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At least 145 records · Page 8Linked to original sources
PubMed2003

Active stromelysin-3 (MMP-11) increases MCF-7 survival in three-dimensional Matrigel culture via activation of p42/p44 MAP-kinase.

Stromelysin-3 (ST3) has the characteristic structure of matrix metalloproteinases (MMP), but its substrate specificity and pattern of expression differ markedly from that of other MMP family members. ST3 was originally isolated on the basis of its expression in primary breast cancers and has been shown to be overexpressed in virtually all primary carcinomas, suggesting that ST3 participates in the initial development of epithelial malignancies. Recent data using murine models reported that ST3 expression was able to increase tumor take by suppressing cell apoptosis. Our present goal was to set up an in vitro model in which we could study this new function. For this purpose, we analyzed survival of MCF-7 transfectants expressing either wild-type or catalytically inactive ST3 (ST3wt or ST3cat-) in three-dimensional (3-D) culture conditions by inclusion in Matrigel. In such conditions, that mimic the in vivo microenvironment, we found a marked decrease in the percentage of cell death when active ST3 was expressed (ST3wt transfectants vs. ST3cat- or vector only transfectants) as assessed by FACS and TUNEL analysis. The addition of batimastat, a broad spectrum MMP inhibitor, reversed the increased cell survival in ST3wt transfectants, confirming that ST3 enzymatic activity was required for this effect. Finally, we analyzed the expression of anti- and pro-apoptotic proteins as well as activation of cell survival pathways and we found that ST3-mediated cell survival was accompanied by activation of both p42/p44 MAPK and AKT. Our data confirm and extend the anti-apoptotic function of ST3 and provide a useful model to dissect this new role and identify new physiological substrates.

Antineoplastic Agents↗
PubMed2004

Glycine-extended gastrin induces matrix metalloproteinase-1- and -3-mediated invasion of human colon cancer cells through type I collagen gel and Matrigel.

The effects of glycine-extended gastrin (G-Gly) on the invasion by colon cancer cells through stromal extracellular matrix and the role of metalloproteinases (MMPs) in this invasion were investigated. We found that 10(-9)-10(-6) M G-Gly significantly increased the invasiveness of 2 human colon cancer cell lines, LoVo and HT-29, both expressing the G-Gly-specific binding site but little gastrin/CCK-B receptor (gastrin receptor). LoVo cells expressed MMP-1, -2, -3 and -9. An amount of 10(-7) M G-Gly enhanced collagenase MMP-1 expression. Overexpression of enhanced green fluorescent protein (EGFP)-fused MMP-1 in LoVo cells, by cDNA transfection, enhanced invasiveness through type I collagen gel. Immunofluorescence study revealed that G-Gly increased the number of cytoplasmic vesicles containing MMP-1, some vesicles being released from the cells. The MMP-1 vesicles contained one of the ubiquitous coat proteins, Golgi-localized, gamma-adaptin ear-containing, ARF-binding proteins-2 (GGA-2). MMP-1 also colocalized with CD147 (EMMPRIN, an extracellular matrix metalloproteinase inducer in adjacent stromal cells). It was suggested that G-Gly increased the number of vesicles containing MMP-1 and that MMP-1 interacted with CD147 to increase invasion. G-Gly significantly enhanced the production of MMP-3, an activator of MMP-1 and -9, as well as gelatinase MMP-9 activity. The G-Gly-mediated MMP-9 increase was inhibited by treatment with anti-MMP-3 IgG and MMP-3 siRNA. Furthermore, G-Gly increased the proMMP-2 level, although no activated MMP-2 was found in conditioned medium in either the presence or the absence of G-Gly. By contrast, gastrin (10(-7) M) had no effect on the levels of these MMPs or the invasiveness of colon cancer cells in type I collagen gel and Matrigel. These effects of G-Gly on the activity and expression of MMPs and the invasiveness of colon cancer cells were inhibited by treating the cells with a broad-spectrum metalloproteinase inhibitor (CGS27023A) and nonselective gastrin/CCK receptor antagonists (proglumide and benzotript). But a gastrin/CCK-B receptor antagonist (YM022) did not inhibit the increased invasion by G-Gly. Together, these results demonstrate that G-Gly renders colon cancer cells more invasive by increasing MMP-1 and MMP-3 expressions via the putative G-Gly receptor and would thus be a good molecular target in a clinical setting.

