Introduction

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Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are ubiquitous environmental pollutants formed during the burning of fossil fuels and combustion of other products, as well as during cooking (Baum, 1978). B[a]P is a complete carcinogen with both tumor-initiating and tumor-promoting properties (Ethier and Ullrich, 1982). For tumor initiation, carcinogenic PAHs seem to require bioactivation by either a family of cytochrome P450s or aldo-keto reductases to form DNA-reactive metabolites (Dipple, 1994; Penning et al., 1999). B[a]P is also known to alter cell-signaling pathways associated with growth factor receptor-dependent cell proliferation and survival (Tannheimer et al., 1997, 1998), as well as cell to cell interactions (Upham et al., 1998). Carcinogenic PAHs are also immunotoxic at high doses. For example, B[a]P and 7,12-dimethylbenz[a]anthracene suppress humoral and cell-mediated immunity in mice (Davila et al., 1996, 1999).

Burchiel and coworkers have shown a correlation between the ability of PAHs to produce immunosuppression and their ability to elevate intracellular Ca²⁺. B[a]P is one of several carcinogenic PAHs that increase intracellular Ca²⁺ in lymphoid (Mounho et al., 1997; Romero et al., 1997; Mounho and Burchiel, 1998) and nonlymphoid cells (Holsapple et al., 1996; Tannheimer et al., 1999). Several mechanisms have been shown to be responsible for altered Ca²⁺ homeostasis. In human T cells, such PAHs as dimethylbenz[a]anthracene have been found to activate src-related proto-oncogenes, leading to a rapid IP₃-dependent Ca²⁺ release from endoplasmic reticulum (Archuleta et al., 1993). However, because this activity has been also seen by some noncarcinogenic PAHs, the specificity of this action is questioned (Davila et al., 1999). Further results indicated that immunotoxic PAHs, such as B[a]P, inhibited ATP-dependent ⁴⁵Ca²⁺ uptake into microsomal vesicles but not by the less immunotoxic compounds anthracene and benzo[e]pyrene. These effects correlated well with decreased SERCA-type Ca²⁺-ATPase catalytic activity but affected neither Na⁺/K⁺-ATPase activity nor plasma membrane Ca²⁺-ATPase activities (Krieger et al., 1995). However, recent work aimed at characterizing the interactions between PAHs and specific SERCA isoforms (including SERCA1, -2a, and -3) demonstrated that PAHs do not have a direct inhibitory effect on the cloned rat SERCAs expressed in human embryonic kidney 293 cells (Zhao et al., 1996). Although thapsigargin (100 nM) and 2,5-di(t-butyl)-1,4-benzohydroquinone (10 µM) completely inhibited each of the heterologously expressed SERCA isoforms, B[a]P and six related PAH structures failed to inhibit any SERCA activity at concentrations as high as 10 µM (Zhao et al., 1996). These results suggest that the Ca²⁺-elevating activity of B[a]P and its known primary metabolites occurs independent of SERCA. Therefore alternate mechanisms may be involved in altered Ca²⁺ homeostasis.

Although B[a]P o-quinones derived from the trans-dihydrodiols have been shown to decrease cell viability and cause cell death, presumably by GSH depletion (Flowers-Geary et al., 1993, 1995), the underlying mechanism(s) are not understood. Because the cytoxicity of B[a]P o-quinones may be mediated via enzymatic one-electron redox cycling, we investigated the activity of B[a]P and five metabolites for their ability to alter Ca²⁺ transport across junctional sarcoplasmic reticulum (SR) membrane vesicles isolated from skeletal muscle. SR preparations serve as a suitable model to elucidate mechanisms of altered Ca²⁺ transport because they have a well-defined Ca²⁺ transport system. The SERCA-1 pump mediates Ca²⁺ sequestration, whereas Ca²⁺-induced Ca²⁺ release (CICR) channels, also known as ryanodine receptors (RyR), mediate the release of stored Ca²⁺. In light of the exquisite sensitivity of RyR to sulfhydryl reagents and redox-active quinones (Feng et al., 1999; Pessah and Feng, 2000) we investigated the hypothesis that RyR rather than SERCA may be a selective target for redox-active B[a]P o-quinones.

