Adult Rat Liver Research Articles

Cell adhesion aside from integrin system can abrogate anoikis in rat liver cells by down-regulation of FasL expression, not by activation of PI-3K/Akt and ERK signaling pathway

Epithelial cells require contact with extracellular matrix (ECM) to inhibit detachment-induced apoptosis (anoikis). The ERK and PI-3K/Akt signaling pathways have been identified to inhibit anoikis. We present here a different story. An adult rat liver cell line, ARLJ301-3, underwent apoptosis within 4 h under suspension conditions even with active forms of Akt and ERK1/2. Once ARLJ301-3 cells are plated on tissue culture plates coated with synthetic polymer, such as poly-( N- p-vinyl benzyl- O-β- d-galactopyranosyl- d-gluconamide) (PVLA), poly- l-lysine or polystyrene, instead of functional ECM such as fibronectin, they could survive and proliferate without activation of Akt and ERK1/2. The expression of Fas receptor ligand (FasL) is specifically detected in cells under suspension conditions or treated with cytochalasin-D. We present here the first report that FasL expression is up-regulated by the cytoskeletal disruption directed by cytochalasin-D treatment or cell detachment from ECM.

Cloning and molecular expression analysis of large and small lecithin:retinol acyltransferase mRNAs in the liver and other tissues of adult rats

Retinyl ester, the most abundant form of vitamin A (retinol), is synthesized by the enzyme lecithin:retinol acyltransferase (LRAT). Previously, we cloned a 2.5 kb LRAT cDNA from rodent liver which codes for functional LRAT activity. However, Northern blots of tissues probed with the 2.5 kb cDNA revealed the presence of a larger transcript of approximately 5 kb as well as several smaller transcripts. To elucidate the nature of the large LRAT transcript, a high-molecular-mass adrenal gland cDNA library was screened. Two similar clones of 3962 and 3187 nt were identified which appeared to be part of the 3'-untranslated region (UTR) of a 5358 nt LRAT mRNA. The 5.3 kb cDNA was then amplified from liver by reverse transcriptase PCR (RT-PCR) and demonstrated to encode functional LRAT activity. The 3'-UTR of the 5.3 kb cDNA contains several AAUAAA polyadenylation signals. Analysis of the 3' ends of LRAT mRNA transcripts from liver, intestine and testis showed the usage of both canonical and non-canonical polyadenylation signals. To further analyse the LRAT mRNAs expressed in vivo, Northern blot analysis was performed using four probes spanning sections from the 5' end to the distal 3' end of the 5.3 kb LRAT cDNA. The results show that the major 5.3 kb transcript uses the canonical signal AAUAAA located at nt 5308, and the major short transcript of approximately 1.5 kb uses the non-canonical signal AUUAAA located at nt 1330. The 5.3 kb LRAT transcript was predominant in the liver of retinoic acid-repleted vitamin A-deficient rats, coincident with increased quantitative expression of LRAT mRNA and enzyme activity. The differential usage of these polyadenylation signals can explain the presence of multiple LRAT mRNA transcripts which are expressed in different tissue-specific patterns.

Heterogeneity of Ductular Reactions in Adult Rat and Human Liver Revealed by Novel Expression of Deleted in Malignant Brain Tumor 1

The regenerative capacity of mammalian adult liver reflects the ability of a number of cell populations within the hepatic lineage to take action. Limited information is available regarding factors and mechanisms that determine the specific lineage level at which liver cells contribute to liver repair as well as the fate of their progeny in the hostile environment created by liver injury. In the present study, we attempted to identify novel molecules preferentially involved in liver regeneration by recruitment of transit-amplifying, ductular (oval) cell populations. With a subtractive cDNA library screening approach, we identified 48 enriched, nonredundant gene products associated with liver injury and oval cell proliferation in the adult rat liver. Of these, only two, namely alpha-fetoprotein and a novel transcript with high homology to human DMBT1 (deleted in malignant brain tumor 1), were specifically associated with the emergence of ductular (oval) cell populations in injured liver. Subsequent cloning and characterization of the rat DMBT1 homologue revealed a highly inducible expression in ductular reactions composed of transit-amplifying ductular (oval) cells, but not in ductular reactions after ligation of the common bile duct. In human liver diseases, DMBT1 was expressed in ductular reactions after infection with hepatitis B and acetaminophen intoxication, but not in primary biliary cirrhosis, primary sclerosing cholangitis, and obstruction of the large bile duct. The expression heterogeneity in ductular reactions and multiple functions of DMBT1 homologues point to intriguing roles in regulating not only tissue repair but also fate decision and differentiation paths of specific cell populations in the hepatic lineage.

