ARTICLE |
Correspondence to: Shinsuke Kanamura, Dept. of Anatomy, Kansai Medical Univ., 10-15 Fumizono-cho, Moriguchi, Osaka 570, Japan..
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Summary |
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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. (J Histochem Cytochem 46:11511160, 1998)
Key Words: cytochrome P-450 2B, phenobarbital, 3-methylcholanthrene, quantitative immunohistochemistry, in situ hybridization, liver, rat
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Introduction |
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Steroids, xenobiotics, and carcinogens induce synthesis of inducer-specific cytochrome P-450 (P-450) isoforms in the liver (
Because animal systems are usually subject to the actions of various inducers, it is probable that inductions of various types of the P-450 isoform occur one after another in the liver. However, effects of an inducer on expression of the P-450 isoform induced antecedently by another inducer are unknown.
We therefore injected first phenobarbital (PB) and then 3-methylcholanthrene (MC) in rats and examined the amount of P-450 2B1/2B2, major PB-inducible P-450 isoforms, in periportal, midzonal, and perivenular hepatocytes by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. Furthermore, expression of P-450 2B1/2B2 proteins and their mRNA were examined by Western blotting and Northern hybridization.
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Materials and Methods |
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A total of 115 male SpragueDawley rats, 812 weeks old (300350 g), were used. The animals were fed laboratory chow and water ad libitum. They were divided into four groups. Animals of one group (PB+MC-treated) received an IP injection of PB dissolved in saline at a dose of 80 mg/kg body weight and then an IP injection of MC dissolved in corn oil at a dose of 25 mg/kg body weight 24 hr after PB injection. Those of the second group (PB-treated animals) received an IP injection of PB (80 mg/kg) and then an IP injection of corn oil instead of MC 24 hr after PB injection. Those of the third group (MC-treated animals) received an IP injection of saline instead of PB and then an IP injection of MC (25 mg/kg) 24 hr after saline injection. Those of the fourth group (control animals) received an IP injection of saline and then an IP injection of corn oil 24 hr after saline injection. The animals were sacrificed under sodium pentobarbital anesthesia 24 hr after the last injection. For in situ hybridization and Northern hybridization, animals injected IP with PB and sacrificed 24 hr after the injection were also used. The animal experiments were performed according to the guidelines of Kansai Medical University.
Immunohistochemical Methods
Under anesthesia, livers of animals (6 animals/group) were briefly perfused with saline, then perfused with cold 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 5 min. Slices cut from the left lobe of the liver were washed at 4C for 6 hr with 0.1 M phosphate buffer (pH 7.4) containing 8% sucrose. Serial frozen sections 6 µm thick were cut and placed on poly-L-lysine-coated glass slides. The thickness of the slides and sections was checked as described previously (
A pair of the serial sections (about 2 x 3 mm) were immersed in 70% (v/v) methanol including 0.3% hydrogen peroxide at room temperature (RT) for 30 min, washed with PBS (three times for 5 min), and soaked for 30 min in normal goat serum (diluted 1:10 with PBS) at RT. Then one section was incubated in PBS containing anti-rat P-450 2B1/2B2 goat IgG (diluted 1:200 with PBS; Daiichi Kagaku, Tokyo, Japan) and the other was incubated with normal goat serum (diluted 1:50 with PBS) at 4C for 12 hr (80 µl/section). After washing with PBS, the sections were incubated in PBS including horseradish peroxidase (HRP)-labeled rabbit anti-goat IgG (Cappel, West Chester, PA; diluted 1:200 with PBS) at 20C for 30 min. The sections were washed again with PBS and stained with PBS containing 3 mg diaminobenzidine and 0.03% (v/v) hydrogen peroxide at 20C for 20 min. Then the sections were dehydrated and mounted.