Biocompatible Materials↗
PubMed1994

Hepatocyte growth factor immobilized onto culture substrates through heparin and matrigel enhances DNA synthesis in primary rat hepatocytes.

Newly prepared phosphatidylethanolamine (PE) conjugates of glycosaminoglycans can be immobilized to solid phase through hydrophobic interaction. Primary rat hepatocytes were cultured on type I collagen or laminin substrates containing heparin-PE and assayed for DNA synthesis initiated by hepatocyte growth factor (HGF). Hepatocytes responded to HGF that had been added to culture media by active DNA synthesis and pronounced cell spreading, regardless of the presence of heparin-PE on the substrates. The preincubation with HGF, but not with epidermal growth factor, of the substrates containing heparin-PE significantly enhanced DNA synthesis and cell spreading in the absence of these mitogens in culture media. The enhancements were abolished by omitting heparin-PE or washing the HGF-treated substrates with 1 M NaCl, suggesting the immobilization of HGF to substrates through heparin. These phenomena were more evident with laminin substrates than with type I collagen substrates. Chondroitin sulfate-PE only partly substituted for heparin-PE, but Matrigel gave results similar to those of the substrates containing heparin-PE. Both type I collagen and laminin substrates containing heparin-PE bound higher amounts of HGF than the respective control substrates, whereas neither of the substrates containing chondroitin sulfate-PE did. However, the increase in HGF binding to the substrates was not as evident as the increase in DNA synthesis, suggesting that the latter is not simply due to the former but due to a concerted action of the immobilized HGF and heparin. Taken together, these results suggest that HGF can be trapped in extracellular matrix, probably through heparan sulfate in vivo, thereby acting as a mitogen for hepatocytes in cooperation with heparan sulfate.

Animals↗
PubMed2001

Reorganization of structural proteins in vascular smooth muscle cells grown in collagen gel and basement membrane matrices (Matrigel): a comparison with their in situ counterparts.

When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in beta-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and alpha-actinin are organized into longitudinally arranged "myofibrils" and the vimentin-containing intermediate filaments form a meshed cytoskeletal network. However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins.

Actinin↗
PubMed1998

Tumor and endothelial cell invasion of basement membranes. The matrigel chemoinvasion assay as a tool for dissecting molecular mechanisms.

The spread of cancer cells from a primary tumor to distant organs is the major cause of death of cancer patients. Metastatic lesions are often resistent to cancer therapy because of the progressive phenotypic changes that they have undergone. Several genetic and epigenetic factors, both in the cell and in the host, contribute to the development of tumor progression towards metastases. In this review we will analyze the steps involved in tumor metastases, which can be potential targets for anti-metastatic therapy. One of the most critical events in cancer metastasis is the invasion of basement membranes. An assay which we developed over ten years ago, the matrigel "chemoinvasion" assay, has been a useful tool for studying the mechanisms involved in tumor and endothelial cell invasion of basement membranes and for the screening of anti-invasive agents. Here we will describe the assay and review some of the major results obtained with it.

Animals↗
PubMed2005

Pomegranate (Punica granatum) pure chemicals show possible synergistic inhibition of human PC-3 prostate cancer cell invasion across Matrigel.

Four pure chemicals, ellagic acid (E), caffeic acid (C), luteolin (L) and punicic acid (P), all important components of the aqueous compartments or oily compartment of pomegranate fruit (Punica granatum), and each belonging to different representative chemical classes and showing known anticancer activities, were tested as potential inhibitors of in vitro invasion of human PC-3 prostate cancer cells in an assay employing Matrigel artificial membranes. All compounds significantly inhibited invasion when employed individually. When C, P, and L were equally combined at the same gross dosage (4 microg/ml) as when the compounds were tested individually, a supradditive inhibition of invasion was observed, measured by the Kruskal-Wallis non-parametric test.

