Ryanodine Receptor-Mediated Rapid Increase in Intracellular Calcium Induced by 7,8-Benzo(a)Pyrene Quinone in Human and Murine Leukocytes

Jun Gao*, Andrew A. Voss{dagger}, Isaac N. Pessah{dagger}, Fredine T. Lauer*, Trevor M. Penning{ddagger} and Scott W. Burchiel*,1

* The University of New Mexico College of Pharmacy Toxicology Program, Albuquerque, New Mexico 87131–0001; {dagger} University of California at Davis, Dept. of VM:Molecular Biosciences, and Center for Children's Environmental Health, Davis, California 95616; and {ddagger} The University of Pennsylvania School of Medicine, Department of Pharmacology, Philadelphia, Pennsylvania 19104–6084

1 To whom correspondence should be addressed at 1 University of New Mexico—MSC09 5360, Albuquerque, NM 87131–0001. Fax: (505) 272-6749. E-mail: sburchiel{at}salud.unm.edu.

Received June 16, 2005; accepted July 14, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzo(a)pyrene (BaP) is an environmentally prevalent polycyclic aromatic hydrocarbon (PAH) known to produce immunotoxicity in murine and human lymphocytes. Previous studies by our lab have shown that certain BaP metabolites increase intracellular Ca2+ in human and murine lymphocytes. The mechanism by which these BaP metabolites increase Ca2+ may involve src kinase activation and mitochondrial oxidative stress. We have implicated a new pathway of Ca2+ elevation in lymphocytes produced by a novel BaP metabolite, BaP-7,8-dione (7,8-BPQ). This ortho quinone is produced from BaP-7,8-dihydrodiol by aldoketoreductase 1C1 (AKR1C) isoforms in human cells. We have previously shown that 7,8-BPQ increases Ca2+ levels in an in vitro rabbit skeletal muscle sarcoplasmic reticulum (SR) vesicle model via interaction with ryanodine receptors (RyR). In the present study, we found that 7,8-BPQ produced a RyR-dependent rapid increase in intracellular Ca2+ in the Daudi human B cell line. However, other BP-diones including 1,6-, 3,6-, and 6,12-BPQs failed to produce a rapid increase in Ca2+. Instead they produced a late increase in intracellular Ca2+, presumably via a redox-cycling-dependent loss of Ca2+ buffering capacity by mitochondria. Functional RyR were detected in Daudi using a 3H-ryanodine binding assay. The studies were extended to normal human peripheral blood and murine spleen cells, where it was found that 7,8-BPQ rapidly elevated intracellular Ca2+ in B cells and T cells in both species. The Ca2+-elevating effect of 7,8-BPQ was prevented by pretreatment with a high concentration of ryanodine (500 µM). Collectively, these results demonstrate a novel mechanism of Ca2+ elevation by an environmentally relevant metabolite of BaP in murine and human lymphocytes.

Key Words: ryanodine receptor; calcium; 7,8-benzo(a)pyrene quinone.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants known to be carcinogenic and immunotoxic in humans and animals (Burchiel and Luster, 2001Go). The immunotoxicity of PAHs is likely due to a combination of genotoxic and nongenotoxic mechanisms, such as alterations in signaling and oxidative stress pathways. Benzo(a)pyrene (BaP), a prototypic PAH, has been widely investigated for many years. Certain metabolites of BaP, most notably BaP-7,8-diol, 9,10-epoxide (BPDE), formed by BaP bioactivation with cytochrome P450 1B1/1A1 (CYP1B1/1A1) and microsomal epoxide hydrolase (EPHX1), appear to be important in immunotoxicity. Our previous studies have shown that BPDE alters cell signaling and Ca2+ homeostasis in lymphoid and nonlymphoid cells (Burchiel and Luster, 2001Go; Davila et al., 1995Go; Mounho et al., 1997Go; Tannheimer et al., 1997Go, 1999Go; Zhao et al., 1996Go). BPDE and other PAHs may act via direct or indirect activation of protein tyrosine kinases that are associated with cell signaling through T- and B-cell antigen receptors (Archuleta et al., 1993Go; Davila et al., 1999Go; Mounho and Burchiel, 1998Go).

