Demonstration by in situ hybridization of the zonal modulation of rat liver cytochrome P-450b and P-450e gene expression after phenobarbital.

Abstract

The various physiological processes that constitute liver function are compartmentalized within the hepatic acinus. The molecular mechanisms modulating the development and maintenance of this hepatocyte heterogeneity have not been defined. The objective of this study was to determine whether transcriptional or posttranscriptional zonal modulation of cytochromes P450b,e gene expression was responsible for the heterogeneous induction of the P450 proteins, which is observed after phenobarbital (PB) administration. The exact localization in liver tissue of hepatocytes responding to PB with induction of either P450b,e mRNA or proteins was established by in situ hybridization and by immunofluorescence, respectively. As demonstrated by quantitative assessment of autoradiographs of -20 hepatocytes located between a terminal portal venule and a hepatic venule, PB induced the P450b,e mRNA up to sixfold in the 12-15 hepatocytes located closer to the hepatic venules (zones 2 and 3). In contrast, there was only a twofold induction in the 4-6 hepatocytes surrounding the terminal portal venules (zone 1). Quantitative immunofluorescence using an MAb showed that the acinar distribution of PB-induced P450b,e proteins was similar to that of the mRNA. This combined approach indicated that, most likely, an increased rate of transcription of cytochromes P450b,e genes in hepatocytes of zones 2 and 3 concomitantly, with a relative lack of activation, or repression, of these genes in hepatocytes of zone 1, were responsible for the heterogeneous phenotype observed after PB administration. Therefore, modulation of gene expression among hepatocytes of the liver acinus is one mechanism by which the functional heterogeneity of hepatocytes is attained. Experiments in which the induction of cytochromes P450b,e genes was studied after administration of either PB or para-hydroxyphenobarbital, a main hepatic metabolite of PB, suggested that the species involved in the inductive process is the parent PB molecule rather than para-hydroxyphenobarbital.

Zonation of cytochrome P450 isozyme expression and induction in rat liver

“…The regulatory level of this zonal differentiation in constitutive and induced expression of P450 genes is poorly understood. It has been shown that the acinar distribution of mRNA and protein for CYP2B1/2 is similar [28] and that also induction by acetone and phenobarbital, which is known to be transcriptionally regulated [14] is similarly located at the mRNA and protein level [28]. A congruent localisation of CYP2E1 mRNA and protein is also observed [lo, 291 and recent evidence suggests that this is due to higher rate of CYP2EI gene expression in nuclei from perivenous as compared to periportal hepatocytes [29].…”

Microengineered cell and tissue systems for drug screening and toxicology applications: Evolution ofin-vitroliver technologies

“…The oxygen tension in the PP zone is 60–65 mmHg and in the PV zone is 30–35 mm Hg 126–128 . These gradients, along with differences in ECM composition, structure, and soluble factors from the PP to PV zones 74 manifest as differences in cellular metabolism, including that of xenobiotics and secreted molecules, as well as changes in the morphology and number and phenotypic characteristics of all cell types along the PP-PV axis 128–130 (see Fig. 2).…”

Nonradioactive in situ hybridization for detection of mRNAs encoding for peroxisomal proteins: heterogeneous hepatic lobular distribution after treatment with a single dose of bezafibrate.

“…1975) and immunocytochemistry of peroxisomal enzymes (Fahimi and Baumgart, 1993). as well as for in situ hybridization (Saber et al, 1990; Wojcik et al, 1988;Tournier et al, 1987). The initial rinsing of the liver with saline removes the blood components from the vascular spaces, exposing the hepatocytes directly to the fixative and preventing the loss of mRNA.…”