Cytochrome P450IA1 Research Articles

GSTM1 polymorphism along with PM 10 exposure contributes to the risk of preterm delivery

We investigated the association between the risk of preterm delivery and each metabolic gene of glutathione S-transferases mu 1 (GSTM1), theta 1 (GSTT1) and cytochrome P450IA1 (CYP1A1) along with exposure to particulate matter <10 μm (PM 10). This study was assumed to identify gene–environment interaction that increases the risk of preterm delivery. A case–control study was carried out on 117 women with preterm deliveries and 118 women with term deliveries in Seoul, Korea. Logistic regression analyses were performed to explore the impact of each gene, PM 10 exposure and their interaction on the risk of preterm birth. The risk of preterm birth was associated with the GSTM1 null genotype only. Exposure to high levels of PM 10 (≥75th percentile) during the third trimester of pregnancy was associated with an increased risk of preterm birth when compared to low-level exposure to PM 10 (<75th percentile). We found that exposure to high levels of PM 10 during the third trimester in the presence of the GSTM1 null genotype is significantly associated with the risk of preterm delivery. This finding is biologically plausible and provides evidence for a gene–environment interaction that increases the risk of preterm birth.

Soot Nanoparticles Promote Biotransformation, Oxidative Stress, and Inflammation in Murine Lungs

We previously described the physicochemical characteristics (particle size, adsorbed polynuclear aromatic hydrocarbons [PAHs], oxygen, and metal content) of butadiene soot (BDS) nanoparticles generated during incomplete combustion of the high-volume industrial petrochemical, 1,3-butadiene. We also demonstrated localization of BDS-delivered PAHs to lipid droplets of murine and human respiratory cells in vitro and up-regulation of biotransformation and oxidative stress responses in these cells. Here, the objective was to determine whether inhalation of BDS nanoparticles promotes up-regulation of Phase I biotransformation enzymes, oxidative stress responses, and inflammation in the lungs of mice. Female Balb/c mice exposed to BDS (5 mg/m(3), 4 h/d, 4 d) were killed immediately or 1 day after final exposure; bronchoalveolar lavage fluid (BALF) was collected from the lungs; total RNA was extracted from one lung and histopathology performed on the other. Histopathology and BALF analysis revealed particle-laden macrophages in airways of BDS-treated mice, accompanied by neutrophilia and epithelial damage. Microarray and qRT-PCR analyses revealed up-regulation of (1) aryl hydrocarbon receptor (AhR)-responsive genes: AhR repressor (Ahrr) and cytochrome P450 IA1 and IB1(Cyp1a1, Cyp1b1); (2) oxidative stress response genes: heme oxygenase 1 (Hmox1), nuclear factor erythroid-derived 2-like 2 (Nfe2l2), NADPH dehydrogenase quinone 1 (Nqo1), and glutathione peroxidase 2 (Gpx2); and (3) pro-inflammatory genes: interleukin-6 (IL-6), C-X-C motif ligand 2 (Cxcl2; analog to human IL-8) and ligand 3 (Cxcl3), and granulocyte chemotactic protein (Cxcl6). Inhalation of PAH-rich, petrochemical combustion-derived nanoparticles causes airway inflammation and induces expression of AhR-associated and oxidative stress response genes, as seen in vitro, plus pro-inflammatory genes.

Oxidative stress-related gene interactions with preterm delivery in Korean women

We hypothesized that the cytochrome P450IA1 (CYP1A1), glutathione S-transferases mu 1 (GSTM1), and theta 1 (GSTT1) polymorphisms are associated with the risk for preterm delivery. This study was undertaken to identify gene-gene interactions and haplotypes that increase the risk of preterm delivery. This case-control study was performed in Korea on 145 women with preterm birth and 120 normal controls. Logistic regression and multifactor dimensionality reduction analysis were used to explore single genes and gene-gene interactions, which have an impact on the risk of preterm delivery, respectively. The GSTM1 null genotype and the interaction between the CYP1A1-I462V and GSTM1 null type conferred a risk of preterm birth. The association between preterm delivery and the CYP1A1-T6235C/I462V haplotype plus GSTM1 null type was verified. The interaction between the CYP1A1-I462V and GSTM1 null genotype were found to increase the risk for preterm birth in Korean women. This finding provides evidence of a gene-gene interaction, which has an impact on preterm delivery.