Some sections were stained with anti-rat P-450 1A1/1A2 antibody (Daiichi Kagaku) instead of anti-rat P-450 2B1/2B2 antibody by the indirect immunoperoxidase method under saturation conditions as described previously (
Microphotometry of P-450 2B1/2B2
Immunostained and mounted sections on the slides were set in a microphotometer (KWSP-1) (
Image Analysis of P-450 2B1/2B2
Sections used for microphotometry were subjected to image analysis with a microscope (Axiovert; Carl Zeiss, Oberkochen, Germany) linked to a video image processor (ARGUS 100-VEC; Hamamatsu Photonics, Hamamatsu, Japan) (
Microphotometry of Total Cytochrome P-450
The content of total P-450 in liver sections was measured microphotometrically as described previously (
In Situ Hybridization of CYP2B2 mRNA
Distilled water used for in situ hybridization was pretreated with diethyl pyrocarbonate (DEPC). Under anesthesia, livers of animals (6 animals/group) were perfused with saline. Serial fresh frozen sections 6 µm thick were cut and placed on aminopropyltriethoxysilane-coated glass slides. The sections were immersed in cold 0.1 M phosphate buffer containing 4% formaldehyde (pH 7.4) for 15 min. After treatment with proteinase K (2 µg/ml in 10 mM Tris-HCl containing 1 mM EDTA, pH 8.0) for 20 min at 37C, the sections were fixed again with buffered 4% paraformaldehyde as above, washed with PBS, immersed in 0.2 M HCl at 20C for 10 min, and washed again with PBS. The sections were acetylated with 0.1 M triethanolamine-HCl (pH 8.0) containing 0.25% acetic anhydride for 10 min, washed with PBS, and hybridized with diluted sodium citratesodium chloride solution (5 x SSC, 0.75M NaCl and 75 mM sodium citrate) containing 2% (w/v) blocking reagent attached to a DIG Detection System Kit (Boehringer Mannheim; Mannheim, Germany), 50 mM sodium phosphate, 7% (w/v) sodium dodecylsulfate (SDS), 0.1% (v/v) N-laurylsarcosine, 200 µg/ml tRNA, 50% (v/v) formamide, and 100 pmol/ml of anti-sense or sense oligonucleotide probe labeled with digoxigenin (DIG) (pH 7.0). The anti-sense sequence of the probe was 3'-CACTAA-CCGAGAGTGTCCGGTGGTAGGGAA-5' for CYP2B2 corresponding to amino acids 331340 (
Western Blotting
Under anesthesia, homogenates and microsomes were prepared from livers of animals (6 animals/group) as described previously (
Northern Hybridization of CYP2B2
Under anesthesia, total RNA was extracted from livers of animals (6 animals/group) with ISOGEN (Nippon Gene; Tokyo, Japan), and mRNA was purified from the total RNA by Poly (A) column chromatography with a Poly (A) Quik mRNA Purification Kit (Stratagene; La Jolla, CA). After denaturation at 65C for 10 min, mRNA (5 µg RNA/lane) was applied to a 1% agarose gel containing 2.2 M formaldehyde, subjected to electrophoresis, and transferred to a nylon membrane (Boehringer Mannheim). The membrane was then hybridized with a DIG-labeled oligonucleotide probe, washed, and stained with the DIG Detection System Kit (Boehringer Mannheim). For dot-blot Northern analysis, denatured mRNA immobilized on a nylon membrane was hybridized with the DIG-labeled oligonucleotide probe, washed, and stained as above. The stained spots on the filters were scanned with a ScanJet IIcx/T scanner (Hewlett Packard Japan; Tokyo, Japan) and analyzed with a personal computer (Macintosh 6100/60av; Apple Japan, Tokyo, Japan) and suitable software (NIH Image version 1.60; Bethesda, MD).
Other Biochemical Methods
Total P-450 content and P-450 2B1/2B2 content in liver homogenates or microsomes were measured by difference spectrophotometry (
Statistical Analysis
Data were subjected to one-way analysis of variance followed by Duncan's multiple-range test. All statistical comparisons were made above a 95% level of confidence.
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Results |
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Effect of Anti-P-450 2B1/2B2 Antibody Dilution on Immunostaining Intensity
When specific absorbance (total absorbance minus absorbance caused by background) resulting from P-450 2B1/2B2 in immunostained spots on antigen-immobilized NC membranes was plotted as a function of the concentration of anti-P-450 2B1/2B2 antibody, the absorbance increased with increasing concentration of the antibody up to a dilution of 1:200 and then leveled off (Figure 1A). When the absorbance in the cytoplasm of perivenular hepatocytes in serial sections cut from a PB-treated rat was plotted as the antibody concentration, the absorbance increased with increasing concentration up to 1:200 and then saturated (Figure 1B). Thus, the antibody dilution of 1:200 used in the present study followed saturation kinetics.