Drug Synergism↗
PubMed2006

Preparation of a tissue-like cortical primary culture from embryonic rats using Matrigel and serum free Start V Medium.

To address the scientific quest for unravelling signalling pathways crucial in CNS development and function, cell culture systems have to be developed that are mimicking the physiological state of brain cells more efficiently. Here, we describe a method for cultivation of a virtual three-dimensional structure consisting of neural stem cell-derived cell types by using Matrigel as surface substrate and Start V as a serum free medium. We demonstrate that free floating dissociated cells form attached neurospheres from which cells start migration to surrounding areas and develop a virtual three-dimensional cell structure composed of neurons, glia and neural stem cells. Neuronal precursor cells differentiate into cholinergic and GABAergic cells and express vesicle proteins. Further, neuronal cells are interwoven with Nestin positive stem cells and GFAP positive astrocytes. Additionally, oligodendrocytes and microglia can also be detected in this neural tissue-like structure. As an example for studying cell migration we added externally microglial cells (BV2) and performed a confocal time lapse study. It revealed, that co-cultivated microglial cells migrated towards neurospheres within 14 h. Thus, the described method provides a serum free, tissue-like primary cell culture system of neural cells useful for the investigations of basic cell-cell interactions under in vitro conditions.

Animals↗
PubMed2004

A new image analysis method based on topological and fractal parameters to evaluate the angiostatic activity of docetaxel by using the Matrigel assay in vitro.

Angiogenesis is regulated both by positive and negative factors that modulate the migration, proliferation, proteolytic activity, and differentiation of endothelial cells. Various in vivo and in vitro models have been developed to test the effect of angiogenic agonists or antagonists. Until very recently, morphometric methods to evaluate the effects of angiogenic inducers and inhibitors were mostly descriptive. Here, we have described a new, automatic, image analysis method to evaluate the angiostatic activity of docetaxel by using the in vitro Matrigel assay. This method allows to establish several parameters, such as dimensional (area % covered by endothelial cells and the total length of the cellular network per field), topological (the number of meshes and the number of branching points per field), and fractal (fractal dimension, lacunarity) of the capillary-like network. Following docetaxel treatment, a reduction of about 25% in the size parameters of cell network, of about 45% in branching points per field, and more 50% in mesh number were observed. The resulting fractal analysis was consistent with these findings and fractal dimension significantly correlated with both the number of branching points and the number of meshes. These results indicate that both topological and fractal parameters allowed a characterization of the spatial texture generated by endothelial cells during in vitro angiogenesis, the former being easier to implement in a computer program and faster to calculate. Finally, topological parameters seem to exhibit a wider dynamic range than the fractal ones.

Angiogenesis Inhibitors↗
PubMed2005

Matrigel: basement membrane matrix with biological activity.

The basement membrane extracellular matrix contacts epithelial, endothelial, fat and smooth muscle cells. Because this extracellular matrix is so thin, it had been hard to study its composition, structure, and function. An extract of a tumor was found to contain all of the components present in basement and to be very biologically active. This extract, termed Matrigel, Cultrex, or EHS matrix, promotes cell differentiation, and is used to measure the invasive activity of tumor cells. In vivo, it is used for measuring angiogenic inhibitors and stimulators, to improve graft survival, repair damaged tissues, and increase tumor growth.

Animals↗
PubMed2001

Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions.

