DISRUPTION OF ENDOGENOUS REGULATOR HOMEOSTASIS UNDERLIES THE MECHANISM OF RAT CYP1A1 MESSENGER-RNA INDUCTION BY METYRAPONE

The transcriptional induction of the cytochrome P-450 1A1 (CYP1A1) gen e by xenobiotics such as polyaromatic hydrocarbons is dependent on the ir interaction with the aryl hydrocarbon receptor. Administration of t he structurally unrelated compounds metyrapone (a cytochrome P-450 inh ibitor) or dexamethasone (a glucocorticoid) to male rats does not indu ce hepatic CYP1A1 mRNA. However, administration of both metyrapone and dexamethasone to male rats results in the induction of hepatic CYP1A1 mRNA expression, The induction response is mimicked in vitro in cultu red rat hepatocytes by the addition of metyrapone and dexamethasone to a serum-free culture medium, suggesting that these compounds act dire ctly on the liver in vivo to effect hepatic CYP1A1 mRNA induction. An examination of the characteristics of CYP1A1 induction by metyrapone a nd dexamethasone in combination in vitro indicate that at least 6 h of treatment is required for detectable levels of CYP1A1 mRNA to accumul ate in hepatocytes. In contrast, beta-naphthoflavone, which is known t o bind to the aryl hydrocarbon receptor to effect CYP1A1 gene expressi on, induces detectable levels of CYP1A1 mRNA within 2 h of treatment, CYP1A1 mRNA is also induced when hepatocytes are treated with metyrapo ne in combination with the protein synthesis inhibitor cycloheximide b ut not with dexamethasone in combination with cycloheximide, indicatin g that CYP1A1 mRNA induction is strictly dependent on the presence of metyrapone and suggesting that the metyrapone-associated induction of CYP1A1 mRNA is dependent on a loss of a constitutively expressed prote in that functions to suppress CYP1A1 gene expression. The role of dexa methasone in metyrapone-associated induction of CYP1A1 is probably med iated through the glucocorticoid receptor since the glucocorticoid rec eptor antagonist RU486 reduces the levels of CYP1A1 mRNA induced by me tyrapone and dexamethasone in combination. Increasing the levels of th e photosensitizer riboflavin present in the culture medium 10-fold and exposure to light increases the levels of CYP1A1 mnNA induced by mety rapone and dexamethasone in combination in vitro, suggesting that phot oactivation of inducing medium constituent(s) might be required for in duction, Failure to induce CYP1A1 mRNA by co-administration of metyrap one and dexamethasone in hepatocytes cultured in a balanced salt solut ion with or without photoactivation indicates that induction is depend ent on a photoactivated component of the culture medium and not on met yrapone or dexamethasone alone. The addition of tryptophan in the pres ence of riboflavin to the balanced salt solution restores CYP1A1 mRNA induction by metyrapone alone and induction is increased when medium i s exposed to light, indicating that induction is dependent on tryptoph an photoactivation in vitro. Metyrapone failed to compete with 2,3,7,8 -tetrachlorodibenzo-p-dioxin for specific binding to the aryl hydrocar bon receptor in rat liver cytosolic fractions. These results suggest t hat CYP1A1 might be induced in rats by metyrapone through an indirect mechanism associated with an elevation in the level of an endogenously generated inducer such as photoactivated product(s) of tryptophan and not because of metyrapone's interacting with the aryl hydrocarbon rec eptor, The dependence of CYP1A1 induction on dexamethasone or cyclohex imide suggests that derepression by a glucocorticoid receptor-modulate d negative-acting factor of CYP1A1 gene expression might be critical t o induction by metyrapone.