Effect of reduced dietary protein intake on hepatic and plasma essential fatty acid concentrations in the adult female rat: effect of pregnancy and consequences for accumulation of arachidonic and docosahexaenoic acids in fetal liver and brain

During pregnancy, the accumulation of long-chain polyunsaturated fatty acids (LCPUFA) in fetal tissues places a substantial demand upon maternal lipid metabolism. As lipid metabolism is intimately linked to aspects of protein metabolism, a reduced protein intake in pregnancy may impair activities of enzymes and transport proteins responsible for supplying LCPUFA to the fetus, thereby compromising fetal development. We have investigated the effect of reduced protein intake on LCPUFA status in the non-pregnant rat and in the pregnant rat, and in fetus at day 20 of gestation. Female rats (n 5 per group) were either mated and fed the control diet (180 g protein/kg) or low-protein diet (90 g protein/kg, LPD) diet throughout pregnancy, or fed the control diet or LPD for 20 d (non-pregnant animals). The fatty acid compositions of maternal liver and plasma, and fetal liver and brain were determined by GC. Feeding the LPD did not lead to any gross changes either in adult or fetal growth, or in total lipid concentrations in adult rat liver. However, the LPD was associated specifically with lower liver (42.6 %) and plasma (19.4 %) phosphatidylcholine (PC), and plasma triacylglycerol (28.6 %) docosahexaenoic acid (DHA) concentrations in pregnant rats and reduced fetal brain PC- (26.1 %) and phosphatidylethanolamine- (25.6 %) DHA concentrations. Together, these results show that variations in maternal dietary protein consumption alter DHA status in pregnancy and modify DHA accumulation into the fetal brain. The present results suggest that lower maternal protein intakes reduce delivery of DHA from the mother to the fetus, which may impair development and function of the fetal brain.

Effect of S-nitrosoglutathione (GSNO) added to the University of Wisconsin solution (UW): II) functional response to cold preservation/reperfusion of rat liver

Livers cold preserved in University of Wisconsin (UW) solution followed by reperfusion suffer ischemia/reperfusion injuries. Microcirculation is the primary target of damage, characterized by sinusoidal perfusion failure due, mainly, to morphological changes of sinusoidal endothelial cells. Here, we demonstrated that the addition of S-nitrosoglutathione (GSNO) to the UW solution before cold storage, as a nitric oxide (NO) donor, attenuated hepatic injuries.Wistar adult rat livers were stored in UW solution (0°C – 48 hs) and then reperfused during 60 minutes using the Isolated Perfused Rat Model (IPRL). We assayed four GSNO concentration (50, 100, 250 and 500 μM). NO concentration was estimated calculating the amount of nitrite (NO2-) generated in the UW solution. Injuries during cold preservation were established measuring lactate dehydrogenase (LDH) released to the UW solution. Meanwhile, intrahepatic resistance (IR), LDH released to the perfusate, the effluent/perfusate ratio for K+, bile flow, liver glycogen content and sinusoidal endothelial cell morphology were studied after 1 hour of reperfusion in the IPRL system.In cold preserved livers without GSNO, glycogen content was dramatically reduced, IR increased markedly, LDH released was high, bile flow diminished and sinusoidal endothelial cells appeared rounded and detached from perisinusoidal matrix after reperfusion. The presence of 100 μM GSNO prevented the IR rise and LDH release, improved bile production and partially reduced endothelial cells damages.In conclusion, the addition of 100 μM GSNO to UW solution improved hemodynamic and function capacity of cold preserved/reperfused livers.

Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary

CYP1B1, a member of the cytochrome P450 superfamily, is expressed constitutively in the steroidogenic tissues of mammals and is inducible by peptide hormones, cAMP and aromatic hydrocarbon receptor (AHR) ligands. The mechanism of induction of this cytochrome P450 is similar to that for CYP1A1, i.e. through the aromatic hydrocarbon receptor (AHR) signaling pathway. We have recently reported that CYP1B1, but not CYP1A1, is expressed in rat granulosa cells (GC) in the absence of any external stimulus. The induction of CYP1B1 mRNA in rat GC by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in vitro was followed by an increase in AHR and estrogen receptor (ER-β) RNA levels. Estrous cycle-dependent expression of AHR, AHR-nuclear translocator (ARNT) and ER-mRNAs in the rat ovary was reported. We suggest that CYP1B1 may play a major role in the regulation of rat ovarian function/cycle but until now this has been unexplored experimentally. The present study was therefore aimed at examining the expression of CYP1A1, CYP1B1 and ER-mRNA in rat ovarian tissues throughout the estrous cycle to establish any correlation in the expressions of these mRNAs in rat ovary. Total RNA was extracted from the ovary and liver of cycling adult rats and the mRNAs were analyzed using relative RT-PCR with gene-specific primers for the target mRNA and for RPL 19 or S16 primers as an internal control. The results indicated that in the ovary, CYP1B1 mRNA increased significantly on the evening of proestrus and dramatically decreased on the morning of estrus, while ER-mRNA remained unaltered throughout the estrous cycle. CYP1A1 mRNA in the ovary and both CYP1A1 and CYP1B1 mRNAs in the liver were undetectable. That the sudden decrease of ovarian CYP1B1 mRNA on the morning of estrus was not an effect of the LH surge was verified in vitro using our short-term GC culture model. GC prepared from rats super-stimulated with equine chorionic gonadotropin (eCG) were cultured for 6 h with or without LH and TCDD. It was observed that both CYP1A1 and CYP1B1 mRNAs were induced by TCDD with no apparent effect of LH. It is suggested that the high level of CYP1B1 mRNA expression on the evening of proestrus in rat ovary might be involved in metabolism of estrogens to catecholestrogen (a known effect of CYP1B1), and that expression is unaffected in GC by LH.

High Dietary Iron Concentrations Enhance the Formation of Cholesterol Oxidation Products in the Liver of Adult Rats Fed Salmon Oil with Minimal Effects on Antioxidant Status

The aim of this study was to investigate the effect of high dietary iron concentrations on the antioxidant status of rats fed two different types of fat. Four groups of male adult Sprague-Dawley rats were fed diets with adequate (50 mg iron supplemented per kg diet) or high (500 mg iron supplemented per kg diet) iron concentrations with either lard or salmon oil as dietary fat at 100 g/kg for 12 wk. The antioxidant status of the rats was profoundly influenced by the type of fat. Rats fed salmon oil diets had higher concentrations of thiobarbituric acid-reactive substances (TBARS) (P < 0.001), various cholesterol oxidation products (COP) (P < 0.001), total and oxidized glutathione (P < 0.05) and a lower concentration of alpha-tocopherol (P < 0.05) in liver and plasma than rats fed lard diets. The iron concentration of the diet did not influence the concentrations of TBARS, the activities of superoxide dismutase and glutathione peroxidase or the concentration of alpha-tocopherol in plasma or liver. The activity of catalase (P < 0.01) and the concentrations of total, oxidized and reduced glutathione (P < 0.05) in liver were slightly but significantly higher in rats fed high iron diets than in rats fed adequate iron diets, irrespective of the dietary fat. Rats fed the high iron diets with salmon oil, moreover, had higher concentrations of various COP in the liver (P < 0.001) than rats fed adequate iron diets with salmon oil. These results suggest that feeding a high iron diet does not generally affect the antioxidant status of rats but enhances the formation of COP, particularly if the diet is rich in polyunsaturated fatty acids.

Morphological changes in lungs, placenta, liver and kidneys of pregnant rats exposed to cigarette smoke.

