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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Toxicol Sci. Author manuscript; available in PMC 2010 February 1.
Published in final edited form as:
PMCID: PMC2739812
NIHMSID: NIHMS112700

Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in breast epithelial cell lines

Abstract

Quantification of polycyclic aromatic hydrocarbons (PAH) and their metabolites within living cells and tissues in real time using fluorescence methods is complicated due to overlaping excitation and/or emission spectra of metabolites. In this study, simultaneous analysis of several metabolites of a prototype carcinogenic PAH, benzo[a]pyrene (BaP) in undifferentiated (MCF10A) and differentiated (MCF10CA1h) breast cancer cells was performed using single-cell multiphoton spectral analysis. The two cell types were selected for this study because they are known to have differences in BaP uptake and metabolism and induction of aryl hydrocarbon receptor-dependent ethoxyresorufin-O-deethylase (EROD) activity. Multiphoton microscopy spectral analysis performed in cells exposed to BaP for 24 hr identified 5 major peaks of fluorescence that were monitored within spectral bands. A comparison of the fluorescence peaks within these bands to those of BaP metabolite standards indicated that a peak in the spectral range of 393–415 nm matched benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(±),(anti) (BPDE), the ultimate carcinogenic BaP metabolite. In addition, the 426–447 nm band matched the major metabolites 3-hydroxybenzo[a]pyrene (3-OH BaP) and 9-hydroxybenzo[a]pyrene (9-OH BaP); the 458–479 nm band corresponded to the secondary metabolite benzo[a]pyrene-3,6-dione (3,6 BPQ); and a peak at 490–530 nm matched the parent compound, BaP. Multiphoton spectral analysis also revealed differences in fluorescence intensities between MCF10A and MCF10CA1h cells within three spectral bands: 393–415 nm, 426–447 nm and 458–479 nm which were partially reversed with cyclosporine A suggesting differences in efflux mechanisms between cell lines. These results demonstrate the feasibility of analyzing BaP metabolism in situ by multiphoton spectral analysis and also identifying cell-type differences in BaP accumulation and metabolism.

Keywords: Benzo[a]pyrene, Breast cancer cells, Aryl hydrocarbon receptor, BaP metabolites, Spectral analysis, Multiphoton microscopy

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are a large group of ubiquitous organic environmental contaminants. Many of them, such as benzo[a]pyrene (BaP) are carcinogens and are formed as products of incomplete combustion of fossil fuels and have been identified in surface water, tap water, rain water, groundwater, waste water, sewage sludge and foodstuffs (Ramesh et al., 2004; Samanta et al., 2002). Exposure to BaP by inhalation results in rapid uptake and distribution to several tissues in rats with the highest levels found in the liver, esophagus, small intestine, and blood within 30 min to 1 hr of exposure (Ramesh et al., 2002; Weyand and Bevan, 1986). BaP is metabolized by cytochrome P450s and other enzymes resulting in the formation of hydroxylated intermediates, quinones and dihydrodiol epoxides in cells (Bolton et al., 2000; Shimada et al., 2002). BaP and its metabolites have both genotoxic (tumor-initiating) and nongenotoxic (tumor-promoting) effects (Naspinski et al., 2008; Jagetia et al., 2003).

Several PAHs and their metabolites fluoresce efficiently in homogeneous media (Dabestani and Ivanov, 1999) and the multi-ring planar PAHs such as BaP are highly fluorescent. This property has been exploited to detect and evaluate environmental PAH contamination (e.g., Wild et al., 2007; Weston et al., 1993; Goryacheva et al., 2005), tissue accumulation of PAHs and their metabolites, and DNA and protein adducts by high performance liquid chromatography and fluorescence detection (Gmur and Varanasi, 1982; Xu and Jin, 1984; Boysen and Hecht, 2003). The fluorescent properties of BaP have also been used to investigate the processes of tissue penetration and metabolism in vivo in a mouse skin model (Lopp et al., 1986). At the cellular level, the characteristic fluorescence properties of BaP have previously been exploited to monitor mixed function oxygenase activity in bulk cell populations by flow cytometry (Miller et al., 1982) and in individual anchored cells in culture (Moore et al., 1994). Using digital fluorescence microscopy (Plant et al., 1985) and laser cytometry, we have evaluated the rapid uptake and partitioning of BaP into the plasma membrane and membranes of intracellular organelles within minutes of addition of the fluorescent genotoxicant (Barhoumi et al., 2000) and analyzed a number of nongenomic effects of BaP on cell signaling in cultured cells (Barhoumi et al., 2002, 2006). The photosensitivity of BaP complicates single cell assessment of BaP uptake and metabolism with conventional fluorescence microscopes or continuous wavelength laser confocal microscopes. However, integration of pulsed femtosecond infrared laser systems in multiphoton microscopes provides high detection sensitivity and minimal fluorophore excitation volumes to reduce photobleaching, thereby providing new opportunities for investigating BaP metabolism in situ (Hornung et al., 2007) as well as the functional consequences of BaP exposure within individual cells.