	Materials and Methods

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Preparation of SR Membranes. Sarcoplasmic reticulum (SR) membrane vesicles enriched in RyR type 1 (RyR1) and SERCA1 were prepared from back and hind limb skeletal muscles of New Zealand White rabbits according to the method of Saito et al. (1984). The preparations were stored in 10% sucrose, 5 mM imidazole, pH 7.4, at 80°C.

Ca²⁺ ATPase Activity. Rates of ATP hydrolysis were determined with a coupled enzyme assay measuring the oxidation of NADH as a linear decrease in absorbance at 340 nm (Schwartz et al., 1969). SR membrane vesicles (50 µg of protein) were added to the temperature-controlled cuvettes at 37°C. The cuvettes contained assay buffer consisting of 5 mM HEPES, pH 7.0, 100 mM KCl, 5 mM MgCl₂, 60 µM EGTA, 100 µM CaCl₂, 0.3 mM sucrose, 2 mM phospho(enol)pyruvate, 0.8 mM NADH, 24 U/ml lactate dehydrogenase, 16.8 U of pyruvate kinase, and 1.5 µg/ml of the Ca²⁺ ionophore A23187 (final volume, 1.2 ml). B[a]P or its derivatives were introduced into test cuvettes 2 min before the start of the reaction, whereas paired control reactions received an equivalent volume of solvent (DMSO). DMSO was  1% in the final assay medium. After zeroing the spectrophotometer, reactions were started by addition of 1 mM Na₂ATP and the total ATPase activity (measured as a linear decline in NADH absorbance) monitored for at least 30 s. The Ca²⁺-independent (non-SERCA) component of ATPase activity was measured by the addition of either 4 mM K₂EGTA or 1 µM thapsigargin to the reaction mixture. Ca²⁺-dependent rates were calculated as the difference between total ATPase and Ca²⁺-independent rates.

Macroscopic Ca²⁺ Transport Measurement. Ca²⁺ transport across SR vesicles was measured with the membrane-impermeant Ca²⁺-sensitive dye antipyrylazo III using a diode array spectrophotometer (model 8452; Hewlett Packard, Palo Alto, CA). Skeletal SR vesicles (42 µg/ml) were added to 1.15 ml of ATP-regenerating buffer consisting of 95 mM KCl, 20 mM MOPS, 7.5 mM sodium pryophosphate (Palade, 1987), 250 µM antipyrylazo III, 12 µg of creatine phosphokinase, 5 µM phosphocreatine, and 1 mM MgATP, pH 7.0 (final volume of 1.2 ml). Transport assays were performed at 37°C in temperature-controlled cuvettes with constant stirring. SR vesicles were loaded with seven sequential additions of 24 nmol of CaCl₂, which constituted approximately 80% of their loading capacity. Net Ca²⁺ fluxes across SR vesicles were measured by monitoring extravesicular changes in free Ca²⁺ by subtracting the absorbance of antipyrylazo III at 790 nm from absorbance at 710 nm at 2- to 4-sec intervals. At the end of each experiment, the total intravesicular Ca²⁺ was determined by addition of 3 µM A23187, a Ca²⁺ ionophore, and the absorbance signals were calibrated by addition of 12 or 24 nmol of CaCl₂ from a National Bureau of Standard stock solution. The actions of B[a]P and its derivatives were studied by adding the compound after the loading phase was complete in the presence or absence of known inhibitors or RyR and in the presence of 0.1 or 5 µM extravesicular free Ca²⁺. None of the PAHs used in this study interfered with the absorbance properties or calibration of the antipyrylazo III dye.

Measurement of [³H]Ryanodine-Binding and Data Analysis. SR membrane vesicles (25 µg of protein/ml) were incubated in the presence or absence of B[a]P-7,8-dione in assay buffer containing 2- mM HEPES, pH 7.1, 250 mM KCl, 15 mM NaCl, 50 µM CaCl₂, and [³H]ryanodine (2 nM). Apparent association kinetics were determined in the presence and absence of B[a]P or B[a]P-7,8-dione and the binding reactions were quenched at times ranging between 5 min and 3 h by filtration through GF/B glass-fiber filters. The filters were washed twice with ice-cold harvest buffer, composed of 20 mM Tris-HCl, 250 mM KCl, 15 mM NaCl, and 50 µM CaCl₂, pH 7.1. Each assay was performed in duplicate and repeated at least twice. Nonspecific binding was determined by incubating SR vesicles in the presence of 1000-fold excess unlabeled ryanodine. Association kinetics were analyzed excluding the inhibition phase (when present) by fitting to a single exponential, and calculating the apparent association rate constant (k_obs) and apparent half-time (T_1/2) (ENZFITTER, Elsevier Biosoft, Cambridge, UK).