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Relationship Between Immunostaining Intensity and Amount of P-450 2B1/2B2 Immobilized on Nitrocellulose Membrane or Between Immunostaining Intensity and Section Thickness
To examine the relationship between immunostaining intensity and the amount of P-450 2B1/2B2, serially diluted liver microsomal preparations containing a known amount of P-450 2B1/2B2 prepared from a PB-treated rat were immobilized as spots on an NC membrane and immunostained under saturation conditions with anti-P-450 2B1/2B2 antibody. When specific absorbance measured in stained spots on the membrane was plotted as a function of the amount of P-450 2B1/2B2, there was a linear relationship between the absorbance and the amount (Figure 2).
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When corresponding portions in the cytoplasm of perivenular hepatocytes in serial sections cut at 4-, 6-, 8-, and 10-µm thickness from a PB-treated animal were analyzed by microphotometry after immunostaining, a linear relationship was also observed between specific absorbance and section thickness (Figure 3). Assuming that the serial sections contain a constant amount of P-450 2B1/2B2, BeerLambert's law holds for the present quantitative immunohistochemical methods. Thus, the absorbance in sections was proportional to the amount of P-450 2B1/2B2.
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Immunohistochemistry of P-450 2B1/2B2
In control and MC-treated animals, immunostaining resulting from P-450 2B1/2B2 was moderate or weak in perivenular hepatocytes and was very weak or negligible in midzonal and periportal hepatocytes (Figure 4). In PB-treated animals, the staining was strong in perivenular hepatocytes, moderate in midzonal hepatocytes, and weak or negligible in periportal hepatocytes. In PB+MC-treated animals, the staining was strong or moderate in perivenular and midzonal hepatocytes and weak or negligible in periportal hepatocytes. No staining was seen in sinusoidal cells, such as endothelial and Kupffer cells, and in the nuclei of hepatocytes from animals of the four groups. When adjacent sections were incubated with normal serum instead of anti-P-450 2B1/2B2 antibody, no staining was observed in hepatocytes and sinusoidal cells.
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Absorbance Resulting from P-450 2B1/2B2 in Sections
In control and MC-treated animals, the absorbance caused by P-450 2B1/2B2 was low in perivenular hepatocytes and very low in midzonal and periportal hepatocytes (Figure 5). In PB-treated animals, the absorbance increased remarkably in perivenular hepatocytes, moderately in midzonal hepatocytes, and did not change in periportal hepatocytes. In PB+MC-treated animals, the absorbance increased moderately in perivenular and midzonal hepatocytes but did not change in periportal hepatocytes. The value in perivenular hepatocytes was about half of that in PB-treated animals, whereas the value in midzonal hepatocytes was twice as much as that in PB-treated animals.
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Conversion of Absorbance to Molar Content
As described above, immunostaining intensity resulting from P-450 2B1/2B2 was proportional to the amount of P-450 2B1/2B2. Thus, the intensity can be converted to an absolute value (molar content) as follows (
Antigen content = (SIPspec/ASIspec) · BC/D · · · · (1),
where SIPspec = specific immunostaining intensity in portions of sections (= SIPab-SIPns), ASIspec = average specific staining intensity in whole section (= ASIab-ASIns), BC = biochemically measured content (nmol/g liver), and D = specific gravity of the liver (1.07) (
Antigen content (nmol/cm3 cytoplasm) = 307 x SIPspec · · · · (2).
The immunostaining intensity shown in Figure 5 (left axis) could thus be converted to molar content of P-450 2B1/2B2 in sections (right axis). For example, if SIPspec is 0.208, P-450 2B1/2B2 content in the portion (nmol/cm3 cytoplasm) is:
P-450 2B1/2B2 content = (307 x 0.208) = 64 nmol/cm3 cytoplasm · · · · · (3).
In Situ Hybridization for P-450 2B2 mRNA
In control and MC-treated animals, hybridization signal for P-450 2B1/2B2 mRNA was sparse in perivenular hepatocytes and negligible in midzonal and periportal hepatocytes (Figure 6). In PB-treated animals, the signal was strong in perivenular hepatocytes and weak or negligible in midzonal and periportal hepatocytes after 24 hr. After 48 hr of PB administration, however, the signal almost disappeared; only a weak reaction was occasionally seen in perivenular hepatocytes. In PB+MC-treated animals, however, the signal was strong in perivenular and midzonal hepatocytes and weak or negligible in periportal hepatocytes.
