Introduction

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The human lung is exposed to various carcinogenic and noncarcinogenic polycyclic aromatic hydrocarbons (PAHs) derived from cigarette smoke, air pollutants, and dietary components. Carcinogenic actions of benzo[a]pyrene (BP), a tobacco-derived PAH, have been attributed to anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a BP metabolite (1, 2). BPDE is thought to exert a genotoxic effect on DNA by forming guanine adducts because the identical DNA adduct formation was observed in rodent lungs treated with BP (3). Other carcinogenic PAH diol epoxides also form guanine adducts (1, 2, 4). Although genotoxic effects of PAHs play an important role in tumor initiation, PAHs also potentiate tumor promotion and progression in rodent models (5). The mechanisms whereby BP and other PAHs promote tumorigenesis are less well understood.

Tumorigenesis requires mutations in key regulatory genes involved in cell death, differentiation, and proliferation. The signaling pathways associated with cell proliferation and death involve intracellular Ca²⁺ signaling that is often altered during tumorigenesis. For example, transformed tumor cell lines became resistant to the apoptotic stimuli of an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i ) (6). It was reported that PAHs increased [Ca²⁺]_i, in part, through mobilization of Ca²⁺ from cytosolic Ca²⁺ stores in both human lymphocytes (7) and primary human mammary epithelial cells (10). However, the precise mechanisms of their action on [Ca²⁺] are virtually unknown because these studies were performed using flow cytometry in cell aggregates and hence the effects of PAH on single-cell [Ca²⁺]_i over time were not addressed. Moreover, there is no data on the effects of PAHs on airway epithelial cell [Ca²⁺]_i, the cells that are most frequently exposed to PAH.

The ability to culture primary human small airway epithelial (SAE) cells (Clonetics, San Diego, CA) and maintain them in culture enabled us to determine the effects of BPDE on SAE cells. Previously, we demonstrated that the cytotoxic effects of BPDE were much greater in SAE cells than in A549 human lung cancer cells (11). More importantly, at noncytotoxic concentrations (1 to 10 nM), BPDE prevented serum-induced SAE cell differentiation into nonciliated epithelial cells, and this BPDE action was mediated through Ras signaling pathways (11). Because Ca²⁺ signaling pathways are crucial in cell proliferation, differentiation, and death, we hypothesized that: (1) BPDE increases SAE [Ca²⁺]_i, and (2) tumorigenicity of PAH diol epoxides is proportional to the effects on SAE [Ca²⁺]_i.

To test our hypotheses, we used dynamic calcium imaging and determined the effects of PAH diol epoxides on subconfluent monolayers of SAE cells. We also determined whether prior exposure of BPDE affects the acute effects of BPDE on SAE [Ca²⁺]_i.

	Materials and Methods

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Analytical Methods

Cell cultures. SAE cells were maintained in a serum-free SAE basal medium (CCMD 160; Clonetics) supplemented with growth factors (11). The medium was changed every 2 d. SAE cells were passaged when confluent (once per week). All the experiments were conducted using cells at less than five passages. SAE cells were passaged by treating cells with trypsin (0.25 g/liter) and ethylenediaminetetraacetic acid (0.1 mg/ml) in Hanks' balanced salt solution (11). SAE cells were grown on 25-mm² glass coverslips placed in a 35 × 35 mm petri dish by seeding 3 × 10³ cells/ml.

