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

The uptake and subcellular partitioning of benzo[a]pyrene (BaP) were examin ed in a rat-liver cell line (Clone 9) using confocal and multiphoton micros copy. Following a 16-h treatment, intracellular accumulation of BaP increas ed with increasing concentration, and cytoplasmic BaP fluorescence reached saturation at 10 mu M. Analysis of the kinetics of BaP uptake at this conce ntration indicated that BaP is rapidly partitioned into all cytoplasmic mem branes within several min, although saturation was not reached until 4 h. B ased upon the rapid uptake of BaP into membranes, the chronology of changes in gap junction-mediated intercellular communication (GJIC), plasma membra ne potential (PMP), and steady state levels of intracellular Ca2+ in relati on to the time-course for induction of microsomal ethoxyresorufin-0-deethyl ase (EROD) activity were examined. EROD activity in Clone 9 cells treated f or 16 h increased with increasing concentrations of BaP and reached the hig hest levels at 40 mu M BaP. In addition, kinetic analysis of EROD activity in Clone 9 cells treated with 10 mu M BaP indicated that significant induct ion of EROD activity was not detected before 3 h, and it reached maximal le vels by 16 h of treatment at this concentration. Both GJIC and PMP were dir ectly 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 mu M BaP for 16 h. Kine tic analysis revealed that suppression of GJIC occurred within 15 min of ex posure of cells to 10 mu M BaP, whereas significant suppression of PIMP 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. Th ese data suggest that early changes in cellular membrane functions occur pr ior to detectable induction of EROD activity, although basal metabolic acti vation of BaP may contribute to these changes.