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1.
Toxicol In Vitro ; 15(4-5): 289-95, 2001.
Article in English | MEDLINE | ID: mdl-11566551

ABSTRACT

The involvement of the NO pathway in several intestinal inflammatory diseases is under investigation. In vitro models may provide a useful approach to better characterise this pathway at the cellular level. For this purpose, we have used Caco-2 cells, which are able to spontaneously differentiate in long-term culture to small intestine enterocytes. The effect of different NO pathway inducers [gamma-interferon (IFN-gamma) and phorbol myristate acetate (PMA)] has been studied. Our results demonstrate that Caco-2 cells constitutively express NOS at very low levels, while the induction with PMA+IFN-gamma triggers the expression of the inducible isoform with a stronger effect starting from day 14 of differentiation. The use of specific inhibitors of gene expression, at transcriptional and translational level, suggests that new synthesis of iNOS mRNA is required, through direct activation of the gene or new synthesis of transcription-required factors, as indicated by CHX inhibition. The morphological alteration induced by PMA+IFN-gamma is reversed by iNOS inhibitor, suggesting that the NO pathway may be involved in the cytoskeletal alterations. The DSP toxins, OA and DTX-1, induce NO production at levels corresponding to their different toxicity, previously detected in Caco-2 cells.


Subject(s)
Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Enzyme Inhibitors/pharmacology , Marine Toxins/pharmacology , Nitric Oxide/biosynthesis , Blotting, Western , Caco-2 Cells/pathology , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Combinations , Enzyme Induction/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Humans , Interferon-gamma/pharmacology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Okadaic Acid/pharmacology , Pyrans/pharmacology , RNA, Messenger/metabolism , Tetradecanoylphorbol Acetate/pharmacology , omega-N-Methylarginine/pharmacology
2.
Chem Biol Interact ; 127(2): 109-24, 2000 Jul 03.
Article in English | MEDLINE | ID: mdl-10936227

ABSTRACT

Thiabendazole (TBZ), an anthelmintic and fungicide benzimidazole, was recently demonstrated to be extensively metabolized by cytochrome P450 (CYP) 1A2 in man and rabbit, yielding 5-hydroxythiabendazole (5OH-TBZ), the major metabolite furtherly conjugated, and two minor unidentified metabolites (M1 and M2). In this study, exposure of rabbit and human cells to 14C-TBZ was also shown to be associated with the appearance of radioactivity irreversibly bound to proteins. The nature of CYP isoforms involved in this covalent binding was investigated by using cultured rabbit hepatocytes treated or not with various CYP inducers (CYP1A1/2 by beta-naphthoflavone, CYP2B4 by phenobarbital, CYP3A6 by rifampicine, CYP4A by clofibrate) and human liver and bronchial CYP-expressing cells. The covalent binding to proteins was particularly increased in beta-naphthoflavone-treated rabbit cells (2- to 4-fold over control) and human cells expressing CYP1A2 (22- to 42-fold over control). Thus, CYP1A2 is a major isoenzyme involved in the formation of TBZ-derived residues bound to protein. Furthermore, according to the good correlation between covalent binding and M1 or 5OH-TBZ production, TBZ would be firstly metabolized to 5OH-TBZ and subsequently converted to a chemically reactive metabolic intermediate binding to proteins. This metabolic activation could take place preferentially in liver and lung, the main biotransformation organs, rather than in intestines where TBZ was shown to be not metabolized. Moreover, TBZ was rapidly transported by passive diffusion through the human intestinal cells by comparison with the protein-bound residues which were not able to cross the intestinal barrier. Consequently, the absence of toxicity measured in intestines could be related to the low degree of TBZ metabolism and the lack of absorption of protein adducts. Nevertheless, caution is necessary in the use of TBZ concurrently with other drugs able to regulate CYP1A2, particularly in respect to liver and lung tissues, recognised as sites of covalent-binding.


