Covalent binding of BP-metabolites to DNA of cultured human hair follicle keratinocytes

Primary cultures of human hair follicle keratinocytes were established by using a basement membrane-like growth substrate, the bovine eye lens capsule. A method was adapted for the isolation of 3H-benzo(a)pyrene (BP)-modified DNA from the cellular outgrowth of only one hair follicle (approximately 2×105 cells). In a routine procedure hair follicle keratinocytes were incubated with 0.5 μM 3H-BP for 24 h. The purified DNA was subjected to enzymic hydrolysis and the adducts were analyzed by Sephadex LH-20 column chromatography followed by HPLC. Only one major adduct, which represented 60–80% of the total radioactivity which can be confined to modified nucleosides in the LH-20 chromatograph, could be identified. This adduct co-chromatographed with the marker adducts resulting from the trans-addition of the N-2-amino group of guanine to the 10-position of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-etrahydrobenzo(a)pyrene.