Distributional variation of P-450 immunoreactive hepatocytes in human liver disorders

Implications of P-450 in human hepatic disorders were immunohistochemically examined. We first confirmed that an antibody against P-450-HM1, an isozyme of cytochrome P-450 which was purified from human livers at autopsy, detects only P-450 on immunoblots. In a study of 79 consecutive autopsied livers using the avidin-biotin-peroxidase complex method, the antibody reacted strongly with fetal hepatocytes, the reaction being more intense in the left lobe than in the right lobe. In normal livers, immunoreactivity was confined to centrilobular hepatocytes, decreasing in the periportal zone. Enhanced expression was occasionally found in scattered hepatocytes and in hepatocytes surrounding sublobular veins; this enhancement was related to longterm steroid therapy. No specific induction was observed in patients with toxic hepatitis. In patients with fibrosis, cirrhosis, or regenerative nodules, however, P-450-positive hepatocytes were observed in the periportal and middle zones as well as in the central zone. In contrast, hepatocellular carcinomas were devoid of P-450 immunoreactivity. These results suggest that P-450-HM1, which is abundant in the fetal liver, is reexpressed in regenerating hepatocytes but not in cancers.     

Effect of a catatoxic steroid pregnenolone-16alpha-carbonitrile, on rat liver microsomal subfractions

Pregnenolone-16α-carbonitrile (PCN), a potent catatoic steroid without known classical hormonal effects, was administered per os to female rats. Its effects were studied on mixed function oxygenases and on various phosphatases in liver microsomal subfractions: rough microsomes, smooth I, and smooth II microsomes. For comparison, phenobarbital (PB) and 3-methylcholanthrene (MC) were also administered. The inducers increased the protein content in total, rough and smooth I microsomes in the following order: PB, PCN, and MC, whereas the protein amount in smooth II microsomal fraction remained unchanged. The content of cytochrome P-450 was about doubled in all subfractions except the smooth II membranes, following treatment with any of the inducers.PCN differed in inducing specificity from PB in increasing benzo(α)pyrene hydroxylase and from MC in stimulating aminopyrine demethylase in total microsomes. PCN also differed from PB in enhancing the capacity not only for rough and smooth I microsomes, but also for smooth II microsomes to demethylase aminopyrine. No major difference in magnitude of effects among the inducers was noted between rough and smooth I microsomes.In contrast to the altered substrate specificities produced in the monooxygenase system by different inducers, a more uniform pattern of specificities was seen in microsomal phosphatases. PCN, MC and PB all increased the activities of nucleoside diphosphatase (IDPase), whereas G6Pase and ATPase activities were little affected. Cycloheximide was partially effective in depressing the increase of both the monooxygenase system (cytochrome P-450 and benzo(α)pyrene hydroxylase) and nucleoside diphosphatase activity. We conclude that (1) treatment with PCN results in different substrate specificity when compared to PB and MC; that (2) PCN is the most potent inducer of the three in stimulating drug hydroxylation in smooth II microsomes, that (3) various smooth microsomal membranes of liver cells are affected differently by inducers of drug metabolism, and finally (4) that drugs also alter some microsomal phosphatase activities but not all.     

A cytochrome P-450 gene family mapped to human chromosome 19

We have recently isolated a cloned cDNA coding for a cytochrome P-450 of human liver microsomal membranes, which corresponds to a major phenobarbital-inducible cytochrome P-450 of rat liver. This human cytochrome P-450 is encoded by a member of a multigene family. DNA extracted from a panel of 12 independent human-rodent somatic cell hybrids was analysed by Southern blot hybridization with the cloned cDNA. The results indicate that all components of this cytochrome P-450 gene family are located on chromosome 19. Evidence from hybrids derived from an individual carrying a balanced translocation suggests a regional localization of 19p13.2→qter. Analysis of human metaphase chromosomes by in situ hybridization localizes this cytochrome P-450 gene family further to the long arm of chromosome 19 in the region q13.1→qter. We propose the designation P450PB for this locus.     