Single Channel Kinetics in Bilayer Lipid Membranes. RyR1 channels were reconstituted into artificial planar lipid bilayer (5:2 phosphatidylethanolamine/phosphatidylcholine, 60 mg/ml in decane) by introducing membrane vesicles to the cis chamber. The cis chamber contained 0.7 ml of 500 mM CsCl, 50 µM CaCl₂, and 10 mM HEPES, pH 7.4, whereas the trans side contained 100 mM CsCl, 50 µM CaCl₂, and 10 mM HEPES, pH 7.4. Upon the fusion of SR vesicle into bilayer, the cis chamber was perfused with the identical solution lacking CaCl₂. Single-channel activity was measured at a holding potential of +30 mV (applied cis relative to the trans ground side) using a patch-clamp amplifier (Dagan 3900). The data was filtered at 1 kHz before acquisition at 10 kHz by a DigiData 1200 (Axon Instruments, Foster City, CA). The data were analyzed using pClamp 7 (Axon Instruments) without additional filtering. Control data were obtained for at least 30 s before addition of the test compound. Once the test compound was present, data were collected for at least 10 min.

Chemical Reagents. Unless otherwise indicated, all reagents, including B[a]P, were obtained from Sigma-Aldrich (St. Louis, MO). Ryanodine was obtained from Calbiochem. B[a]P-7,8-diol, (±)anti-BPDE, epoxide, and diones were obtained from the National Cancer Institute's Chemical Repository at the Midwestern Research Institute (Kansas City, MO), except for the B[a]P-7,8-dione, which was synthesized according to published methods (Sukumuran and Harvey, 1980). All PAHs were assessed for purity by high-performance liquid chromatography and were greater than 95% pure at the time of use. Stock (1000×) PAH were prepared in anhydrous DMSO and were stored at 80°C under nitrogen until used.

	Results

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B[a]P-7,8-dione Selectively Mobilizes Ca²⁺ from SR. Ca²⁺ fluxes were measured across SR vesicles under conditions of active loading in the presence of ATP and a regenerating system. Once Ca²⁺ loading approached 70 to 80% of the empirically determined capacity of the vesicles, B[a]P and its metabolites (Fig. 1) were added singly to test their ability to release accumulated Ca²⁺. B[a]P-7,8-dione was found to have a high efficacy toward mobilizing stored Ca²⁺, giving an efflux rate of 2.2 µmol Ca²⁺ mg¹ min¹ (Fig. 2). By contrast, B[a]P, B[a]P-3,6-dione, B[a]P-6,12-dione, B[a]P-7,8-diol, and (±)-anti-BPDE failed to mobilize Ca²⁺ from actively loaded SR vesicles under the assay conditions used.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Structures of benzo[a]pyrene and relevant metabolites.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Among related B[a]P structures, only B[a]P-7,8-dione induces net Ca2+ efflux from actively loaded junctional sarcoplasmic reticulum. Ca2+ fluxes were measured with antipyrylazo III in cuvettes containing 40 µg/ml of microsomal protein in transport buffer consisting of MgATP (1 mM) and coupling enzyme as described under Materials and Methods. Vesicles were actively loaded by seven sequential additions of CaCl2 (20 µM). Upon completion of the loading phase, B[a]P-7,8-dione, B[a]P-3,6-dione, B[a]P-6,12-dione, B[a]P-7,8-diol, (±)-anti-BPDE, B[a]P (each at a final concentration of 10 µM), or DMSO (10 µL) were added and the change in extravesicular Ca2+ monitored at 710 to 790 nm. The rate giving 100% was 2.17 µmol of Ca2+ mg1 min1. The data is the average of two determinations.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   B[a]P-7,8-dione induces microsomal Ca2+ release by selective activation of ryanodine receptor type 1 (RyR1). Cuvettes containing microsomal vesicles (40 µg/ml protein) in Ca2+ transport buffer were actively loaded by seven sequential additions of CaCl2 (20 µM each). Top trace, when loading was complete, a saturating concentration of B[a]P-7,8-dione (10 µM) was introduced into the cuvette (first arrow). RR (2 µM), a selective blocker of RyR1, was added at the peak of the B[a]P-7,8-dione response (second arrow), and the vesicles actively reaccumulated the released Ca2+. Middle trace, when loading was complete, the alkaloid ryanodine (Ry; 500 µM) was introduced to the reaction mixture (first arrow), causing a characteristic sequential activation and irreversible inhibition of RyR1. After the RyRs were blocked, an addition of B[a]P-7,8-dione (10 µM) was unable to induce release of the accumulated intravesicular Ca2+ (second arrow). Bottom trace, when the Ca2+ loading phase was completed, RR (2 µM) was first introduced (first arrow). Approximately 1 min later, B[a]P-7,8-dione (10 µM) was added (second arrow) although it was ineffectual in releasing microsomal Ca2+. The effects of B[a]P-7,8-dione were consistently inhibited by known blockers/inactivators of RyR. Each experiment was repeated at least three times with similar results.

