Modulation of ethanol effect on hepatocyte proliferation by polyamines.

An occurrence and a magnitude of alcoholic liver diseases depend on the balance between ethanol-induced injury and liver regeneration. Like ethanol, polyamines including putrescine, spermidine, and spermine modulate cell proliferation. Thus, the purpose of this study was to evaluate the relationship between effect of ethanol on hepatocyte (HC) proliferation and polyamine metabolism using the HepaRG cell model. Results showed that ethanol effect in proliferating HepaRG cells was associated with a decrease in intracellular polyamine levels and ornithine decarboxylase (ODC) activity. Ethanol also induced disorders in expression of genes coding for polyamine-metabolizing enzymes. The α-difluoromethyl ornithine, an irreversible inhibitor of ODC, amplified ethanol toxicity on cell viability, protein level, and DNA synthesis through accentuation of polyamine depletion in proliferating HepaRG cells. Conversely, putrescine reversed ethanol effect on cell proliferation parameters. In conclusion, this study suggested that ethanol effect on HC proliferation was closely related to polyamine metabolism and that manipulation of this metabolism by putrescine could protect against the anti-proliferative activity of ethanol.

Bacteroides fragilis prevents Salmonella Heidelberg translocation in co-culture model mimicking intestinal epithelium.

Salmonella Heidelberg is one of the most common serovar causing foodborne illnesses. To limit the development of digestive bacterial infection, food supplements containing probiotic bacteria can be proposed. Commensal non-toxigenic Bacteroides fragilis has recently been suggested as a next-generation probiotic candidate. By using an original triple co-culture model including Caco-2 cells (representing human enterocytes), HT29-MTX (representing mucus-secreting goblet cells), and M cells differentiated from Caco-2 by addition of Raji B lymphocytes, bacterial translocation was evaluated. The data showed that S. Heidelberg could translocate in the triple co-culture model with high efficiency, whereas for B. fragilis a weak translocation was obtained. When cells were exposed to both bacteria, S. Heidelberg translocation was inhibited. The cell-free supernatant of B. fragilis also inhibited S. Heidelberg translocation without impacting epithelial barrier integrity. This supernatant did not affect the growth of S. Heidelberg. The non-toxigenic B. fragilis confers health benefits to the host by reducting bacterial translocation. These results suggested that the multicellular model provides an efficient in vitro model to evaluate the translocation of pathogens and to screen for probiotics that have a potential inhibitory effect on this translocation.

Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism.

OBJECTIVE: Iron loading has been observed to have a hyperproliferative effect on hepatocytes in vitro and on tumour cells in vivo; removal of this iron being required to induce antitumour activity. MATERIAL AND METHODS: Antiproliferative effects of orally active tridentate iron chelator ICL670 (deferasirox) and bidentate iron chelator CP20 (deferiprone), mediated through the chelation of intracellular iron, were compared in rat hepatoma cell line FAO and human hepatoma cell line HUH7. RESULTS: In FAO cell cultures, we have shown that ICL670 decreased cell viability and DNA replication and induced apoptosis more efficiently than an iron-binding equivalent concentration of CP20. Moreover, ICL670 decreased significantly the number of the cells in G(2)-M phase. In the HUH7 cell cultures, ICL670 and a four-time higher iron-binding equivalent concentration of CP20, decreased cell viability and DNA replication in the same range. CP20 increased the number of the cells in G(2)-M phase. However, ICL670 inhibited polyamine biosynthesis by decreasing ornithine decarboxylase mRNA level; in contrast, CP20 increased polyamine biosynthesis, particularly putrescine level, by stimulating spermidine-spermine N(1)-acetyl transferase activity that could activate the polyamine retro-conversion pathway. By mass spectrometry, we observed that ICL670 cellular uptake was six times higher than CP20. CONCLUSIONS: These results suggest that ICL670 has a powerful antitumoural effect and blocks cell proliferation in neoplastic cells by a pathway different from that of CP20 and may constitute a potential adjuvant drug for anticancer therapy.

Ultraviolet A-induced lipid peroxidation and antioxidant defense systems in cultured human skin fibroblasts.

