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Keywords:

  • P450 gene expression;
  • stable transformation;
  • reporter assay;
  • mapping regulatory elements;
  • xenobiotic induction

Abstract

Many Drosophila cytochrome P450 or Cyp genes are induced by caffeine and phenobarbital (PB). To understand the induction mechanism, we created Drosophila S2 cell lines stably transformed with different luciferase reporter plasmids carrying upstream DNAs of Cyp6a8 allele of the resistant 91-R strain, and the 1.1-kb upstream DNAs of Cyp6g1 of the 91-R and the susceptible 91-C strains. Following 24 h treatment with dichlorodiphenyltrichloroethane (DDT), caffeine or PB, luciferase activity of all cell lines was determined. Results showed that the 0.1-kb DNA of Cyp6a8 and the upstream DNAs of Cyp6g1 from both strains are not induced by these chemicals in S2 cells. However, the 0.2-, 0.5- and 0.8-kb DNAs of Cyp6a8 showed 13–24-, 4–5- and 2.2–2.7-fold induction with caffeine, PB and DDT, respectively. These DNAs also showed a 2–3-fold synergistic effect of caffeine and PB but not of caffeine and DDT. The results suggest that the cis-regulatory elements for all three chemicals are located within the -11/-199 DNA of Cyp6a8. Furthermore, caffeine and PB inductions appear to be mediated via different cis-elements, whereas caffeine and DDT induction may involve common regulatory elements. These stably transformed cell lines should help understand the mechanism of resistance-associated Cyp gene overexpression in Drosophila.