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View larger version (26K): [in this window] [in a new window]   Fig. 4.   B[a]P-7,8-dione induces microsomal Ca2+ release in a dose- and Ca2+-dependent manner. Vesicles (40 µg/ml protein) were actively loaded by seven sequential additions of CaCl2 (20 µM each). After the active loading phase, B[a]P-7,8-dione was added to the cuvette. Rates were taken as the difference between the B[a]P-7,8-dione-containing reaction vessel and a control reaction vessel, which was measured in parallel. A, dose responses were performed either in the presence of 100 nM () or 5 µM () free extravesicular Ca2+. Data shown are the average and range of two trials () or the mean and S.D. of three trials (). Ionophore A23187 was added at the end of each experiment to assure equal loading and to calibrate the dye signal. B, dose-response data were analyzed by logit-log plots. Linear regression was performed to determine the slope and EC50 value. The presence of 5 µM free extravesicular Ca2+ (), a concentration that sensitizes RyR1 to CICR, produced a threshold and EC50 value for 7,8 BPQ of 400 nM and 2 µM, respectively. The logit slope was 0.82. By contrast, in the presence of 100 nM extravesicular Ca2+ (), a concentration that minimizes CICR, gave a threshold and EC50 value of 2 µM and 6 µM, respectively. The logit slope was 2.0. The maximum rates of release with saturating B[a]P-7,8-dione were 1.92 and 0.12 µmol Ca2+ mg1 min1 in the presence of 5 µM and 100 nM free extravesicular Ca2+, respectively, a difference in efficacy of 16-fold.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   B[a]P-7,8-dione mobilizes microsomal Ca2+ in a time-dependent manner. Cuvettes containing microsomal vesicles (40 µg/ml protein) were actively loaded by seven sequential additions of CaCl2 (20 µM each). Following active loading, B[a]P-7,8-dione was added to the cuvette in the presence of 5 µM final free Ca2+. The time to release ~15% (20 nmol) of the total stored Ca2+ (140 nmol) was measured as a function of B[a]P-7,8-dione concentration. The data shown are mean ± S.D. of three determinations.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   B[a]P-7,8-dione does not significantly alter the rate of Ca2+-dependent ATP hydrolysis by SERCA pumps at concentrations that maximally stimulate RyR1. Microsomes (10.4 µg/ml protein) were added to an ATPase buffer containing coupling enzymes, substrates, NADH, and Ca2+ ionophore as defined under Materials and Methods. Ca2+-dependent oxidation of NADH was monitored at 340 nm. After 1-min equilibration, Na2ATP (1 mM) was added to the reaction to commence measurement of total ATPase activity. After 3 min, EGTA (4 mM) was added to determine the Ca2+-independent rate of ATP hydrolysis. Rate of ATP hydrolysis by SERCA pump was reported as the difference between total and Ca2+ independent rates of hydrolysis. In these microsomal preparations, SERCA activity accounted for 95% of the total ATPase activity. Each condition was repeated at least twice and the bar graph reports the mean value with the corresponding S.D.