Measurement of [Ca²⁺]_i. Cells were loaded with 5 nM of the Ca²⁺-sensitive fluorophore fura-2-AM (the acetoxymethyl derivative of fura-2; Molecular Probes, Eugene, OR) plus 0.2 µg/ml pluronic acid (Molecular Probes) for 15 min at room temperature in a Ca²⁺ free solution (RPMI 1640) (12, 13). Cells were then bathed in calcium-containing recording solution (RPMI 1640 with 1 mM CaCl₂) for 20 min before the start of each experiment to prevent depletion of cytosolic Ca²⁺ stores. The coverslip was then placed in a metal holder on the stage of an inverted microscope (Nikon Eclipse TE200). Cells were then treated with BPDE in either low- or high-Ca²⁺ medium. Given the short duration of exposure to low-concentration calcium-containing solution, there was no effect on basal levels of SAE [Ca²⁺]_i. Ratiometric imaging was performed using excitation wavelengths of 340 and 380 nm and emission wavelength of 560 nm. Images were recorded with an extended ISIS intensified ICCD camera from Photonic Science (Robertsbridge, UK) using Axon Instruments (Foster City, CA) image capture and analysis software. Calcium calibration was achieved by measuring a maximum (with 50 nM ionomycin) and a minimum (with 5 mM ethyleneglycol-bis-(-aminoethyl ether)- N,N'-tetraacetic acid [EGTA]) for each cell, assuming the dissociation constant (Kd) of 224 (12). Measurement was done in the area containing 3 or 4 cells. [Ca²⁺]_i was calculated using the following equation; [Ca²⁺]_i = Kd [(R  R_min)/(R_max  R)]Sf2/Sb2 (12); where R is the 340/380 excitation ratio of the sample; R_max was calculated after permeabilizing the cells with 50 nM ionomycin and represents maximal [Ca²⁺]_i; R_min was obtained using 5 mM EGTA and is the minimal [Ca²⁺]_i; Sf2 is the maximum 380-nm signal intensity; and Sb2 is the minimum 380-nm signal intensity (12).

Reagents

BPDE, anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (5MeCDE), anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-methylchrysene (6MeCDE), and anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPDE) were gifts from Dr. Shanto Amin (American Health Foundation, Valhalla, NY) and Stephen S. Hecht (University of Minnesota Cancer Center, Minneapolis, MN). These compounds are all racemic forms (purity > 99%). They were dissolved in dimethyl sulfoxide (DMSO), aliquotted, and kept at 20°C until the time of the experiments. BcPDE is a fjord region diol epoxide in which the benzylic carbon of the epoxide ring is located in a sterically congested four-sided fjord region (14, 15). BcPDE is one of the most potent carcinogenic PAH diol epoxides tested in rodents (15). 5MeCDE has a sterically hindered bay region; its carcinogenicity is similar to that of BPDE (16). 6MeCDE is structurally related to 5MeCDE without a hindered bay region, and its carcinogenicity in rodents is less than those of the other PAH diol epoxides used in this study (16). The final concentration of DMSO in the SAE cell cultures was < 0.5%. The DMSO used for dissolving PAH diol epoxides did not affect cell viability, proliferation, or apoptotic changes in our culture system (11). BP (Aldrich, Milwaukee, WI), thapsigargin (Molecular Probes), ryanodine (Molecular Probes), 2-aminoethosydiphenyl borate (2-APB; Calbiochem-Novabiochem, La Jolla, CA), and xestospongin C (Xest C) were dissolved into DMSO, aliquotted, and kept at 20°C.

Experimental Design

Exp. 1: BPDE induced increase in SAE [Ca²⁺]_i. Fura-2-loaded SAE cells were treated with BPDE at a starting concentration of 0.05 nM in a medium with 1 or 10 nM Ca²⁺. If there was no change for 100 s, cells were treated with BPDE at the doubling concentrations until > 10-fold increase in SAE [Ca²⁺]_i was seen. This concentration was defined as the threshold BPDE concentration for changes in SAE [Ca²⁺]_i. Duration of SAE [Ca²⁺]_i change was also determined. We defined the duration of SAE [Ca²⁺]_i as time required for SAE [Ca²⁺]_i to return to < 10% above the baseline [Ca²⁺]_i. To determine whether BPDE caused Ca²⁺ mobilization from intracellular Ca²⁺ stores, a low-Ca²⁺ (10 nM) medium and a medium without Ca²⁺ were used. We also examined the effects of BP, a parent PAH of BPDE, on SAE [Ca²⁺]_i at concentrations of 0.05 to 2 nM. This was to determine whether binding to arylhydrocarbon receptor (AhR) ligand by BPDE and BP is associated with their actions on [Ca²⁺]_i, inasmuch as both compounds have equivalent affinity to AhR ligand (5). Lastly, to determine the subcellular mechanisms whereby BPDE increased SAE [Ca²⁺]_i, SAE cells were treated with either thapsigargin, an inhibitor of Ca-adenosine triphosphatase (ATPase) (6); ryanodine, a plant alkaloid that causes release of calcium from specific intracellular stores through ryanodine receptor (RyR) (17); and inhibitors of inositol 1,4,5-triphosphate receptor (InsP₃R) (2-APB or Xest-C) (18) before treatment with BPDE. In these experiments, the total amount of BPDE stock solution added to the chamber (volume; 1.5 ml) was less than 7.5 µl. Vehicle controls (DMSO) were performed for each experimental protocol.

