The microsomal monooxygenase system, composed of the cytochrome P-450s as the terminal oxidase, play a central role in the metabolism of thousands of compounds including drugs, xenobiotics and carcinogens. The capacity of this system to handle such a diverse array of compounds reflects both the large number of distinct P-450s as well as the broad and overlapping substrate selectivity. These enzymes usually provide a defense against undesirable chemicals that gain access to the cell. However, certain forms are also capable metabolizing compounds such as polycyclic aromatic hydrocarbons to highly reactive intermediates that are capable of initiating a carcinogenic event. Many of the different forms are regulated differentially according age, tissue, inheritance and their ability to be induced in response to different types of agents. Therefore, variations in the expression of different P-450s depending on factors such as age, the level of induction following exposure to certain agents, or the genetic events that contribute to heritable differences, can alter the balance between the formation of toxic and non-toxic products. Differences in the expression of the P-450s can most be likely attributed to transcriptional or post transcriptional events. The critical aspects of the process are the trans acting factors that interact with cis acting regulatory sites in a manner which modulates the expression of the various genes. To understand the molecular events that control the expression of the different P- 450s, we have developed cDNA and genomic clones to a number of differentially regulated rabbit P-450s. Clones have been developed to rabbit P-450 4 and P-450 6. Both of these P-450s are inducible by polycyclic aromatic hydrocarbons and are regulated differentially with respect to age and tissue. We have also identified cDNA and genomic clones to rabbit P-450 1, which is expressed constitutively but reflects heritable differences governing the amount of P-450 1 that is expressed in various inbred rabbits and genetic crosses. In addition, a cDNA clone that encodes rifampicin inducible P-450 3c has also been identified. Molecular approaches will be pursued to determine if cis and trans acting factors can be identified that contribute to the differential expression of the P-450 genes. The employment of nuclear extracts will be used in DNA footprint and in vitro transcription analysis to determine the potential role of negative and positive regulators of transcription in response to induction, tissue specificity and temporal control. In addition, experiments will be outlined to determine the events that underlie the heritable differences responsible for the regulation of P-450 1. Combined, we anticipate that a clear understanding of the regulatory events as they relate to tissue specificity, age, inducibility and inheritance of the different P-450 genes will evolve from these studies.
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