Cultured human skin fibroblasts from healthy donors were irradiated with 180 kJ.m-2 ultraviolet (UV) A (320-400 nm) and assayed for thiobarbituric acid-reactive substances (TBARS), taken as an indicator of lipid peroxidation. Antioxidant defenses, including total glutathione (GSH) levels, superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase (Cat) activities were simultaneously assayed before and after irradiation. For the various donors, with different activities of these antioxidant systems before irradiation, TBARS correlated positively with SOD activity and negatively with Cat activity, whereas no correlation with GSH level or GSHPx activity was found. These data support the view that O2- is generated by UVA irradiation. They also suggest that H2O2, arising from O2- dismutation by SOD is not completely removed by Cat. Thus, the sensitivity of human fibroblasts to UVA-induced lipid peroxidation depends on a balance between SOD and Cat activities. After UVA irradiation, Cat activity was strongly inhibited, whereas GSH level was slightly decreased. By contrast, GSHPx and SOD activity remained unchanged after UVA irradiation.

Spectroscopic study of the dar interaction and of the photoreaction between a new monofunctional psoralen: 3-carbethoxy psoralen, and DNA.

3-carbethoxypsoralen (3-CPs) is a new linear psoralen derivative. Its dark interaction and photoreaction with DNA has been studied and compared with that of a well known bifunctional psoralen : 8-methoxypsoralen (8-MOP). 3-CPs is able to form in the dark a non covalent complex with native DNA. After irradiation of this complex with UV-A light (365 nm) 3-CPs is able to link covalently to DNA. Heat denaturation and renaturation patterns of treated DNA clearly show that, in contrast to 8-MOP, 3-CPs does not form DNA interstrand cross links. Fluorescence studies show that the photobinding of 3-CPs gives rise to the formation of monoadducts involving the 4',5' double bond of this molecule.

Chemical structure of 3-carbethoxypsoralen-DNA photoadducts.

The enzymatic digest from salmon sperm DNA photochemically modified by the monofunctional 3-carbethoxypsoralen was analyzed by high-performance liquid chromatography. The modified nucleosides extracted from DNA were compared with model compounds obtained from irradiation in the dry state of mixtures of 3-carbethoxypsoralen with 2'-deoxyribonucleosides whose chemical structures had previously been characterized. The main photoadducts formed in DNA are two cis-syn diastereoisomers formed via a C4-cycloaddition reaction involving the 4', 5' double bond of 3-carbethoxypsoralen and the 5,6 double bond of 2'-deoxythymidine. Among them, the most polar one accounts for 72%. Under the same conditions, photoadducts formed between 3-carbethoxypsoralen and 2'deoxycytidine account for less than 1%.

Physico-chemical properties of the heat-induced 'superaggregates' of amphotericin B.

The aggregation state of amphotericin B (AmB) was previously reported to modulate its therapeutic efficiency. As a preliminary study to test the biological effects of 'superaggregates' generated by heat treatment, we present spectroscopic data related to their formation in aqueous solutions. Drastic changes in the AmB aggregation state in water were shown to occur on heating at 50-60 degrees C. The concentration of the aggregates formed at high (A(t)) or room (A) temperature, and the concentration of the monomeric form (M) of AmB were calculated by processing absorption data. The thermally induced conversion from A to A(t) depends on the AmB concentration. Rayleigh scattering measurements suggest that the A(t) aggregates are larger than the A aggregates. At room temperature, the condensation rate of A with M-leading to the 'superaggregated' form A(t)-was slower and depended on the concentration of M. The superaggregated species A(t) was shown to be the most chemically stable species. Physico-chemical properties of these superaggregates are discussed as a potential new solution to improve the therapeutic efficacy of AmB.

Membrane damage induced in cultured human skin fibroblasts by UVA irradiation.

Irradiation of cultured human skin fibroblasts with ultraviolet light from 320 to 400 nm (UVA) leads to a decrease in the membrane fluidity exemplified by an enhanced fluorescence anisotropy of the lipophilic fluorescent probe 1-[4-trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. This UVA-induced decrease in fluidity is associated with lactate dehydrogenase leakage in the supernatant. Vitamin E, an inhibitor of lipid peroxidation, exerts a protective effect on both phenomena. Therefore, this UVA-induced damage in membrane properties may be related to lipid peroxidation processes. Moreover, exponentially growing cells are more sensitive to these UVA-induced alterations than confluent cells.

