The main thrust of this proposal is based upon the hypothesis that the cannabinoids interact with the cytochrome P-450s at the molecular genetic level. In support of this hypothesis, experimental data is presented showing that the cannabinoids do indeed modulate the mRNA levels of specific cytochrome P-450s.
The specific aims are to: 1) Determine which of the cannabinoids and compounds with cannabinmimetic activity) modulate the level of the cytochrome P-450 mRNAs. 2) Study the mechanism by which the cannabinoids affect the levels of mRNA (activation or repression of gene transcription; stabilization or destabilization of mRNA) and 3) Relate the changes found at the level of a specific cytochrome P-450 genes to the activity of the corresponding enzymes. Cannabinoids are widely used in our society (20 million Americans smoke marijuana). In addition, THC is used clinically (Dronabinol) for nausea and new man made analogs of the cannabinoids, some of which are 100 times more potent than THC, have been synthesized by various groups. Some of these congeners are also candidates for therapeutic agents (as analgesics, in movement disorders, as antiasthmatics, as secondary antiepileptics, etc.). Knowledge of the cannabinoids' role in drug interactions at the molecular genetic level is important since this interaction will influence the metabolism of endogenous substances, such as steroids, as well as exogenous substances such as therapeutic drugs and potential carcinogens. Cloning techniques have resulted in the synthesis of DNA complementary (cDNA) to the cytochrome P-450 mRNAs allowing quantitation of constitutive and inducible forms of the messengers. Using these probes, the novel observation has been made that co-administered of a cannabinoid with phenobarbital results in the superinduction of certain cytochrome P-450 mRNAs. The 1st set of experiments are designed to determine which of the cannabinoids (and compounds with cannabinmimetic activity) modulate the level of the cytochrome P-450 mRNAs in the rat. In these experiments hepatic mRNA levels of the cytochromes will be determined by northern and dot-blot analysis after isolation of RNA from rat liver. The 2nd set of experiments are designed to study the mechanism by which the cannabinoids affect the levels of mRNA (activation or repression of gene transcription; stabilization or destabilization of mRNA). Nuclear run-off transcription, kinetic analysis of mRNA stability, and gel-retardation analysis will be employed. Cloned fragments of the P-450 genes, containing upstream regulatory regions, which are transcribed in freeze-thawed rat liver nuclei, will provide a method to study the mechanism by which the cannabinoids regulate gene expression. The 3rd set of experiments are designed to relate the changes found at the level of a specific cytochrome P-450 genes to the activity of the corresponding enzymes employing isozyme specific substrates.
