Comparison of DNA adduct formation by means of synchronous scanning and by isotope tracers: in-vitro study on formation of DNA adducts in human lymphocytes exposed to benzo(a)pyrene.
The P-450 complex transforms polyaromatic hydrocarbons (PA) into active intermediates that may cause mutations due to DNA adduct formation. The present communication compares the rate of DNA adduct formation in human lymphocytes incubated for varying times with different concentrations of benzo(a)pyrene (B(A)P). In the presence of B(A)P the cultures were standardized as to medium and lectin concentration. Then the cells were exposed to increasing levels of cold and 3H-labelled B(A)P for varying times. B(A)P adducted to DNA was estimated both by synchronous scanning (SS) and by counting radioactivity. SS revealed a synchronous signal at 382 nm, corresponding to data available from the literature, and the peak height declined linearly with dose of B(A)P. However, it appeared that the signal height decreased if the DNA was successively extracted with chloroform. Five times extraction gave rise to a stable content of B(A)P of about 25% of the B(A)P originally found in the DNA. This DNA could only be traced with the radioactive tracer, since the concentration of adducted B(A)P was below the lower level of detection by the SS method. Even at low B(A)P levels the carcinogen exists in two forms in DNA: as 'free' non-adducted (extractable with lipid solvents) and an adducted form. Time variation showed that the DNA was incorporated linearly with both forms of B(A)P for up to 2 hours, then the take-up was constant. Concentration variation showed linear incorporation up to 1 microM B(A)P. The present data may explain conflicting data on the extent to which lymphocytes adduct B(A)P to DNA. The lipid-soluble planar B(A)P of DNA may, like photosensitizers, intercalate between the two DNA strands. Like the intercalation of acridines into DNA, which is known to cause frameshift mutations, the intercalation of B(A)P may also have mutagenic consequences.
Clausen
J
J
Institute for Life Sciences and Chemistry, Roskilde University Centre, Denmark.
D001564:Benzo(a)pyrene / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D011865:Radioisotope Dilution Technique; D014316:Tritium
D014316:Tritium; D001564:Benzo(a)pyrene; D004247:DNA