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

  1. Isaac N. Pessah1,
  2. Chris Beltzner1,
  3. Scott W. Burchiel2,
  4. Gopishetty Sridhar3,
  5. Trevor Penning3 and
  6. Wei Feng1
  1. 1Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California (I.N.P., C.B., W.F.);2College of Pharmacy Toxicology Program, The University of New Mexico, Albuquerque, New Mexico (S.W.B.); and 3Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania (G.S., T.P.)

    Abstract

    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+ min−1 mg−1. 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.

    Footnotes

    • Send reprint requests to: Dr. Isaac N. Pessah, Department of Molecular Biosciences, School of Veterinary Medicine, University of California Davis, Davis, CA 95616. E-mail:inpessah{at}ucdavis.edu

    • Supported by National Institutes of Health Grants ES05707and ES10173 (I.N.P.); ES07259 and ES05495 (S.W.B.), and CA39504 (T.M.P.).

    • Abbreviations:
      PAH
      polycyclic aromatic hydrocarbon
      B[a]P
      benzo[a]pyrene
      SERCA
      sarcoplasmic/endoplasmic reticulum ATPase
      CICR
      Ca2+-induced Ca2+ release
      SR
      sarcoplasmic reticulum
      RyR
      ryanodine receptor
      DMSO
      dimethyl sulfoxide
      MOPS
      potassium 3-(N-morpholino)propanesulfonic acid
      B[a]P-7,8-diol
      (±)-trans-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene
      (±)-anti-BPDE
      (±)-anti-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene
      RR
      ruthenium red
      AKR
      aldo-ketoreductase
      CPM
      7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin
      NQ
      naphthalene-1,4-dione
      • Received August 31, 2000.
      • Accepted November 7, 2000.
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    1. Molecular Pharmacology March 1, 2001 vol. 59 no. 3 506-513
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