The objective of the current study was to extend previous single cell analysis studies of BaP uptake and partitioning into cells in order to evaluate the feasibility of investigating BaP metabolism in situ by multiphoton microscopy spectral analysis. Two human mammary epithelial cell lines were used in this study: MCF10A, a spontaneously immortalized, nontumorigenic, growth factor-dependent cell line (Soule et al., 1990; Tait et al., 1990) and MCF10CA1h, a Ras-transformed malignant variant that produces rapidly growing carcinomas with invasive potential (Strickland et al., 2000). MCF10A and MCF10CA1h cells have been used in breast cancer progression studies and MCF10A cells have previously been extensively utilized as a model system for investigating the nongenomic effects of BaP metabolites on growth factor signaling, cell proliferation, and altered intracellular Ca2+ homeostasis (Tannheimer et al., 1997; Burdick et al., 2003, 2006). MCF10CA1h cells were also investigated because these Ras-transformed cells derived from MCF10A cells have previously been shown to exhibit reduced aryl hydrocarbon receptor (AhR) function and cytochrome P450 1A1 induction (Reiners et al., 1997). These cells lines were therefore expected to exhibit differences in BaP accumulation, partitioning, and metabolism. Results of this study support the feasibility of spectral analysis of BaP and metabolites in situ, including cell-type specific differences in BaP accumulation and metabolism.

MATERIALS AND METHODS

Materials

Culture media, Dulbecco's phosphate buffered saline (PBS), Janus green, BaP, horse serum, doxorubicin, resorufin ethyl ether and 3,3'-methylene-bis(4-hydroxycoumarin) (dicumarol) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Benzo[a]pyrener-7,t-8-dihydrodiol-t-9,10-epoxide(±),(anti) (BPDE), benzo[a]pyrene-3,6-dione (3,6 BPQ), benzo[a]pyrene-1,6-dione (1,6 BPQ) 3-hydroxybenzo[a]pyrene (3-OH BaP) 9- hydroxybenzo[a]pyrene (9-OH BaP), were purchased from Midwest Research Institute (Kansas City, MO, USA). Tissue culture flasks were purchased from Corning Inc. (Kennebunk, ME, USA), and 2-well Lab-Tek chambered coverglass slides and 96 well Greiner glass plates were purchased from Nunc, Inc. (Naperville, IL, USA). Prolong antifade media was purchased from Invitrogen Inc. (Carlsbad, CA, USA). Primary antibody against AhR was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). BaP was prepared as 10 mM stock in dimethyl sulfoxide (DMSO). Resorufin ethyl ether was prepared at 7 mM stock in methanol and diluted to 7 µM solution for ethoxyresorufin-O-deethylase (EROD) activity measurement. Doxorubicin was prepared as 5 mM stock in ethanol. Cyclosporine A was prepared as 10 mM stock in ethanol and diluted to 10 µM for treatment. Janus green was prepared in PBS at 1 mg/ml. BaP and 3-hydroxy BaP were prepared as 10 mM stock in DMSO and ethanol respectively while BaP quinones were prepared as 5 mM stock in ethanol. BPDE was prepared as 10 mM stock in ethanol.

Cell Culture

The human breast cancer cell lines MCF10A (Soule et al., 1990) and MCF 10CA 1h (Santner et al., 2001) were purchased from Karmanos Cancer Institute (Detroit, MI, USA) at passages 89 and 36, respectively, and were maintained in Dulbecco's modified Eagle Medium (DMEM; Invitrogen, Grand Island, NY, USA) supplemented with 10% horse serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. All experiments were performed with MCF10A between passage 91 and 100 and MCF10CA1h between passage 38 and 48.