Calcium is an important second messenger that regulates various biological functions in cells (Berridge, 2001Go). Calcium signaling in lymphocytes and other cells is influenced by such properties as the duration, intensity, or frequency of oscillations (Lewis, 2003Go). We have found that several BaP metabolites alter Ca2+ signaling in Daudi human B cells, albeit by different mechanisms. Cytochrome P450 activation of BaP appears to be critical for immunotoxicity to human peripheral blood cells (Davila et al., 1996Go). Our previous studies also found that BaP-7,8-diol alters intracellular calcium levels after several hours of treatment of normal human peripheral blood mononuclear cells (HPBMC) (Mounho and Burchiel, 1998Go). Using Daudi human B cells as an in vitro model, we found increases in Ca2+ levels through activation of Src-like protein tyrosine kinases (Mounho and Burchiel, 1998Go). Recently, we found that quinones of BaP (BPQs) also signal Ca2+, but through several different pathways. BP-quinones, including 1,6-BPQ, 3,6-BPQ, and 6,12-BPQ, are formed through cytochrome P450, peroxidase, and photochemical (UV) reactions (Reed et al., 2003Go). BPQs are known to redox-cycle, leading to oxidative stress and energy depletion in mitochondria followed by loss of Ca2+ buffering capacity in cells (Zhu et al., 1995Go). More recently we discovered another potential mechanism of Ca2+ elevation in cells due to a unique interaction of an ortho-BPQ (7,8-BPQ) with ryanodine receptors (RyR) (Pessah et al., 2001Go). In these studies, we found that 7,8-BPQ causes Ca2+ release in rabbit skeletal muscle sarcoplasmic reticulum (SR) vesicles through a selective interaction with RyR.

RyR are homotetrameric calcium release channels of intracellular stores, which were first described on the sarcoplasmic reticulum (SR) in mammalian skeletal muscle cells and cardiac muscle cells (Fill and Copello, 2002Go; McPherson and Campbell, 1993Go). More recently, RyR have been found on the endoplasmic reticulum of nonexcitable cells (Sei et al., 1999Go). Mammalian RyRs are expressed as three widely distributed isoforms: RyR1, RyR2, and RyR3. Recent studies have shown that RyR1 and RyR2 are expressed in nonexcitable cells, such as lymphocytes (Sei et al., 1999Go). RyR3 is widely expressed in many types of cells (Ogawa et al., 2000Go). To investigate the role of RyR, ryanodine binding, which occurs only with the open channel, is frequently used as a probe of RyR function (Pessah et al., 1986Go). Low concentrations of ryanodine (~1 µM) activate RyRs, whereas high concentrations (~100 µM) irreversibly inhibit RyR calcium release.

Based on previous studies of calcium responses produced by BPQs and other BaP metabolites in lymphoid and nonlymphoid cells, as well as rabbit muscle SR vesicles, we hypothesized that 7,8-BPQ would selectively (as compared to other BPQs) elevate intracellular Ca2+ in human and murine B and T cells via a RyR-dependent mechanism. In this report, we utilized Daudi human B cells, HPBMC, and murine spleen cells to analyze the immediate and delayed Ca2+ responses following exposure to 7,8-BPQ and various other BPQ and BaP metabolites. Our data show that only 7,8-BPQ produces a significant rapid increase in intracellular Ca2+. We found that this increase in Ca2+ was blocked by high concentrations of ryanodine. Therefore, we conclude that Ca2+ elevation by 7,8-BPQ in human and murine lymphoid cells is due to activation of RyR.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies.
Benzo(a)pyrene (BaP, Sigma Cat. No. B1760) (purity >97%) and ryanodine (purity >95%) (Sigma Cat. No. R6017) were obtained from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO). All BaP metabolites used in this paper were purchased from Midwestern Research Institute (Kansas City, MO), with the exception of 7,8-beno(a)pyrene quinone (7,8-BPQ) that was provided by the Penning laboratory. Tissue-culture grade (purity >95%) dimethyl sulfoxide (DMSO) (Sigma Chemical Co., St. Louis, MO) was used to dissolve and dilute BaP and its metabolites, and the final concentration of DMSO used was adjusted to 0.1% (v/v). Fluo-3/AM was purchased from Molecular Probes (Eugene, OR). Fico/Lite LymphoH (Cat. No. 140150) was purchased from Atlanta Biological (Lawrenceville, GA). Phycoerythrin (PE)-conjugated mouse anti-human CD3 and mouse anti-human CD19 monoclonal antibodies were from BD Pharmingen (San Diego, CA).