Histopathological examination of changes in foetal and newborn rats, and histopathological and morphometric assessments of changes in lungs, placenta, liver and kidneys of adult rats exposed to cigarette smoke were performed. Non-pregnant and pregnant Wistar female rats were exposed to cigarette smoke at a carbon monoxide concentration of 1500 mg/m(3) for 6 h per day, 5 days per week, for 3 weeks. Levels of urine nicotine and cotinine were used as measures of exposure. Paraffin-embedded, haematoxylin and eosin (HE)-stained sections were used for examination. Morphometry of studied organs was performed using a computer image analyser. Applied smoke dose and exposure time produced dramatic histopathological changes in lungs of exposed rats (emphysema, emphysematous, inflammatory, metaplastic changes) and reduction in height of respiratory-bronchiole epithelium, and considerably less-marked morphological changes in hepatic (number of apoptotic hepatocytes) and renal (height of proximal convoluted tubule epithelium) cells as well as in the placenta (for example, size of giant cells in labyrinth, height of epithelium covering yolk sac ). Exposure to cigarette smoke did not result in histopathological changes in lungs and liver of surviving foetuses. The duration of pregnancy was not changed but a tendency for a decrease in the mothers' fertility indices as well as some changes in foetal and newborn parameters was observed. Taking into account the morphological changes observed in adult rat tissues and placentae which can result in definite hormonal and trophic effects on the foetus, the possibility of early or late physiological effects in progeny under the influence of cigarette smoke must be taken into consideration.

A Role for CCAAT/Enhancer-binding Protein in Hepatic Expression of Thrombin-activable Fibrinolysis Inhibitor

Thrombin-activable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase B-like zymogen that upon activation by thrombin, thrombin-thrombomodulin, or plasmin attenuates fibrin clot lysis by inhibiting positive feedback in the fibrinolytic cascade. The concentration of TAFI in plasma varies in the human population and thus may constitute a risk factor for thrombotic disorders. In addition, TAFI has been reported to be a positive acute phase reactant in mice. We have initiated molecular analysis of the human TAFI promoter to understand the mechanisms underlying regulation of TAFI gene expression. We identified a putative C/EBP-binding site between -53 and -40 of the promoter. Mutations in this site that abolish C/EBP binding decrease TAFI promoter activity in human hepatoma (HepG2) cells by approximately 80%. Gel mobility shift analyses indicated that C/EBP-beta present in HepG2 nuclear extracts and C/EBP-alpha and -beta present in adult rat liver nuclear extracts bind to the C/EBP site. C/EBP-alpha, -beta, and -delta isoforms are all capable of binding to the C/EBP site and activating the TAFI promoter. The identification of a functional C/EBP-binding site in the human TAFI promoter may have important implications for the regulation of expression of this gene during development and in response to inflammatory stimuli.

Effect of dietary fatty acids on lipoprotein lipase gene expression in the liver and visceral adipose tissue of fed and starved red sea bream Pagrus major

Juvenile red sea bream Pagrus major were fed either a commercial diet (diet 1) or diets supplemented with 10% oleate (diet 2), 5% oleate+5% linoleate (diet 3) or 5% oleate+5% n-3 polyunsaturated fatty acid mixture (diet 4) for 4 weeks. Following the conditioning period, the effects of dietary fatty acids on lipoprotein lipase (LPL) gene expression in the liver and visceral adipose tissue of fed (5 h post-feeding) and starved (48 h post-feeding) fish were investigated by competitive polymerase chain reaction. Fish liver showed substantial LPL mRNA expression that is not found in adult rat liver. When compared with diet 1, diets 2–4 tended to increase the LPL mRNA level in the liver, but tended to decrease it in the visceral adipose tissue under the fed condition. The reciprocal regulation of the liver and visceral adipose LPL mRNA abundance by dietary fatty acids was comparable to that of rat brown and white adipose tissue, respectively. The change in the LPL mRNA level by fatty acids was not completely consistent with the degree of fatty acid unsaturation. Our results indicate that the regulatory effect of dietary fatty acids on LPL gene expression was tissue-specific and related to feeding conditions, but was not solely dependent on the degree of unsaturation of fatty acids.

