Vol. 59, Issue 3, 506-513, March 2001

A Bioactive Metabolite of Benzo[a]pyrene, Benzo[a]pyrene-7,8-dione, Selectively Alters Microsomal Ca²⁺ Transport and Ryanodine Receptor Function

Isaac N. Pessah, Chris Beltzner, Scott W. Burchiel, Gopishetty Sridhar, Trevor Penning, and Wei Feng

Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California (I.N.P., C.B., W.F.); College of Pharmacy Toxicology Program, The University of New Mexico, Albuquerque, New Mexico (S.W.B.); and Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania (G.S., T.P.)

Polycyclic aromatic hydrocarbons are environmental pollutants known to be carcinogenic and immunotoxic. In intact cell assays, benzo[a]pyrene (B[a]P) disrupts Ca²⁺ homeostasis in both immune and nonimmune cells, but the molecular mechanism is undefined. In this study, B[a]P and five metabolites are examined for their ability to alter Ca²⁺ transport across microsomal membranes. Using a well-defined model system, junctional SR vesicles from skeletal muscle, we show that a single o-quinone metabolite of B[a]P, B[a]P-7,8-dione, can account for altered Ca²⁺ transport across microsomal membranes. B[a]P-7,8-dione induces net Ca²⁺ release from actively loaded vesicles in a dose-, time-, and Ca²⁺-dependent manner. In the presence of 5 µM extravesicular Ca²⁺, B[a]P-7,8-dione exhibited threshold and EC₅₀ values of 0.4 and 2 µM, respectively, and a maximal release rate of 2 µmol of Ca²⁺ min¹ mg¹. The mechanism by which B[a]P-7,8-dione enhanced Ca²⁺ efflux was further investigated by measuring macroscopic fluxes and single RyR1 channels reconstituted in bilayer lipid membranes and direct measurements of SERCA catalytic activity. B[a]P-7,8-dione ( 20 µM) had no measurable effect on initial rates of Ca²⁺ accumulation in the presence of ruthenium red to block ryanodine receptor (RyR1), nor did it alter Ca²⁺-dependent (thapsigargin-sensitive) ATPase activity. B[a]P-7,8-dione selectively altered the function of RyR1 in a time-dependent diphasic manner, first activating then inhibiting channel activity. Considering that RyR1 and its two alternate isoforms are broadly expressed in mammalian cells and their important role in Ca²⁺-signaling, the present results reveal a mechanism by which metabolic bioactivation of B[a]P may mediate RyR dysfunction of pathophysiological significance.