Tissue Array Substratum Composed of Histological Sections: A New Platform for Orienting Differentiation of Embryonic Stem Cells Towards Hepatic Lineage

Culture technologies for differentiating embryonic stem cells (ESCs) into hepatic cells generally require some growth factors for several days. Here we present a new technology for efficiently differentiating mouse ESCs into hepatocyte-like cells in a supplement-free basal medium by using a tissue array substratum composed of histological sections. The substratum was prepared from liver tissues in various stages after administration of carbon tetrachloride to mice. The substrata derived from regenerating livers enhanced cell attachment, supported growth as clusters, and induced differentiation into cells expressing albumin, although the substrata from injured livers did not. In particular, the cells cultured on the most-proliferative regenerating liver-derived section substratum reconstructed the hepatic cord-like structures with bile canaliculus-like aspects in which some binucleated cells were involved, secreted albumin, and expressed cytochrome P450IA1 activity within a few days. This culture technology also provides a novel concept for Histomics, categorizing the features of different tissues based on the behavior of a cell line cultured on their array substratum (i.e., tissuome).

Three-Dimensional Primary Hepatocyte Culture in Synthetic Self-Assembling Peptide Hydrogel

Drug metabolism studies and liver tissue engineering necessitate stable hepatocyte cultures that express liver functions for a minimum of 4 days to 3 weeks. Current techniques, using different biomaterials and geometries, that maintain hepatocellular function in vitro exhibit a low cell density and functional capacity per unit volume. Herein we investigated a well-defined synthetic peptide that can self-assemble into three-dimensional interweaving nanofiber scaffolds to form a hydrogel, PuraMatrix, as a substrate for hepatocyte culture. Freshly isolated primary rat hepatocytes attached, migrated, and formed spheroids within 3 days after seeding on PuraMatrix. Hepatocytes expressed the apical membrane marker dipeptidyl peptidase IV at cell-cell contacts. Compared to the collagen sandwich, albumin and urea secretion on PuraMatrix were higher for the first week, and cytochrome P450IA1 activity was higher throughout the culture period. Mitochondrial membrane potential 1 day after seeding was higher on PuraMatrix than in the collagen sandwich, suggesting better preservation of the metabolic machinery. PuraMatrix and Matrigel showed similar albumin and urea production. PuraMatrix is an attractive system for generating hepatocyte spheroids that quickly restore liver functions after seeding. This system is also amenable to scale-up, which makes it suitable for in vitro toxicity, hepatocyte transplantation, and bioartificial liver development studies.

Contribution of Cytochrome P450IA1 Haplotypes Along With Air Pollution Exposures to Reduced Birth Weight

Contribution of Cytochrome P450IA1 Haplotypes Along With Air Pollution Exposures to Reduced Birth Weight

Cytochrome P450IA1 polymorphisms along with PM 10 exposure contribute to the risk of birth weight reduction

We explored the effects of particulate matter <10 μm (PM 10) exposure along with CYP1A1 polymorphisms of MspI (T6235C) and NcoI (Ile462Val) on reduced birth weight (BW). A prospective cohort study was done with women who delivered from 2001 to 2004 at Ewha Womans University Hospital, Seoul, Korea. We compared the estimated least squares means of BW in the generalized linear model, after adjusting for controlling factors. High PM 10 exposure at the 90th percentile level and above during the 1st trimester conferred a significant risk for reduced BW, compared with low PM 10 exposure below the 90th percentile level. The effect of high PM 10 exposure during the 1st trimester of pregnancy compared with low PM 10 exposure was greater for women with MspI TC/CC and NcoI IleVal/ValVal genotypes than for those with MspI TT and NcoI IleIle genotypes. In conclusion, high PM 10 exposure during the 1st trimester increased the risk for reduced BW in concert with MspI TC/CC and NcoI IleVal/ValVal genotypes in Korean women.

