Immunofluorescent determination of the lobular distribution of a constitutive form of hepatic microsomal cytochrome P‐450

We have used an immunohistochemical approach to study the lobular distribution of constitutive liver microsomal cytochrome P‐450. Cytochrome P‐450 isolated (10.3 nmoles per mg protein) from hepatic microsomes from untreated, mature male Sprague‐Dawley rats was used to produce antisera in rabbits. The IgG fraction produced single precipitin lines of identity with liver microsomes after double immunodiffusion, precipitated 80% of the total microsomal cytochrome P‐450 and inhibited three cytochrome P‐450‐dependent enzyme activities. By these criteria, the IgG appeared to be specific for a constitutive form (or immunochemically related family) of liver microsomal cytochrome P‐450. The pattern of fluorescence after indirect immunofluorescent labeling of liver sections depended on the route of tissue preparation and the concentration of primary antibody. In frozen sections, the labeling was uniform throughout the lobule, whereas in “antigen‐depleted” paraffin‐embedded sections it was heaviest in the centrilobular and midzohal regions. Increasing the concentration of primary antibody to 500 μg per ml inhibited the centrilobular labeling in frozen sections in a concentration‐dependent manner. When specific isozymes of cytochrome P‐450 were induced with phenobarbital or 3‐methylcholanthrene, the constitutive cytochrome P‐450 was localized predominantly in the periportal region. Decreases in cytochrome P‐450 in rats treated with carbon tetrachloride or 1,2‐dibromo‐3‐chloropropane were associated with antigen loss only in necrotic cells. Regional differences in the loss of antigen in paraffin sections and the inhibition of fluorescence in frozen sections establish that the lobular distribution of constitutive hepatic microsomal cytochrome P‐450 is qualitatively heterogeneous and may be altered during hepatocellular responses to chemical treatment.