B[a]P-7,8-dione Induces Direct Biphasic Actions on RyR1 Function. Recently it has been shown that nanomolar concentrations of redox-active naphthalene-1,4-dione enhance SR Ca²⁺ release by modifying RyR1 activity, whereas low micromolar concentrations produce a time-dependent activation and subsequent inhibition of RyR1 function (Feng et al., 1999). We examined whether a direct modification of RyR1 function could account for B[a]P-7,8-dione-induced changes in microsomal Ca²⁺ fluxes described above. Single RyR1 channels were reconstituted in bilayer lipid membranes and the influence of B[a]P-7,8-dione on channel gating kinetics studied under voltage clamp. Figure 7A shows the gating kinetics of a typical RyR1 channel in the presence of 7 µM Ca²⁺ on the cytoplasmic (cis) side of the bilayer lipid membrane chamber. Under these conditions, channel transitions from closed to open are extremely fast, but rare, giving an open probability (P_o) of 0.011. After addition of 1 µM B[a]P-7,8-dione to the cis chamber, the P_o increased 7-fold to 0.079. However, within 10 min of introducing B[a]P-7,8-dione, the channel ceased to gate. Figure 7B shows the same type of experiment performed on another channel with higher (100 µM) Ca²⁺ in the cis chamber. Although the channel initially has a higher P_o because of the optimal cis Ca²⁺ for channel activation, addition of 1 µM B[a]P-7,8-dione enhanced P_o by approximately 6-fold. As seen with lower cis Ca²⁺, the activating effect of B[a]P-7,8-dione was transient, culminating in persistent inhibition of the channel within 10 min. Figure 7C shows 12 min of continuous channel recording, highlighting the biphasic actions of B[a]P-7,8-dione on RyR1. In separate experiments, addition of B[a]P to 10 µM failed to alter channel activity over a period of 10 min (not shown).

View larger version (51K): [in this window] [in a new window]   Fig. 7.   B[a]P-7,8-dione modifies RyR1 channel gating kinetics in a biphasic manner. Single RyR1 channels were reconstituted in planar bilayer lipid membranes and current transitions measured under voltage clamp as described under Materials and Methods. A, a typical channel exhibiting low Po in the presence of suboptimal cytoplasmic Ca2+ (7 µM). After addition of 1 µM B[a]P-7,8-dione to the cytoplasmic side of the channel, transiently increased Po by 7-fold. Within 10 min of addition, B[a]P-7,8-dione induced channel closure. B, a typical channel in the presence of optimal cytoplasmic Ca2+ exhibits higher Po (more frequent transitions to the open state). Addition of B[a]P-7,8-dione to the cytoplasmic side of the channel further increases Po 6-fold; again, this activation is transient. Within 10 min, B[a]P-7,8-dione induces channel closure. C, a summary showing the fluctuation of channel Po from a complete 12-min record. Note the characteristic delay in the onset of B[a]P-7,8-dione-induced channel activation and subsequent inhibition. These results were repeated on four separate channels.

	Discussion

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Polycyclic aromatic hydrocarbons are environmental pollutants that are known to be carcinogenic and immunotoxic. In intact cell assays, B[a]P in particular has been shown to disrupt Ca²⁺ homeostasis in both immune and nonimmune cells, but the molecular mechanism has not been defined. Using a well-defined junctional SR model system, the present study identifies for the first time that a single o-quinone metabolite of B[a]P, B[a]P-7,8-dione, can account for altered Ca²⁺ transport across the microsomal membrane. The key findings in this study are: 1) metabolic bioactivation of B[a]P to B[a]P-7,8-dione is required for deregulation of Ca²⁺ transport. Other metabolites, including the B[a]P-3,6- and -6,12-diones, the B[a]P-7,8-diol, and the (±)-anti-BPDE, lack activity at the concentrations tested. 2) B[a]P-7,8-dione disrupts Ca²⁺ transport by influencing the activity of RYR1 without any measurable influence on SERCA or membrane integrity. 3) The actions of B[a]P-7,8-dione are biphasic, involving a time-dependent activation and subsequent inactivation of channel function.