Single Cell Multiphoton Spectral Analysis of BaP Fluorescence

Cells were cultured for 24 hr in normal culture medium in 2-well coverglass slides. Cells were then washed and treated for 24 hr with 2 µM BaP only or 2 µM BaP and 10 µM cyclosporine A in serum-free and epidermal growth factor (EGF)-free medium. Treatment chemicals were then removed by washing cells in serum- and phenol red-free DMEM and transferred to the stage of a Zeiss 510 META NLO (Carl Zeiss Microimaging, Thornwood, NY, USA) laser scanning microscope and spectral analysis of an area of 143 × 143 µm (typically containing 25 to 40 cells was performed using a Chameleon tunable Ti:Sapphire laser (Coherent Inc., Santa Clara, CA, USA) at an excitation wavelength of 740 nm (which is roughly equivalent to 370 nm in single photon excitation with a continous wavelength laser system). Using the lambda stack algorithm available with Zeiss 510 META NLO, an emission spectrum ranging from 395–600 nm that covers major emission spectra of BaP and its metabolites observed in cultured cells (Moore and Cohen, 1978) was recorded for the BaP-treated cells to identify major emission peaks. Once major peaks were identified, wavelength bands corresponding to these peaks were selected for collection of data for statistical analysis and also for identification of metabolites. Images were collected with a C-APO 40X/1.2 NA water immersion objective designed for viewing specimens in an aqueous medium. Fifteen images per treatment were recorded and at least 3 experiments were performed, each on a different day.

Bulk Spectral Analysis of BaP Fluorescence

Cells were cultured in 96 Greiner glass plates for 24 hr. Cells were then washed and serum-free medium was added with or without treatment (depending on the treatment time) for the next 24 hr. At least 8 wells per treatment (BaP, 3,6 BPQ, 3-OH BaP, 9-OH BaP, BPDE) were analyzed. All wells were sequentially read with the BioTek Synergy 4 plate reader (BioTek Instruments, Inc., Winooski, VT, USA) at an excitation wavelength of 370 nm and the emission spectra from 395 nm to 600 nm were recorded.

Immunofluorescence Microscopy

Cells grown on 2-well Lab-Tek chambered coverglass slides were washed 3 times with ice-cold PBS, fixed in ice-cold methanol for 10 min at −20°C, washed three times in PBS, and incubated with 1:20 goat serum solution at room temperature for 1 hr. Primary antibody against AhR (Santa Cruz Biotechnology) was added 1:100 in antibody dilution buffer overnight at 4°C After washing with 0.3% Tween in 0.02 M PBS (PBST), Alexa 488-conjugated goat anti-rabbit secondary antibody (1:200) was added and cells were then incubated in the dark at room temperature for 1 hr. Slides were mounted with Prolong Antifade Gold mounting media following several washes. Negative controls involved substituting IgG for primary antibodies. Confocal images were collected with Zeiss 510 Meta NLO laser scanning microscope with 488 nm excitation and 530 nm emission wavelengths. At least 8 cells were identified per image and fifteen images per treatment were collected. To analyze the AhR data, the ratio of nuclear to total AhR was computed and used to statistically compare the different treatments.