Exp. 2: Effects of other carcinogenic PAH diol epoxides on SAE [Ca²⁺]_i. The sensitivity of SAE [Ca²⁺]_i to other carcinogenic PAH diol epoxides was determined. PAH diol epoxides tested included BcPDE, 5MeCDE, and 6MeCDE. These PAH diol epoxides were added to the medium at a starting concentration of 0.033 nM. The threshold concentration on SAE [Ca²⁺]_i was determined as described in Exp. 1 in media with high (1 mM) and low (10 nM) Ca²⁺. Vehicle controls (DMSO) were performed for each compound.

Exp. 3: Effects of prior BPDE exposure on the acute effect of BPDE on SAE [Ca²⁺]_i. SAE cells at 50 to 60% confluence (usually 4 d after subcultivation) were treated with BPDE (1 nM) or control vehicle (DMSO) in SAE cell medium (low Ca²⁺, 4 nM) for 1 h. The medium was changed, and cells were then cultured for an additional 36 to 48 h. We chose to use this time point secondary to good cell viability. At the end of culture, BPDE threshold concentration that caused an increase in SAE [Ca²⁺]_i was determined in both low (10 nM)- and high (1 mM)-Ca²⁺ media as described in Exp. 1. We chose to treat SAE cells with 1 nM BPDE because this BPDE dose was not toxic but significantly altered serum-induced SAE cell differentiation (11).

Statistics

For comparison of test values with control values, the Mann-Whitney test or Chi-Square test was used. A value of P < 0.05 was considered significant.

	Results

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Effects of BPDE on SAE Cell [Ca²⁺]_i

The first part of the experiments was done to determine whether BPDE affects SAE [Ca²⁺]_i by affecting cytosolic Ca²⁺ mobilization and/or extracellular Ca²⁺ influx. BPDE (0.2 to 2 nM) increased SAE [Ca²⁺]_i in both low (10 nM)- and high (1 mM)-Ca²⁺ media (Figures 1 and 2) within 20 s. The threshold concentration of BPDE was 0.2 nM (Figures 1 and 2). The duration of [Ca²⁺]_i changes induced by BPDE was greater in a high-Ca²⁺ medium (Figures 1 and 2). BP, a parent PAH of BPDE, did not affect SAE [Ca²⁺]_i at concentrations of 0.2 to 2 nM (Figure 1). Thapsigargin (50 nM), which depletes cytosolic Ca²⁺ stores, abolished the effects of BPDE in a low-Ca²⁺ medium but not in a high-Ca²⁺ medium (Figure 3). To determine the mechanisms of BPDE action on [Ca²⁺]_i, we tested the effects of ryanodine that induce Ca²⁺ release from cytoslic stores through RyR and the effects of selective inhibitors of InsP₃R. Neither baseline SAE [Ca²⁺]_i nor the BPDE action on [Ca²⁺]_i was altered by ryanodine (1 to 50 µM) (Figure 4). In contrast, 2-APB (25 to 50 µM), an inhibitor of InsP₃R-gated Ca channel, abolished the BPDE action in a low-Ca²⁺ medium (Figure 5). Xest-C (5 µM), another InsP₃R inhibitor, inhibited the acute BPDE action on SAE [Ca²⁺]_i when cells were pretreated with Xest-C for 15 min (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 1.   The effects of BPDE (0.2 nM), BP (1 nM), and solvent (DMSO) on SAE [Ca2+]i in a high-Ca2+ medium (RPMI 1640 with 1 mM CaCl2). These are representative results obtained in a single cell in six replicated experiments.

