particularly in liver cells, TCDD binds to the aryl hydrocarbon receptor. This binding induces increased production of cytochrome P4501A1 by increasing expression of the CYP1A1 gene that manufactures it. Cytochrome P4501A1 is involved in metabolizing foreign compounds, such as aromatic hydrocarbons, in humans.

HepG2 cells as seen through the infrared microscope on Beamline 1.4.3. Spot sizes for spectromicroscopy were 10 µm or less, allowing study of individual cells.

In the study, three experimental groups of human hepatocellular carcinoma (HepG2) cells (cells from a liver tumor) were exposed to TCDD at different concentrations for 20 hours, and HepG2 cells in a control group were kept in an incubator for 20 hours with no TCDD exposure. Spectra from cells in the experimental groups and in the control group were then obtained with the infrared microscope. In addition, some cells from each group were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) to track the effect of TCDD on expression of the CYP1A1 gene. A correlation between spectroscopic and RT-PCR data would demonstrate that the spectroscopic changes were indeed related to the pathway of CYP1A1 expression.

Measuring Something Real

In their research with dioxin, Holman et al. have shown that infrared light from a synchrotron can be used to study changes in single, living human cells. This is a new application of synchrotron light that opens up possibilities for its eventual use in medical diagnostics or environmental health research. Some day, it may be routine to study a tiny sample of tissue from a patient and, by using synchrotron infrared spectroscopy, identify a single cell that has been subtly altered by exposure to a chemical.

Of course, using a scientific technique in a way in which it hasn't been used before calls for determining that you are indeed measuring what you think you are measuring. This proof is particularly necessary when measuring aspects of biological systems, since they are inherently complex. The work reported here is a good example of correlating observed effects (changes in infrared spectra) with known effects (an increase in the expression of the CYP1A1 gene) to show that what is being measured has a bona fide basis in the systems under study. The strong correlation shows that the group was indeed measuring real changes associated with the dioxin.

Absorption spectra were obtained at wavenumbers between 4000 and 650 cm^-1. Significant absorption differences between the treated samples and the control samples were evident for wavelengths associated with stretching vibrations in two bond types, phosphate and C-H. For phosphate, the intensity for the symmetric stretch band increased relative to that for the asymmetric stretch band with increased TCDD concentration. This finding agrees with previous studies in cancer cells. One thing, however, was different about the phosphate bands in the present study. They did not shift in wavelength. Such a shift would have indicated increased or decreased hydrogen bonding with exposure to TCDD. (Hydrogen bonding is important for maintaining the three-dimensional structure of a protein.) The wavelengths at which the phosphate vibrations were found and the fact that they did not vary show that phosphate has weak hydrogen bonding in HepG2 cells and that this does not change with TCDD exposure. This behavior is different from that seen in the early response to oxidative stress. The band representing methylene (CH₂) stretch decreased and that for methyl (CH₃) stretch increased with increasing TCDD concentration. Thus, the number of methyl groups relative to the number of methylene groups increased with greater TCDD exposure. This may indicate increased DNA methylation, which some researchers have suggested may cause gene inactivation--one possible mechanism by which TCDD could take its toxic toll.

Infrared spectra near phosphate bands at 1236 cm-1 (asymmetric stretch) and 1082 cm-1 (symmetric stretch) show differences for cells treated with varying concentrations of TCDD.

The RT-PCR results showed the expected increase in CYP1A1 gene expression with increasing exposure to TCDD. Comparing these results with the increases in intensity for the symmetric versus asymmetric phosphate bands gave excellent correlation (r² = 0.96). This strong correlation shows that the spectromicroscopy measurements succeeded in tracking real changes that are associated with induction of the CYP1A1 gene.