Western Blot Analysis

Cells were extracted in lysis buffer (10 mM Tris, pH 7.4, 4% sodium dodecyl sulfate (SDS)) supplemented with two inhibitor cocktails - Phosphatase Inhibitor Cocktail Set II and III (Calbiochem, Gibbstown, NJ, USA) and protein content was quantified by the bicinchoninic acid (BCA) method (Pierce Biotechnology, Rockford, IL, USA). Samples were then diluted in 2x sample buffer (250 mM TrisHCl, pH 6.8, 4% SDS, 10% glycerol, 2% β-mercaptoethanol, 0.006% bromophenol blue) and boiled for 5 min before electrophoresis by 8% SDS-polyacrylamide gel electrophoresis. Separated proteins were transferred onto a Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham BioSciences, Piscataway, NJ, USA) in transfer buffer (25 mM Tris, 200 mM glycine, 20% methanol, and 1% SDS). Nonspecific binding was blocked by incubation with Tris-buffered saline plus Tween 20 (TBST) blocking buffer (0.1% Tween 20, 10 mM Tris, pH 7.5, 100 mM NaCl) supplemented with 5% nonfat dry milk for 1 hr at room temperature. A primary antibody against AhR (Santa Cruz Biotechnology) was diluted in the same buffer and incubated at 4°C overnight. After subsequent washes with TBST, membranes were incubated with secondary antibody (anti-rabbit IgG: horseradish peroxidase, 1:20,000 in TBST: 5% nonfat dry milk) for 1 hr at room temperature. The blots were washed 3x in TBST and proteins were detected with the Amersham ECL system and exposed to X-ray film (Burnette, 1981). Three replicate experiments per cell type were performed on different days.

EROD Activity

EROD activity is a biomarker of exposure to planar halogenated and polycyclic aromatic hydrocarbons (PHHs and PAHs, respectively) and provides evidence of receptor-mediated induction of cytochrome P450-dependant monooxygenases (Donato et al, 1993). In this assay cells were plated on 96 well-plate at 25K/well for 24 hr prior to treatment. Cells were then treated with BaP (0–20 µM), cyclosporine, BaP and cyclosporine in serum-free medium for 24 hr. For timed experiments, cells were treated with 2 µM BaP for different time periods (0–24 hr). Following treatments, cells were then washed twice with PBS and loaded with 7 µM resorufin ethyl ether and 10 µM dicumarol for 30 min. EROD activity was measured using a BioTek Synergy 4 plate reader with an excitation wavelength of 540 nm and an emission wavelength of 590 nm. For comparison of EROD activity between multiple treatments within the same cell type, cell number per well was determined using the Janus green assay (as described below) and EROD fluorescence intensities measured were corrected accordingly. Eight samples per treatment were collected and at least 3 experiments were preformed on different days.

Cell Counts and Viability Assessment

For cell counting in wells, cultures were washed twice with PBS and fixed with methanol for 30 min at room temperature. Methanol was then completely removed and 1 mg/ml Janus green was added to the cultures for 3 min. Following removal of Janus green, cultures were washed twice with PBS and 100 µl of 50% methanol was added to each well. Cell counts were then determined with a BioTtek Synergy 4 plate reader set to an absorbance of 654 nm (Raspotnig et al, 1999).

Statistical Analysis

BaP data collected by multiphoton microscopy were reported as mean fluorescence intensities +/− S.E. of at least 15 images per treatment. Data were analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni test at P < 0.05. Measurements of AhR fluorescence with AhR antibody were reported as mean ratio of nuclear to total AhR fluorescence intensity per cell. Data from the Biotek Synergy 4 plate reader for measuring EROD activity were presented as mean fluorescence intensities +/− S.E. of 8 wells per treatment and were analyzed statistically by ANO VA followed by Dunnett's or Bonferroni's multiple comparison test at P < 0.05.

RESULTS

Evaluation of AhR in MCF10A and MCF10CA1h

Both cell lines were tested for BaP cytotoxicity with the Janus green assay and showed no evidence of cytotoxicity or a decrease in cell number associated with BaP treatment for 24 hr. Immunofluorescence microscopy analysis of the AhR revealed that AhR in both MCF10A and MCF10CA1h cells translocate to the nucleus upon BaP exposure, however, by 24 hr there was an identical ratio of nuclear to total cellular AhR in control and BaP-treated cells (2 µM for 24 hr) (Fig. 1). However, western blot analysis identified si10nificantly higher AhR protein levels in untreated MCFCA1h compared to MCF10A cells (Fig. 2, left panel). AhR protein levels were similar in MCF10CA1h cells treated with solvent control or 2 µM BaP for 24 hr whereas treatment of MCF10A cells with 2 µM BaP for 24 hr significantly decreased AhR protein levels compared to untreated MCF10A cells (Fig. 2, right panel).

Fig. 1
A) Typical AhR fluorescence intensity obtained by immunofluorescence microscopy in MCF10CA1h Control (left panel) and BaP-treated cells (right panel) using a primary antibody against AhR. B) Comparison of the ratio of nuclear to total fluorescence intensity ...
Fig. 2
A) Comparison of AhR protein in control MCF10A and MCF10Ca1h cells. B) Changes in AhR protein levels in MCF10A and MCF10CA1h following treatment with 2 µM BaP for 24 hr.