Identification of the two cis-syn [2 + 2] cycloadducts resulting from the photoreaction of 3-carbethoxypsoralen with 2'-deoxycytidine and 2'-deoxyuridine.

Near-ultraviolet photolysis of 2'-deoxycytidine (dCyd) and 3-carbethoxypsoralen (3-CPs) in the dry state was found to generate two main stable photoadducts which were separated by thin-layer and high-performance liquid chromatography. Fast atom bombardment and plasma desorption mass spectrometry analyses suggested that the bound molecule to 3-CPs is dCyd. These two compounds were found to produce the corresponding 2'-deoxyuridine (dUrd) derivatives through a deamination process when left in aqueous solutions with a lifetime close to 24 h at 20 degrees C. The chemical structure of the deaminated photoadducts was confirmed by photochemical synthesis using dUrd as the substrate. UV and fluorescent measurements indicated that the furan moiety of 3-CPs is involved in the photobinding reaction. The cyclobutane type structure of the modified dUrd derivatives was established on the basis of its photoreversibility and detailed 1H NMR analysis. The cis-syn stereoconfiguration of the two photocycloadducts was inferred from coupling constant considerations and on the basis of the complete assignment of the cyclobutyl protons, requiring the synthesis of deuterated nucleosides at pyrimidine carbon C(6). Further confirmation of the diastereoisomeric relationship between the two cis-syn dUrd <54' 65'> 3-CPs was provided by circular dichroism measurements.

Wall current probe: a non-invasive in situ plasma diagnostic for space and time resolved current density distribution measurement.

In the context of low temperature plasma research, we propose a wall current probe to determine the local charged particle fluxes flowing to the chamber walls. This non-intrusive planar probe consists of an array of electrode elements which can be individually biased and for which the current can be measured separately. We detail the probe properties and present the ability of the diagnostic to be used as a space and time resolved measurement of the ion and electron current density at the chamber walls. This diagnostic will be relevant to study the electron transport in magnetized low-pressure plasmas.

Studies on antibacterial dressings obtained by fluorinated post-discharge plasma.

Surface modification of wool, polyamide 6 and cotton fabrics was investigated with an Ar-CF(4) post-discharge plasma. The radical F, as determined by optical emission spectroscopy, is considered to be the main active species acting on the fabrics and producing different effects as a function of the textile substrate. Fluorination of the surface is achieved on the three materials studied, but only wool and polyamide 6 fluorinated surfaces become hydrophobic at long treatment times, and show antibacterial properties. The treatment conditions used are mild enough so as not to alter surface topography, as confirmed by scanning electron and atomic force microscopy.

Genetic hemochromatosis update.

Hereditary Hemochromatosis is an autosomal recessive disease, characterized by chronic iron overload. It is mainly due to mutations of the HFE-1 gene. In the large majority of patients, the substitution of tyrosine for cysteine at amino acid 282 (C282Y) is found at the homozygous state. Since the HFE-1 hemochromatosis identification, several other entities of iron overload have been individualized. In the present article, the frequency, penetrance and pathophysiology of HFE-1 hemochromatosis as well as various clinical presentations resulting from different mutations affecting different proteins involved in iron metabolism are described.

Isolation and characterization of psoralen photoadducts to DNA and related model compounds.

The main products of the photoreaction of 3-carbethoxypsoralen and 8-methoxypsoralen with 2'-deoxyribonucleosides have been isolated and characterized by various spectroscopic measurements involving proton nuclear magnetic resonance and mass spectrometry (fast atom bombardment and 252Cf plasma desorption techniques). Near ultraviolet photolysis of frozen aqueous solutions of thymidine containing 3-carbethoxypsoralen gives rise to two furan-side photocycloadducts having cis-syn stereochemistry. The corresponding thymine mean value of 3-carbethoxy-psoralen monoadduct has been shown to be the major photoproduct in DNA. The main cis-syn diastereoisomeric [2+2] photocycloadducts which arise from the photoreaction of 8-methoxypsoralen and thymidine in frozen aqueous solutions were shown to involve either the 4',5' furan ring or the 3,4 pyrone moiety and the 5,6-pyrimidine bond. Photobinding of 8-methoxypsoralen to 2'-deoxyadenosine also occurs, with covalent bond formation between carbon 3 or 4 of the pyrone ring and the sugar moiety of the nucleoside.