Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line

Rola Barhoumi, Youssef Mouneimne, Kenneth S. Ramos, Stephen H. Safe, Timothy D. Phillips, Victoria E. Centonze, Clay Ainley, Mona S. Gupta, Robert C. Burghardt

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

The uptake and subcellular partitioning of benzo[a]pyrene (BaP) were examined in a rat-liver cell line (Clone 9) using confocal and multiphoton microscopy. Following a 16-h treatment, intracellular accumulation of BaP increased with increasing concentration, and cytoplasmic BaP fluorescence reached saturation at 10 μM. Analysis of the kinetics of BaP uptake at this concentration indicated that BaP is rapidly partitioned into all cytoplasmic membranes within several min, although saturation was not reached until 4 h. Based upon the rapid uptake of BaP into membranes, the chronology of changes in gap junction-mediated intercellular communication (GJIC), plasma membrane potential (PMP), and steady state levels of intracellular Ca2+ in relation to the time-course for induction of microsomal ethoxyresorufin-0-deethylase (EROD) activity were examined. EROD activity in Clone 9 cells treated for 16 h increased with increasing concentrations of BaP and reached the highest levels at 40 μM BaP. In addition, kinetic analysis of EROD activity in Clone 9 cells treated with 10 μM BaP indicated that significant induction of EROD activity was not detected before 3 h, and it reached maximal levels by 16 h of treatment at this concentration. Both GJIC and PMP were directly affected by the partitioning of BaP into cellular membranes. The most sensitive index of BaP-induced changes in membrane function was GJIC which revealed a 25% suppression in cells exposed to 0.4 μM BaP for 16 h. Kinetic analysis revealed that suppression of GJIC occurred within 15 min of exposure of cells to 10 μM BaP, whereas significant suppression of PMP was not detected prior to 30-min exposure at this concentration. Elevation of basal Ca2+ level was also detected simultaneously with PMP at this dose. These data suggest that early changes in cellular membrane functions occur prior to detectable induction of EROD activity, although basal metabolic activation of BaP may contribute to these changes.

Original languageEnglish (US)
Pages (from-to)264-270
Number of pages7
JournalToxicological Sciences
Volume53
Issue number2
DOIs
StatePublished - 2000
Externally publishedYes

Keywords

  • Benzo[a]pyrene
  • Clone 9 cells
  • Confocal microscopy
  • Cytotoxicity
  • Fluorescence
  • Gap junctional intercellular communication
  • Intracellular Ca homeostasis
  • Multiphoton microscopy
  • Plasma membrane potential
  • Rat liver cell line

ASJC Scopus subject areas

  • Toxicology

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