EROD Activity in MCF10A and MCF10CA1h Cells

Measurement of EROD activity in cells treated with 2 µM BaP at multiple time points revealed a small but significant increase in EROD activity startin1 from 3 hr of exposure in both MCF10A and MCF10CAh cells (Fig. 3A). However, following a 24 hr exposure, MCF10A exhibited higher EROD activity than MCF10CA1h cells (Fig. 3B). EROD activity in MCF10CA1h was enhanced by co-treating cells with 2 µM BaP plus 10 µM cyclosporine A, an inhibitor of the P-glycoprotein mediated drug efflux pump (Fig. 4).

Fig. 3
EROD activity in arbitrary fluorescence intensity units (FI) in MCF10A and MCF10CA1h cells treated with 2 µM BaP for different time points. A significant increase in EROD activity was observed after 3 hr in both cell types (A). However, a larger ...
Fig. 4
EROD activity in arbitrary fluorescence intensity units (FI) in MCF10A and MCF10CA1h cells treated with 2 µM BaP for 24 hr in the presence or absence of 10 µM cyclosporine A (Cyc A). Cyclosporine A in combination with BaP significantly ...

Multiphoton Microscopy Spectral Analysis of BaP Fluorescence

Preliminary characterization of basal and BaP-inducible EROD acitivity in MCF10A and MCF10CA1h cells and AhR levels identified functional differences between cells that should result in differences in BaP accumulation, partitioning, and metabolism that can be analyzed by multiphoton microscopy. Multiphoton microscopy spectral analysis of BaP fluorescence in MCF 10A cells treated with 2 µM BaP for 24 hr at an excitation wavelength of 740 nm resulted in an emission spectrum illustrated in Fig. 5. In this spectrum, five major peaks were identified at wavelengths of 410 nm, 436 nm, 468 nm, 520 nm and 545 nm. Based on this information, and due to the photosensitivity of BaP which results in rapid photobleaching with high intensity laser irradiation, five wavelength bands in the ranges of 393–415, 426–447, 458–479, 500–522, and 532–565 nm were selected from which to collect spectral images as rapidly as possible and thereby obtain a BaP and metabolite fluorescence signature from cells. As shown in Fig. 5 (right panel), accumulation/overlay of images from each of the five wavelength bands gives the distribution of BaP metabolites in both cell types. MCF10A cells treated with 2 µM BaP for different time periods (0.5 hr–24 hr) exhibited significant changes in fluorescence emission in the first three spectral bands (Fig. 6). Similar multiphoton spectral analysis of MCF10CAh revealed qualitatively similar spectra. Statistical analyses of spectral data obtained from the five bands in MCF 10A and MCF10CA1h cells showed that significant differences in normalized fluorescence intensity between the two cell types were observed in the two bands at 426–447 nm and 458–479 nm.

Fig. 5
Typical multiphoton emission spectrum (left panel) and resulting spectral images (right panel) recorded between 400 nm and 600 nm with MCF10A and MCF10CA1h cells loaded with 2 µM BaP for 24 hr and excited with the multiphoton laser at 740 nm. ...
Fig. 6
Multiphoton emission spectra from 5 wavelengh bands (1 = 393–415 nm, 2 = 426–447 nm, 3 = 458–479 nm, 4 = 500–522 nm, and 5 = 532–565 nm) obtained with MCF10A cells treated with 2 µM BaP for different time ...

Cell context-dependent differences in fluorescence intensities were further investigated in cells co-treated with 2 µM BaP plus 10 µM cyclosporin A for 24 hr. Co-treatment with cyclosporine A resulted in an increase in the BaP fluorescence signal in MCF10CA1h cells (Fig. 7, right panel) whereas in MCF10A cells which do not express P-glycoprotein, the fluorescence intensity remained unchanged (Fig. 7, left panel). The increase in MCF10CAlh cells is mainly due to an increase in fluorescence of the 393–415 and 426–447 nm bands (Fig. 8), since changes in the other wavelength bands were not detected. It is noteworthy that MCF10CA1h cells accumulate less doxorubicin than MCF10A cells and this is also reversed in the presence of cyclosporine A (data not shown) suggesting that the differences in fluorescence intensity between the two cell lines after treatment with BaP was due, in part, to differences in efflux mechanisms.