View larger version (17K): [in this window] [in a new window]   Figure 2.   The effects of BPDE on SAE [Ca2+]i in a low-Ca2+ medium (RPMI 1640 with 10 nM CaCl2). These are representative results obtained in a single cell in six replicated experiments.

View larger version (15K): [in this window] [in a new window]   Figure 3.   The effects of thapsigargin on SAE [Ca2+]i. Thapsigargin (50 nM) abolished the effects of BPDE in a low-Ca2+ medium. These are representative results obtained in a single cell in four replicated experiments.

View larger version (16K): [in this window] [in a new window]   Figure 4.   The effects of ryanodine on baseline and BPDE- induced SAE [Ca2+]i. Ryanodine (1 to 50 µM) did not alter baseline SAE [Ca2+]i. BPDE increased SAE [Ca2+]i after ryanodine treatment. This is a representative result observed in a single cell. These are representative results obtained in a single cell in three replicated experiments.

View larger version (19K): [in this window] [in a new window]   Figure 5.   The effects of 2-APB on baseline and BPDE-induced SAE [Ca2+]i. 2-APB (25 to 50 µM) did not alter baseline SAE [Ca2+]i but abolished the acute BPDE action on SAE [Ca2+]i. These are representative results obtained in a single cell in three replicated experiments.

Effects of BcPDE, 5MeCDE, and 6MeCDE on SAE Cell [Ca²⁺]_i

All the carcinogenic PAH diol epoxides tested increased SAE [Ca²⁺]_i in high (1 mM)- and low (10 nM)-Ca²⁺ media (Figure 6). The threshold concentrations on SAE cell [Ca²⁺]_i were 0.067 nM for BcPDE (a most potent carcinogenic PAH diol epoxide in rodents), 0.2 nM for 5MeCDE, and 0.4 nM for 6MeCDE (a least potent carcinogen among PAH diol epoxides tested) (Figure 6). The duration of SAE [Ca²⁺]_i changes was greater in a high-Ca²⁺ medium but did not differ among carcinogenic PAH diol epoxides tested.

View larger version (17K): [in this window] [in a new window]   Figure 6.   The effects of carcinogenic PAH diol epoxides (BPDE, BcPDE, 5MeCDE, and 6MeCDE) on SAE [Ca2+]i in high (A)- and low (B)-Ca2+ media. The data are expressed as a ratio of the highest SAE [Ca2+]i induced by PAH diol epoxides against baseline SAE [Ca2+]i. Each data point represents a mean value observed in 12 to 18 cells ± standard deviation (SD). The threshold concentrations of SAE [Ca2+]i were 0.067 nM for BcPDE, 0.2 nM for BPDE and 5MeCDE, and 0.4 nM for 6MeCDE in both high- and low-Ca2+ media. At these concentrations, PAH diol epoxides increased SAE [Ca2+]i significantly (P < 0.01; Mann- Whitney test). These are aggregated data obtained in eight replicated experiments.

Effects of Short-Term Prior Exposure of BPDE on the BPDE Action on SAE [Ca²⁺]_i

Short-term (1-h) exposure of SAE cells with BPDE (1 nM) 36 to 48 h before the experiment did not alter the baseline SAE [Ca²⁺]_i. However, SAE cells became less sensitive to the acute action of BPDE on SAE [Ca²⁺]_i, requiring 323 ± 108 and 282 ± 105% higher threshold concentrations in high- and low-Ca²⁺ media, respectively (Figure 7). This decrease in sensitivity to BPDE was observed up to 96 h after BPDE exposure; we were unable to determine SAE cell sensitivity to BPDE beyond that time period due to declining SAE cell viability. Such BPDE action was not observed when SAE cells were pretreated at BPDE concentrations of 0.1 to 0.5 nM. Short-term prior exposure of control vehicle (DMSO) did not alter the BPDE action on SAE [Ca²⁺]_i; all the cells tested increased [Ca²⁺]_i at a BPDE concentration of 0.2 nM. In addition, as opposed to the acute effects of BPDE on [Ca²⁺]_i, the presence of 2-APB during prior treatment of BPDE did not prevent decreased SAE cell sensitivity to BPDE 36 to 48 h after BPDE treatment.