Subject(s)
Antinematodal Agents/metabolism , Cytochrome P-450 CYP1A2/metabolism , Intestinal Absorption/physiology , Proteins/metabolism , Thiabendazole/analogs & derivatives , Thiabendazole/metabolism , Animals , Antinematodal Agents/pharmacokinetics , Antinematodal Agents/toxicity , Biological Transport , Bronchi/drug effects , Bronchi/enzymology , Caco-2 Cells/drug effects , Caco-2 Cells/enzymology , Cells, Cultured , Colony-Forming Units Assay , Cytochrome P-450 CYP1A2/biosynthesis , Enzyme Induction/drug effects , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Intestinal Mucosa/drug effects , Intestinal Mucosa/enzymology , Intestinal Mucosa/metabolism , Isoenzymes/biosynthesis , Isoenzymes/metabolism , Liver/drug effects , Liver/enzymology , Liver/metabolism , Male , Protein Binding , Rabbits , Thiabendazole/pharmacokinetics , Thiabendazole/toxicity
3.
Int J Dev Neurosci ; 18(1): 93-9, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10708910

ABSTRACT

Antennapedia homeobox peptide has been reported to enhance neurite outgrowth and branching. Thus it is of interest to investigate whether antennapedia peptide is capable of modulating the expression of genes related to different events of neuronal development. In this paper we report the enhancement of a 68 KDa neurofilament subunit, choline acetyltransferase and acetylcholinesterase expression in spinal cord neurons, elicited by antennapedia peptide. Modulation of gene expression is different with respect to each gene product analyzed, suggesting a specific action of the peptide on diverse genes controlling different events of neuronal differentiation.


Subject(s)
Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Neurites/enzymology , Neurons/ultrastructure , Nuclear Proteins , Transcriptional Activation/physiology , Acetylcholinesterase/metabolism , Animals , Antennapedia Homeodomain Protein , Blotting, Northern , Blotting, Western , Cells, Cultured , Chick Embryo , Choline O-Acetyltransferase/genetics , Gene Expression Regulation, Enzymologic , Homeodomain Proteins/analysis , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Neurites/chemistry , Neurofilament Proteins/analysis , Neurofilament Proteins/genetics , Neurons/chemistry , Neurons/enzymology , RNA, Messenger/analysis , Spinal Cord/cytology , Spinal Cord/embryology , Transcription Factors/analysis , Transcription Factors/genetics
4.
Mol Cell Biochem ; 215(1-2): 73-80, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11204458

ABSTRACT

Neurological impairment is a common feature of Acquired Immunodeficiency Syndrome (AIDS); functional alterations have been reported both in central and peripheral nervous system and the Human Immunodeficiency Virus (HIV) envelope glycoprotein gp120 has been proposed as a neurotoxin acting through a calcium-dependent mechanism. On the other hand it has been reported that gp120 treatment also induce about a 20% decrease in the cerebral glucose utilization and in the cellular ATP levels. The reported observations were performed on experimental system where also non-neuronal cells where present; in order to evaluate whether a direct interaction between HIV proteins and neuronal cells takes place, we used a neuroblastoma cultures where only neuronal cells are present. We analysed the effects of gp120 on the N18TG2 neuroblastoma clone. Treatments were performed both on growing and confluent cultures. Short time treatment with gp120 of confluent cultures causes a 25% reduction in the level of neuron-specific enolase, resulting in a similar decrease of oxygen consumption. Long time exposure of growing cells also causes a reduction in cell survival. Furthermore, using a membrane-specific fluorescent probe we observed that gp120 produces an increase of membrane trafficking. These observations suggest a direct interaction between the viral envelope protein and neuronal cells, which results in an alteration of glycolytic metabolism. This alteration may be related to the neurologic impairments observed in AIDS patients.