Resistance of different forms of cytochrome P-450 of rat liver to factors destabilizing the microsomal membrane

The effect of factors destabilizing the membrane of the liver microsomes on the spectral properties of cytochrome P-450 (P-448) was investigated in intact rats and rats receiving phenobarbital (PB) or 3-methylcholanthrene (MC). Considerable resistance of microsomes induced by PB and MC to enzymic and nonenzymic peroxidation of polyunsaturated fatty acids of membrane phospholipids was discovered. A clear difference was shown in the sensitivity of cytochrome P-448 and cytochrome P-450 of intact rats and rats receiving PB to in vitro treatment with sodium deoxycholate. The results indicate structural changes in the microsomal membrane during induction by PB and MC, which are two different types of inducers of the monooxygenases of the liver.Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 553–555, May, 1977.     

Selective enhancement of cytochrome p-450 activity in rat hepatocytes by in vitro heat shock

We investigated the effect of heat shock on cytochrome P-450 activity in rat hepatocytes and report a significant, selective, and time-dependent enhancement of cytochrome P-450 activity in heatshocked hepatocytes. Stable long-term cultures of rat hepatocytes were heat shocked (42.5 degrees C) for 1 to 3 h and allowed to recover at 37 degrees C. Cytochrome P-450-dependent ethoxyresorufin O-dealkylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were measured up to 48 h after heat shock treatment. In general, the optimal heat shock exposure time was between 2 and 3 h. BROD activity (induced by sodium phenobarbital) increased approximately 6-fold in hepatocytes heat shocked for 3 h in comparison with hepatocytes maintained at 37 degrees C. EROD activity (induced by 3-methylcholanthrene) increased 2-fold on exposure to heat shock for 2 h. The expression of inducible heat shock proteins Hsp70 and Hsp32 was verified by Western immunoblot analyses. In the absence of the appropriate inducer, heat shock treatment did not enhance cytochrome P-450 activity. Furthermore, enhanced P-450 enzyme activity was delayed for heat-shocked hepatocytes. It is hypothesized that heat shock treatment attenuates the negative effects triggered by the addition of the toxic inducers and possibly stabilizes the levels of cytochrome P-450 proteins. These results suggest that heat shock treatment may be used to enhance the functionality of hepatocytes, specifically, in bioartificial liver assist devices.     

Dexamethasone modulation of in vivo effects of endotoxin, tumor necrosis factor, and interleukin-1 on liver cytochrome P-450, plasma fibrinogen, and serum iron

Treatment of mice with endotoxin (lipopolysaccharide, LPS) and the two LPS-induced monokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), caused a depression of liver cytochrome P-450 and related drug-metabolizing enzymes, as well as other acute-phase changes including increase in plasma fibrinogen levels and hypoferremia. However, only IL-1, not TNF or LPS, depressed cytochrome P-450 in cultured hepatocytes, suggesting that the effect of TNF in vivo might be mediated by a second mediator. TNF- or LPS-stimulated monocytes released a factor capable of depressing cytochrome P-450 in cultured hepatocytes. This factor was inhibited by anti-IL-1 antiserum, and its synthesis, like that of IL-1, was inhibited by dexamethasone (DEX). Pretreatment of mice with DEX protected against the depression of liver cytochrome P-450 by LPS or TNF but not by IL-1, suggesting that IL-1 directly depresses cytochrome P-450 and that DEX acts by inhibiting IL-1 synthesis in vivo induced by LPS or TNF. However, DEX did not inhibit two other effects of LPS and TNF in vivo: increase of plasma fibrinogen levels and decrease of plasma iron, suggesting that these might not be mediated by IL-1. Therefore, the effect of DEX in vivo, although supporting the hypothesis that depression of liver cytochrome P-450 by LPS and TNF is mediated by IL-1, indicates the existence of IL-1-independent pathways in the acute-phase response.     

