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Binding of Benzo(a)pyrene and Dibenz(a,h)anthracene to the Ah Receptor in Mouse and Rat Hepatic Cytosols¹

Division of Clinical Pharmacology, Department of Paediatrics, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8

Binding of the polycyclic aromatic hydrocarbon (PAH) carcinogens [³H]benzo(a)pyrene (BP) and [³H]dibenz(a,h)anthracene [DB(a,h)A] to components in rodent hepatic cytosols was characterized by sucrose density gradient centrifugation and by gel permeation chromatography on Sephacryl S-300. In hepatic cytosols from Sprague-Dawley rats, [³H]BP and [³H]DB(a,h)A bound to a component which sedimented at 8 to 9S under lowionic-strength conditions, and had a Stokes' radius of 4.7 nm. The 8 to 9S component also bound [³H]-2,3,7,8-tetrachlorodi-benzo-p-dioxin (TCDD) and [³H]-3-methylcholanthrene (MC), two compounds previously established as Ah receptor ligands. In hepatic cytosols from C57BL/6J mice [a strain genetically "responsive" for induction of aryl hydrocarbon hydroxylase (AHH, cytochrome P₁-450) by PAHs], [³H]DB(a,h)A bound to a component sedimenting at 8 to 9S which had a Stokes' radius of 6 nm. The 8 to 9S component also bound [³H]TCDD and [³H]MC, but no direct binding of [³H]BP could be detected at 8 to 9S in hepatic cytosol from C57BL/6J mice, nor could specific [³H]BP binding to the 6 nm component be detected in C57BL/6J cytosol by Sephacryl S-300 chromatography. The 8 to 9S component was not detectable in hepatic cytosol from DBA/2J mice (genetically "nonresponsive" to AHH induction by PAHs) with [³H]BP or [³H]DB(a,h)A, nor with the previously established Ah receptor ligands [³H]TCDD or [³H]MC. In addition to binding to the 8 to 9S-6 nm component in C57BL/6J cytosols, [³H]BP, [³H]-DB(a,h)A, and [³H]MC also bound extensively to a cytosolic component sedimenting at 4 to 5S with a Stokes' radius of 3 nm. The 4 to 5S-3 nm component was present in hepatic cytosols from genetically nonresponsive DBA/2J mice, as well as in genetically responsive C57BL/6J mice. Binding of [³H]BP, [³H]-DB(a,h)A, and [³H]MC to the 8 to 9S component in rodent hepatic cytosols was eliminated by incubation in the presence of a 100-fold molar excess of nonradioactive TCDD, whereas TCDD had no effect on binding of [³H]BP, [³H]DB(a,h)A, or [³H]MC to the 4 to 5S-3 nm component. The 8 to 9S component segregates with the Ah^b allele in genetic crosses with mice, but the 4 to 5S component is not associated with the Ah^b allele which governs AHH responsiveness. Specificity of chemicals that bind to the 8 to 9S component and segregation of the 8 to 9S component with the Ah^b allele identify it as the Ah receptor which functions to regulate AHH induction in animals treated with PAHs. The 4 to 5S-3 nm component does not segregate with the Ah^b allele, and binding of [³H]BP, [³H]DB(a,h)A and [³H]MC to the 4 to 5S component is not inhibited by the most potent Ah receptor agonist, TCDD. Thus, the 4 to 5S-3 nm component is not a receptor involved in induction of AHH. Binding of PAH carcinogens to the 4 to 5S-3 nm component may play some other important role in carcinogenesis. These experiments have demonstrated that the Ah receptor can be detected and characterized with [³H]BP (in rat hepatic cytosols), and with [³H]DB(a,h)A (in mouse and rat hepatic cytosols), provided that techniques are used which separate the Ah receptor from other cytosolic components which also bind PAH carcinogens.

¹ Supported by a grant from the National Cancer Institute of Canada. A preliminary report of this work was presented at the 74th Annual Meeting of the American Association for Cancer Research, San Diego, CA, May 1983 (20).

² To whom requests for reprints should be addressed, at Division of Clinical Pharmacology, Department of Paediatrics, Research Institute, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8.

Received 8/15/83. Accepted 12/20/83.

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