Several different reactive metabolites of B[a]P are derived from P450-dependent pathways. The P450 pathways represented by CYP1A1 or CYP1B1 form phenolic metabolites and epoxides and, in the presence of epoxide hydrolase, produce the 7,8-dihydrodiol of B[a]P (B[a]P-7,8-diol). B[a]P-7,8-diol can enter another round of metabolism by CYP1A1 or CYP1B1 to form 7,8-diol-9,10-epoxide of B[a]P [(±)-anti-BPDE] (Conney, 1982). In addition to the metabolism of diols and diol-epoxides, cytochrome P450 enzymes can also catalyze the production of hydroxylated metabolites leading to the B[a]P-6,12-, 1,6-, and 3,6-diones. The cytochrome P450-peroxidase activity will also give rise to reactive radical cations (Cavalieri and Rogan, 1995). Recent studies have shown that o-quinones can also be formed from the B[a]P-7,8-diol in the presence of dihydrodiol dehydrogenase, members of the aldo-ketoreductase (AKR) superfamily (Smithgall et al., 1988; Penning et al., 1999). B[a]P o-quinones are of interest because they undergo redox-cycling, leading to the production of oxygen free radicals that can exert oxidative stress in cells. Within this metabolic scheme, enzymatic oxidation of B[a]P-diol leads to the formation of a catechol that undergoes autoxidation to form B[a]P-7,8-dione. Conversion to the o-quinone results in production of reactive oxygen species. Once formed, the o-quinone can either enter futile redox cycles to generate reactive oxygen species or it can form thioether conjugates. In support of one or more "bioactivation" steps in B[a]P-induced Ca²⁺ mobilization from microsomal stores, Burchiel and coworkers have shown that certain oxidative and electrophilic metabolites of B[a]P increase intracellular Ca²⁺ in human B cells (Mounho and Burchiel, 1998) and human mammary epithelial cells (Tannheimer et al., 1999). Dimunition of B[a]P-induced increases in intracellular Ca²⁺ by the AhR partial antagonist and cytochrome P450 inhibitor, -naphthaflavone, suggests that cytochrome P450 metabolism is required for altered Ca²⁺ homeostasis in lymphoid and nonlymphoid cells. Elevation of intracellular Ca²⁺ may be associated with altered cell proliferation in human mammary epithelial cells (Tannheimer et al., 1997) and the induction of apoptosis in human B cells (Salas and Burchiel, 1998).

It is not easy to assess which tissues may be affected by B[a]P-7,8-dione. Five different human AKRs are responsible for its formation from the corresponding BP-7,8-diol. These are AKR1C1 through AKR1C4 and AKR1A1 (which is aldehyde reductase) (Burczynski et al., 1998). Tissue distribution studies show that AKR1C4 is hepatic specific, whereas the remaining isoforms each show their own patterns of distribution and differ in their catalytic efficiencies for BP-7,8-diol (Penning et al., 2000). Liver has been shown to possess specific [³H]ryanodine-binding protein (Shoshan-Barmatz et al., 1991). Moreover, breast cancer cells (MCF10A) and such immune cells as B-lymphocytes, also suspected targets of B[a]P and its metabolites, have been shown to express RyR (S. W. Burchiel and I. N. Pessah, unpublished observations). However, further confounding this issue is whether these isoforms are coexpressed with CYP1A1 and epoxide hydrolase, which are required for the production of the diol substrate. Also, of these human isozymes, AKR1C1 can be robustly induced by planar aromatics and reactive oxygen (Burczynski et al., 1999).

Within the metabolic scheme involving AKRs, B[a]P-7,8-dione is the only compound that exhibits potent and efficacious disruption of microsomal Ca²⁺ transport. Furthermore toxicologically relevant concentrations (nanomolar to low micromolar) of B[a]P-7,8-dione alter Ca²⁺ transport by a highly selective mechanism involving RYR1 without measurable changes in SR membrane integrity or decreased SERCA pump activity. Considering the emerging role of hyper-reactive sulfhydryl chemistry in regulating RyR complexes (Pessah and Feng, 2000), this finding is not surprising. A small number of hyper-reactive thiols have been shown to exist within the RyR complex (Liu et al., 1994; Liu and Pessah, 1994). Their functional role does not seem to directly affect overt aspects of channel gating. Rather hyper-reactive Cys moieties may represent biochemical components of a redox sensor that conveys information about localized changes in redox potential produced by physiologic (e.g., glutathione, nitric oxide) and pathophysiologic (e.g., quinones, reactive oxygen species) channel modulators to the Ca²⁺ release process.

