Benzo(a)pyrene (BP) and other polycyclic aromatic hydrocarbons are important environmental carcinogens and are undoubtedly involved in the etiology of many human cancers. These compounds are not carcinogenic per se but are metabolised to active forms in the body. Aryl hydrocarbon hydroxylase (AHH) and epoxide hydrase (EH) are the principal enzymes involved. The former is inducible by its substrates and tetra-chlorodibenzo-p-dioxin (TCDD). We have isolated a number of mutants of the mouse hepatoma line, Hepa-1, that are resistant to BP and deficient in AHH. Cell/cell hybridization experiments carried out so far indicate that they belong to a minimum of three complementation groups (genes). One class is defective in the TCDD-receptor protein that mediates PAH induction of AHH. We shall continue with the complementation tests in order to ascertain exactly how many classes our mutants fall into. We shall carry out a number of studies directed towards determining the biochemical defects in each class. These include: investigations on various aspects of TCDD-receptor functioning; determination of the amount of cytochrome P-450 in the various mutants; application of two dimensional polyacrylamide gel electrophoresis to ascertain whether the cytochrome P-450 spot, or any other spot is changed in intensity or location in particular mutants; and the isolation of revertants of the mutants. In addition we shall attempt to isolate mutants that have high activities of AHH even when grown without inducer and to isolate epoxide hydrase deficient mutants. We shall attempt to restore AHH activity to representatives of each of complementation groups, using DNA from wild-type Hepa-1 cells, and also attempt to transfect wild-type Hepa-1 with DNA from a dominant AHH-deficient mutant. If successful in the transfection experiments we shall attempt to clone the AHH genes using an established procedure that is based on this process. The ability to clone these genes will open up whole new lines of research on their regulation and structure. Finally we shall map the human counterparts to each of our complementation groups, either using the cloned Hepa-1 genes as DNA/DNA hybridization probes, or by monitoring the loss of AHH activity from hybrids formed between human cells and representative mutants.
NIH ExPorter
NIH ExPorter