Subject(s)
Glycolysis , HIV Envelope Protein gp120/metabolism , HIV Envelope Protein gp120/pharmacology , Neurons/metabolism , Animals , CHO Cells , Cell Membrane/metabolism , Cell Survival/drug effects , Cricetinae , Fluorescent Dyes/pharmacology , Galactosylceramides/metabolism , Immunoblotting , Mice , Microscopy, Fluorescence , Neuroblastoma/drug therapy , Neurons/drug effects , Neurons/enzymology , Oxygen Consumption/drug effects , Phosphopyruvate Hydratase/metabolism , Time Factors , Tumor Cells, Cultured
5.
Xenobiotica ; 29(11): 1157-69, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10598749

ABSTRACT

1. The metabolism and cytotoxicity of the antimicrobial nitrofuran drug furazolidone have been studied in Caco-2, HEp-2 and V79 cell lines. Free radical production, metabolite pattern, formation of bound residues, inhibition of cellular replication and protection by the antioxidant glutathione were compared for the three cell lines. 2. All three cell lines produced the same nitro-anion radical with similar kinetics. Little further metabolic breakdown was observed in V79 cells, whereas Caco-2 and HEp-2 cells showed extensive degradation of furazolidone, but with different end patterns. 3. Under hypoxic conditions, the colony-forming ability was extensively impaired in HEp-2 cells whereas the other two cell lines were less affected, suggesting that irreversible damage to DNA occurred prevalently in HEp-2 cells. In V79 cells the absence of oxygen caused a 25-fold increase in the formation of protein-bound residues. 4. Brief exposure to furazolidone caused a 50% loss of endogenous glutathione in Caco-2 cells, but no loss could be detected in V79 and HEp-2 cells. Consistently, when glutathione was depleted by buthionine-[S,R]-sulphoximine (BSO) and diethylmaleate (DEM) treatment, the viability of V79 and HEp-2 cells was minimally affected by furazolidone, whereas that of Caco-2 cells was substantially reduced. 5. It is concluded that the cytotoxicity of furazolidone in these cell lines can be exerted by a number of different mechanisms, possibly related to different metabolic pathways. The cytotoxicity of nitrofuran drugs, therefore, cannot be ascribed to a single toxic intermediate, but in Caco-2 cells furazolidone is extensively metabolized and detoxified by GSH, in V79 is only partially activated and then bound to proteins, whereas in HEp-2, once activated, may react with DNA.


Subject(s)
Anti-Infective Agents, Local/metabolism , Anti-Infective Agents, Local/toxicity , Furazolidone/metabolism , Furazolidone/toxicity , Biotransformation , Caco-2 Cells , Cell Division/physiology , Cell Hypoxia/physiology , Cell Line , Colony-Forming Units Assay , Electron Spin Resonance Spectroscopy , Free Radicals/metabolism , Glutathione/metabolism , Humans , Protein Binding
6.
Xenobiotica ; 29(6): 641-54, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10426562

ABSTRACT

1. The gastrointestinal fate of protein-bound residues of the model compound furazolidone (FZD) was investigated in vitro and ex vivo. Protein-bound residues were generated in rat liver microsomes, isolated by solvent extraction and digested with 0.5% hydrochloric acid and Pronase E. 2. During digestion, 3-amino-2-oxazolidinone (AOZ), the side chain of furazolidone, was partly released from bound residues. 3. The absorption of free AOZ and digested protein-bound residues was tested in isolated perfused rat gut segments (IPGS) and in the intestinal cell line Caco-2. Free AOZ was transfered both in the IPGS model and in Caco-2 monolayer cultures, while no indications for passage of bound residues were obtained. 4. No acute toxicity of AOZ or digested food residues respectively was observed in gut segments and Caco-2 cells at concentrations that were substantially above maximum residue levels to be expected in food of animal origin after administration of therapeutic doses. 5. The results demonstrate that digestive processes can alter the chemical nature of drug residues and yield degradation products that may be bioavailable for the consumer. Thus, the covalent binding of xenobiotics to macromolecular tissue constituents cannot necessarily be regarded as an irreversible endpoint of residue bioavailability and toxicity.


