Nongenotoxic Effects of Polycyclic Aromatic Hydrocarbons and Their Ozonation By-Products on the Intercellular Communication of Rat Liver Epithelial Cells

Since polycyclic aromatic hydrocarbons (PAHs) are known to have epigenetic effects, we evaluated the effect of the parent chemical and the ozonated products on in vitro cell to cell communication bioassays which measures a nongenotoxic event. The scrape loading/dye transfer (SL/DT) technique was used to determine the effect of the following PAHs on gap-junction intercellular communication (GJIC): fluorene, 1-methyl-fluorene, fluoranthene, anthracene, 9-methyl-anthracene, phenanthrene, pyrene, benzo(a)pyrene, and benzo(e)pyrene. The methylated PAHs were more inhibitory to GJIC than the unmethylated counterparts. Fluoranthene, which has an additional ring added to fluorene, was more effective in inhibiting GJIC than fluorene. The three-ringed PAHs were also more inhibitory than the four-and five-ringed PAHs. A time-course study of fluoranthene and of pyrene resulted in maximal inhibition occurring within 30 min of incubation with the cells. The cells recovered from the inhibition within 1 hr after fluoranthene and pyrene were removed from the cell culture medium. Pyrene, benzo(a)pyrene, fluorene, and fluoranthene were ozonated until the parent compound was completely eliminated as determined by reversephase high-pressure liquid chromatography (RP-HPLQ. An increased level of inhibition of GJIC was observed for the ozonated mixtures of by-products of pyrene, fluoranthene, and benzo(a)pyrene, but not for fluorene, as monitored with the SL/ DT technique. The products of the ozonated pyrene mixture were fractionated and collected by RP-HPLC. Each fraction was found to be inhibitory to GJIC as monitored by fluorescence recovery after photobleaching. In conclusion, current treatment technologies, such as ozonation or biologically based oxidations and methylations, do not necessarily eliminate toxicity. Therefore, it is imperative that toxicological studies be used to complement traditional chemical detection techniques used to monitor the fate of a pollutant in environmental treatment systems.