Immunohistochemical determination of inducibility phenotype with a monoclonal antibody to a methylcholanthrene-inducible isozyme of cytochrome P-450.
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 (AhbAhd). 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.
Anderson
L M
LM
Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701-1013.
Ward
J M
JM
Park
S S
SS
Jones
A B
AB
Junker
J L
JL
Gelboin
H V
HV
Rice
J M
JM
D000818:Animals; D000911:Antibodies, Monoclonal; D001711:Biotransformation; D003433:Crosses, Genetic; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D004790:Enzyme Induction; D005260:Female; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000166:cytology / Q000201:enzymology*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D007668:Kidney / Q000166:cytology / Q000201:enzymology*; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008168:Lung / Q000166:cytology / Q000201:enzymology*; D008297:Male; D008748:Methylcholanthrene / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D010641:Phenotype; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System