Fig. 7
(A) The five wavelength band spectra obtained with MCF10CA1h cells treated with 2 µM BaP in the presence or absence of 10 µM cyclosporine A (Cyc A). Cells treated with 2 µM BaP and cyclosporine A exhibited a higher signal than ...
Fig. 8
Bulk emission spectra in obtained with MCF10A (left panel) and MCF10CA1h cells (right panel) treated with 2 µM 3,6 BPQ (top panel), 2 µM BPDE (middle panel), 2 µM 3-OH BaP (bottom panel) for different time intervals and excited ...

Bulk Spectrofluorimetric Analysis of BaP and Metabolites

Identification of BaP metabolites in MCF10A and MCF10CA1h cells that might correspond to the major fluorescence bands was evaluated by incubating cells with 3,6 BPQ, BPDE, 9-OH BaP or 3-OH BaP, each at 2 µM, for up to 6 hr followed by spectrofluorimetric analysis at an excitation wavelength of 370 nm. 3,6 BPQ produced a time-dependent increase at 460 nm in both cell types (Fig. 8, top panel) suggesting that part of the BaP spectrum (458–479 nm) observed when cells were treated with BaP may be due to 3,6 BPQ and/or metabolites generated by 3,6 BPQ. However 3,6 BPQ did not change the fluorescence intensity signal at 405 nm (measured in the first band at 393–415 nm). On the other hand, when cells were treated with BPDE, the spectrum exhibited a time-dependent increase at 405 nm suggesting that the 393–415 band is associated with BPDE and/or its subsequent major metabolites (Fig. 8, middle panel). The band 426–447 nm observed with cells that were treated with BaP corresponds to the fluorescence observed for the primary metabolites 9-OH BaP or 3-OH BaP (Fig. 8, bottom panel). However, more work is needed to identify the metabolites generating the fifth spectral band.

Further analysis of the parent compound and its metabolites was obtained from individual spectra of BaP, BPDE, 9-OH BaP, and 3,6 BPQ in cell free medium (Fig. 9). These spectra identified a concentration-dependent peak for BaP, BPDE and 9-OH BaP at 520, 405 and 435 nm respectively. However, a spectrum for 3,6 BPQ or 1,6 BPQ was not observed in cell free medium (data not shown). Binary mixtures of BaP and individual metabolites revealed peaks proportional to the corresponding metabolite of the mixture (data not shown). Further validation of the utility of multiphoton microscopy spectral analysis of BaP and major metabolites was performed by analyzing the spectrum of a mixture of three metabolites BPDE, 9-OH BaP and 3,6 BPQ (Fig. 10) within 30 min after addition to MCF10A and followed by addition of BaP for 15 min prior to significant generation of metabolites. The mixture of the three metabolites exhibited three bands with each band proportional to the corresponding metabolites (Fig. 10, symbol [triangle]). The addition of BaP to this mixture produced an extra band at 522 nm proportional to the corresponding parent compound (Fig. 10, symbol ♦).

Fig. 9
Bulk emission spectra in cell free medium obtained with different concentrations (0 to 20 µM) of BaP (A), BPDE (B) and 3-OH BaP (C). BaP was detected at 525 nm, BPDE at 410 nm, 3-OH BaP at 435 nm while neither 3,6 BPQ nor 1,6 BPQ weret detected ...
Fig. 10
Multiphoton emission spectrum obtained with MCF10A cells incubated with a mixture of 2 µM BPDE, 2 µM 9-OH BAP, 2 µM 3,6 BPQ for 30 min (symbol [triangle]) followed by addition of 2 µM BaP (symbol ♦). The mixture ...

DISCUSSION

Metabolism of BaP is complex and involves biological activation through oxidative metabolism by cytochrome P450s and other enzymes. The proposed ultimate carcinogen, BPDE results from metabolic activation by cytochrome P450 1A1 and 1B1 enzymes and hydrolysis by epoxide hydrolase (Thakker et al., 1984). Numerous additional metabolites are also generated including epoxides, phenols, dihydrodiols, quinones, triols, tetrols and diol epoxides (Weeks et al., 1991; Kim et al., 1998) and these metabolic products can affect a wide variety of cellular responses.

