Psoralen adducts in a shuttle vector plasmid propagated in primate cells: high mutagenicity of DNA cross-links.

A Simian virus 40-driven shuttle vector plasmid (pZ189) was treated with 8-methoxypsoralen plus a split dose of long-wavelength UV (UVA) radiation in order for a large number of psoralen cross-links, as compared with monoadducts, to be formed in the plasmid DNA. The shuttle vector was then transfected into monkey Vero or human Raji cells. Plasmids replicated in the primate host cell were extracted 2 days later and analysed for mutations in the vector suppressor tRNA (supF) gene. A spontaneous mutation frequency of 0.7 X 10(-3) was 15-fold elevated in vectors exposed to psoralen plus two UVA irradiations. Most of these mutations were considered to be dependent on DNA cross-link adducts, since psoralen plus only a single UVA dose (producing mainly monoadducts) did not appreciably affect the mutation rate. DNA sequence analysis revealed a hot spot at an AT repeat constituting a potential site for psoralen cross-link formation, whereas the second hot spot noted did not contain any sequence where psoralen adduct formation is likely to occur. Since the AT repeat hot spot was not represented in previous studies with pZ189 exposed to other genotoxic agents, the results indicate that a specific mutational pattern may be resulting from induction of DNA cross-links.