The immunocytochemical detection of cytochrome P-450 monooxygenase in the lungs of fetal, neonatal, and adult hamsters

Antibodies against rabbit cytochrome P-450 reductase (reductase), cytochrome P-450 isozyme 2 (P-450 IIB), and cytochrome P-450 isozyme 5 (P-450 IVB) were used to detect homologous enzymes in the developing lung of the Syrian golden hamster. No immunocytochemical labeling was observed on gestational days 11, 12, and 13. On gestational day 14, light immunoperoxidase labeling for reductase and P-450 IIB was observed over cells lining the trachea and cranial portions of lobar bronchi. On gestational day 15, these enzymes were detected in conducting airways at all anatomic levels, and in the media of the pulmonary vein and its branches. Light labeling for P-450 IVB was first observed over cells lining the trachea and lobar bronchi on gestational day 15, but the smallest bronchioles and the media and endothelium of the pulmonary vein did not label for this enzyme until gestational day 16 (neonatal day 1). Type II pneumocytes and the pleural mesothelium first labeled for each of the three enzymes on neonatal day 1. Although the mesothelium no longer labeled for reductase or P-450 IIB in hamsters 3.5 wk old, the other labeling sites persisted in adult hamsters. Because cytochrome P-450 enzymes are associated with the endoplasmic reticulum, an ultrastructural examination of differentiating secretory cells was made to detect its appearance. At each conducting airway level, smooth endoplasmic reticulum was present in the cells 2 d before cytochrome P-450 enzymes could be detected immunocytochemically. The appearance of these enzymes paralleled the development of the hamster lung; they were first present in the trachea and lobar bronchi, then in the bronchioles, and finally in the alveoli.     

Biochemical properties of carcinogen-metabolizing enzymes in cultured hepatoma cells

We have previously demonstrated the inducibility of both cytochrome P-448- and P-450-dependent monooxygenases in the differentiated rat hepatoma cell line MH1C1. Further experiments with these cells on the expression of different forms of cytochrome P-450, inducible not only by phenobarbital (PB) and 3-methylcholanthrene (MC), but also by metyrapone (MP), ethanol (E), and beta-naphthoflavone (BNF) are reported here. The effects of the in vitro addition of the inhibitors alpha-naphthoflavone and beta-naphthoflavone on the aryl hydroxylase activity (AHH) and the influence of protein synthesis on the induction of cytochrome P-450 were also assessed. Cultures were exposed to the inducers PB, MC, BNF, and MP during the last 6 days of culture and to E for 10 days. The inhibition of protein synthesis was obtained by adding cycloheximide (CY) to the cultured cells during the last 24 hr. The exposure of MH1C1 cells to various concentrations of MP resulted in a dose-dependent increase in AHH activity. The treatment of MH1C1 cells with different concentrations of ethanol produced a significant dose-dependent increase of monooxygenases. AHH activity, induced by the various treatments, was inhibited in a dose-dependent way by alpha-naphthoflavone and beta-naphthoflavone. Cy reduced the concentration of cytochrome P-450 and the AHH activity induced by the various treatments, thus indicating an implication of the protein synthesis in the mechanism(s) of induction.     

Turnover of hepatic cytochrome P-450 in experimental cholestasis

In mechanical experimental chllestasis, hypertrophy of smooth microsomal membranes was observed. In contrast to typical induction, the membranes were deficient in cytochrome P-450. The total cytochrome P-450 content of the liver, however, as determined in the liver homogenate remained unchanged. To clarify the mechanism of the development of cytochrome P-450 deficient membranes in cholestasis, the half life of the heme portion of cytochrome P-450, and the initial rate of synthesis of cytochrome P-450 and b₅ hemes were compared in bile duct ligated rats and in control animals after labeling the heme by injection of the precursor δ-[4-¹⁴C]aminolevulinic acid. The half lives were not significantly different, which eliminates the possibility that selective destruction of cytochrome P-450 has occurred. Depression of cytochromal heme synthesis was not observed. During mechanical cholestasis, the relative cytochrome P-450 deficiency is probably caused by proliferation of components of the endoplasmic reticulum other than the hemoprotein.     