A novel formulation of oxygen‐carrying matrix enhances liver‐specific function of cultured hepatocytes

Oxygen is an important component of the cellular microenvironment, mediating cell survival, differentiation, and function. Oxygen supply is a limiting factor during culture of highly metabolic cells such as hepatocytes. Here we present a simple formulation of a fluorocarbon-based oxygen carrier embedded in collagen gel that increases oxygen concentration in culture 6-fold. Rat hepatocytes cultured on oxygen carrier-collagen showed a significant increase in viability and function. Cytochrome P450IA1 activity was increased by 140% in serum-free cultures and by 820% in serum-containing cultures. The significantly higher hepatocellular function on oxygen carrier-collagen matrix persisted and increased during long-term culture. Long-term albumin secretion was increased by 350% in serum-free cultures and by 166% in serum-containing culture. Long-term urea secretion was increased by 79% in serum-free cultures and by 76% in serum-containing cultures. We conclude that oxygen supply may limit hepatocyte function in vitro. This limitation can be overcome by addition of an oxygen carrier to the extracellular matrix. Culture of hepatocytes on oxygen-carrying matrix mimics the oxygen-rich environment of the liver and provides a simple method for enhanced long-term function.

Evidence for a new human CYP1A1 regulation pathway involving PPAR-α and 2 PPRE sites

Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction. The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer. We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.

Comparison of Activities Dependent on Glutathione S-Transferase and Cytochrome P-450 IA1 in Cultured Keratinocytes and Reconstructed Epidermal Models

There is an increasing need for in vitro testing of compounds for topical application. Reconstructed epidermal models may provide a suitable and relevant model for screening compounds that may affect the activities of phase I and II enzymes involved in epidermal detoxification. In this study, we measured the activity of a phase I enzyme, cytochrome P450 IA1, i.e. 7-ethoxyresorufin-O-deethylase (EROD) and 7-ethoxycoumarin-O-deethylase (ECOD) activities, and that of a phase II enzyme, glutathione S-transferase (GST). The enzyme activities were determined in cultured keratinocytes, reconstructed epidermal models and samples of human epidermis or hair follicle. EROD activity was detected in cultured keratinocytes and was induced by 3-methylcholanthrene (3-MC) and β-naphthoflavone. The level of induction increased with increasing confluence. Induced EROD activity could be inhibited by clotrimazole in a dose-dependent manner. However, EROD activity was not detected in either hair follicles or untreated epidermal models but could be induced by 3-MC. The ability to induce EROD activity in epidermal models was batch dependent, and clotrimazole was able to inhibit the induced EROD activity. ECOD activity was detected in untreated models and paralleled EROD activity. GST activity was detected in cultured keratinocytes and all epidermal models. GST activity in models was equal or higher than the activity in epidermal samples. Reconstructed skin models may be useful to study the effects of non-water-soluble topical formulations on xenobiotic metabolism.

Propofol-induced cytochrome P450 inhibition: An in vitro and in vivo study in rats

Propofol, a widely used anesthetic drug, is known to inhibit cytochrome P450 activities in vitro. The goal of this study was to compare cytochrome P450 activities in vitro and in vivo in presence of propofol. In vitro (liver microsomes and freshly isolated hepatocytes), cytochrome P450 IA2 and IIB1 activities were measured as the production of resorufin from ethoxy- and pentoxyresorufin, respectively, in presence of various concentrations of propofol. In vivo, cytochrome P450 activities were assessed as the production of 13CO 2 from 13C-aminopyrine injected intravenously, during acute administration or after chronic treatment. In vitro results confirmed the dose-dependent inhibitory effect of propofol on cytochrome P450 activities, both on liver microsomes and isolated hepatocytes. In vivo, the acute administration of propofol induced a significant decrease of 13C-aminopyrine metabolism. Chronic treatment with propofol induced a significant inhibition of 13C-aminopyrine metabolism only after 2 weeks. No enzyme induction was observed. In conclusion, our results demonstrate that propofol inhibits cytochrome P450 also in vivo. Drug interactions may thus occur during propofol administration.

