Detecting exposure to environmental organic toxins in individual cells: towards development of a micro-fabricated device Page: 2 of 8
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SPIE Vol. 3606, p. 55 (1999)
region (4000-400 cm') as a tool to identify IR radiation signals (from individual cells) that are specific to the
intracellular response after their exposure to PAHs or OCs. SR FTIR spectromicroscopy was used because it has
been proven to be a sensitive analytical technique capable of providing molecular information in a biological
system quickly (within minutes) at dilute concentrations and a spatial resolution of 10 microns (3-4). The
potential use of the new method to screen for the exposure to PAHs and OCs at environmentally relevant
concentrations was tested using human cells in an environmental medium for which contaminant types and levels
are known. In these tests, HepG2 (human hepatoma derived) cells modeled the human epithelial cells that would
activate the test PAHs and OCs; benzo(a)pyrene (BaP) modeled the PAHs, and 2,3,7,8-tetrachlorodibenzo-p-
dioxin (TCDD) modeled the OCs. HepG2 cells metabolize PAHs efficiently. They also show CYPIAI induction
after their treatment with PAHs and OCs. The potential use of our marker signals for detecting intracellular
response to mixtures of PAHs was also demonstrated in an environmental medium with dilute concentrations of
coal tar, a complex mixtures of different PAHs.
2. EXPERIMENTAL
2.1. Cells and Cell Treatment
HepG2 cells were obtained from the American Tissue Culture Collection (Rockville, MD). Cells were
maintained in MEM (Minimum Essential Medium, Eagle) with non-essential amino acids and Earle's BSS
supplemented with 10% fetal calf serum, 1 mM L-glutamine, 10 mM Hepes and antibiotics. Cells were sub-cultured
every 7 days. Cells were treated for 2 - 20 hours with various amounts of BaP, coal tars, and TCDD dissolved in
DMSO.
2.2. SR FTIR spectromicroscopy
The infrared spectromicroscope in conjunction with Beamline 1.4.3 at the Advanced Light Source (ALS) at
Lawrence Berkeley National Laboratory was used to monitor intracellular changes in response to TCDD, BaP, and
coal tars exposure. Figure 1 depicts the experimental setup for this direct measurement. As shown in the diagram, the
infrared microprobe uses a synchrotron source that has much higher brightness than a conventional thermal IR
source. The synchrotron light is focused into a Nicolet Magna 760 FTIR bench, then passes through a Nic-Plan IR
microscope. As detailed in [3] the spot size of the unmasked synchrotron beam focused through an infrared
microscope is 10 gm, nearly diffraction-limited, and significantly smaller than the 100-pm spot size of a
conventional thermal IR source. This leads to an improved signal to noise level and finer spatial resolution than is
possible for conventional source FTIR spectromicroscopy. Since the synchrotron experimental procedure is non-
destructive to the biological materials being studied, SR FTIR spectromicroscopy is extremely useful in detecting
subtle intracellular changes as the cells are exposed to environmental stimuli.
All SR FTIR spectra were recorded in the 4000-650 cm infrared region. This region was selected because it is
the region that contains unique molecular fingerprint-exhibiting absorption features of intact biomolecules. For each
IR measurement, 128 spectra were co-added at a spectral resolution of 4 cm. All spectra were obtained in the
reflection geometry and were ratioed to the reflectance spectrum of a gold-coated slide to produce absorbance values
(see Figure 1).
2.3. Semi-quantitative RT-PCR
After the treatment of cells (as described in 2.1), total RNA was isolated using TRI Reagent. Total RNA was
reverse transcribed using oligo dT, MMLV reverse transcriptase. CYPIAI expression was measured by determining
the CYP1A1 transcript level relative to a constantly expressed internal control gene (2). Primers designed to span an
intron were used to generate PCR products. PCR conditions and cycle numbers were optimized for each target
sequence to ensure the reaction is in the linear phase of product accumulation. After amplification, the products were
separated by electrophoresis on a polyacrylamide gel. The gel was stained with SYBR Gold fluorescent stain
(Molecular Probes) and the gels were scanned on a Molecular Dynamics STORM 860 laser scanner. The fluorescent
signal for each band was quantified using ImageQuant software.56
LBNL-43874
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Holman, Hoi-Ying N.; Zhang, Miqin; Goth-Goldstein, Regine; Martin, Michael C.; Russell, Marion; McKinney, Wayne R. et al. Detecting exposure to environmental organic toxins in individual cells: towards development of a micro-fabricated device, article, January 10, 1999; Berkeley, California. (https://digital.library.unt.edu/ark:/67531/metadc722234/m1/2/: accessed April 23, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.