Effects of phospholipid-containing hepatoprotectors on cytochrome P-450-dependent antitoxic function of the liver during experimental toxic hepatitis

Phospholipid-containing hepatoprotectors essentiale and eplir inhibited conversion of cytochrome P-450 into cytochrome P-420 and restored aminopyrine N-demethylase and anilinen-hydroxylase activities of cytochrome P-450 in rats during acute hepatitis induced by CCl₄ and allyl alcohol. The polyphenol phytopreparation legalon did not prevent degradation of cytochrome P-450. Differences in the effects of hepatoprotectors on the impaired antitoxic function of the liver are probably associated with the abilities of essentiale and eplir to provide phospholipids for regeneration of endoplasmic reticulum membranes of hepatocytes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 4, pp. 392–394, April, 1999     

Role of interleukin-1 in the depression of liver drug metabolism by endotoxin.

Endotoxin-resistant C3H/HeJ mice were used to test the hypothesis that a macrophage product, possibly interleukin-1, might mediate the depression of liver cytochrome P-450-dependent drug metabolism in endotoxin-treated mice. Depression of liver drug metabolism by endotoxin was observed in normal mice (C3H/HeN) but not in C3H/HeJ mice. Serum transfer experiments demonstrated that a serum factor was responsible for the depression of liver drug metabolism. Experiments of passive transfer of peritoneal macrophages showed that this endotoxin-induced factor might be a macrophage product. In vitro experiments showed that endotoxin-stimulated monocytes produced a factor that depressed cytochrome P-450-dependent metabolism in cultured hepatocytes. Homogeneous human monocyte and recombinant interleukin-1 also depressed liver drug metabolism both in vivo and in vitro, suggesting that this macrophage product might be involved in the regulation of liver function by the immune system.     

Effect of phenobarbital on hepatic injury induced by chronic carbon tetrachloride treatment

Rats were given 1 ml CCl4 per kg body weight subcutaneously 2 times a week, for 16 weeks. The effects of simultaneous phenobarbital (PB) treatment (0.05% in drinking water) on the hepatotoxicity of CCl4 was studied during 16 weeks of treatment. The retardation of growth, the increase in liver weight and mortality were greater in animals receiving both PB and CCl4 than those given CCl4 alone. Cirrhosis was apparent only in animals treated by PB + CCl4. The potentiating effect of PB on CCl4 hepatotoxicity was also seen in hexobarbital sleeping time, the rate of hexobarbital metabolism, and the cytochrome P-450 content in liver microsomes. The inducing effect of PB alone decreased with time both in vivo and in vitro, which suggests an adaptation or some kind of exhaustion of liver to the effects of PB.     

Protection from N-nitrosodimethylamine-mediated liver damage by indole-3-carbinol

Indole-3-carbinol (I-3-C) was examined for its ability to protect mice against 24-hr N-nitrosodimethylamine (NDMA)-mediated hepatotoxicity. NDMA (20 mg/kg body weight) alone produced extensive hemorrhagic and centrolobular necrotic lesions, with a necrotic severity index of 3.0 +/- 0.4 (scale of 0-5). Treatment with 50 mg/kg body weight of I-3-C by gavage, 1 hr prior to NDMA, substantially protected against hemorrhagic lesions. Furthermore, I-3-C lowered the NDMA-mediated tissue necrotic index to 1.5 +/- 0.3, by reducing the extent of tissue necrosis rather than the severity in the necrotic region. Release of liver enzymes into the blood correlated with the histopathology; I-3-C reduced NDMA-mediated elevated activities of plasma alanine transaminase and ornithine transcarbamylase by 84 and 51.3%, respectively. Although no changes in nonprotein sulfhydryls were evident at 24-hr after NDMA, ascorbate levels were reduced to 40% of control values. However, treatment with I-3-C prior to NDMA prevented the decline in tissue ascorbate concentrations. In vitro, I-3-C was found to be a type II ligand for cytochrome P-450, with a Ks value of 237 microM. However, if such binding occurs in vivo, it does not protect against the approximately 60% decrease in hepatic cytochrome P-450 or the 80% decrease in NDMA demethylase I activity produced by NDMA. Since I-3-C slightly enhances cytochrome P-450 content and NDMA demethylase activity, the histopathologic protection by I-3-C must be due to factors other than inhibiting metabolic activation of NDMA.     

