Cytotoxicity of benzo[a]pyrene diol epoxide is associated with premature arrest of nascent strand elongation in serum-stimulated monkey kidney cells.

S M Treon,J C Bartholomew

doi:10.1016/s0021-9258(18)89524-2

Abstract

The relationship between the density of adducts on the DNA of monkey kidney cells caused by benzo[a] pyrene diol epoxide (BaP-DE) and the amount of cytotoxicity and DNA synthesis inhibition was examined. Above 1.7 microM, BaP-DE was maximally cytotoxic; whereas, at concentrations below 0.8 microM, colony-forming efficiency was the same as controls even though DNA adducts and DNA synthesis inhibition could be seen at these lower concentrations. Adduct analysis showed a linear relationship between DNA binding and applied BaP-DE concentration with no evidence of parental strand nicking over the concentrations at which adducts were measured. Analysis of nascent DNA synthesis following BaP-DE treatment showed that premature arrest of strand elongation and inhibition of DNA segment maturation were both factors contributing to the decreased level of DNA synthesis caused by BaP-DE. Above 1.7 mM, BaP-DE arrested strand elongation at subreplicon sizes which correlated with the average interadduct distances estimated from DNA binding studies. Lower, nontoxic concentrations permitted the completion of replicons, but inhibited the maturation of these nascent segments. Flow cytometric analysis of the cell cycle distributions of cells after BaP-DE treatment at concentrations which inhibited proper maturation of newly synthesized DNA showed that cells did progress through the G2 phase of the cell cycle and did divide with fragmented newly synthesized DNA. The results of these studies suggest that growth-dependent toxicity is not simply proportional to template adduct level but rather that major toxicity results when adduct levels exceed one/strand/replicon due to the inability of the replication apparatus to complete genome segments containing multiple adducts with replicons.

Highlights

Benzo[a]pyrene diol epoxide (BaP DE1)is an ultimate carcinogen whose mutagenicity, cytotoxicity, and ability to induce transformation have been well characterized [1,2,3]
6 replication in CV-l monkey kidney cells using autoradiography. Their findings show that this primate cell line possesses the same general patterns of replication seen in other eukaryotic cell types including: rate of fork migration, synchrony of initiation of tandem RU, and bidirectionality of elongation from initiation sites~ monkey kidney cells have been used as a model system in which to characterize the .type and effects of BaP DE lesions on DNA repl ication and cytotoxicity
The concentration dependence of SaP DE cytotoxicity on serum stimulated TC-7 monkey kidney cells was determined in order to better evaluate this possibility

Summary

EXPERIMENTAL PROCEDURES

The TC-7 line of African green monkey kidney cells is a subclone of CV-l cells obtained from J. SaP DE (3.3mM in Me 2S04) was applied at the indicated concentrations in 2.0ml of media for 60 minutes at 18 hours after transfer. Labelling was terminated by washing the dishes 4 times with dilute Tris buffer and harvesting the cells by trypsinization. Parental DNA was labelled by growing stimulated cells in the presence of [ 14C] dThd (O.02~Ci/ml) for 48 hours, changing the media and allowing' the cel; s to grow to confl uency. Nascent DNA of repl icating cells was pulse labelled by applying 100J£i [3H] dThd in 2.0ml of media for 5 minutes, washing the cells twice with cold, dilute Tris buffer and applying 10ml of fresh media. DETERMINATION OF BaP DE BINDING TO PARENTAL DNA Cells prelabelled ,-lith [ 14C] dThd were transferred with serum stimulation as previously described. Final DNA concentrati ons I'lere determined from the UV spectra of the fi nal preparations

RESULTS

DISCUSSION

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