OBJECTIVES: It has been shown that circulating human non-adherent CD34+ cells coexpressing vascular endothelial growth factor (VEGF)-R2 and AC133 have the capacity to differentiate into adherent mature endothelial cells. However, prior studies have demonstrated that a much bigger subset of primary adherent mononuclear cells can also form endothelial progenitor cells (EPC). To determine the origin of the latter cell population we tested the hypothesis: do monocytes as a firmly adherent and plastic cell type have the potential to differentiate into an endothelial phenotype. METHODS: CD34-/CD14+ monocytes were isolated from human peripheral blood by adherence separation and magnetic bead selection (purity >90%) and cultured on fibronectin-coated plastic dishes (medium containing VEGF 10 ng/ml, basic fibroblast growth factor (bFGF) 2 ng/ml, insulin like growth factor (IGF-1) 1 ng/ml, 20% fetal calf serum). RESULTS: After 2 weeks of culture, using fluorescence activated cell analysis we observed a new expression of the endothelial markers von Willebrand factor (vWf), VE-cadherin (VE) and ec-NOS in 45.2, 12.4 and 9.8% of the cells, respectively. The proportion of cells expressing these markers further increased after 4 weeks (94.2, 89.7 and 58.8% of these cells, respectively). The proportion of CD45 expressing cells remained unchanged during this period. However, after 14 days the specific macrophage antigen CD68 was newly expressed in 62% of the analysed cells with a further increase to 90% after 28 days of culture. In three-dimensional gel (Matrigel the formation of cord- and tubular-like structures was observed. CONCLUSION: The present data indicate that under angiogenic stimulation macrophages develop an endothelial phenotype with expression of specific surface markers and even form cord- and tubular-like structures in vitro suggesting that this cell population may be recruited for vasculogenesis.

Antigens, CD↗
PubMed1996

Inhibitory effects of retinoids on development of squamous metaplasia in rat mammary epithelial organoids cultured in Matrigel.

Squamous metaplasia (SQM) developed in cultures of rat mammary organoids in reconstituted basement membrane, Matrigel, under either a complete hormone medium (CHM) or a serum-free mammary epithelium growth medium (MEGM). Organoids cultured in CHM gave rise to fewer such SQM (approximately 5%) than those in MEGM (approximately 16%). Formation of SQM was completely suppressed when retinoids were added to CHM. However, a few SQM were still observed in cultures in MEGM with added retinoids. Addition of 5% fetal bovine serum suppressed development of SQM cultured in MEGM. Delayed addition of retinoids also inhibited further development of SQM. Development of SQM from mammary epithelial cells is not common, and regulatory molecules other than retinoids apparently are involved in their formation and prevention.

Animals↗
PubMed2002

Comparative study of tube assembly in three-dimensional collagen matrix and on Matrigel coats.

This study compares phenotypic changes of human umbilical endothelial vein cells cultured in three-dimensional collagen matrixes in the presence of basic fibroblast growth factor or on Matrigel coats. Under both conditions, endothelial cells rapidly assembled into an irregular network of tubular structures with a high frequency of intercellular or lumen-like spaces. Tubular structures were characterized and compared by phase-contrast, confocal and electron microscopy. The dominant mechanism of lumen-like formation was highly model-dependent. Ultrastructural analyses of capillary-like structures and the mechanism of lumen-like formation indicated that the in vivo angiogenesis was better reproduced in the collagen model.

Animals↗
PubMed2005

Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine.

Tissue fibrosis is one of the characteristics of chronic pancreatitis and pancreatic adenocarcinoma. Activated pancreatic stellate cells (PSC) play a central role in this process. However, analysis of the molecular mechanisms leading to PSC activation is hampered by the lack of an established human PSC line. To overcome this problem, we immortalized and characterized primary human PSC. The cells were isolated by the outgrowth method and were immortalized by transfection with SV40 large T antigen and human telomerase (hTERT). Primary human PSC served as controls. An immortalized line, RLT-PSC, was analyzed for the expression of stellate cell markers. Moreover, the effects of transforming growth factor beta 1(TGFbeta1) or platelet-derived growth factor stimulation and of cultivation on basement membrane components or N-acetylcysteine (NAC) treatment on gene and protein expression and proliferation were analyzed. Immortal RLT-PSC cells retained the phenotype of activated PSC proven by the expression of alpha-smooth muscle actin (alphaSMA), vimentin, desmin and glial fibrillary acidic protein (GFAP). TGFbeta1 treatment upregulated the expression of alphaSMA, collagen type I (Col I), fibronectin and TGFbeta1. Incubation of RLT-PSC cells and primary human activated PSC on Matrigel plus NAC treatment resulted in a deactivated phenotype as evidenced by a decrease of alphaSMA, connective tissue growth factor and Col I expression and by a decreased proliferation of the cells. Moreover, this treatment restored the ability of the cells to store vitamin A in cytoplasmic vesicles. In conclusion, we have established an immortal pancreatic stellate cell line, without changing the characteristic phenotype. Importantly, we were able to demonstrate that besides soluble factors, the matrix surrounding PSC plays a pivotal role in the maintenance of the activation process of PSC. Cultivation of activated PSC on a reconstituted basement membrane plus treatment with NAC was able to deactivate the cells, thus pointing to the possibility of an antifibrosis therapy in chronic pancreatitis.