Quantitative analysis of BaP and BaP metabolites and binding to macromolecules within cells and tissues has been performed with high-performance liquid chromatography (HPLC) methods with fluorescence detection (Stampfer et al., 1981; Miles et al., 1996; Ramesh et al., 2001) following tissue isolation and extraction. Time course studies of BaP metabolism in extracted tissues have also utilized spectrofluorimetry at excitation and emission wavelengths specific for each metabolite of BaP (Moore and Cohen, 1978). However, quantitative analysis of BaP metabolism in real time within viable cells and tissues by fluorescence methods is sometimes complicated due to overlap in excitation and/or emission spectra of metabolites and this limits opportunities to simultaneously identify all major metabolites. Recently, multiphoton laser scanning microscopy has been used to identify the tissue distribution of BaP and some of its metabolites in medaka embryos and post-hatch larvae by taking advantage of differences in excitation spectral properties of the parent compound and metabolites (Hornung et al., 2004, 2007). An excitation below 830 nm and emission at 450/80 nm was used to identify the presence of the parent compound, whereas excitation at 840 nm or 860 nm indicated the presence of conjugated metabolites (BaP-3-glucuronide, BaP-3-SO4) and excitation at 880 nm indicated the presence of 3OH-BaP only.

The current study utilized a different approach to investigate BaP uptake, partitioning and metabolism in MCF10A and MCF10CA1h cells that involved multiphoton microscopy spectral analysis of BaP and metabolite emission wavelengths. This approach employed a single excitation wavelength of 740 nm, and rapid image scanning of spectral bands at multiple time points making it possible to follow up the changes in the BaP spectrum over time to monitor metabolite generation. The MCF10A and MCF10CA1h cell lines were expected to exhibit differences in BaP accumulation, partitioning, and metabolism due to differences in AhR function, Cyp1A1 enzyme inducibility and expression of P-glycoprotein which results in differences in BaP efflux (Reiners et al., 1997). This was confirmed as both cell lines exhibited comparable AhR protein distribution within cells as determined by immunofluorescence microscopy prior to and following treatment with BaP (Fig. 1). Also both cell lines showed some basal P450 1A1 activation through AhR activation suggesting an endogenous role for P450 1A1 (Puga et al., 2002; Backlund et al., 2005; Ikuta et al., 2004). Higher AhR protein levels were detected in untreated MCF10CA1h compared to untreated MCF10A cells, whereas AhR turnover was greater in MCF10A cells treated with 2 µM BaP for 24 hr (Fig. 2) (Joiakim et al., 2004). Catalytic measurement of cytochrome P4501A1-dependent induction of EROD identified greater activity in MCF10A compared to MCF10CA1h cells (Fig. 3). These functional differences made them suitable for the study of BaP metabolism by multiphoton spectroscopy where differences in emission spectra were expected.

Multiphoton microscopy spectral analysis identified 5 major emission bands in MCF10A and MCF10CA1h cells following 24 hr exposure to 2 µM BaP (Fig. 5). Four emission peaks were monitored within spectral wavelength bands that included 393–415 nm, 426–447 nm, 458–479 nm, and 500–522 nm. Changes in the shortest 3 wavelength emission bands were observed in both MCF10A and MCF10CA1h over the 24 hr exposure period; and this spectral shift to lower wavelengths was due to the generation of BaP metabolites (Fig. 6).

Evidence that the spectral band detected at 458–479 nm was due to quinones was obtained by direct addition of 3,6 BPQ metabolites of BaP to cells followed by spectrofluorimetric assessment which identified a peak in the same spectral band (Fig. 9, top panel). Similarly, the band at 393–415 nm was identified as BPDE and/or subsequent metabolites of BPDE because addition of BPDE directly to cells identified a 405 nm peak (Fig. 9, middle panel). Further, the 425–447 nm band appears to be due to the presence of hydroxy BaP isomers as 3-OH BaP and 9-OH BaP major metabolites of BaP added directly to cells produced peak fluorescence within this band (Fig. 9, lower panel). BaP was identified as the spectral band at 500–522 nm because addition of BaP directly to culture medium in the presence or absence of cells resulted in a concentration-dependent peak in this spectral region (Fig. 9, left panel) by spectrofluorimetry In addition, a mixture of 9-OH BaP, 3,6 BPQ and BPDE added directly to cells resulted in a spectrum with 3 bands (393–415, 425–447, 458–479 nm) supplemented with a fourth band (500–525 nm) upon addition of BaP and prior to any increase in P450 activity (Fig. 10).