View larger version (54K): [in this window] [in a new window]   Figure 7.   The effects of prior short-term exposure of BPDE on the acute effect of BPDE on SAE [Ca2+]i. SAE cells were treated with control solvent (DMSO) or BPDE (1 nM) and cultured for an additional 36 to 48 h. Then the effects of BPDE were tested in both low- and high-Ca2+ media. Total numbers of cells tested in each setting were 28 to 30, and the results are expressed as means ± SD% increase of threshold concentration. These are aggregate data obtained in 10 replicated experiments. BPDE-treated cells increased threshold concentrations when pretreated with BPDE (P < 0.005 in a high-Ca2+ medium and P < 0.02 in a low-Ca2+ medium; Chi-Square test), whereas there was no change in threshold in control cells.

	Discussion

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Although the mechanisms whereby PAH exposure causes lung cancer remain poorly defined, the present study provides data that BPDE, a major tobacco-derived carcinogen, acutely increases [Ca²⁺]_i in untransformed human SAE cells at a threshold concentration of 0.2 nM. Further evidence that elevation in SAE [Ca²⁺]_i plays a role in carcinogenesis derives from the observation that the effects of carcinogenic PAH diol epoxides on SAE [Ca²⁺]_i are directly proportional to the ability of these compounds to induce tumors in rodents. The most potent carcinogenic PAH diol epoxide tested caused an increase in SAE [Ca²⁺]_i at the lowest threshold concentration. Short-term prior exposure of BPDE rendered SAE cells less sensitive to the acute action of BPDE on SAE [Ca²⁺]_i. This is the first report revealing the direct effects of BPDE on cellular functions in normal human lung cells, suggesting that short-term exposure of SAE cells to BPDE exerts a prolonged effect on intracellular SAE cell calcium homeostasis.

To determine the subcellular mechanism whereby BPDE causes an elevation in SAE [Ca²⁺]_i, SAE cells were treated with BPDE in both high and low Ca²⁺. Because the extracellular calcium had no effect on the response of SAE cells to BPDE, influx of extracellular calcium is not the primary source of the BPDE-induced transient change in SAE [Ca²⁺]_i. The observation that thapsigargin, an inhibitor of Ca-ATPase activity in the endoplasmic reticulum (ER), blocked the effects of BPDE on SAE [Ca²⁺]_i indicates that BPDE causes calcium release from the ER. Because ryanodine, a plant alkaloid that causes release of intracellular calcium stores through a RyR (17), had no effect on either baseline SAE [Ca²⁺]_i or BPDE-induced changes in SAE [Ca²⁺]_i, we conclude that calcium release from the ryanodine-sensitive stores does not play a role in the BPDE- induced increase in [Ca²⁺]_i. In contrast, InsP₃R blockade with 2-APB and Xest-C (18) abolished the BPDE-induced increase in SAE [Ca²⁺]_i. Taken together, these findings indicate that BPDE induces transient Ca²⁺ release from InsP₃-sensitive intracellular stores via InsP₃-gated Ca²⁺ channels (18).

Carcinogenic PAH diol epoxides examined in this study include BcPDE (a fjord region diol epoxide), 5MeCDE (a hindered bay-region diol epoxide), and 6MeCDE. One of the striking findings is that the effects of these PAH diol epoxides on SAE [Ca²⁺]_i were directly proportional to their tumorigenicity observed in rodents and the potency of these PAH diol epoxides on guanine adduct formation (4, 14). BcPDE, one of the most potent carcinogenic PAH diol epoxides (14), altered SAE [Ca²⁺]_i at the lowest threshold concentration of 0.067 nM. 6MeCDE differs from 5MeCDE at only a single methylation site, but has less tumorigenicity in rodents than either 5MeCDE or BPDE (16). 6MeCDE had the highest threshold concentration at 0.4 nM on SAE [Ca²⁺]_i. These concentrations of PAH diol epoxides are attainable in vivo (5). These findings indicate that the effects of PAH diol epoxides on SAE [Ca²⁺]_i can theoretically play a role in tumorigenesis caused by PAH diol epoxides.