Iron Ore Mines Leachate Potential for Oxyradical Production

The ecotoxicological effects of mining effluents is coming under much greater scrutiny. It appears necessary to explore possible health effects in association with iron ore mining effluents. The present results clearly demonstrate that iron-ore leachate is not an inert media but has the potential to induce lipid peroxidation. Peroxidation was assessed by measuring oxygen consumption in the presence of a reducing agent such as ascorbate or NADPH and a chelator such as EDTA. Labrador iron ore is an insoluble complex crystalline material containing a mixture of metals (Fe, Al, Ti, Mn, Mg,…, ) in contrast to the iron sources used for normal lipid peroxidation studies. The metal of highest percentage is iron (59.58%), a metal known to induce oxyradical production. Iron ore powder initiated ascorbic acid-dependent lipid peroxidation (nonenzymatic) in liposomes, lipids extracted from rat and salmon liver microsomes, and intact salmon liver microsomes. It also revealed an inhibitory effect of NADPH-dependent microsomes lipid peroxidation as well as on NADPH cytochrome c reductase activity. However, nonenzymatic peroxidation in rat liver microsomes was not significantly inhibited. Cytochrome P450 IA1- and IIB1-dependent enzymatic activities as well as P450 levels were not affected. The inhibition could be due to one of the other components of iron ore leachate (Mn, Al,…, ). These effects of iron-ore leachate indicate that a potential toxicity could be associated with its release into lakes. Further studies are necessary to explore in vivo effects on aquatic animals.

Theoretical investigation of substrate specificity for cytochromes P450 IA2, P450 IID6 and P450 IIIA4.

Three-dimensional models of the cytochromes P450 IA2, P450 IID6 and P450 IIIA4 were built by means of comparative modeling using the X-ray crystallographic structures of P450 CAM, P450 BM-3, P450 TERP and P450 ERYF as templates. The three cytochromes were analyzed both in their intrinsic structural features and in their interaction properties with fifty specific and non-specific substrates. Substrate/enzyme complexes were obtained by means of both automated rigid and flexible body docking. The comparative analysis of the three cytochromes and the selected substrates, in their free and bound forms, allowed for the building of semi-quantitative models of substrate specificity based on both molecular and intermolecular interaction descriptors. The results of this study provide new insights into the molecular determinants of substrate specificity for the three different eukaryotic P450 isozymes and constitute a useful tool for predicting the specificity of new compounds.

Toxicity evaluation of organic sediment extracts resolved by size exclusion chromatography using rainbow trout hepatocytes

The (geno)toxicity of sediment dichloromethane extracts and fractions obtained by size exclusion chromatography were evaluated to investigate effects based on size fractionation. In this study, three sediments were selected according to their incremental contamination in PAHs and in PCBs: Hamilton harbour, Toronto bay and lake St. Clair sediments. Heavy metals, total sulfur and elemental sulfur (S 8) were also determined in the (un)fractionated sediment extracts. The liver cells were exposed to concentrations of sediment extracts and fractionated samples for 24 h at 15°C, afterwhich cell viability, cytochrome P450IA1 activity, available free Zn, DNA damage and oxidative stress were determined. The results showed that the sediment extracts contained high levels of sulfur most of which was found in the low molecular weight (LMW) region, i.e., the 2000-50 atomic mass unit (amu) fraction. Elemental sulfur (S 8) accounted for 14–41 % of extractable sulfur and were found to elute in the post-column volume (PCV) fraction despite its molecular weight of 256 amu. Heavy metals were found mainly in the HMW (i.e the >2000 amu) fraction and LMW fractions and very few or none were observed in the PCV fractions. In sediment extracts, sublethal effects were present principally by the HMW and LMW fractions suggesting that some chemicals were also associated with high molecular weight compounds of extractable organic matter. Less toxicity or effect was sometimes found in the extract indicating an antagonistic effect of the contaminants. We found that cell viability and genotoxicity evaluations could be performed on the unfractionated extracts while EROD, available Zn and oxidative stress measurements should be performed on the LMW fractions because of possible antagonist or shielding effects. Considering the cytotoxic responses, the best toxicity ranking in respect to contaminant levels in sediment extract was obtained with the LMW and PCV fractions which accounted for most of the toxic responses in the chromatographic fractions. Moreover, the shielding effect could be explained, in part, by the association of LMW contaminants to large macromolecules.

