Youko Asaka

Postnatal development and sublobular distribution of cytochrome P-450 in rat liver: a microphotometric study.

To study the process of expression of cytochrome P-450 (P-450) in hepatocytes during development, we measured microphotometrically the P-450 content in periportal and perivenular hepatocytes of male rats during peri- and postnatal growth. From Day 19 of gestation to Day 5 after birth, P-450 content in both periportal and perivenular hepatocytes increased markedly (periportal 1046%; perivenular 819%). The content in periportal hepatocytes remained unchanged from 5 to 20 days of age, and increased slightly (24%) from 20 to 45 days of age. However, the content in perivenular hepatocytes increased progressively (105%) between 5 and 45 days of age. The difference in P-450 content became apparent between periportal and perivenular hepatocytes after 7 days of age. The content in periportal or perivenular hepatocytes reached the adult level at 45 days of age. Therefore, the perinatal period is the time at which a marked increase in P-450 occurs in hepatocytes throughout the liver lobule. The subsequent period before weaning is the time at which the sublobular heterogeneous distribution of P-450 appears. The period after weaning is the time at which a slight increase in P-450 content in periportal hepatocytes and a marked increase in the enzyme in perivenular hepatocytes takes place.

Densities of NADPH-ferrihemoprotein reductase and cytochrome P-450 molecules in the endoplasmic reticulum membrane of rat hepatocytes.

In hepatocytes, NADPH-ferrihemoprotein reductase (reductase) has been hypothesized to exist as aggregates or micelles in endoplasmic reticulum (ER) membrane. However, if the number of reductase molecules per unit area of ER is low, this hypothesis cannot explain how a few reductase molecules efficiently reduce many P-450 molecules. To test this hypothesis, we estimated the numbers of reductase and P-450 molecules per unit ER area (reductase and P-450 densities) by microphotometry of the two enzymes in conjunction with morphometry of ER in periportal, midzonal, and perivenular rat hepatocytes. The reductase density in periportal, midzonal, and perivenular hepatocytes (107-179 molecules/microns 2 of ER) was high enough to efficiently reduce all P-450 molecules in the ER, although the value in perivenular hepatocytes was lowest owing to the relatively greater amount of ER in this region. The pattern of sublobular gradient in the reductase density was similar to that in the P-450 density. Consequently, the molar ratio of P-450 to reductase in ER was similar (about 40:1) in hepatocytes regardless of their positions within the liver lobule.

Relationship between immunostaining intensity and antigen content in sections.

We studied the relationship between staining intensity of immunohistochemical reaction and antigen content in sections. Alpha-fetoprotein (AFP) and albumin in sections cut from livers of newborn, 5-, 10-, 20-, and 60-day-old rats were examined as examples. First, we compared average immunostaining intensity (sum of specific absorbance in pixel/number of pixels) measured by image processing (IP), with antigen content measured by immunochemical assay to determine whether the intensity is proportional to antigen content. The intensity of AFP was proportional to the antigen content, whereas that of albumin was not. Subsequently, the antigen preservation test was carried out to determine whether the intensity was decreased by fixation and, if so, which type of decrease (proportional or disproportionate) occurred. Thereafter, antigen content in the same portion in the same immunostained section was measured by the microphotometric (MP) method followed by the IP method, because the MP method gives a low average antigen content when a decrease in antibody binding occurs in sections, whereas the average antigen content measured by the IP method is unchanged. The intensity of AFP decreased primarily by a proportional decrease in antigenicity during fixation. However, the intensity of albumin decreased not only by a proportional decrease during fixation but also by a disproportionate reduction in antibody binding during immunostaining or before fixation. The results indicate that AFP content in sections is measurable by quantitative immunohistochemical methods, whereas albumin content is not.

Measurement of NADPH-cytochrome P-450 reductase content in rat liver sections by quantitative immunohistochemistry with a video image processor.

We have a quantitative light microscopic immunohistochemical method using video image processing. First, an antigen (NADPH-cytochrome P-450 reductase) content in homogenates of livers of rats was measured by enzyme immunoassay. Then frozen sections from rat livers were incubated with the anti-NADPH-cytochrome P-450 reductase antibody under saturation conditions by the indirect immunoperoxidase method. Subsequently, relative staining intensities in small portions and those in wide areas in the sections were measured with a video image processor. Finally, the resulting relative values obtained from the small portions were converted into absolute NADPH-cytochrome P-450 reductase contents using the results of enzyme immunoassay and the average relative staining intensity obtained from the wide areas in the sections. The reductase content in sections from rat livers measured by the image processing method coincided with the content measured by the microphotometric method using a nitrocellulose model system. The present image processing method is applicable to measurement of contents of antigens that can not be immobilized in model systems.