Subject(s)
Drug Residues/toxicity , Furazolidone/toxicity , Jejunum/drug effects , Veterinary Drugs/toxicity , Animals , Biological Transport , Caco-2 Cells/cytology , Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Drug Residues/metabolism , Furazolidone/metabolism , Glucose/metabolism , Humans , In Vitro Techniques , Intestinal Absorption/drug effects , Jejunum/metabolism , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Rats , Rats, Wistar , Safety , Veterinary Drugs/metabolism
7.
J Neurosci Res ; 51(1): 109-18, 1998 Jan 01.
Article in English | MEDLINE | ID: mdl-9452315

ABSTRACT

The presence and distribution of dystrophin was studied in selected areas of the chick embryo nervous system and in primary cultures. Dystrophin was examined at the protein level by immunocytochemistry and at the transcriptional level by a semiquantitative reverse transcriptase-polymerase chain reaction analysis. Immunofluorescence staining shows that dystrophin is present early during embryogenesis in dorsal root ganglia, spinal cord, and ciliary ganglia and colocalizes with neurofilament subunits. Cultured dorsal root ganglion, spinal cord, and ciliary ganglion neurons show immunoreactivity for dystrophin, both in cell bodies and along fibers. Dystrophin mRNA level in ciliary and dorsal root ganglia is higher than in spinal cord throughout development and shows a tissue-specific pattern of expression. In primary cultures of dorsal root ganglia and ciliary ganglia, dystrophin mRNA level increases with time in vitro. However, in spinal cord cultures, dystrophin mRNA drastically decreases with time in vitro, but it is significantly increased when embryonic muscle extract is added to the cultures. Our results show that dystrophin is present in neurons from different areas of embryonic chick nervous system and that its mRNA level is developmentally regulated both in vivo and in vitro.


Subject(s)
Dystrophin/analysis , Gene Expression Regulation, Developmental/physiology , Nervous System/metabolism , Animals , Cells, Cultured , Chick Embryo , Fluorescent Antibody Technique , Ganglia, Parasympathetic/metabolism , Ganglia, Spinal/metabolism , Nervous System/embryology , Polymerase Chain Reaction/methods , Spinal Cord/metabolism , Transcription, Genetic
8.
J Neurosci Res ; 49(2): 186-96, 1997 Jul 15.
Article in English | MEDLINE | ID: mdl-9272641

ABSTRACT

We investigated the level and characteristics of "low Km" 3'-5' cyclic nucleotide phosphodiesterase (PDE) activity in adult and embryo chick spinal cord. The DEAE cellulose chromatography elution profile of Triton X-100 extracts showed a single peak of calmodulin-dependent cAMP/cGMP PDE activity. After two additional purification steps, this activity showed a five-fold activation by calmodulin (Ka = 1.5 nM) for cGMP hydrolysis, and a linear kinetic behaviour with a Km of 1.3 microM. Conversely, the activity showed a biphasic behaviour for cAMP hydrolysis, with Km values of 3.1 and 18.5 microM. The enzyme showed a Stokes radius of 4.5 nm. Western blot analysis of the purified enzyme revealed two immunoreactive bands with molecular mass of 59 and 65 kDa, respectively. Immunohistochemical staining showed motoneuron decoration both on cell soma and fibres. The developmental pattern of Ca2+-calmodulin-dependent PDE expression in spinal cord was also studied; the hydrolytic activity for both substrates has been found to increase constantly from E5 to post-hatching stages, when it appears 5.6-fold higher as compared to the early embryo levels. Furthermore, in cultured spinal cord neurons from E8 embryos, muscle extract has been shown to induce a two-fold increase of Ca2+-calmodulin-dependent cGMP activity. In conclusion, the studies reported here present three relevant findings: (1) the presence in adult and embryo chick spinal cord of PDE activities with characteristics similar to those of the mammalian PDE I enzyme; (2) its localization in the ventral horn motoneurons; (3) its regulated expression during embryogenesis that is possibly related to soluble epigenetic factors produced by the target cells.