The Existence of the 4S Polycyclic Aromatic Hydrocarbon-Protein Binding in 14-Day-Old Chick Embryo Liver

Cytochrome P-450IA1, the isozyme most closely associated with aryl hydrocarbon hydroxylase (AHH), is regulated by two high-affinity binding proteins, the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein which primarily binds PAHs and the 8S Ah (dioxin) receptor which binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and like congeners. The present study was conducted to determine whether the 4S protein existed in 14-day-old chick embryo liver when AHH activity is maximal to determine if they are linked as is the 8S Ah receptor and to confirm the existence of the dioxin receptor by investigating their ligand binding characteristics in the presence and absence of sodium molybdate, an agent that stabilizes steroid hormone receptors and partially stabilizes the dioxin receptor. Competitive ligand binding studies were performed with liver cytosol from livers of male 14-day-old chick embryos using [3H]-benzo[a]pyrene (B[a]P) or [3H]-TCDD in the presence and absence of a 200-fold excess of B[a]P, benzo[e]pyrene (B[e]P), 3-methylcholanthrene (3-MC), and tetrachlorodibenzofuran (TCDBF). Specific PAH-binding activity was assayed using sucrose gradient analysis. In the absence of molybdate, the 4S PAH-binding protein had high affinity for B[a]P, B[e]P, 3-MC, but very low affinity for TCDBF; the Ah receptor exhibited high affinity for TCDBF. In the presence of sodium molybdate, the Ah receptor was stabilized while the 4S PAH-binding protein was relatively unaffected. These results affirm the existence of two distinct PAH-binding proteins in 14-day-old chick embryo liver cytosol and suggest a linkage of the 4S protein to AHH.

Enhanced cytochrome P450 IA1 activity of self-assembled rat hepatocyte spheroids.

Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes. Spheroids have been observed to exhibit enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures on dry collagen. With confocal scanning laser microscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. This highly fluorescent molecule is the P450-mediated product of 7-ethoxyresorufin O-dealkylation (EROD). Significantly higher P450 activity was observed in spheroids compared to monolayers on collagen upon induction with 50 microM beta-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measuring microsomal EROD activity. The distribution of CYP1A1 activity within spheroids was heterogeneous, with higher activity localized to the hepatocytes in the interior. During the process of spheroid formation, cells were initially seen to attach and spread out as a monolayer. This stage was associated with relatively low CYP1A1 activity. As cells formed multicellular structures and aggregated into spheroids, the level of CYP1A1 activity increased over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The higher P450 activity within spheroids may be associated with their ability to maintain a greater degree of differentiation compared to monolayers. These studies demonstrate the potential of hepatocyte spheroids as a model system for investigating drug metabolism, tissue engineering, and tissue self-assembly.

Inhibitory effects of isopropyl-2-(1,3-dithietane-2-ylidene)-2-[ N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439) on benzo[ a]pyrene-induced skin carcinogenesis and micronucleated reticulocyte formation in mice

Recently, a great deal of attention has been devoted to organosulfur compounds with potential cancer chemopreventive properties. Many sulfur-containing substances present in Brassica plants have been reported to possess striking anticarcinogenic and antimutagenic activities. Besides naturally occurring organosulfur compounds, certain synthetic sulfur-containing pharmaceuticals, such as oltipraz and sulindac, are known to exert substantial chemopreventive or chemoprotective effects. Isopropyl-2-(1,3-dithietane-2-ylidene)-2-[ N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439) was initially developed for its possible use as a hepatoprotectant. The compound has been found to up-regulate the expression of cytochrome P-450 IA1 [I.J. Lee, K.S. Jeong, B.J. Roberts, A.T. Kallarakal, P. Fernandez-Salguero, F.J. Gonzalez, B.J. Song, Transcriptional induction of the cytochrome P-450 1A1 gene by a thiazolium compound YH439, Mol. Pharmacol. 49 (1996) 980–988.] which plays a pivotal role in metabolism of the majority of polycyclic aromatic carcinogens and mutagens, such as benzo[ a]pyrene (B[ a]P). In the present study, we found that oral administration of YH439 to CD-1 mice significantly suppressed B[ a]P-initiated skin tumorigenesis. B[ a]P-induced formation of micronuclei in mouse peripheral reticulocytes was also attenuated by YH439 pretreatment. Likewise, diallyl sulfide, a major volatile thioether present in garlic, also protected against B[ a]P-induced skin tumorigenesis and micronucleated reticulocyte formation in mice.