Immunohistochemical localization of cytochrome P-450 in rat brain

The presence of cytochrome P-450 in rat brain was studied by immunohistochemistry, using antibodies to cytochrome P-450 purified from livers of phenobarbital- or 3-methylcholanthrene-treated rats. Immunoreactive nerves were observed only in brain sections incubated with immunoglobulin-G to 3-methylcholanthrene-induced cytochrome P-450. This immunoreactivity was abolished by preabsorption of the antibody with highly purified rat liver cytochrome P-450c, the major cytochrome P-450 isozyme induced by 3-methylcholanthrene, but was not affected by other cytochrome P-450 isozymes induced by phenobarbital, isosafrole or pregnenolone-16-carbonitrile.

The most abundant concentration of nerve fibers with cytochrome P-450 immunoreactivity was observed in the globus pallidus. Immunoreactive fibers were also observed in the caudate putamen, amygdala, septum, ventromedial nucleus of the hypothalamus, medial forebrain bundle, ansa lenticularis, and ventromedial portion of the internal capsule and crus cerebri. Cell bodies with cytochrome P-450 immunoreactivity were observed in the caudate putamen and in the perifornical area of the hypothalamus. The cytochrome P-450 immunoreactive fibers in the globus pallidus and caudate putamen do not appear to emanate from cell bodies in the substantia nigra, since there was no reduction in the density of these fibers after unilateral stereotaxic electrolytic destruction of the substantia nigra (zona compacta and reticulata). Our data suggest that these striatal nerve processes are derived from cell bodies within the caudate putamen itself.

The present results indicate that rat brain contains a form of cytochrome P-450 with antigenic relatedness to the hepatic 3-methylcholanthrene-inducible cytochrome P-450c. This cytochrome P-450 isozyme was detected in brain areas which metabolize morphine and convert estradiol and estrone into catecholestrogens, which suggests an important role for this enzyme in the metabolism of both ex´ogenous and endogenous compounds in brain.     

Drug metabolism in rats with arthritis induced by 6-sulfanilamidoindazole (6-SAI)

Hexobarbital sleeping time was prolonged and ethylmorphine N-demethylation was inhibited after a single dosage or seven administrations of 6-SAI to old rats. These effects were independent of the development of arthritis. Changes in cytochrome P-450 concentration after 6-SAI treatment were insignificant and thus not responsible for the decrease in drug metabolism.In vitro 6-SAI inhibited ethylmorphine N-demethylation; the inhibition was of a mixed type. 6-SAI bound to cytochrome P-450 and induced a type II spectrum. The magnitude of hexobarbital-induced type I spectral changes was diminished by 6-SAI.It is concluded that 6-SAI inhibits cytochrome P-450-dependent drug metabolism by binding to cytochrome P-450.     

Influence of age-related changes in rodent liver morphology and physiology on drug metabolism--a review