Subject(s)
Phosphoric Diester Hydrolases/metabolism , Spinal Cord/enzymology , Animals , Cells, Cultured , Chick Embryo , Chickens , Cyclic Nucleotide Phosphodiesterases, Type 1 , Immunoblotting , Immunohistochemistry , Kinetics , Logistic Models , Spinal Cord/embryology , Spinal Cord/growth & development
9.
J Neurochem ; 64(4): 1528-35, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7891079

ABSTRACT

We have observed that cultured neurons from chick spinal cord and the neuroblastoma hybrid line 108CC15 released lower amounts of acetylcholinesterase (AChE) when compared with the parental line, N18TG2. AChE activity extracted by hypotonic buffer, which can be regarded as the source of the released enzyme, was considerably higher in the parental than in the hybrid 108CC15 (respectively, approximately 80% and approximately 40% of cellular activity). On the other hand, evaluation of ectocellular, with respect to total, AChE activity showed that in N18TG2 cells only 7% of AChE was localized on the plasmalemma, whereas in the hybrid line the percentage of ectocellular activity was 3.7 times higher than in the parental line. We have also examined the effect of cytochalasin B and nocodazole. In the N18TG2 line, the former did not affect AChE release, which was significantly reduced by the latter. High K+ level in the culture medium, of both N18TG2 and hybrid 108CC15 cultures, induced an increase in AChE secretion; Ca2+ presence was required for high K(+)-induced release. Muscle extracts increased AChE secretion in both the hybrid 108CC15 and the spinal cord neurons. The present data suggest that AChE secretion during neuronal development is modulated by depolarizing stimuli and by soluble factors produced by target cells and may be involved in the control of neuronal differentiation.


Subject(s)
Acetylcholinesterase/metabolism , Neurons/enzymology , Potassium/pharmacology , Animals , Cell Membrane/enzymology , Cells, Cultured , Mice , Muscles/chemistry , Neuroblastoma/enzymology , Neuroblastoma/pathology , Spinal Cord/cytology , Spinal Cord/enzymology , Tissue Extracts/pharmacology , Tumor Cells, Cultured
10.
J Neurosci Res ; 39(5): 535-44, 1994 Dec 01.
Article in English | MEDLINE | ID: mdl-7891389

ABSTRACT

The cellular distribution of synapsin I in chick spinal cord has been examined during embryo development and in cultured neurons from different developmental stages. Using immunocytochemical methods we have observed that synapsin I appears lightly detectable in spinal cord of embryonic day (E)5-E8 embryos when the motor neurons have already established functional contacts with muscle fibers, and increases at E9. Until E8 synapsin I immunoreactivity appeared mainly localized in the gray matter of spinal cord; immunostaining of white matter becomes clearly evident only at E9. These observations indicate that synapsin I expression and possibly its transport to the nerve terminals may be stimulated by sequential signals. The cellular distribution of synapsin I observed in vivo is maintained in E8 and E9 spinal cord neuron cell cultures. In fact, in E8 cultured neurons, synapsin I immunostaining is observed only in the cell body, while in E9 cultured neurons both cell body and fibers are stained. The addition of muscle extracts to E8 cultures induces synapsin I decoration of fibers similar to that observed in E9 cultured neurons. Indeed Western and Northern blot analysis and in situ hybridization demonstrate an increase of synapsin I and its mRNA in spinal cord neurons kept in the presence of muscle extracts. These data suggest that synapsin I expression, as previously reported for other neuronal markers, can be modulated by soluble factors present in target cells.


Subject(s)
Gene Expression Regulation, Developmental , Neurons/metabolism , Spinal Cord/cytology , Synapsins/biosynthesis , Animals , Blotting, Northern , Blotting, Western , Cells, Cultured , Chick Embryo , In Situ Hybridization , RNA, Messenger/analysis , Spinal Cord/embryology , Spinal Cord/metabolism , Synapsins/genetics
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