Cell line.
The Daudi human B cell line was obtained from American Type Culture Collection (Rockville, MD) and was grown in a humidified, 37°C, 5% CO2 incubator in complete RPMI 1640 medium containing 20% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 100 µg/ml streptomycin (Cambrex), and 2 mM L-glutamine.

Preparation of human peripheral blood mononuclear cells (HPBMC).
Human mononuclear cells were isolated from healthy blood donors under an approved IRB protocol using Fico/Lite LymphoH density gradient centrifugation method. Briefly, collected peripheral blood was placed in 6 x 10 ml Vacutainer® plasma tubes with heparin (VWR Cat. No.VT6480). Heparinized blood was diluted with equal volume of sterile phosphate-buffered saline (PBS) (without calcium and magnesium salts) (Sigma Chemical Co., St. Louis, MO), and then 30 ml of this mixture was overlayed on 15 ml Fico/Lite LymphoH at room temperature (RT). After density gradient centrifugation for 30 min at 200 x g without brake at RT, peripheral blood mononuclear cells were collected from the white layer between the plasma fraction and the Fico/Lite LymphoH fraction. The HPBMC were washed twice with PBS, and the cell pellet was then resuspended in 3 ml complete RPMI 1640 medium containing 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 100 µg/ml streptomycin (Cambrex), and 2 mM L-glutamine. Cells were counted and adjusted to 20 x 106 cells/ml.

Preparation of mouse spleen cells.
Spleens from female wild-type C57BL/6N mice (13–14 weeks old) (Harlan laboratories, Indianapolis, IN) were aseptically removed and were dissociated in RPMI 1640 complete medium supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, 100 unit/ml penicillin, and 2 mM L-glutamine. Cells were centrifuged at 280 x g for 10 min, and pellets were resuspended and maintained in 2 ml RPMI 1640 complete medium on ice. The nucleated spleen cells were counted using the trypan blue (Sigma Chemical Co., St. Louis, MO) exclusion method.

Intracellular calcium measurement using flow cytometry.
The analysis of intracellular Ca2+ levels in Daudi human B cells, HPBMC, and mouse spleen cells was performed by flow cytometric methods (Burchiel et al., 1999Go, 2000Go; Rethi et al., 2002Go). For all experiments, cells were loaded with in 200 µl of 2 µM Fluo-3/AM (Molecular Probes) in FACS tubes and incubated at 37°C, 5% CO2 for 1 h, with cells being resuspended approximately every 20 min to ensure even dye loading. Then 800 µl of Dulbecco's phosphate buffered saline (DPBS) (with calcium and magnesium) were added to yield a 1 ml solution. Ca2+ responses were observed in two ways: (1) For rapid real-time analysis, Fluo-3 loaded cells were treated with various PAHs and continuously monitored for up to 30 min. (2) For longer incubations periods (1 h or more), after treatment with PAHs, cells were loaded with Fluo-3, and the fluorescence intensity was measured by flow cytometry. For Daudi human B cell experiments, cells were resuspended in complete RPMI medium at 2 x 105 cells/ml, and 1-ml cell aliquots were placed into three 12 x 75 mm tubes. After the centrifugation and aspiration, the cells were loaded with Fluo3. For HPBMC studies, 10 µl of 20 x 106 cells/ml HPBMC were loaded with Fluo-3 and stained with PE-conjugated mouse anti-human CD3 or mouse anti-human CD19 to label T or B cells, respectively. For murine spleen cell studies, 10 µl of 20 x 106 cells/ml murine spleen cells were put in 12 x 75 mm tubes and loaded with the addition of 200 µl of Fluo-3AM. After 1 h, 800 µl of DPBS solution was added to yield 2 x 105 cells/ml. Tubes were capped and covered with aluminum foil to transport to Flow Cytometry Facility for analysis. Fluorescence intensities were measured in the resuspended cells by FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA) to obtain baseline readings. Mean channel fluorescence intensities were calculated using CellQuest software.