Although the present study did not identify the receptors that cause the effects of PAH diol epoxides on SAE cells, our data indicate that BPDE may increase SAE [Ca²⁺]_i through binding a cytosolic AhR ligand. However, PAHs and PAH diol epoxides bind to AhR ligand at equal affinity. If BPDE acts through binding of the 95-kD ligand that binds to a subunit of AhR in the cytosol (9), both BPDE and BP should cause an elevation in SAE [Ca²⁺]_i. However, BP, a parent PAH of BPDE, did not alter [Ca²⁺]_i in SAE cells. It is thus unlikely that BPDE increases SAE [Ca²⁺]_i through binding to a cytosolic AhR ligand.

To determine whether prior exposure to BPDE might affect calcium homeostasis of airway epithelial cells, we treated SAE cells with BPDE after exposure to BPDE (< 1 h). Despite the fact that prior exposure occurred more than 36 h before the study, we found that SAE cell sensitivity to the BPDE-induced increase in [Ca²⁺]_i was significantly attenuated. The half-life of BPDE is very short and BPDE per se is very unlikely to occupy any ligand sites more than 10 min (21). Thus, if we observed any significant changes they may more likely be due to the DNA/RNA/protein adduct formation caused by BPDE and not associated with acute change in [Ca²⁺]_i through InsP₃R. This assumption is consistent with our finding that 2-APB failed to prevent this BPDE action. Moreover, this possibility is further supported by our recent preliminary finding that the prior BPDE exposure also affects sensitivity to reagents (adenosine triphosphate and acetylcholine) that exert their actions through InsP₃R (unpublished observations). Such BPDE action may have an implication for BPDE tumorigenicity because Ca²⁺ signaling pathways are often altered in tumor cells, rendering them less sensitive to external growth/apoptotic signals (6). Previously, we observed that prior short-term exposure of BPDE prevented serum-induced SAE cell differentiation into nonciliated epithelial cells and that this was partially blocked by inhibitors of phosphatidylinositol (PI)-3K and extracellular regulated kinase (Erk)1/Erk2 (11). Given the close relationship between Ca²⁺ signaling pathways and PI-3K and Erk1/Erk2 activation, the BPDE action on SAE [Ca²⁺]_i may be associated with the BPDE inhibitory action on SAE cell differentiation (11). Further study of this aspect will be important for addressing the effects of BPDE on tumor promotion.

Our findings raise an important question of how the BPDE action on SAE [Ca²⁺]_i affects SAE cell functions and/or homeostasis in association with its tumorigenicity. BPDE is thought to induce lung tumorigenesis in A/J mice, exerting actions on K-ras oncogene and Ras-mediated signaling pathways that are activated by external growth factors (22). We also observed recently that BPDE activates Erk1/Erk2, downstream protein kinases of Ras-mediated signaling pathways in SAE cells (unpublished observations). Intracellular Ca²⁺ signaling is closely associated with PI-3K and Ras-mediated signaling pathways (26, 27). Our results suggest that the BPDE action on [Ca²⁺]_i could lead to activation of Ras-mediated signaling pathways and resultant change in cellular homeostasis.

In summary, our results reveal that BPDE and other carcinogenic PAH diol epoxides acutely increase [Ca²⁺]_i in SAE cells, untransformed human lung epithelial cells, inducing Ca²⁺ release from intracellular IP₃-sensitive Ca²⁺ stores via IP₃R-gated Ca²⁺ channels. The effects of carcinogenic PAH diol epoxides were proportional to their tumorigenicity observed in rodents. Moreover, prior short exposure of BPDE more than 36 h before the study attenuated the acute BPDE action on SAE [Ca²⁺]_i, suggesting that even short-term exposure to BPDE may have long-lasting effects on SAE cell calcium homeostasis. Whether this observation has any implications for the carcinogenic effects of PAH exposure remains unknown. This experimental system may be valuable for determining BPDE action on initiation and progression of tumorigenesis in normal human lung cells in relation to its action on Ca²⁺-mediated intracellular signaling.