Effect of 3-Methylcholanthrene Administration on Expression of Cytochrome P-450 Isoforms Induced by Phenobarbital in Rat Hepatocytes

Abstract

The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.

Amplification of Immunostaining Intensity by the Peroxidase-Antiperoxidase, Avidin-Biotin-Peroxidase Complex or Catalyzed Signal Amplification Method Gives Erroneous Antigen Content in Sections.

“…Under the incubation conditions, effects of antibody trapping and steric hindrance are shown to be negligible in the sections [9,18]. The sections were washed with PBS and mounted in glycerol.…”

“…When the relationship between immunostaining intensity of and antigen content in sections is linear, molar content of the antigen in sections (nmol/cm3 cytoplasm) can be estimated as follows [9,15,17] Antigen content (nmol/cm3) = (SIPspec/ASIspec). BC/D......1), where BC = content in cell lysate measured by the quantitative radial immunodiffusion, and D = specific gravity of the liver (1.07) [17].…”

“…7B, C). The extinction coefficient of the IPO method was calculated to be 300 [9,18]. Thus, CYP2B1/2 content in hepatocyte cytoplasm of PB-treated animals was 93 ± 7.4 (nmoles/cm3 cytoplasm; means + S.E.…”

“…Immunostaining intensity of cytochromes P-450 2B1/2 (CYP2B 1 /2) stained by using the indirect immunoperoxidase (IPO) method under saturation conditions is shown to be proportional to the content of the enzymes in sections [9,16,18]. Thus, molar content of the enzymes can be measured by quantitative immunohistochemistry [9,18] .…”

“…Thus, molar content of the enzymes can be measured by quantitative immunohistochemistry [9,18] . However, a sensitive immunohistochemical method is necessary to measure small amounts of antigens in sections by quantitative immunohistochemistry, because the sensitivity of the IPO method is low [16].…”