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The absence of changes in the overall hepatic cytochrome P450 content after administration of 3-methylcholanthrene (MC) to congenitally jaundiced Gunn rats is believed to be related to a limited heme availability in this strain of rat. The amount of available heme, estimated by tryptophan pyrrolase activity, shows a substantial decrease in control Gunn versus control Wistar rats. This reduction is moderately enhanced by MC treatment in Gunn rats but is abolished after phenobarbital administration. Heme oxygenase activity is diminished in Gunn rats and consequently is not responsible for the decrease in the hepatic heme availability. These data point out that the depletion of the intracellular heme can lead to a limitation in the synthesis of cytochrome P450 isoenzymes in the MC-induced Gunn rat.

Catalytic activities and substrate specificity of flavin-containing monooxygenase were examined in human tissues. During incubation with imipramine, human hepatic microsomes efficiently carried out cytochrome P450-dependent reactions but not the formation of N-oxide, while in kidney imipramine N-oxide was the only metabolite formed during in vitro incubation. The production of imipramine N-oxide was essentially due to flavin-containing monooxygenase as shown by thermal inactivation. In contrast, thiobenzamide and dimethylaniline were actively transformed by both human liver and kidney flavin-containing monooxygenase. Neither the modification of pH nor the solubilization of microsomal membranes increased imipramine N-oxidation in human liver. Kinetic analysis indicated a poor affinity (about 7 mM) of human liver microsomes for imipramine versus 0.3 mM in kidney. Immunological studies were undertaken to support enzymatic data. Antibodies raised against rat liver flavin-monooxygenase reacted strongly with human kidney microsomes but extremely weakly with liver microsomes. The relative amount of immunochemically determined protein correlated well with imipramine N-oxidation activity. A dose-dependent inhibition of imipramine N-oxidation by anti-flavin-monooxygenase antibodies was observed in human kidney, as well as in rat kidney and liver. Taken together, the results can be interpreted by the possible existence in human tissues of distinct flavin-containing monooxygenases exhibiting a partial overlapping substrate specificity. The protein involved in imipramine N-oxidation is missing from human liver but actively carries out the reaction in kidney, while another protein catalyzes the oxidation of thiobenzamide and dimethylaniline in both tissues.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces aryl hydrocarbon hydroxylase (AHH) activity and human cytochrome P1-450 mRNA in the human breast carcinoma MCF-7 and hepatoblastoma HepG2 tissue culture cell lines. Although AHH activities induced by 100 nM TCDD are comparable in these cell lines, the EC50 values for TCDD differ: EC50 approximately equal to 1 nM for HepG2; EC50 greater than 20 nM for MCF-7. In order to determine the mechanism responsible for this difference in EC50, we have examined putative regulatory factors such as the intracellular TCDD receptor as well as the kinetics of mRNA transcription and accumulation in these cells. TCDD increases transcription of hP(1)450 mRNA in both MCF-7 and HepG2 cells; however, MCF-7 cells require higher concentrations of TCDD to produce transcriptional activation comparable to that observed for HepG2 cells. These data indicate that the difference in EC50 is determined at an early step in the induction of hP(1)450 mRNA. With the use of a sensitive assay based on high-performance anion-exchange liquid chromatography, an intracellular protein which binds TCDD with high affinity was detected in HepG2 cytosolic fractions but not in MCF-7 cells. Thus, the difference in EC50 for TCDD can be correlated to the differences in TCDD binding. We postulate that MCF-7 cells contain a "defective" receptor with decreased affinity for TCDD.