Benzo(a)pyrene-Induced Alterations in Growth-Related Gene Expression and Signaling in Precision-Cut Adult Rat Liver and Kidney Slices

Benzo(a)pyrene (BaP) and related aromatic hydrocarbons are suspected carcinogens; however, the molecular basis underlying tumorigenesis remains unclear. To identify acute molecular targets of BaP within the liver and kidney, precision-cut slices harvested from naive, adult female Sprague–Dawley rats were challenged with BaP (0.3–30 μM) for 0.5 to 24 h. BaP did not elicit cytotoxicity, as assessed by intracellular K+and ATP content and histological evaluation over the 24-h period. To determine if molecular signaling pathways were maintained in precision-cut slices, induction of the aryl hydrocarbon receptor (AhR) pathway was assessed following BaP challenge. Induction of cytochrome P450IA1 (P450IA1) mRNA and protein expression was observed in both liver and kidney slices. c-fosand c-Ha-rasgene expression was enhanced in liver, but not kidney, slices by BaP. c-junmRNA levels were decreased in liver and kidney slices, although the effect was earlier (0.5 h) in liver slices compared to kidney slices. BaP increased the DNA binding of nuclear proteins to the AP-1 consensus recognition element in liver, but decreased DNA binding in kidney slices. In contrast, DNA binding of NF-κB was not affected by BaP in either liver or kidney slices. These results suggest that acute BaP challenge is associated with altered expression of several growth-related genes and AP-1 signaling and establish precision-cut slices as a usefulin vitrosystem to investigate the molecular basis of BaP-induced tumorigenesis, including organ-specific differences.

Effects of vegetables–fruit extracts and indole-3-carbinol on stearic acid-modulated intercellular communication and cytochrome P450-IA activity

Modulatory effects were investigated of extracts of a vegetables–fruit mixture and indole-3-carbinol (I3C) on stearic acid-modulated gap junctional intercellular communication (GJIC) and cytochrome P450-IA activity (EROD). In V79 cells, pure water and hexane extracts of a vegetables–fruit mixture and 25 μg/ml I3C significantly protected against decreased GJIC caused by 10 μM stearic acid. Furthermore, pure, 10× and 100× diluted vegetables–fruit extracts significantly maintained their capacity to induce EROD activity in Caco-2 cells, but only when these extracts were added to the cells in media already containing 500 μM stearic acid for 48 h. Stearic acid itself did not induce EROD activity. I3C (10, 25, and 50 μg/ml) clearly induced EROD activity in Caco-2 cells, irrespective of the order at which I3C and stearic acid were added to the cells. In conclusion, the present in vitro study showed that vegetables–fruit extracts and I3C modulate effects of stearic acid on intercellular communication and cytochrome P450-IA activity.

Colonial growth and differentiation of epithelial cells derived from abattoir adult porcine livers.

The parenchymal cell fraction was isolated from abattoir adult porcine livers and cultured in Dulbecco's Modified Eagles' medium/Ham's F12 medium (DMEM/F12; 1:1) medium supplemented with 5% foetal calf serum, 10 ng/mL glucagon, 10 µg/mL insulin, 60 ng/mL hydrocortisone and eight other factors (NAIR-1 medium). The fraction contained a number of epithelial cells other than hepatocytes, some of which attached to the culture plates as cell clusters and began to grow after 3 days in culture. These epithelial cells growing as colonies were found to express cytokeratin 18 by immunocytochemistry. After 7-8 days, duct-like structures emerged in the central parts of the colonies. The cells constituting the duct-like structures and some cells located outside the structures were positive for cytokeratin 19 and γ-glutamyltransferase (GGT). The albumin-positive cells were located in the outer parts of the colonies rather than their central parts. Albumin was also detectable in the cells surrounded by the duct-like structures. Moreover, cytochrome P450 IA1 was induced by 3-methylcholanthrene (3-MC) on day 16. These results suggest that porcine liver epithelial cell clusters may contain stem-like cells which can differentiate into mature hepatocytes or bile duct epithelial cells.