Acetylcysteine↗
PubMed1998

Evidence that platelets promote tube formation by endothelial cells on matrigel.

The involvement of platelets in neovascularization was investigated in the matrigel tube formation assay, an in vitro model of angiogenesis. Platelets promoted the formation of capillary-like structures (expressed as relative tube area) number- and time-dependently. Relative tube area increased from 0.98+/-0.02 (n = 8) in the presence of 6.25 x 10(4), to 3.21+/-0.12 (n=8) in the presence of 10(6) platelets/well compared to 0.54+/-0.04 (n=8) in their absence. This increase was unaffected by acetyl salicylic acid (ASA), apyrase, and hirudin. Photographs from representative experiments, showed that platelets adhered along the differentiating endothelium. Addition of alpha-thrombin (0.1-1 i.u. ml(-1)), the nitric oxide (NO) donor sodium nitroprusside (SNP; 1-100 microM) or the NO synthase inhibitor, L-NG-arginine-methylester (L-NAME, 30-300 microM) to the assay, had no effect on tube formation compared to that seen with platelets alone. Neuraminidase (0.01 i.u./10(7) platelets), which strips sialic acid residues from membrane glycoproteins, abolished the promoting effect of platelets on tube formation. The relative tube area in the presence of neuraminidase-treated platelets was 0.81+/-0.03 (n = 8), in the presence of untreated platelets 1.69+/-0.09, P<0.001 (n=8) and in the absence of platelets, 0.80+/-0.04 (n=8). The tetrapeptide Arg-Gly-Asp-Ser (RGDS; 20-200 microM) which inhibits von Willebrand factor, fibrinogen and fibronectin-mediated adhesion, had no effect on the promoting effect of platelets on tube formation. These results indicate that platelets promote angiogenesis in vitro. This effect is largely independent from activation by alpha-thrombin, is not modified by manipulating NO and prostaglandin metabolism and proceeds possibly through adhesion of the platelets to the differentiating endothelium.

Biocompatible Materials↗
PubMed1997

Suppression of anchorage-independent growth and matrigel invasion and delayed tumor formation by elevated expression of fibulin-1D in human fibrosarcoma-derived cell lines.

Using differential display, we identified an mRNA that is markedly down-regulated in cell line 6A/SB1, derived from a fibrosarcoma formed in an athymic mouse following injection of carcinogen-transformed MSU-1.1 cells. The nontumorigenic parental cell strain, MSU-1.1, expresses high levels of this mRNA. Sequencing of the corresponding cDNA fragment revealed that it corresponded to an expressed sequence tag, which ultimately led to its identification as the fibulin-1D gene. Fibulin-1 is a cysteine-rich, calcium-binding extracellular matrix and plasma protein, which has four isoforms, A-D, derived from alternative splicing. Northern and Western blotting analysis of 16 cell lines established from tumors formed in athymic mice by MSU-1.1-derived cell strains independently transformed in culture showed that 44% exhibited low level or lack of expression of fibulin-1D mRNA and protein. In a similar analysis of 15 malignant cell lines derived from patients, 80% showed low level or no expression. To study the role of fibulin-1D in transformation, we transfected 6A/SB1 cells and a human fibrosarcoma-derived cell line (SHAC) with a fibulin-1D cDNA expression construct. Transfectants displaying high levels of fibulin-1D were isolated and characterized. Elevated expression of fibulin-1D led to reduced ability to form colonies in soft agar and reduced invasive potential as tested in a matrigel in vitro invasion assay. Furthermore, expression of fibulin-1D resulted in a markedly extended latency in tumor formation in athymic mice. These results indicate that low expression of fibulin-1D plays a role in tumor formation and invasion.

