The effect of UV photoproducts or
benzo[a]pyrene-diol-
epoxide-I (
BPDE-I) adducts in
DNA on the transient expression of a reporter gene was measured in mammalian cells. The plasmid pRSVCAT was UV irradiated or treated with
BPDE-I in vitro and co-transfected with undamaged pRSVBGAL into mouse and human fibroblasts. Variations in transfection efficiency among different cell lines were corrected by adjusting the volumes of
cell extracts used in the
chloramphenicol acetyl
transferase (CAT) assays to contain equal
beta-galactosidase (BGAL) activity. The expression of the CAT gene was found to decrease exponentially after transfection of pRSVCAT containing increasing numbers of DNA lesions per molecule. The average number of
BPDE-I adducts per plasmid molecule was measured by ELISA; the average number of
pyrimidine dimers was estimated from the dose kinetics for the disappearance of the supercoiled form of irradiated plasmid
DNA treated with Micrococcus luteus
UV endonuclease. By expressing the inhibition of CAT activity in terms of the average number of lesions per gene, we were able to compare directly the effects of two different
carcinogen lesions on transient transcription. We observed comparable kinetics of inhibition of gene expression by
BPDE-I adducts and
pyrimidine dimers in
DNA. D0 values determined by linear regression analysis of dose-response curves for inhibition of CAT activity were 4.9
BPDE-I adducts or 6.6
pyrimidine dimers per gene in excision-proficient human fibroblasts; the corresponding values in mouse cells were 4.4
BPDE-I adducts or 5.5
pyrimidine dimers. Similar threshold densities of
BPDE-I adducts and
pyrimidine dimers were observed before inhibition of transcription from pRSVCAT was detected. No threshold was observed in experiments with human fibroblasts deficient in excision repair (
xeroderma pigmentosum group A); calculated D0 values were 1.2
pyrimidine dimers of 2.1
BPDE-I adducts. Our results permit direct comparisons of the magnitude of inhibition of gene transcription by distinct DNA lesions, and suggest that
BPDE-I adducts and UV-induced
cyclobutane pyrimidine dimers in template
DNA block transcription with similar efficacy.