Recently, the functional role of hyper-reactive sulfhydryl moieties within the RyR complex in Ca²⁺ deregulation by redox active quinones such as naphthalene-1,4-dione (Feng et al., 1999). 7-Diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM, a fluorogenic maleimide) was used to measure the reactivity of hyper-reactive sulfhydryl moieties on SR membranes in the presence and absence of naphthalene-1,4-dione by analyzing the kinetics of forming CPM-thioether adducts and localization of fluorescence by SDS-PAGE. Naphthalene-1,4-dione (NQ) selectively and dose dependently (EC₅₀ = 0.3 µM) interacts with a class of hyper-reactive sulfhydryl groups localized on RyR1 and its associated protein triadin. Similar to B[a]P-7,8-dione in the present study, nanomolar NQ enhanced the association of [³H]ryanodine for its high-affinity binding site and directly enhanced channel P_o in bilayer lipid membrane measurements in a reversible manner. By contrast, micromolar NQ produced a time-dependent biphasic action on channel function, leading to irreversible channel inactivation. Most importantly, the sensitivity to redox-active quinones was eliminated upon formation of thioether adducts between CPM and the most reactive thiols of the RyR channel complex. The Ca²⁺-dependent cytotoxicities observed with reactive quinones formed at the microsomal surface by oxidative metabolism may be related to their ability to selectively modify hyper-reactive thiols regulating normal functioning of microsomal Ca²⁺ release channels. Importantly, these results raise the possibility that microsomal Ca²⁺ channels may actually utilize hyper-reactive sulfhydryl chemistry in "sensing" localized changes in redox environment. B[a]P-7,8-dione undergoes 1,4-Michael addition with thiols to yield catechol conjugates which then autooxidize to give o-quinone thiol-ether conjugates with the production of reactive oxygen species (Smithgall et al., 1988). In addition, measurements of oxygen metabolism during the AKR-dependent oxidation of B[a]P-7,8-diol to B[a]P-7,8-dione showed that hydrogen peroxide was formed before undergoing arylation reactions with GSH or protein acceptors. Thus the biphasic effects observed with B[a]P-7,8-dione could be explained by its redox and electrophilic properties. The early activation phase could be the direct result of redox cycling with hyper-reactive (redox sensing) Cys within the RyR1 complex, whereas the subsequent inhibition phase may result from arylation of the receptor at one or more sites. Importantly hydrogen peroxide has been shown to activate RyR1 and microsomal Ca²⁺ release (Favero et al., 1995). Exposure of intact cells to B[a]P could also influence redox sensing indirectly by local production of hydrogen peroxide and/or changes in the ratio of GSH to GSSG (Zable et al., 1997; Feng et al., 2000), provided the appropriate oxidative and AKR1C enzymes are expressed. What drives redox cycling in these in vitro measurements that lack any exogenously added reducing equivalents (e.g., NADPH, GSH)? One possible mechanism is that the primary electron donor is the RyR1 complex itself. Alternatively, Michael addition of either thiols or buffer nucleophiles to the quinone will produce a ketol, which will rearrange to a catechol conjugate. The catechol conjugate will autoxidize in air to produce reactive oxygen species on route to the o-semiquinone anion radical and eventually the quinone conjugate. If contaminating transition metal ions were present, they would catalyze the 1-electron redox-cycle between the o-semiquinone anion radical and the catechol conjugate with concomitant production of reactive oxygen (Flowers et al., 1997). This situation will exist unless all solutions used in the assay were Chelex treated.

In conclusion, RyR1 and its two alternate isoforms are broadly expressed in mammalian cell types. For example, RyR1 has been recently identified in B lymphocytes (Sei et al., 1999) and RyR protein is expressed MCF10A breast cancer cells (I. N. Pessah and S. W. Burchiel, unpublished observations). Considering the important role of RyR in Ca²⁺-signaling, the observation of a selective mechanism mediating RyR dysfunction by B[a]P-7,8-dione may be of pathophysiological significance.