The adverse biological effects of most chemical carcinogens, including polycyclic aromatic hydrocarbons (PAH's), are dependent on the formation of reactive metabolites. The oxidative metabolism of such compounds are dependent upon induction of a specific group of cytochromes P-450. In the rabbit, cytochrome P-450 form 4 and cytochrome P-450 form 6 are iduced differentially as a function of age by PAH's in various tissues. By the use of recombinant DNA techniques, DNA sequences coding for rabbit liver form 4 and form 6 mRNA will be isolated and used to investigate regulatory events involved in the induction process. Clones ds-cDNAs complimentary to mouse cytochrome P1-450 and cytochrome P-448 mRNA, postulated to be equivalent to rabbit forms 4 and 6, will be used as probes to hybridize to rabbit liver ds-cDNA libraries. The recombinants that anneal will be subjected to hybrid selection-translation experiments to verify that the sequences are immunologically associated with form 4 and form 6. Following treatment with PAH's at different times during development, form 4 and form 6 mRNA and protein levels will be concommitantly examined in various tissues. The rates of invitro transcription, accumulation of intranuclear pre-mRNA forms, cytoplasmic mRNA and synthesis of membrane associated protein will be quantitated. These determinations will help to establish at what level the induction process of form 4 and form 6 is regulated in the various tissues as a function of age. The ds-cDNA clones will also be used to isolate specific genomic DNA sequences from a cosmid rabbit genomic library. The DNA sequences will be extensively characterized to determine the organization of coding sequences and important 5' flanking sequences responsible for the initiation of transcription. Cloned DNA fragments representative of 5' and 3' portions of the genes, will be employed to investigate if alterations in the DNA methylation status of the genes plays a role in modulating the expression of form 4 and form 6 genes during development and induction in various tissues. These studies, in combination with other ancillary recombinant DNA techniques, will aid in our understanding of the regulatory events involved in the expression of these cytochromes, and those events that contribute to the onset of chemical carcinogenesis.
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