The cytochrome P1(450) (P450IA1) cDNA has been isolated and sequenced from liver of 3-methylcholanthrene-treated rainbow trout (Salmo gairdneri). The cDNA hybridizes to a 2.8-kb mRNA that is induced at least 10-fold by 3-methylcholanthrene. Southern blot analysis suggests the presence of a single gene or a very small number of genes. An open reading frame of the 2573-bp cDNA encodes a 522-residue protein (Mr = 59,241) that is more similar to the mammalian P450IA1 than the mammalian P450IA2 proteins. The aromatic hydrocarbon (Ah) receptor, responsible for mammalian P450IA1 and IA2 inducibility, was detected in trout liver cytosol by specific binding to [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro. Comparison of the fish P450IA1 protein with human, mouse, rat and rabbit P450IA1 and P450IA2 proteins reveals the presence of a remarkably large number of single amino acids and stretches of 2-6 residues in a row that are invariant among these nine P450 proteins. These conserved regions may participate in the binding of the NADPH-P450 oxidoreductase flavoprotein, substrate, or heme.

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The murinc Ah complex represents a 'duster' of genes controlling the induction of numerous cytochrome P-450-mediated monooxygenase 'activities' by polycyclic aromatic compounds. These forms of cytochrome represent structural gene products. A major regulatory gene product is a cytosolic reccptor to which radiolabeled 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) binds with very high affinity. Developmcntal differences in mause, rat and rabbit liver have been previously reported, indicating that some form of temporal control exists during the induction of enzymes associated with the ah locus. Tue cytosolic receptor levels were determined by sucrose density gradient cen trifugation after dextran-charcoal trcatment. Hepatic receptor levels, ß-naphthoflavone-inducible and control aryl hydrocarbon hydroxylase (EC 1.14.14.2) and acetanilide 4-hydroxylasc activities and total P-450 content were studied in the Sprague-Dawley rat, C57BL/6N mause, New Zealand White rabbit and Sigmoden hispedis (cotton rat) as a function of age. 3-Mcthylcholanthrene-inducible and control aryl hydrocarbon hy droxylase and acetanilide 4-hydroxylase activitics in nonhepatic tissues of the neonatal and adult rabbit were examined. ß-Naphthoflavone-inducible hepatic microsomal proteins detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were also studied in the four species. Taken altogether, the developmental data indicate that: (i) the form of P-450 responsible for induced aryl hydrocarbon hydroxylase activity is not the same as that for induced acetanilide 4-hydroxylase, and neither onc is the same as induced cytochromc P-448; (ii) the presence of the TCDD-specific cytosolic receptor per se (detected as a distinct high-specificity saturable peak on sucrose density gradients) does not guarantee the expression of inducible aryl hydrocarbon hydroxylase or acetanilide 4-hydroxylase activity; (iii) although interesting developmental differences exist among all four species examined, the TCDDspecific receptor is maximal between the neonatal and weanling period, is considerably decreased in the adult, and is suppressed even more during the latter half of pregnancy; (iv) in general, the times at which the cytosolic receptor is highest or lowest parallels quite closely the well-known increases in inducible drug-metabolizing enzymes that have been commonly observed in the rat, mouse and other laboratory animals, and (v) more studies are necessary before we understand what the 'TCDD-specific binding peak ' (as obscrved on the sucrose density gradients) actually represents. These data show that some form of temporal control cxists, but at present we cannot distinguish between a direct effect on structural genes and a direct effect on regulatory genes. The latter possibility would suggest that multiple receptors are associated with the Ah cornplex. The regulation of P-450-mediatcd monooxygenase activities by tissue-specific regulatory gencs, and modification of these by some sort of temporal control, may play an important role in carcino...

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