Identification and quantification of a messenger ribonucleic acid induced by polynuclear aromatic hydrocarbons - using a cloned human cytochrome P-450 gene

Abstract

We have isolated four overlapping human genomic clones associated with the polynuclear aromatic hydrocarbon‐induced form of cytochrome P‐450. The form of P‐450 most closely associated with polynuclear aromatic hydrocarbons induction has been defined as P₁‐450. These four overlapping genomic clones span a total of 31.0 × 10³ base pairs in length with the coding sequence lying in the center of these clones. Translation in vitro of 3‐methylcholanthrene‐induced mRNA, selected with the human P₁‐450 genomic clone, detect a protein with M_r 52000, which is immunoprecipitable by the anti‐(mouse P₁‐450) antibody. The isolated human P₁‐450 genomic clone hybridizes to 3‐methylcholanthrene‐induced mRNA from monkey liver, benzanthracene and 3‐methylcholanthrene‐treated human mammary tumor cells (MCF‐7), but not to isosafrole‐treated human cells. Upon treatment with polynuclear aromatic hydrocarbons there is a positive correlation between induced arylhydrocarbon hydroxylase (flavoprotein‐linked monoxygenase) activity and the amount of mRNA that hybridizes to the isolated human genomic clone for P₁‐450. The size of mRNA, induced from human cells and monkey liver by polynuclear aromatic hydrocarbons, is around 3.3 × 10³ base pairs, which is the same as the larger of two mRNA induced by polynuclear aromatic hydrocarbons in the inbred strain of mouse (C57BL/6N). Our data also showed that the isolated DNA clone can detect a mRNA size of 3.3 × 10³ base pairs from phytohemagglutinin‐activated, benzanthracene‐treated human lymphocytes. Densitometer scanning indicated the presence of a 3.6‐fold variation (highest—lowest) in the levels of lymphocyte P₁‐450 mRNA contents among six individuals studied.