Superfund Research Program
Human Cell Culture Studies of Mutagens in the Aberjona Basin
Project Leader: William G. Thilly
Grant Number: P42ES004675
Funding Period: 1995 - 2000
Studies of Aberjona Basin sediments continued to identify PAHs associated with incomplete combustion with marked mutagenicity in the human lymphoblastoid cell mutation assays at the HPRT and TK gene loci. Among those found were cyclopenta(c,d)pyrene and dibenzo(a,h)pyrene. These PAHs were assayed using long term low dose conditions in the AHH-1 cell line.
As with benzo(a)pyrene, it was found that for the first five days of exposure mutant fractions increased rapidly, but after five days through twenty days of exposure the increase in mutant fractions was markedly lower, although significantly greater than observed in untreated cultures. One PAH could induce resistance to mutation attempted by subsequent exposure to other PAH mutagens. Earlier studies with radioactive benzo(a)pyrene indicated that this shift in mutation rates was not due to a shift in metabolic rate because the instantaneous rate of DNA adduct formation remained constant from day two through day twenty.
To extend this observation and test the conclusion that changes in metabolism were not responsible for this shift in mutation rate at day five, researchers used the metabolite benzo(a)pyrene diol epoxide (racemic mixture) and found it, too, induced mutations rapidly for five days and then at a distinctly lower rate thereafter. The relative rates of mutation before and after the five day point were approximately the same for benzo(a)pyrene for which cellular metabolism by cytochrome P450 1A1 is necessary and by the benzo(a)pyrene chemically reactive metabolite which requires no enzymatic activity to act as a mutagen. With these new data scientists have extended the previous conclusion that after 5 days a new cellular condition is induced characterized by a lower mutation rate and independent of changes in the xenometabolic machinery of the AHH1 cells.
To discover if this "mutagen resistant state" is a general property of human cells, the investigators subjected the widely used TK6 cell line to daily exposure to low concentrations of benzo(a)pyrene diol epoxide. For the first five days of exposure, the induced mutant fractions in TK6 cells and AHH1 cells were essentially the same. However, the TK6 cells did not demonstrate induction of a mutagen resistant state at day five but increased in mutant fraction each day as in the first five days through twenty days.