Relationship between DNA adduct formation and unscheduled DNA synthesis (UDS) in cultured mouse epidermal keratinocytes

Abstract

Primary cultures of mouse epidermal keratinocytes from SENCAR mice were treated with 7,12‐dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene [B(a)P], (±)7β‐8α‐dihydroxy‐9α,10α‐epoxy‐7,8,9,10‐tetrahydrobenzo(a)pyrene [(±)anti‐BPDE], and (±)7β,8α‐dihydroxy‐9β,10β‐epoxy‐7,8,9,10‐tetrahydrobenzo(a) pyrene [(±)syn‐BPDE] to examine the relationship between DNA adduct formation and the induction of unscheduled DNA synthesis (UDS). DNA adducts were measured as pmol hydrocarbon bound per mg of DNA, and UDS was quantitated autoradiographically as net grains per nucleus. A good correlation was observed between the levels of UDS detected and the amount of DNA adducts present in the cell population when comparing similar compounds within the linear dose‐response range of 0.005 μg/ml‐0.25 μg/ml. A higher rate of UDS for a given level of DNA adducts was interpreted as an increased efficiency of DNA repair. In some cases, an increase in the efficiency of DNA excision repair correlated with lower tumor‐initiating activity. For this family of PAH, the concentration below which UDS could no longer be detected was approximately 0.01 μg/ml. However, DNA adducts were measurable at concentrations of 0.01 and 0.005 μg/ml. The limits of detection of the current UDS assay in the SENCAR MEK culture system occurred at hydrocarbon adduct levels of approximately 10 pmol/mg DNA, or approximately 1 adduct per 3 × 10⁵ bases. Additionally, the UDS assay was unable to detect DNA repair induced by the weakly carcinogenic PAHs, dibenz(aj)anthracene and 7‐methyl‐dibenz(aj)anthracene. The UDS assay did detect DNA repair by the more strongly carcinogenic PAH, 6‐methylcholanthrene. These results suggest that the present UDS assay with MEKs is a useful assay for the rapid screening of potential genotoxic agents. However, the limits of sensitivity are such that the current assay may be unable to detect a low level of DNA damage induced by some weakly genotoxic (carcinogenic) agents. In addition, while the limits of sensitivity determined in these experiments apply to the polycyclic aromatic hydrocarbon class, other classes of genotoxic compounds such as alkylating agents or crosslinking agents may exhibit different thresholds of detection.