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Immunohistochemical Determination of Inducibility Phenotype with a Monoclonal Antibody to a Methylcholanthrene-inducible Isozyme of Cytochrome P-450¹

Laboratory of Comparative Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Frederick, Maryland 21701-1013 [L. M. A., J. M. W., A. B. J., J. L. J., J. M. R.], and Laboratory of Molecular Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892 [S. S. P., H. V. G.]

A monoclonal antibody (MAb) to a methylcholanthrene (MC)-induced cytochrome P-450, designated MAb 1-7-1, was used for immunohistochemical staining of formalin-fixed tissues from oil- and MC-treated C57BL/6, DBA/2, and [(C57BL/6 x DBA/2) F₁ x DBA/2] F₂ mice. An avidin-biotin-peroxidase complex immunohistochemical technique was used. For controls, the tissues were also exposed to MAbs 1-48-5 and HyHel-9 (to egg white lysozyme). In liver, MAb 1-7-1 specifically stained the cytoplasm of centrilobular hepatocytes of C57BL/6 mice treated with MC (80 mg/kg) 48 h before kill; staining was not observed with vehicle-treated C57BL/6 mice, with oil- or MC-treated DBA/2 mice, or with comparable antibody concentrations of control MAbs 1-48-5 or HyHel-9. In the F₂ mice, about 50% were expected to be MC inducible (Ah^bAh^d). Inducibility phenotype was determined by measuring the conversion of [¹⁴C]MC to oxidized and conjugated products by liver homogenates. In freshly fixed material from MC-treated mice, those livers shown by the determination of phenotype to be inducible also stained with MAb 1-7-1, whereas those not induced were immunohistochemically negative. Furthermore, there was a significant positive correlation between degree of staining and the level of MC-metabolizing activity measured biochemically. The immunohistochemical procedure was also accurate in determination of inducibility phenotype of livers that had been in paraffin blocks for up to 2 yr if more concentrated antibody was used. In lung, MAb 1-7-1 stained specifically the alveolar walls and endothelium of blood vessels in MC-induced C57BL/6 mice only; the control MAbs and other mice gave negative results. Similarly, in kidney MAb 1-7-1 stained only glomeruli and interstitial tissue of MC-induced C57BL/6 mice and only endothelium of blood vessels in the colons of these mice. These observations are consistent with induction of the cytochrome P-450 recognized by MAb 1-7-1 in the endothelial cells of extrahepatic tissue. Immunohistochemical staining with MAb thus shows great promise for highly specific localization of particular species of cytochromes P-450 in tissues, for in situ quantification of these enzymes, and for determination of inducibility phenotype with fixed material.

¹ Supported in part by Public Health Service Contract N01-C0-23910 to Program Resources, Inc., Frederick, MD.

² To whom requests for reprints should be addressed.

Received 10/14/86. Revised 1/ 2/87. Revised 7/17/87. Accepted 8/17/87.

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