Measurement of 3H-ryanodine binding.
High affinity binding of 3H-ryanodine (57 Ci/mmol; New England Nuclear, Boston, MA) to crude microsomal membrane extracts (0–360 µg/ml) from cultured Daudi cells was performed in the presence of 500 mM KCl, 20 mM HEPES, 100µM CaCl2, 1 mM DTT, 100 µM PMSF, 100 µg/ml BSA, 1 µg/ml leupeptin, pH 7.4, and 10 nM 3H-ryanodine (Pessah et al., 1987Go). The binding reaction was initiated by the addition of microsomal membranes to the medium, and the mixture was permitted to equilibrate at 37°C for 2 h. Nonspecific binding was assessed in the presence of 10 µM unlabeled ryanodine. Separation of bound and free ligand was performed by rapid filtration through Whatman GF/B glass fiber filters using a Brandel cell harvester (Gaithersburg, MD). Filters were washed with two volumes of 0.5 ml ice-cold wash buffer containing 20 mM Tris–HCl, 250 mM KCl, 15 mM NaCl, 50 µM CaCl2, pH 7.1, and placed into vials with 5 ml scintillation cocktail (Ready Safe; Beckman Instruments, Inc., Fullerton, CA). The 3H-ryanodine remaining on the filters was quantified by liquid scintillation spectrometry.

Statistical design and analysis.
All of the data reported in this paper were analyzed by SigmaStat software (SPSS, Point Richmond, CA). The statistical differences were determined by one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test, Dunnett's test and Student's t-test. A p-value of ≤0.05 between DMSO control and PAH-treated samples was considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immediate Ca2+ Response Produced by 7,8-BPQ in Daudi Human B Cells
The immediate calcium responses produced by BaP and various metabolites were evaluated using real-time flow cytometry. As shown in Figure 1A, 7,8-BPQ produced a statistically significant increase in intracellular Ca2+ in Daudi human B cells within 6 min. Neither BaP nor any of the other BaP metabolites studied produced such rapid increase in Ca2+, including 1,6-BPQ, 3,6-BPQ, 6,12-BPQ, and BPDE. The Ca2+ response produced by 7,8-BPQ in Daudi was continually measured for 30 min (Figure 1B). A 1 µM concentration of 7,8-BPQ increased intracellular Ca2+ levels with a time to peak of 10 min. The Ca2+ level then declined during the next 20 min. DMSO control samples did not produce any measurable effect on intracellular Ca2+ response. This result shows that 7,8-BPQ produces a rapid increase of intracellular free Ca2+ in Daudi human B cells.