Age-related changes in weight, morphology and physiology of the rodent liver influencing hepatic drug metabolism are reviewed. Next to the changes in liver weight/body weight ratio with age, the spontaneous occurrence of neoplastic and non-neoplastic lesions may be of particular importance. In addition, the decrease in liver blood blow with age diminishes the biotransformation capacity of the total liver. However, the albumin concentration in plasma and drug uptake do not play important roles, since they are unchanged or only slightly lower in old rats or mice. Drugs are generally metabolized by the liver in two phases: the so-called phase I and phase II metabolism. For most drugs, the phase I reaction is an oxidation. This reaction is catalyzed by cytochrome P-450, cytochrome b5 and NADPH-cytochrome c reductase. In microsomes, a decrease with age is generally observed in the cytochrome P-450 concentration and the NADPH-cytochrome c reductase activity, while there is no change in cytochrome b5. In addition, most microsomal drug-metabolizing enzymes decrease with age in male rats but not in females. The changes in enzyme activities in the male and female mouse are more complex. In fact, increases, decreases and no changes were found. Important phase II reactions are glutathione conjugation and glucuronidation; changes in both reactions with age seem to be of minor importance. Studies with hepatocytes isolated from male rats of different ages reveal that the monooxygenase system mediated metabolism of digitoxin and aflatoxin B1 decreases with age. It can be concluded that the observed decrease in the functional capacity of the monooxygenase system greatly determines the decrease in drug metabolism with age. However, it should always be kept in mind that, among others, the age-related changes in drug metabolism in rats are strongly sex dependent, which is not the case in man. Therefore, caution should be exercised in transferring these data to the human situation.     

Liver tumor promoting effects of fenbendazole in rats.

In order to examine whether fenbendazole has tumor-promoting activity, a total of 70 male Fischer 344 rats were initiated with a single intraperitoneal injection of 100 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone; beginning 1 wk later, rats were given a diet containing 3,600; 1,800; 600; 200; 70; or 0 ppm of fenbendazole for 8 wk. Subgroups of 5 rats each from the DEN+ 1,800; DEN+0; 1,800; and 0 ppm groups were euthanatized after 1 wk of fenbendazole treatment, and the remaining animals were euthanatized at 8 wk. After 1 wk, relative liver weights (ratios to body weights) were significantly increased in the DEN+ 1,800 and 1,800 ppm groups, and based on light microscopy, periportal hepatocellular hypertrophy was evident in these groups. After 8 wk, relative liver weights were significantly increased in the groups given > or =600 ppm with or without DEN initiation. Periportal hepatocellular hypertrophy, characterized by a marked increase in smooth endoplasmic reticulum, was observed in the groups given > or =600 ppm with or without DEN initiation. Induction of cytochrome P-450 (CYP) 1A2, 2B1, or 4A1 was noted in the fenbendazole-treated groups with or without DEN initiation; that associated with CYP 1A2 was most marked. Positive immunostaining for anti-CYP 1A1/2 or CYP 2B1/2 was observed diffusely in the livers of animals in the DEN+1,800 and DEN+3,600 ppm groups. The numbers and areas of connexin 32 (Cx32)-positive spots per square centimeter in centrilobular hepatocytes were significantly decreased in an almost dose-dependent manner with fenbendazole treatment after DEN initiation. In situ hybridization for Cx32 mRNA revealed a remarkable decrease in its expression in the centrilobular hepatocytes in the DEN+70 ppm group. The numbers of glutathione S-transferase placental-form positive single cells (plus mini foci) were significantly increased in the DEN+ 1,800 and DEN+3,600 ppm groups. Since those agents that induce CYP 2B1/2 isozymes and reduce Cx32 in centrilobular hepatocytes have been suggested to be liver tumor promoters, the present results indicate that fenbendazole may be a liver tumor promoter.     

The effect of nanofibrous galactosylated chitosan scaffolds on the formation of rat primary hepatocyte aggregates and the maintenance of liver function