Relation between cytochrome P-450 increase and endoplasmic reticulum proliferation in hepatocytes of mice treated with phenobarbital: a microphotometric and morphometric study.

To obtain detailed information on phenobarbital (PB)-induced cytochrome P-450 (P-450) increase and endoplasmic reticulum (ER) proliferation in hepatocytes, we estimated microphotometrically the amount of P-450 per unit cytoplasmic volume and morphometrically the area of ER per unit cytoplasmic volume in hepatocytes adjacent to the portal area or central venule (1 periportal or 1 perivenular cells) and in the second and third layers from the portal area or central venule (2, 3 periportal or 2, 3 perivenular cells) from mice injected with 35, 50, 100, or 150 mg/kg PB once a day for 3 days. By dividing the P-450 amount by the ER area, the number of P-450 molecules per unit ER area was also calculated. In 1 and 2, 3 perivenular cells, except for 2, 3 perivenular cells after injection of 150 mg/kg PB, the amount of P-450 increased with ER proliferation and the number of P-450 molecules in ER remained unchanged after injection of 50, 100, or 150 mg/kg PB. In 2, 3 periportal cells, however, the P-450 amount and the number of P-450 molecules in ER increased markedly without or with some ER proliferation after injection of 50, 100, or 150 mg/kg PB; the P-450 increase appears to be generally independent of ER proliferation. The 1 periportal cells are probably exceptional hepatocytes that usually did not respond to PB stimulation.

Localization of Xenobiotic-responsive Element Binding Protein in Rat Hepatocyte Nuclei After Methylcholanthrene Administration as Revealed by In Situ Southwestern Hybridization

Xenobiotic-responsive element binding protein (XRE-BP), a heterodimer of aryl hydrocarbon receptor (AhR) and its nuclear translocator (Arnt), regulates the transcription of cytochrome P-450 1A1 gene (CYP1A1) through XRE in response to xenobiotic inducers. For a better understanding of the regulatory mechanism of CYP1A1 through XRE, localization of XRE-BP was examined in liver sections or isolated hepatocyte nuclei from control and 3-methylcholanthrene (MC)-treated rats by in situ Southwestern hybridization, using synthetic XRE as a probe, and was observed by confocal laser scanning microscopy and electron microscopy. Gel mobility shift assay and competitive binding assay showed specificity of the synthetic XRE probe. XRE-BP was exclusively localized in hepatocyte nuclei in liver sections from animals 3 hr after MC injection, whereas the protein was absent in hepatocyte cytoplasm in MC-treated animals and in hepatocyte nuclei and cytoplasm in control animals. In isolated hepatocyte nuclei, XRE-BP began to accumulate in the central region between 0.5 and 3 hr, showed a peak between 3 and 6 hr, decreased gradually between 6 and 72 hr, and disappeared at 72 hr after MC injection. The protein was scarce in peripheral and nucleolar regions of the nucleus. Therefore, XRE-BP is formed in the nuclei of hepatocytes after MC stimulation. In addition, XRE-BP was found in isolated hepatocyte nuclei from control animals after preincubation with cytoplasmic lysate from MC-treated animals, although the protein was absent in the nuclei before the preincubation. These findings strongly suggest that AhR translocates from hepatocyte cytoplasm to the nucleus and forms XRE-BP in the nucleus after MC stimulation.

PERI- AND POSTNATAL CHANGES IN REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE-CYTOCHROME P-450 REDUCTASE CONTENT IN HEPATOCYTES OF RATS

