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 carcinogen effects, we studied hepatocytes transfected with nonreplicating plasmids, adducted covalently with BPDE-I, varying in promoter structure and encoded reporter gene (beta-galactosidase or luciferase). BPDE inactivated gene expression as a first-order function of BPDE concentration in adduction reactions. No evidence of cytotoxicity, diminished coprecipitation and availability, enhanced nicking of supercoiled forms and reduced cellular uptake, or instability of adducted plasmids was observed. At low BPDE:plasmid ratios, inactivation occurred with 1 adduct/plasmid within a target 23-27% of plasmid bases. Using nuclear extracts 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 approximately 3 adducts were distributed among 48% of plasmid bases. These observations suggest that carcinogens such as BPDE block mRNA transcription along DNA templates by forming limited numbers of persistent adducts at coding or noncoding sites.
Koch
K S
KS
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Fletcher
R G
RG
Grond
M P
MP
Inyang
A I
AI
Lu
X P
XP
Brenner
D A
DA
Leffert
H L
HL
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000494:pharmacology*; D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism / Q000494:pharmacology*; D004247:DNA / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018736:DNA Adducts; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010957:Plasmids / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic; D014162:Transfection
D001580:Benzopyrenes; D018736:DNA Adducts; D004278:DNA, Superhelical; D012333:RNA, Messenger; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C072019:benzo(a)pyrene diolepoxide I; D004247:DNA