Animals↗
PubMed2001

DNAzymes to beta 1 and beta 3 mRNA down-regulate expression of the targeted integrins and inhibit endothelial cell capillary tube formation in fibrin and matrigel.

A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of beta(1) and beta(3) integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in beta(1) and beta(3) mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated beta1-1053 and beta3-1243, significantly inhibited expression of beta(1) and beta(3) integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, beta1-1053 and beta3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to beta(1) and beta(3) mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for beta(1) and beta(3) DNAzymes, respectively. Both DNAzymes in the presence of Mg(2+) specifically cleaved their substrates, synthetic beta(1) and beta(3) mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2'-O-methyl groups at both the 5' and 3' ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to beta(1) and beta(3) mRNAs with 2'-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.

Base Sequence↗
PubMed2000

Time lapse phase contrast video microscopy of directed migration of human microvascular endothelial cells on matrigel.

Migration of microvascular endothelial cells is an early and critical step in angiogenesis. Formation of branching and polygonal cellular aggregates by endothelial cells on matrigel has often been considered to be an in vitro model for angiogenesis, although formation of lumens has not always been confirmed. The dynamics of migration of living cells of a human dermal microvascular endothelial cell line (HMEC-1) on a reconstituted basement membrane matrix have been captured in real time using time lapse video microscopy. The cells exhibit periods of quiescence and directed rapid migration by formation of extensions towards a specific target cell. Cells repeatedly extend flexible protrusions from the cell body both within the plane of the matrix and out of the plane of the matrix into the incubation medium. Connections between protrusions and target cells are made frequently, but not all cells which start to form protrusions achieve connections with other cells. Some of these migrating cells which do not connect arrest before reaching the target, or arrest and retract to their origin. After formation of multicellular polygonal structures, the structures contract to form amorphous clusters of fused cells without visible effects on the underlying matrix. The study demonstrates that time lapse video microscopy is a simple but very useful approach to monitor the dynamics of movements which vary in speed and frequency during migration of living cells.

Basement Membrane↗
PubMed1997

Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)-phenol) induces cytochrome P450s and inhibits haem biosynthesis in rat hepatocytes cultured on Matrigel.

1. The effect of Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)phenol) on cytochrome P450 (P450) induction and haem biosynthesis was studied in rat hepatocytes cultured on Matrigel. 2. Irgasan DP 300 significantly induced 7-benzyloxyresorufin O-debenzylase activity, followed by 7-pentoxyresorufin O-depentylase and 7-ethoxyresorufin O-deethylase activities. 4-Nitrophenol hydroxylase, testosterone 6 beta-hydroxylase and methoxyresorufin O-demethylase activities were also slightly increased. The maximum induction of these enzyme activities was obtained at the same concentration of 125 microM in the culture medium. 3. Immunochemical blots using anti-rat cytochrome P450 antibodies revealed that Irgasan DP 300 preferably induced CYP2B1/2 along with a slight increase in 3A. These results indicate that Irgasan DP 300 is a phenobarbital-type inducer. 4. In the absence of exogenous 5-aminolevulinic acid (ALA), slight increases in protoporphyrin IX (2.6-fold) and coproporphyrin III (1.3-fold) were observed in the Irgasan DP 300-treated cultures. In contrast, when 75 microM ALA was present, Irgasan DP 300 (250 microM) caused an extensive accumulation of uroporphyrin I (13-fold). 5. Irgasan DP 300 inhibited rat hepatic uroporphyrinogen III synthase in vitro. 6. These results indicate that Irgasan DP 300 produced accumulation of hydroxymethylbilane in rat hepatocytes by inhibiting uroporphyrinogen III synthase, and consequently an accumulation of uroporphyrin I.

Aminolevulinic Acid↗