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FIG. 1. Flow cytometric analysis of the rapid calcium release by BaP- quinones in Daudi Human B cells. Daudi human B cells (2 x 105 cells) were loaded in 200 µl, 2 µM Fluo-3/AM at 37°C, 5% CO2 for 1 h; 800 µl complete RPMI medium was then added to yield 2 x 105 cells/ml. Fluorescence intensities were measured in real-time using a FACSCalibur. (A) After loading with Fluo-3, Daudi cells were dosed with 1 µM PAHs, and the fluorescence intensities were monitored for 6 continuous min. (B) after the baseline measurement, Daudi cells were treated with 1 µM BaP-7,8-quinone (7,8-BPQ), and measurement of the change of fluorescence intensities continued for 30 min. Mean channel fluorescence intensities were calculated by CellQuest software. The plots represent the mean ± standard error of triplicate measurements. Asterisks (*) indicate statistically significant differences from the DMSO vehicle control (p < 0.05 by Dunnett's test).

 
BaP Metabolites Induce Intracellular Ca2+ Elevation at Different Time Points
To further investigate the Ca2+ effect by BaP and various metabolites, Daudi human B cells were treated with 1 µM of various PAHs for 30 min, 2, 4, 8, and 18 h. Treated cells were loaded with Fluo-3/AM and incubated at 37°C for 1 h. 3,6-BPQ increased Ca2+ within 2 h, BPDE increased Ca2+ within 4 h, whereas, 1,6-BPQ and 6,12-BPQ increased Ca2+ at later time points (8–18 h) (Fig. 2A). 7,8-BPQ did not show any Ca2+ effect at these late time points. These results suggest that increases in intracellular Ca2+ by BaP metabolites may be associated with different metabolic pathways and mechanisms. BaP itself does not possess significant Ca2+-elevating activity at early time points in Daudi human B Cells. After an 18 h exposure to 1 µM PAH treatments, BPDE, 1,6-BPQ, 3,6-BPQ, and 6,12-BPQ significantly increased intracellular Ca2+ in Daudi cells (Fig. 2B). We noted that 3,6-BPQ fluorescence (no Fluo-3 loading) in the green channel was significantly higher than DMSO controls, and therefore controls must be run with and without the Ca2+ dye to correct for the fluorescence produced by the test compound; however, none of the other PAHs demonstrated significant fluorescence at the wavelength pairs used to measure Fluo-3 (not shown). Therefore, only background fluorescence associated with 3,6-BPQ needed to be corrected for analysis with Fluo-3.



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FIG. 2. Effect of PAHs on Ca2+ responses in Daudi cells at later time points. Changes in intracellular Ca2+ measured at different time points after treatment with PAHs. (A) Daudi B cells were treated with 1 µM various PAHs for 30 min, 2, 4, 8, and 18 h. Treated cells were then pelleted and resuspended in complete medium containing 2 µM Fluo-3 and incubated at 37°C for 1h. The flow cytometric method was used to measure the change of Fluorescence. (B) Change in intracellular Ca2+ produced by BaP and its various metabolites measured at 18 h by flow cytometry. There was no significant cell death, as assessed using propidium iodide, following 18 h treatment of Daudi human B cells with 1 µM BPQs.

 
Detection of RyRs in Daudi Human B Cells
Because ryanodine specifically binds RyR in the open state, high-affinity binding of 3H-ryanodine can be used to verify the expression of functional RyR (Pessah et al., 1986Go). Figure 3 shows the specific binding of 3H-ryanodine to Daudi microsomal membranes. RyRs are large (~560 kDa) homotetrameric receptors that mediate Ca2+ release from the endoplasmic reticulum stores. Our previous studies show that 7,8-BPQ induces RyR-mediated Ca2+ release from microsomal fractions (Pessah et al., 2001Go). Thus, this result, coupled with our Ca2+ studies above, demonstrates that functional RyRs are expressed in human lymphocytes.



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FIG. 3. 3H-Ryanodine binding to Daudi microsomes. Daudi microsomes were incubated with 3H-ryanodine as described in Materials and Methods. Data were fit by linear regression (r2 = 0.9624).