Results of multiphoton microscopy single cell spectral analysis of BaP uptake, partitioning into cells and in situ metabolism indicate that MCF10CA1h cells produce lower levels of BaP metabolites than MCF10A cells (Fig. 7). This conclusion was supported by analysis of EROD activity in both cell types which showed that EROD activity was lower in MCF10CAlh than MCF 10A after treatment with BaP for different time intervals (Fig. 3). MCF10CA1h exhibited lower uptake of doxorubicin than MCF10A cells (data not shown) and when MCF10CA1h cells were treated with BaP in combination with cyclosporine A, the P-glycoprotein pump inhibitor, greater EROD activity was detected albeit at a lower level than with MCF 10A cells (Fig. 4). This finding suggests that although inhibition of P-glycoprotein resulted in accumulation of the parent compound, there were differences in Cyp1A1-dependent activity that decreased metabolism of BaP in MCF10CA1h cells compared to MCF10A cells.

These data derived from multiphoton microscopy spectral analysis of living cells can provide additional insights into the mechanisms of cellular injury caused by BaP. Adverse nongenomic effects of BaP and metabolites on growth factor signaling, cell proliferation, and altered intracellular Ca2+ homeostasis have been reported in MCF10A cells (Tannheimer et al., 1997; Burdick et al., 2003, 2006). BaP alters intracellular Ca2+ homeostasis and agonist-induced Ca2+ oscillations in rat liver and in human uterine smooth muscle cells through a mechanism that involves alterations of gap junction mediated intercellular communication, membrane channels, actions on protein kinase C and receptor kinase pathways (Barhoumi et al., 2000, 2002 and 2006). Multiphoton analysis of BaP in conjunction with assessment of alterations of intercellular homeostasis should provide important insights into the cellular and molecular mechanisms by which these functional alterations are propagated.

This methodology can also be readily adapted for analysis of BaP uptake and metabolism in conjunction with assessment of cellular and tissue injury in precision-cut liver tissue slices from toxicant-exposed animals. The combination of multiphoton microscopy/spectral analysis with precision cut tissue slices has the potential to provide a powerful tool to bridge the gap between in vitro and in vivo models for mechanistic analysis of cellular injury caused by BaP and other intrinsically fluorescent polycyclic aromatic hydrocarbons. This approach is advantageous because tissue level organization is maintained including three dimensional cell-cell and cell-matrix relationships, functional heterogeneity of cell types, and maintenance of intermediary metabolic control over xenobiotic metabolism. Furthermore, the range of optimal slice thicknesses (which is a function of the oxygen consumption rate of the tissue) varies from 200–250 µm for liver and kidney (Parrish et al., 1995), and is well within the optimal performance range for multiphoton microscopy (Helmchen and Denk, 2002).

The present study demonstrates the validity of multiphoton spectral analysis for simultaneous detection and identification of the major metabolites of BaP in living cells. Future studies will determine the kinetic analysis of different mixtures of metabolites in order to establish reference spectra. Reference spectra of BaP and other PAHs will be collected in a database and will be used for comparison purposes with exposed tissues to identify metabolites and to assess the role of Cyp1A1 in PAH-induced cytotoxicity mechanisms.

ACKNOWLEDGMENTS

Confocal and multiphoton microscopy was performed in the Texas A&M University College of Veterinary Medicine & Biomedical Sciences Image Analysis Laboratory, supported by NIH-NCRR (1 S10 RR22532-01), and NIH-NIEHS grants P30-ES09106, P42-ES04917 and T32 ES07273. This research was performed in part using compounds provided by the National Cancer Institute's Chemical Carcinogen Reference Standards Repository operated under contract by Midwest Research Institute, No. N02-CB-07008.

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