SummaryTo study the process of the expression of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase (EC 1.6.2.4) in the liver during development, the amount of enzyme in the cytoplasm of periportal and perivenular hepatocytes in sections cut from livers of male rats was measured during peri- and postnatal growth by quantitative immunohistochemistry with a video image processor. In livers of 19-day-old foetuses, the reductase content in the cytoplasm of periportal and perivenular hepatocytes was 0.16 μM and 0.20 μM, respectively. From the 19th day of gestation to 5 days after birth, the enzyme content increased markedly in the cytoplasm of periportal (288%) and perivenular hepatocytes (301%). Subsequently, the content in the cytoplasm of periportal hepatocytes increased slightly (46%) from 5 to 20 days of age, remained unchanged from 20 to 45 days of age, and increased slightly (15%) from 45 to 90 days of age. However, the content in the cytoplasm of perivenular hepatocytes increased progressively (125%) between 5 and 90 days of age. Thus, the amount of cytochrome P-450 reductase increases markedly in periportal and perivenular hepatocytes during the perinatal period, and subsequently the enzyme content increases gradually in periportal hepatocytes and progressively in perivenular hepatocytes. The present results also suggest that the divergence between cytochrome P-450 expression and the cytochrome P-450-dependent drug metabolic activity in hepatocytes during the perinatal period, found in previous studies, can be attributed to a low cytochrome P-450 reductase density in the membrane of endoplasmic reticulum of periportal and perivenular hepatocytes.

Postnatal changes in sublobular distribution of NADPH-cytochrome P-450 reductase in rat liver

SummaryImmunohistochemical distribution of NADPH-cytochrome P-450 reductase (NADPH-ferrihaemoprotein reductase; EC 1.6.2.4.) in the liver lobule was examined during development of the rat. From the 19th day of gestation to 4 days after birth, the enzyme was distributed uniformly throughout the lobule. The immunostaining for the enzyme was weak before birth, and became slightly stronger after birth. A slightly uneven distribution of immunoreactivity, stronger in perivenular zones, appeared at 5 days after birth. Then, the staining intensity in perivenular zones became progressively stronger with age, except for a slight increase between 10 and 20 days of age. The intensity in periportal zones also increased gradually, although it remained weaker than that in perivenular zones. Around 30 days of age, the distribution of the immunostaining, stronger in perivenular than in periportal zones, was similar to that seen in the lobules of adult animals. thus, heterogeneity among hepatocytes with respect to the enzyme content is not present in fetal and newborn rats but develops gradually during postnatal development; the postnatal growth of the liver is accompanied by a change in the pattern of the distribution of this enzyme within the lobule.

High glucose-6-phosphatase activity in non-pigmented epithelial cells of rabbit ciliary body.

For study of the origin of glucose in the aqueous humor, glucose-6-phosphatase (G6Pase) and hexokinase activities, and glycogen, were cytochemically examined in the ciliary body (CB) of rabbit. G6Pase activity was also assayed biochemically. The staining reaction for G6Pase activity was strong in the non-pigmented epithelium (NPE) in the pars plana and tips of ciliary processes in the region containing large ciliary pockets within the pars plicata. NPE cells contained abundant reaction product for G6Pase activity in the endoplasmic reticulum (ER) and nuclear envelope. However, NPE in other regions of the CB and pigmented epithelium (PE) of CB, and other areas surrounding the anterior and (PE) of CB, and other areas surrounding the anterior and posterior chambers, showed weak or no G6Pase staining reaction. Biochemical G6Pase activity in the whole ciliary body was relatively high. Both NPE and PE in the pars plana and the tips showed strong staining reaction for hexokinase activity but no staining for glycogen. Furthermore, NPE cells in the tips bore large aggregates of smooth ER and many Golgi apparati. These suggest that the high G6Pase activity in NPE cells in the pars plana and the tips is related to glucose release into the aqueous humor.

Significance of high glucose-6-phosphatase activity in rat oviduct epithelium.

To study the origin of glucose in the oviduct fluid, we cytochemically examined glucose-6-phosphatase (G6Pase) activity in rat oviduct. The activity in the whole oviduct was also assayed biochemically. During proestrous, estrous, and metestrous phases, staining reaction for the activity was moderate in the epithelium of the caudal isthmus (CaI) and uterotubal junction (UJ), whereas it was weak in that of the ampulla (A) and cephalic isthmus (CeI). In the diestrous phase, staining reaction in the epithelium of CaI and UJ became strong although it remained weak in that of A and CeI. Reaction product for the activity was localized in the endoplasmic reticulum and nuclear envelope of all cell types in the epithelium. The amount of reaction product in secretory cells was small to moderate in CaI and UJ, and small in A and CeI during proestrus, estrus, and metestrus. In diestrous the amount became abundant in CaI and UJ and moderate in A and CeI. However, the amount in ciliated cells remained small in the four segments during the four phases. The biochemical activity in diestrous was greater than that in proestrus, estrus, or metestrus. This shows that the activity is high in secretory cells in the epithelium of CaI and UJ in the diestrous phase and suggests that the role of the high activity is to release glucose into the oviduct fluid for use by the embryo passing down the CaI and UJ to the uterus.