Concentrations of Seven Iodothyronine Metabolites in Brain Regions and the Liver of the Adult Rat

The concentrations of the iodothyronine metabolites T4, T3, 3,5-diiodothyronine (3,5-T2), 3,3′-diiodothyronine (3,3′-T2), reverse T3 (rT3), 3,3′-T2 sulfate (3,3′T2S), and T3 sulfate (T3S) were measured in 12 regions of the brain, the pituitary gland, and liver in adult male rats. Quantification of iodothyronine was performed by RIA following a newly developed method of purification and separation by HPLC. 3,5-T2, 3,3′-T2, rT3 and T2S were detectable in the low femtomolar range (20–200 fmol/g) in most areas of the rat brain. T3S was detectable only in the hypothalamus. The concentrations of T3 and T4 were approximately 20- to 60-fold higher, ranging between 1 and 6 pmol/g. There was a significant negative correlation between the activities of inner-ring deiodinase and T3 concentrations across brain areas. In the liver, 3,5-T2, rT3, and T3S were measurable in the low femtomolar range, whereas 3,3′-T2 and 3,3′T2S were not detectable. 3,5-T2 and 3,3′-T2 were not detectable in mitochondrial fractions of the brain regions. Tissue concentrations of 3,5-T2 exhibited a circadian variation closely parallel to those of T3 in the brain regions and liver. T3 was not a substrate for outer-ring deiodination under different experimental conditions; thus, it remains unclear which substrate(s) and enzyme(s) are involved in the production of 3,5-T2. These results indicate that five iodothyronine metabolites other than T3 and T4 are detectable in the low femtomolar range in the rat brain and/or liver. The physiological implications of this finding are discussed.

Concentrations of seven iodothyronine metabolites in brain regions and the liver of the adult rat.

The concentrations of the iodothyronine metabolites T(4), T(3), 3,5-diiodothyronine (3,5-T(2)), 3,3'-diiodothyronine (3,3'-T(2)), reverse T(3) (rT(3)), 3,3'-T(2) sulfate (3,3'T(2)S), and T(3) sulfate (T(3)S) were measured in 12 regions of the brain, the pituitary gland, and liver in adult male rats. Quantification of iodothyronine was performed by RIA following a newly developed method of purification and separation by HPLC. 3,5-T(2), 3,3'-T(2), rT(3) and T(2)S were detectable in the low femtomolar range (20-200 fmol/g) in most areas of the rat brain. T(3)S was detectable only in the hypothalamus. The concentrations of T(3) and T(4) were approximately 20- to 60-fold higher, ranging between 1 and 6 pmol/g. There was a significant negative correlation between the activities of inner-ring deiodinase and T(3) concentrations across brain areas. In the liver, 3,5-T(2), rT(3), and T(3)S were measurable in the low femtomolar range, whereas 3,3'-T(2) and 3,3'T(2)S were not detectable. 3,5-T(2) and 3,3'-T(2) were not detectable in mitochondrial fractions of the brain regions. Tissue concentrations of 3,5-T(2) exhibited a circadian variation closely parallel to those of T(3) in the brain regions and liver. T(3) was not a substrate for outer-ring deiodination under different experimental conditions; thus, it remains unclear which substrate(s) and enzyme(s) are involved in the production of 3,5-T(2). These results indicate that five iodothyronine metabolites other than T(3) and T(4) are detectable in the low femtomolar range in the rat brain and/or liver. The physiological implications of this finding are discussed.

Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells

The liver is believed to contain stem cells that can differentiate into either hepatocytes or biliary epithelial cells. In the present study, we established a nonhepatocytic epithelial cell line from the normal livers of adult rats. The established cells, designated HSL cells, were immunoreactive against α-fetoprotein, but neither albumin nor cytokeratin 19. To demonstrate the differentiation potential of HSL cells in vitro, the cells were cocultured with hepatic stellate cells as a mixture or separately using insert wells. Consequently, although coculture with hepatic stellate cells rendered HSL cells able to produce albumin, the mixed coculture system mimicking the hepatic environment elicited this phenomenon more effectively than the separated coculture system. In conclusion, HSL cells have immature properties and the potential to differentiate into mature cells. Not only the extracellular matrices but also soluble factors, which are produced by hepatic stellate cells, induce this maturation, demonstrating the importance of the hepatic environment for hepatocyte differentiation.