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In animals of the four groups, hybridization signal for P-450 2B1/2B2 mRNA was negligible in sinusoidal cells. When adjacent sections were incubated with sense probe or unlabeled anti-sense probe, the signal was negligible or very weak in hepatocytes and sinusoidal cells.
Total P-450 Content in Hepatocyte Cytoplasm
In control animals, total P-450 content measured by difference microphotometry was greater in perivenular or midzonal than periportal hepatocytes (Figure 7). In PB-treated animals, the content of P-450 increased about 1.2-fold in periportal hepatocytes, 1.3-fold in midzonal hepatocytes, and 2.2-fold in perivenular hepatocytes. In PB+MC-treated animals, the content increased about 1.3-fold in periportal hepatocytes, 1.6-fold in midzonal hepatocytes, and 2.1-fold in perivenular hepatocytes. In MC-treated animals, the content increased about 1.2-fold in periportal hepatocytes, 1.3-fold in midzonal hepatocytes, and 1.5-fold in perivenular hepatocytes.
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Proportion of P-450 2B1/2B2 to Total P-450 in Hepatocyte Cytoplasm
The proportion of P-450 2B1/2B2 to total P-450, calculated by dividing P-450 2B1/2B2 content by total P-450 content, increased in perivenular (from 12 to 58%) or midzonal hepatocytes (from 5 to 29%) in PB-treated animals. The value also increased in perivenular (to 28%) or midzonal hepatocytes (to 45%) in PB+MC-treated animals. The content of P-450 forms other than 2B1/2B2, calculated by subtracting 2B1/2B2 content from total P-450 content (45, 44, or 33 nmol/cm3 liver tissue for perivenular, midzonal, or periportal hepatocytes in control animals), remained unchanged in PB-treated animals and increased in PB+MC (77 or 44 nmol/cm3 for perivenular or periportal hepatocytes) or MC-treated animals (71, 59, or 41 nmol/cm3 for perivenular, midzonal, or periportal hepatocytes) except for midzonal hepatocytes in PB+MC-treated animals (43 nmol/cm3).
Immunohistochemistry of P-450 1A1/1A2
In control animals and PB-treated animals, immunostaining caused by P-450 1A1/1A2 was weak in perivenular hepatocytes and very weak in midzonal and periportal hepatocytes (Figure 8). In MC-treated animals, the staining was strong in perivenular hepatocytes and very weak in midzonal and periportal hepatocytes. In PB+MC-treated animals, the immunostaining was moderate in perivenular and midzonal hepatocytes and weak or very weak in periportal hepatocytes. No staining was seen in sinusoidal cells and in the nuclei of hepatocytes in animals of the four groups. When adjacent sections were incubated with normal serum instead of anti-P-450 1A1/1A2 antibody, no immunostaining was observed in hepatocytes and sinusoidal cells in animals of the four groups.
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Biochemical Results
When homogenates or microsomes were applied to SDS-PAGE followed by Western blotting with anti-P-450 2B1/2B2 antibody, two stained bands (2B1 and 2B2) were identified (data not shown). This is in accord with the result of
Northern blot analysis (Figure 9) revealed a weak hybridization signal for CYP2B2 mRNA in control (OD = 0.070.13) and MC-treated animals (0.050.09). Although the signal was strong after 24 hr of PB injection (0.570.93), it became weak after 48 hr of injection (0.100.19). A strong signal for the message was found in PB+MC-treated animals (0.450.84).
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Total P-450 content in homogenates and microsomes (37 ± 3.1 nmol/g liver, 1.3 ± 0.14 nmol/mg protein in control animals; means ± SE for four animals) increased in PB- (61 ± 5.8 nmol/g liver, 2.2 ± 0.33 nmol/mg protein), PB+MC- (63 ± 9.9 nmol/g liver, 2.3 ± 0.31 nmol/mg protein), or MC-treated animals (51 ± 4.4 nmol/g liver, 1.8 ± 0.15 nmol/mg protein).