Liver tissue engineering requires a perfect extracellular matrix (ECM) for primary hepatocytes culture to maintain high level of liver-specific functions and desirable mechanical stability. The aim of this study was to develop a novel natural nanofibrous scaffold with surface-galactose ligands to enhance the bioactivity and mechanical stability of primary hepatocytes in culture. The nanofibrous scaffold was fabricated by electrospinning a natural material, galactosylated chitosan (GC), into nanofibers with an average diameter of ～160 nm. The GC nanofibrous scaffolds displayed slow degradation and suitable mechanical properties as an ECM for hepatocytes according to the evaluation of disintegration and Young's modulus testing. The results of morphology characterization, double-staining fluorescence assay and function detection showed that hepatocytes cultured on GC nanofibrous scaffold formed stably immobilized 3D flat aggregates and exhibited superior cell bioactivity with higher levels of liver-specific function maintenance in terms of albumin secretion, urea synthesis and cytochrome P-450 enzyme than 3D spheroid aggregates formed on GC films. These spheroid aggregates could be detached easily during culture period from the flat GC films. We suggest such GC-based nanofibrous scaffolds could be useful for various applications such as bioartificial liver-assist devices and tissue engineering for liver regeneration as primary hepatocytes culture substrates.     

Cytological and quantitative cytochemical changes in the hepatocyte population of newborn rats following hydrocortisone administration

Changes have been assessed in cytological and quantitative cytochemical parameters of the hepatocyte population of newborn rats under glucocorticoid stimulation. Administration of hydrocortisone-acetate at the dose of 25 micrograms/g b.w./d during the 2nd week of postnatal life, caused: 1. an increase of the liver weight and of average dry mass, protein content, and volume of the hepatocytes; 2. a decrease of the number of hepatocytes per mg of liver tissue; 3. a reduction of the mitotic activity in liver parenchyma; 4. a gain in number of hepatocytes per liver lower than under normal conditions; 5. an increase of frequency of binuclear cells; 6. an increase of DNA-Feulgen per hepatocyte nucleus; 7. an increase per cell, greater than the mean protein increase per cell, in activity of arylhydrocarbonmonooxygenase and 7-ethoxycoumarin 0-deethylase, 2 enzymes dependent on cytochrome P-450. Induction of arylhydrocarbonmonooxygenase activity was prevalent in centrolobule. All the examined parameters, except that of DNA-Feulgen per nucleus and that of mitotic activity, changed strictly correlated with the duration of hormonal treatment. The values of a number of hepatocyte parameters (particularly: mean cell dry mass and volume, frequency of binuclear cells, enzymic activity) detected in the 12 d old rats after a 5 d long hormonal pretreatment, were in the range of those of animals 1 to 2 weeks older.     

Microarray analysis on CYPs expression in pregnant rats after treatment with pregnenolone-16alpha-carbonitrile and phenobarbital

We previously reported the protein expression profiles of nine cytochrome P450 isozymes (CYPs) in pregnant rat's liver, fetal liver, and placenta after treatment with pregnenolone-16alpha-carbonitrile (PCN), dexamethasone (DEX), or phenobarbital (PB). In this study, the gene expression of 40 CYPs and 2 orphan nuclear receptors for CYP inducers, that is, Nr1i2 (CYP3A subfamily inducible by PCN) and Nr1i3 (CYP2B subfamily inducible by PB), in pregnant rat's liver, fetal liver, and placenta was investigated at one time. Fischer 344 (F344) pregnant rats were daily treated intraperitoneally with 50 mg/kg of PCN or 80 mg/kg of PB from 13 to 16 days of gestation (DG). They were sacrificed on 17 DG, and microarray analysis using Affymetrix Rat Expression Array 230A was performed. Ten genes expression significantly increased in dam's liver in PCN group, and seven genes expression in PB group. On the other hand, four genes expression increased in fetal liver in PCN group, and three genes expression increased in PB group. Being common to dam's and fetal livers, the gene expression of Cyp3A1 (CYP3A subfamily) and cytochrome P-450e (CYP2B subfamily) increased in both PCN and PB groups. In placenta, the expression of Cyp3A1 gene was significantly induced in PB group, and it also showed a tendency to increase in PCN group. The expression of Nr1i2 gene was significantly elevated only in dam's liver of PCN group, while the expression of Nr1i3 gene showed no changes in all groups. The results of the present study of 40 CYPs gene expression mostly corresponded to our previous reports on 9 CYPs protein expression.