 
The Immediate Calcium Response Induced by 7,8-BPQ Is Inhibited by Ryanodine in Daudi Cells
Previous investigators found that RyRs are expressed in Jurkat T-lymphocytes (Hohenegger et al., 1999Go) and play an important role in the regulation of intracellular Ca2+. To test whether RyRs are responsible for 7,8-BPQ-induced Ca2+ elevation, we used ryanodine (50 and 100 µM) to specifically block the RyRs. As reported earlier, high concentrations of ryanodine (>100 µM) irreversibly inhibit RyR-mediated Ca2+ release (Zimanyi et al., 1992Go). Daudi human B cells were pretreated with 100 µM ryanodine and loaded with the Fluo-3 dye for 1h. PAH treatments were added after the baseline reading, and samples were monitored for 20 min by flow cytometry (Fig. 4). The intracellular Ca2+ level in the 100 µM ryanodine-treated group was significantly decreased compared with non-ryanodine-treated control group. This finding suggests that RyRs regulate the rapid Ca2+ response of Daudi to 7,8-BPQ.



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FIG. 4. The effect of ryanodine on Daudi human B cells. Daudi cells were pretreated for 1 h with 50 µM or 100 µM ryanodine (Ry) and were then loaded with Fluo-3/AM dye. Immediate intracellular Ca2+ was measured by flow cytometry as described in Materials and Methods. Results are shown as the change in Mean Channel Fluorescence for triplicate samples ± SEM.

 
Immediate Calcium Response in HPBMC
To investigate the immediate Ca2+ response in normal HPBMC, freshly isolated HPBMC from healthy blood donors were treated with 1,6-BPQ, 3,6-BPQ, 7,8-BPQ, or DMSO in vitro. We observed that the 7,8-BPQ increase in intracellular Ca2+ was maximal at about 10 min, which was a result quite similar to that obtained with Daudi (Fig. 5A). Unlike Daudi, however, the rise in intracellular Ca2+ in HPBMC was maintained for much longer periods of times (data not shown). Because HPBMC are composed of both T and B lymphocytes, we wondered whether these cells have the same intracellular Ca2+ response to 7,8-BPQ. Mouse anti-human CD3 and CD19 monoclonal antibodies were used to study the human T- and B-cell subsets. Results showed that 7,8-BPQ significantly increased intracellular Ca2+ in both cell populations (Fig. 5B). Interestingly, the Ca2+ response to 7,8-BPQ in B cells appeared to be higher than in T cells.



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FIG. 5. Rapid intracellular Ca2+ response in HPBMC. (A) HPBMC were isolated from healthy donors by Ficoll-hypaque method as described in Material and Methods. Cells were loaded with Fluo-3/AM dye for 1 h. After the baseline reading, HPBMC were treated by 7,8-BPQ, 1,6BPQ, 3,6BPQ, and DMSO control. Results are shown as the Mean Channel Fluorescence. (B) Surface-marker-defined T cells, B cells, and monocytes in HPBMC were treated with 7,8-BPQ and DMSO control. The immediate intracellular Ca2+ response was continuously monitored for 15 min. Results are shown as the change in Mean Channel Fluorescence ± SEM. The numbers shown in this figure were the averages of triplicate determinants.

 
To examine the role of the RyR in the 7,8-BPQ-induced immediate Ca2+ response, HPBMC were pretreated with 50 µM or 500 µM ryanodine for 1 h. We observed that both concentrations of ryanodine reduced the Ca2+ response produced by 7,8-BPQ (Fig. 6). Inhibition of the Ca2+ response was not complete, however, suggesting that other mechanism may also be involved in the response to 7,8-BPQ.



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FIG. 6. HPBMC pretreatment with ryanodine to block RyRs. HPBMC were pretreated for 1 h with 50 µM or 500 µM ryanodine (Ry) and loaded with Fluo-3/AM dye at 37°C in a 5% CO2 incubator. Immediate intracellular Ca2+ responses were monitored in real time by flow cytometry as described in Methods.

