Induction of the cytochrome P450 gene, Cyp6a8, of Drosophila melanogaster by caffeine

Cytochrome P450 monooxygenases (CYPs) are enzymes involved in the metabolism of a variety of endogenous and xenobiotic compounds including steroids and drugs. In insects, CYPs are involved in conferring resistance to insecticides. In mammals, CYP1A1/1A2 enzymes have been shown to metabolize the commonly consumed xenobiotic, caffeine. In addition, the CYP1A1/1A2 gene is induced by caffeine in both rat kidney and liver. Although common drugs such as barbital and phenobarbital induce CYP genes in insects, it is not known whether these genes are also induced by caffeine. In the present investigations, the effect of caffeine on the promoter of a Drosophila Cyp gene, Cyp6a8, was examined. For this purpose transgenic Drosophila carrying a luciferase (luc) reporter transgene, under the control of 0.8-kb (- 11/-766) or 0.2-kb (-11/-199) upstream DNA of the Cyp6a8 gene, were examined. Adult female flies were treated with Vivarin® containing caffeine or pure caffeine, and activity of the luc reporter gene was monitored at the enzyme level as well as at the RNA level. Different tissues of the treated and untreated female flies were also examined. Various other caffeine-related compounds were examined for their ability to induce the Cyp6a8 promoter. The results showed that (a) both Vivarin and caffeine induce the Cyp6a8-luc transgene activity, (b) transgene activity was not induced by other purines (adenine, adenosine, hypoxanthine, and uric acid) tested, (c) the 0.8-kb construct gave higher constitutive expression as compared to the 0.2-kb construct, (d) the level of induced expression for both the 0.2-kb line and the 0.8-kb line was similar, (e) caffeine induced the transgenes at the RNA level, (t) the endogenous Cyp6a8 gene was induced by caffeine, as well as, the related Cyp6a2 gene, (g) caffeine induction occurred in different tissues of Drosophila at varying levels, and (h) the highest level of constitutive reporter gene expression was found in the malpighian tubules with a 512 fold increase in transgene expression in the 0.8-kb line as compared to the 0.2-kb line. These results suggest that the 0.8- and 0.2-kb upstream DNA have sequences that can support caffeine-mediated induction of the Cyp6a8 gene. Analysis of the Cyp6a8 upstream DNA identified the presence of several putative binding sites for the transcription factor APl (Activator Protein 1). In rat striatum, caffeine treatment has been shown to increase mRNA levers of two members of APl complex, c-fos and junB, and their target genes. Therefore, we propose that a possible mechanism for caffeine induced expression of Cyp6a8 may be via caffeine-induced expression of D-fos and Jra, Drosophila homologues of c-fos and junB. Future analysis of D-fos and Jra expression in caffeine treated flies may reveal evidence to support this hypothesis