This heterodimer acts as a transcription factor of the basic helixloophelix family of DNA binding proteins.This gene encodes the enzyme cytochrome P A, which catalyzes the oxidative catabolism of PAH.This reaction generates genotoxic metabolites that can enter the nucleus and bind to specific residues of DNA, leading to mutagenesis. Natural endogenous or exogenous ligands have been postulated but remain to be identified.Curcumin has been shown to be effective in inhibiting azoxymethaneinduced colon tumorigenesis and in inhibiting O tetradecanoylphorbolacetate induced tumor promotion in rat skin following initiation by DMBA. The mechanism of curcumins action, however, was not identified.Curcumin has been shown to inhibit cytochrome P A A activity in rodent hepatic microsomes pyrene or aflatoxingenerated DNA adducts in rodent liver. Furthermore, curcumin decreases DMBA activation by inhibiting CYPA activity in a competitive manner.Curcumin used in all experiments was dissolved in DMSO at a concentration of mM and stored at. Following PCR, mL of highdensity sample buffer was added to the samples, and they were subjected to electrophoresis on a TBE gel in TBE running buffer.The binding reactions were carried out for min and contained mg of nuclear protein, mg of poly, ng of salmon sperm DNA, and, cpm of labeled probe in a final volume of mL of binding buffer. The cells were washed once in PBS, harvested by trypsinization, and pelleted by centrifugation at gfor min at. The cytosol was divided into aliquots and stored at. Cytosolic protein, mM of anaphthoflavone, or mM of curcumin in a total volume of mL of HEDGM for hr at. The sample was applied to a hydroxyapatite column, bed volume mL, equilibrated in HEDGM without protease inhibitors.AhR with bound TCDD was eluted with a step gradient of sodium phosphate in HEDGM every mM from to mM.The radioactivity of the fractions was counted by liquid scintillation.Specific binding eluted in three fractions, and mM of phosphate.At the end of the incubation, the medium was removed and the wells were washed two times with fresh medium.The sonicate was centrifuged at, gfor min at, and the supernatant was subjected to centrifugation at, gfor min at. The nuclei were pelleted by centrifugation at gfor min at. Treatment of cells with mM of DMBA for hr caused a fold increase in CYPA mRNA accumulation. Surprisingly, treatment with curcumin alone caused an approximately sixfold increase in CYPA mRNA.We therefore examined the effect of curcumin in the absence of other treatment on CYPA mRNA.Treatment with curcumin for hr resulted in an increase in CYPA mRNA accumulation in a concentrationdependent manner from to mM that was maximal after hr of incubation but was still elevated after hr of incubation.Actinomycin D completely blocked curcumininduced CYPA mRNA accumulation. MCF cells were treated with DMSO, mM of DMBA, mM of curcumin, or mM of DMBA and mM of curcumin for hr.In the presence of mM of curcumin, this increase was partially inhibited. Based on this result, we performed another EMSA on nuclear extracts of MCF cells treated with mM of curcumin at different time points in the absence of other treatment.