 
7,8-BPQ Produces Immediate Ca2+ Response in Mouse Splenocytes
To further extend our findings in HPBMC to murine lymphocytes, we investigated the response of mouse spleen cells to 7,8-BPQ. As shown in Figure 7A, the immediate Ca2+ increase was only observed in the 7,8-BPQ-treated group of spleen cells. The mouse spleen cell response (~80 channel fluorescence shift) was somewhat lower than that seen in HPBMC (over 120 channel fluorescence shift). 1,6-BPQ and 3,6-BPQ treatments did not produce significant changes in intracellular Ca2+ in spleen cells compared with DMSO control. To determine if this Ca2+ increase was mediated by RyR, we pretreated murine spleen cells with different concentrations of ryanodine (50 µM or 500 µM) for 1 h. At the 500 µM concentration of ryanodine, we found that Ca2+ elevation was significantly reduced in mouse spleen cells (Fig. 7B). These results strongly suggest that murine splenocytes also utilize RyR in response to 7,8-BPQ to cause a rapid Ca2+ signal.



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FIG. 7. The immediate intracellular Ca2+ elevation in murine splenocytes. Mouse spleens were aseptically removed and single cell suspensions were prepared in complete RPMI 1640 medium as described under Material and Methods. Cells were loaded with Fluo-3/AM dye for 1 h. After the baseline reading, murine splenocytes were treated with 7,8-BPQ in DMSO, with DMSO vehicle serving as a control. (A) The rapid Ca2+ response to 7,8-BPQ, 1,6-BPQ, and 3,6-BPQ was measured continuously for 15 min. (B) Experiments were performed as in (A), except cells were pretreated with 50 or 500 µM ryanodine (Ry). Results are shown as the change in Mean Channel Fluorescence ± SEM for triplicate samples.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our laboratory is interested in the mechanisms by which BaP and other environmental polycyclic aromatic hydrocarbons (PAHs) produce immunomodulation and immunotoxicity. It is known that BaP can be metabolized by cytochrome P450 and other enzymes to produce bioactive metabolites (Burchiel and Luster, 2001Go). These bioactive metabolites include 1,6-BPQ, 3,6-BPQ, 6,12-BPQ, 7,8-BPQ, BaP-7,8-Diol, and BaP-7,8-Diol-9,10-epoxide (BPDE). In the present study we evaluated BaP and six metabolites for their ability to alter Ca2+ levels in Daudi human B cells, human peripheral blood mononuclear cells (HPBMC), and murine splenocytes using flow cytometry. 7,8-BPQ is a particularly interesting BaP metabolite that is derived from the metabolism of BaP-7,8-dihydrodiol under the influence of a dihydrodiol dehydrogenase (DD1), recently identified as AKR1C1 (Burczynski et al., 1998Go, 1999Go). We have previously shown that 7,8-BPQ produces a rapid increase in Ca2+ by activation of ryanodine receptors (RyR) in a rabbit skeletal muscle SR vesicle model system (Pessah et al., 2001Go). Concentrations of 0.1–1 µM 7,8-BPQ were required in our studies to elevate intracellular Ca2+, which may be at the high end of environmentally relevant concentrations. However, we have no accurate way to determine the actual in vitro concentration of 7,8-BPQ, and it is well known that this agent extensively binds to proteins and other macromolecules, suggesting that the effective concentration may be far less.

Our previous studies demonstrated that two P450-derived metabolites of BPDE and BaP-7,8-Diol increased tyrosine phosphorylation of Lyn and Syk (Mounho and Burchiel, 1998Go). This phosphorylation is known to result in the activation of PLC{gamma}1 and PLC{gamma}2 leading to cleavage of PIP2 and release of inositol-1,4,5-trisphosphate (IP3) (Winslow et al., 2003Go). IP3-sensitive Ca2+ release receptors are located on the ER in lymphocytes. The current studies also demonstrated the presence of ryanodine receptors on microsomes (ER fraction) obtained from Daudi human B cells. Thus, there are apparently two closely related Ca2+ channels that control Ca2+ release from the ER of lymphoid cells, the IP3R and RyR (Grafton and Thwaite, 2001Go). It has been proposed that IP3 induces a small amount of Ca2+ release from ER, which then triggers a Ca2+-induced Ca2+ release (CICR) mechanism (Fill and Copello, 2002Go). RyR are also activated by increased cytosolic Ca2+ (Pessah et al., 2001Go). Therefore, RyR may play an important role in lymphocytes to induce or maintain Ca2+ signaling responses.

