INACTIVATION OF PLASMID REPORTER GENE-EXPRESSION BY ONE BENZO(A)PYRENE DIOL-EPOXIDE DNA ADDUCT IN ADULT-RAT HEPATOCYTES

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene diol-epoxide ( BPDE-I) cause hepatocellular carcinoma. To identify short-term carcino gen effects, we studied hepatocytes transfected with nonreplicating pl asmids, adducted covalently with BPDE-I, varying in promoter structure and encoded reporter gene (beta-galactosidase or luciferase). BPDE in activated gene expression as a first-order function of BPDE concentrat ion in adduction reactions. No evidence of cytotoxicity, diminished co precipitation and availability, enhanced nicking of supercoiled forms and reduced cellular uptake, or instability of adducted plasmids was o bserved. At low BPDE:plasmid ratios, inactivation occurred with 1 addu ct/plasmid within a target 23-27% of plasmid bases. Using nuclear extr acts and BPDE-adducted G-free cassette-encoding plasmids, the fraction of full-length RNA polymerase II-initiated transcripts also declined as a first-order function of BPDE concentration when almost-equal-to 3 adducts were distributed among 48% of plasmid bases. These observatio ns suggest that carcinogens such as BPDE block mRNA transcription alon g DNA templates by forming limited numbers of persistent adducts at co ding or noncoding sites.