In vitro mutational spectrum of cyclopenta[cd]pyrene in the human HPRT gene

Cyclopenta[cd]pyrene (CPP) is a widely distributed polycyclicaromatic hydrocarbon with potent mutagenic and carcinogenicactivity. In order to acquire an understanding of the mutagenicpathways of CPP, we studied mutations induced by this chemicalin human cells. Four independent cultures of a human cell lineexpressing cytochrome P450 CYP1A1 (cell line MCL-5) were treatedwith CPP, and mutants at the hypoxanthine phosphoribosyltransferase(HPRT) locus were selected en masse by 6-thioguanine (6TG) resistance.The kinds and positions of the mutations were analyzed usingthe combination of high-fidelity polymerase chain reaction (hifi-PCR)and denaturing gradient gel electrophoresis (DGGE). The thirdexon of the HPRT gene was amplified from the 6TG-resistant cellsusing the hifi-PCR and the amplified fragment was subsequentlyanalyzed by DGGE to separate mutant sequences from the wild-typesequence. Mutant bands were excised from the gel, amplifiedusing PCR and sequenced. Sixteen different mutations were identifiedand consisted mostly of the G to T and A to T transversions.Other mutations identified included G to A and A to G transitions,a G to C transversion, and a single G deletion. Of these mutations,six occurred within a run of six guanines. The predominanceof transversions involving a guanine or an adenine observedwith CPP is similar to the data previously reported for theracemic mixture of benzo[a]pyrene (B[a]P), suggesting that themechanisms of mutation induced by CPP may be similar to thoseinduced by B[a]P.