The present study demonstrated that 7,8-BPQ produces a rapid increase in intracellular Ca2+ in Daudi human B cells, human peripheral blood B and T cells, and murine spleen cells. By contrast, the Ca2+ response of other BaP metabolites, including 1,6-BPQ, 3,6-BPQ, and BPDE, occurred at later time points (2–18 h). Therefore, our data suggest that 7,8-BPQ utilizes different signaling pathways to elicit early Ca2+ effects. Because we detected the presence of RyRs in Daudi microsomes, and we found that high concentrations of ryanodine prevented the 7,8-BPQ Ca2+ response in Daudi human B cells, human peripheral blood B and T cells, and murine spleen cells, we believe that RyR are responsible for the Ca2+ response of lymphoid cells to 7,8-BPQ. In our previous work discussed above with BaP-7,8-Diol, we presumed that BaP-7,8-diol was converted to BPDE to produce its effects on src kinases. However, we now know that Daudi and other human lymphocytes express the AKR1C1, and thus they have the ability to make the 7,8-BPQ. Other BPQs also increase intracellular Ca2+, but we believe that these much later effects are caused by redox-cycling and loss of Ca2+ buffering capacity by mitochondria due to ATP depletion, although this hypothesis was not tested in our current studies (Zhu et al., 1995Go). Because we found that 7,8-BPQ induces rapid increases in intracellular Ca2+ levels in human and murine B and T cells, we are now examining pathways for endogenous 7,8-BPQ formation, as well as the regulation of the human AKR1C1 gene in human cells and its homologue in mice.

In summary, we found that 7,8-BPQ is a potent and specific activator of RyR in the present studies leading to a rapid rise in intracellular Ca2+ in human and murine B and T cells. We believe that the early and rapid Ca2+ response produced by 7,8-BPQ may be immunomodulatory and may play a role in the immunotoxicity induced by BaP. BPQs have previously been found to activate murine splenic T cells following in vitro treatment at concentrations of 0.1 and 1 µM (Burchiel et al., 2004Go), although human leukocytes are inhibited following in vitro treatment with BPQs. Ca2+ elevation by 7,8-BPQ may also play a role in immune activation, in that many Ca2+-sensitive pathways regulate cell activation, such as adhesion molecular activation, enzymatic activation, exocytosis, migration and response to other ion channels activation (Panyi et al., 2004Go). Changes in intracellular Ca2+ are also likely important in specific gene regulation, perhaps through PKCs (Lewis, 2003Go). Pomorski et al. (2004)Go found that intracellular calcium changes regulate cytokine expression, such as IL-1ß, and IL8, and effect the monocytes migration. However, this calcium-regulated specific gene expression occurs at relatively late time points. Thus, the early and rapid increase in Ca2+ produced by 7,8-BPQ suggests that RyR may play an important role in lymphocyte activation and may be a target of environmental agents.


    NOTES
 
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.


    ACKNOWLEDGMENTS
 
This study was funded in part by NIEHS RO1-ES04595 (SWB) with flow cytometry support funded in part by the New Mexico NIEHS Center P30-ES012072 (SWB); other support included NIEHS P01-11269 (INP), RO1-CA39S04 (TMP), P01-CA092537 (TMP), and UNM HSC GCRC Grant M01RR-00997. A.A.V. was supported by NIH Training Grant ES07059.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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