Low-Dose Responses to 2,3,7,8-Tetrachlorodibenzo- p-dioxin in Single Living Human Cells Measured by Synchrotron Infrared Spectromicroscopy HOI-YING N. HOLMAN,* ,† REGINE GOTH-GOLDSTEIN, ‡ MICHAEL C. MARTIN, § MARION L. RUSSELL, ‡ AND WAYNE R. MCKINNEY § Center for Environm ental Biotechnology, Environm ental Energy Technologies Division, and Advanced Light Source Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720 Synchrotron radiation (SR)-based Fourier transform infrared (FTIR) spectromicroscopy measurements are presented of HepG2 cells exposed to an environmental contaminant that is a known ligand for the aryl hydrocarbon (Ah) receptor. Measurements were made on cells treated with an Ah receptor-binding model compound 2,3,7,8- tetrachlorodibenzo- p-dioxin (TCDD).Compared to untreated control cells, treated cells displayed unique spectral changes with TCDD concentrations of 0.01-10.0 nM. Key spectral changes involved PdO, C-O, and C-H stretching bands. The first changes are related to the environment of the phosphate backbone of nucleic acids. The C-H stretching bands data show a relative increase in the number of methyl to methylene groups. An excellent correlation was found between spectral changes and an increase in CYP1A1 expression measured by the reverse transcriptase polymerase chain reaction (RT-PCR) technique demonstrating that the SR-FTIR method is observing cellular biochemical changes related to this gene expression pathway. Finally, the potential use of SR- FTIR spectromicroscopy is discussed as a diagnostic tool for monitoring cellular exposure and early molecular responses to environmental pollutants. Introduction Exposure to polychlorinated aromatic compounds can lead to various health effects including cancers, alteration of hormone levels, and reproductive defects in animals (1-6) andhumans(7-14).Amongthisfamilyofpollutants,2,3,7,8- tetrachlorodibenzo- p -dioxin (TCDD) is one of the most potentand moststudied “man-made”toxins,causingharmful effects at exposure levels ofhundreds or thousands oftimes lower than most chemicals of environmental concern (15). TCDD acts by binding to the aryl hydrocarbon (Ah) receptor (16, 17).Bindingtriggersinduction ofvariousgenesinvolved in xenobiotic metabolism includingthe cytochrome P4501A1 (CYP1A1) gene (16-20). In the risk management of poly- chlorinated aromatic compounds, frequently the induction of cellular responses (such as the induced expression of CYP1A1)are measured usingslot blot,Northern blot,Western blot (21), or the reverse transcription polymerase chain reaction (RT-PCR) technique that employs gene-specific primers (6, 22, 23). This approach involves techniques that require fairly large numbers of cells, is destructive to the cells, and requires lengthy sample preparations including DNA or RNA purification and amplification and/or other chemicaland enzymatictreatmentsbefore theirfinalanalysis. An alternative to the above analytical, chemical, and biochemical techniques is microcopy methods where pro- cesses in the intact cells or tissues are investigated. These include electron microscopies (24-26), soft X-ray micro- scopies (27, 28), optical fluorescence microscopy (29-31), and its recent extension using multiphoton excitation (32- 40). However, many of these methods are harmful to living cells, and most require treatments with exogenous dyes, fluorescent labels,or stains.Synchrotron radiation (SR)-based Fourier transform infrared (FTIR) spectromicroscopy does not share the requirement for labels,while the method rapidly and nondestructively probes individual living cells and provides, in addition, chemical information from the IR spectrum (41). Conventionalnon-SR-based FTIRspectromicroscopyhas been widely used as a diagnostic tool for characterizing the composition and structure of cellular components within intact tissues (42-45) and for measuring tumor tissue responses to therapy(46).However,the spatialresolution of traditionalFTIRspectromicroscopyislimited to ∼75 µm with sufficient signal-to-noise (47, 48). SR-FTIR spectromicros- copy,on the contrary,providesseveralhundred timeshigher brightness at a diffraction-limited spatial resolution of 10 µm or better and is therefore a sensitive analyticaltechnique capable of providing molecular information on biological specimens (47-51). In a recent example, Jamin et al. (41) used SR-FTIR to map the distribution of functional groups of biomolecules such as proteins, lipids, and nucleic acids in individual live cells with a spatial resolution of a few microns. In this study, we use SR-FTIR spectromicroscopy to measure directly intracellular responses to TCDD. Experimental Details The SR-FTIR spectromicroscopy experiments began with exposingHepG2cells (derived from a human hepatocellular carcinoma)to various concentrations ofTCDD.Afraction of the exposed cells were investigated by acquiring SR-FTIR spectra from individual live cells. The remaining cells were analyzed for CYP1A1 gene expression, using the RT-PCR technique. Observed changes in the SR-FTIR spectral measurements were compared with those from RT-PCR results. Cells and Cell Treatment. HepG2 cells were selected for use in this study as their ability to metabolize polyaromatic compounds is well-characterized (52). HepG2 cells were obtained from the American Tissue Culture Collection (Rockville, MD). They were maintained in Dulbecco’s minimum essential medium supplemented with 10% fetal calfserum,nonessentialamino acids,1mM L-glutamine, 10 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),and antibiotics.Cellswere subcultured every7days. For TCDD experiments,subconfluent cultureswere exposed for 20 h to 10 -11 , 10 -10 , and 10 -9 M TCDD initially dissolved in pure dimethylsulfoxide (DMSO)(Sigma,USA,99.9%pure). *Corresponding author e-mail: hyholman@lbl.gov; telephone: (510)486-5943; fax: (510)486-7152. † Center for Environmental Biotechnology. ‡ Environmental Energy Technologies Division. § Advanced Light Source Division. Environ. Sci. Technol. 2000, 34, 2513-2517 10.1021/es991430w CCC: $19.00  2000 American Chemical Society VOL. 34, NO. 12, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2513 Published on Web 05/11/2000