Delta-aminolevulinate dehydratase inhibition by phenyl selenoacetylene: effect of reaction with hydrogen peroxide.

The effect of phenyl selenoacetylene and its selenoxide on delta-aminolevulinate dehydratase from liver of adult rats (mammalian source) and from cucumber leaves (plant source) was investigated. In vivo, selenides can be oxidized to selenoxides by flavin-containing monooxygenases and selenoxides can regenerate selenides by thiol oxidation. The compound phenyl selenoacetylene was converted to selenoxides by reaction with hydrogen peroxide. Phenyl selenoacetylene inhibited mammalian and plant delta-aminolevulinate dehydratase with an IC50 about 250 microM and >400 microM, respectively. Its selenoxide inhibited the enzyme more strongly, with IC50 values of 45 microM and 100 microM for the mammalian and plant source, respectively. The selenoxide inhibitory action was antagonized by dithiothreitol suggesting the involvement of -SH groups. Moreover, delta-aminolevulinate dehydratase from a plant source was inhibited by the selenoxide, suggesting a possible involvement of -SH groups located at a site distinct from the region implicated in Zn2+ binding in mammalian delta-aminolevulinate dehydratase. The results of the present study suggest that (i) delta-aminolevulinate dehydratase is a potential molecular target for phenyl selenoacetylene, due to the oxidation of enzyme sulfhydryl groups, and that (ii) the monooxygenation of this selenocompound, which in vivo could be possibly mediated by flavin-containing monooxigenases, increases its inhibitory effect.

Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats

Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction. (HEPATOLOGY .)

In vivo cell lineage analysis in cyproterone acetate–treated rat liver using genetic labeling of hepatocytes

Genetic labeling using recombinant retroviruses is a powerful strategy for the study of cell lineage in the liver. However, this type of vector is only able to infect dividing cells. The synthetic steroid cyproterone acetate (CPA) is mitogenic and carcinogenic in the adult rat liver. In this study, we used retroviral vectors carrying the nuclear targeted β-galactosidase gene to selectively label and follow the fate of hepatocytes dividing on administration of CPA. Labeled cells as well as those in mitosis were preferentially located around the portal tract, whereas apoptotic bodies were predominant in the pericentral area. Labeled hepatocytes did not disappear after apoptosis, suggesting a preferential elimination of nontransduced cells. The presence of labeled binucleated hepatocytes showed the persistence of a binucleation process. Finally, we performed long-term analysis of labeled cells in transgenic animals tolerant for β-galactosidase and treated with 2-acetylaminofluorene (2-AAF) to promote the growth of CPA-initiated hepatocytes. The presence of β-galactosidase–positive hepatocyte clones showed that hepatocytes divided during treatment with 2-AAF. Only 3% of β-galactosidase clones were positive for the placental form of glutathione S-transferase (GSTp), indicating the absence of a preferential appearance of preneoplastic foci in the population of β-galactosidase–labeled hepatocytes. In conclusion, our results show that the mitogenic and tumor-initiating activities of CPA are directed toward different hepatocyte populations. (HEPATOLOGY .)

Developmental increases in rat hepatic microsomal UDP-glucuronosyltransferase activities toward xenoestrogens and decreases during pregnancy.

Xenoestrogens, such as bisphenol A and diethylstilbestrol, are glucuronidated by an isoform of UDP-glucuronosyltransferase named UGT2B1 in the livers of adult male rats. In this study, we found that nonylphenol and octylphenol are also conjugated with glucuronic acid by adult rat liver microsomes. Although UDP-glucuronosyltransferase activities toward these xenoestrogens were not detected in the fetal rat liver, a linear increase in enzymatic activities during neonatal development was observed. At 3 weeks after birth, the activities had reached the same level as that of adult rats. The protein and mRNA contents of UGT2B1 also were not detected in the fetal rat liver, but a developmental increase in newborn rat liver was detected by Western and Northern blotting analysis. Additionally, rat hepatic microsomal UDP-glucuronosyltransferase activities toward these xenoestrogens were reduced by about half during pregnancy of mother rats. The results suggest that the reproductive organs of fetal and early-stage neonatal rats, which are sensitive to sex hormones, face a high risk of exposure to free active xenoestrogens.

Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes.

Small hepatocytes (SHs), which are known to be hepatic progenitor cells, were isolated from an adult rat liver. SHs in a colony sometimes change their shape from small to large and from flat to rising/piled-up. The aim of the present study is to clarify whether the alteration of cell shape is correlated with the maturation of SHs and whether extracellular matrix (ECM) can induce the morphological changes of SHs. We used liver-enriched transcription factors (LETFs) such as hepatocyte nuclear factor (HNF) 4 alpha, HNF6, CCAAT/enhancer binding proteins (C/EBP) alpha, and C/EBP beta, tryptophan 2,3-dioxygenase (TO), and serine dehydratase (SDH) as markers of hepatic maturation. To enrich the number of SH colonies, the colonies were isolated from dishes and replated. Replated colonies proliferated and the average number of cells per colony was about five times larger at day 9 than at day 1. When the cells were treated with laminin, type IV collagen, a mixture of laminin and type IV collagen, Matrigel or collagen gel (CG), only the cells treated with Matrigel dramatically changed their shape within several days and had reduced growth activity, whereas the cells treated with other ECM did not. HNF4 alpha, HNF6, C/EBP alpha, C/EBP beta, and TO were well expressed in the cells treated with Matrigel. Furthermore, addition of both glucagon and dexamethasone dramatically induced the expression of SDH mRNA and protein in the cells treated with Matrigel. In conclusion, morphological changes of SHs may be correlated with hepatic maturation and basement membrane (BM)-like structure may induce the morphological changes of SHs.

Liver epithelial cell culture.

The establishment of liver epithelial cell lines or strains from newborn or adult rat liver has been reported by many investigators (-). The cells of these lines are smaller in size and morphologically more simple than hepatocytes, have a considerable growth potential, and are easy to passage. These features are different from normal rat hepatocytes, which usually do not proliferate without addition of growth factors (), as well as from biliary epithelial cells that quickly proliferate in culture, but cannot simply be subcultured in the absence of factors such as hybridoma growth factor (HGF) (,). The behavior is also different from that of isolated oval cells, which are much more reluctant to proliferate in culture (). Despite this fact, a relationship with oval cells has been claimed (), but is still not proven. Since other nonparenchymal cells do not show an epithelial morphology, it is rather unlikely that such cells could be the origin of these epithelial cells. Thus, the ultimate precursors of these cell lines are still not known, despite many efforts to characterize them using enzyme patterns and other antigens (,,,).

Displacement of histamine from liver cells and cell components by ligands for cytochromes P450.

Intracellular histamine (HA) and cytochrome P450 monooxygenases (P450) each have been proposed as mediators of cell function, growth, and proliferation. The P450 family of heme enzymes is found in virtually all cells and generates, transforms, or inactivates steroids and other lipids that participate in cell regulation. We previously demonstrated a second messenger role for HA in blood platelets and the formation of a HA-P450 heme complex when exogenous HA was added to microsomes isolated from rat liver cells or to purified human P450 isozymes. Employing a radioimmunoassay, we now demonstrate that rat liver slices, microsomes derived from the livers of adult male rats and mast cell-deficient mice, and hepatoma cells, all contain endogenous HA. HA release from microsomes into the incubation medium, as determined by radioimmunoassay, is enhanced in the presence of carbon monoxide, steroids, and certain drugs, all agents that unite either directly with the iron atom or bind elsewhere within the heme cavity. Rat liver slices preincubated with (3)H-HA release labeled amine into the medium in the presence of those same ligands. These findings provide evidence of an in situ HA-P450 complex and offer further support that the imidazole, HA, is a physiological, intracellular modulator of cytochromes P450 in liver cells, and perhaps of these and other heme proteins in tissues in general.