P-450 2B1/2B2 content in homogenates or microsomes in control animals was low (2 nmol/g liver or 0.1 nmol/mg microsomal protein, means for four animals). The content in homogenates or microsomes increased in PB- (22 nmol/g liver or 0.9 nmol/mg microsomal protein) or PB+MC-treated animals (19 nmol/g liver or 0.8 nmol/mg microsomal protein) but was unchanged in MC-treated animals. Content of P-450 forms other than 2B1/2B2 in homogenates or microsomes, calculated by subtracting 2B1/2B2 content from total P-450 content (35 nmol/g liver or 1.2 nmol/mg microsomal protein in control animals), remained unchanged in PB-treated animals and increased in PB+MC and MC-treated animals (4449 nmol/g liver or 1.51.7 nmol/mg microsomal protein).
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Discussion |
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As revealed in the present study, the PB-induced increase in P-450 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes in PB+MC-treated animals (48 hr after PB injection and 24 hr after MC injection). The suppression observed in perivenular hepatocytes could be due to an increase in turnover of the isoforms caused by an increase in the rate of degradation induced by MC administration, or to inhibition of the synthesis of the isoforms by the action of MC. The promotion found in midzonal hepatocytes probably relates to an increase in the synthesis of the isoforms. Thus, the pattern of intralobular expression of PB-inducible P-450 isoforms induced antecedently by PB is drastically changed by the action of MC. The promotion of increase in the isoforms in midzonal hepatocytes appears to compensate for the suppression of induction in perivenular hepatocytes.
The hybridization signal for P-450 2B2 mRNA increased exclusively in perivenular hepatocytes after 24 hr of PB injection and almost disappeared after 48 hr of injection in PB-treated animals. In PB+MC-treated animals, however, a strong hybridization signal for the mRNA was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. This suggests that transcription of the P-450 2B2 gene, induced by PB injection, continues to be activated by MC, or that degradation of mRNA is suppressed by MC. However, it is unlikely that the retention of P-450 2B2 mRNA in perivenular hepatocytes in PB+MC-treated animals is due to an activation of transcription of P-450 2B2 gene, because P-450 2B2 mRNA in perivenular hepatocytes did not increase in MC-treated animals. Therefore, the retention of P-450 2B2 mRNA in perivenular hepatocytes in PB+MC-treated animals is probably due to suppression of mRNA degradation.
In MC-treated animals, the intensity of immunostaining for P-450 1A1/1A2 increased markedly in perivenular hepatocytes, although the intensity for P-450 2B1/2B2 did not increase. In PB+MC-treated animals, however, the intensity for P-450 1A1/1A2 increased moderately in both midzonal and perivenular hepatocytes. This suggests that induction of P-450 1A1/1A2 is suppressed in perivemular hepatocytes but activated in midzonal hepatocytes. This is similar to the change seen in the immunostaining for P-450 2B1/2B2 in PB+MC-treated animals. The induction mechanism for P-450 1A1/1A2 has been considered as follows (
The increase in P-450 2B1/2B2 content seen in PB-treated animals was suppressed despite the retention of a large amount of P-450 2B2 mRNA in perivenular hepatocytes in PB+MC-treated animals. There appears to be a divergence between the intensity of hybridization signal for P-450 2B2 mRNA and P-450 2B1/2B2 content. The divergence can be explained by suppression of translation in P-450 2B2 mRNA in perivenular hepatocytes from PB+MC-treated animals. It is possible that degradation of mRNA by ribonuclease after translation is repressed and that untranslated mRNA is retained in the cytoplasm (
As alternative reason for the divergence, suppression of an increase in P-450 2B1/2B2 by promotion of degradation of P-450 2B1 can be considered. However, a decrease in the staining intensity for the P-450 2B1 band in microsomes from PB+MC-treated animals was not observed in Western blots. This indicates that promotion of degradation of P-450 2B1 is negligible.
The relationship between immunostaining intensity resulting from P-450 1A1/1A2 and section thickness was curvilinear (
There appear to be two types of increase in amount of P-450 isoforms after drug administration: an additive increase without reduction in other constitutive forms and a substitutional increase with reduction in other constitutive forms (
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Acknowledgments |
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Supported by grants-in-aid from the Japanese Ministry of Education, Science and Culture (grants 06670041 and 09470005).
Received for publication January 21, 1998; accepted June 9, 1998.
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