BENZO(A)PYRENE-DNA ADDUCT FORMATION AND REMOVAL IN MOUSE EPIDERMIS INVIVO AND INVITRO - RELATIONSHIP OF DNA-BINDING TO INITIATION OF SKIN CARCINOGENESIS

Abstract

An antiserum specific for the major benzo(a)pyrene (BP) adduct formed with deoxyguanosine in vivo was used by enzyme-linked immunosorbent assay [ELISA] to monitor the formation and removal of DNA-bound products in BALB/c mouse epidermis exposed topically to intitiating doses of BP and in BALB/c mouse keratinocytes exposed in vitro to BP or its activated derivatives. In mouse epidermal DNA, formation of antibody-recognizable products increased proportionally between doses of 50 and 250 nmol of BP, giving 2.3-6.0 fmol/.mu.g of DNA, respectively, and reached a plateau of 10-11 fmol/.mu.g of DNA at doses beween 1000 and 1500 nmol. Antibody-recognizable adducts comprised roughly 1/2 of the total BP-DNA binding, since a 250-nmol dose of [3H]BP yielded 6 fmol/.mu.g of DNA by ELISA and 12.9 fmol/.mu.g of DNA by radiolabeling. Removal of trans-(7R)-N2{10-[7.beta.,8.alpha.,9.alpha.-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]-yl}-deoxyguanosine adducts was monitored in epidermal DNA of mice exposed for DNA turnover in the skin, about 1/2 of the adducts formed by 24 h were removed 3 days later and only 10% remained at the end of a week. BP-DNA binding and removal were also studied in cultured mouse keratinocytes, where proliferating basal cells and terminally differentiating cells can be selectively studied by modulating the Ca2+ concentration of the medium. BP dose-response studies showed that, in cells of different maturation states, BP-DNA adduct levels were similar. Adduct formation > 10-11 fmol/.mu.g (the highest obtained in vivo) was associated with extensive cytotoxity and cell death. The kinetics of adduct removal was followed in culture under conditions in which dilution by DNA synthesis or cell loss could be monitored. Initial removal of BP-DNA adducts apparently was more rapid in the differentiating population although, in both populations, 50% of the adduct was removed by 24 h. The formation of foci resistant to Ca2+-induced terminal differentiation was associated previously with carcinogen treatment in cultured keratinocytes. Exposure to BP or the anti-diol-epoxide, at concentrations producing low cytotoxicity, yielded frequencies of differentiation-altered foci proportional to the dose of the compound used and to the number of DNA adducts formed. The anti-diol-epoxide was .apprx. 5 times more effective than was BP in inducing transformed foci and this difference correlated directly with the quantity of DNA adducts formed. Adduct levels associated with induction of altered foci in the hydrocarbon-exposed cultured cells were similar to those found in mouse epidermal DNA after exposure to initiating doses of BP. Striking similarities evidently exist between mouse epidermis in vivo and in vitro in the formation and removal of BP adducts. In this tissue, an alteration in normal biological programming can be associated with relatively low levels of BP-DNA adduct formation.