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Biomedical subjects

J C Béani

Publications and source records attributed to J C Béani.

At least 19 recordsLinked to original sources
PubMed2001

Induction of thioredoxin by ultraviolet-A radiation prevents oxidative-mediated cell death in human skin fibroblasts.

The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) system in UVA-irradiated human skin fibroblasts. Irradiation increases the intracellular level of Trx and a time-dependent increase of Trx mRNA is observed. Our data indicate that Trx translocates from the cytoplasm to the nucleus. In addition, UV exposure results in an increase in TR synthesis. In order to evaluate the function of Trx/TR system, we investigated the antioxidant role of Trx in transient transfected cells. The ROS accumulation in UVA irradiated cells was assessed using flow cytometry. A 3-fold decrease in ROS production was observed in transiently transfected fibroblasts. These results indicate that Trx acts as an antioxidant protein in UVA irradiated fibroblasts. As ROS are inducers of cell death, this raises the question as to whether Trx is able to protect cells from apoptosis and/or necrosis induced by UVA. Six hours after UVA-irradiation, 29.92% of cells were annexin-V positive. This population was significantly reduced in Trx-transfected cells (8.58%). Moreover, this work demonstrates that Trx prevents the loss of the membrane potential of the mitochondria, the depletion of cellular ATP content, and the loss of cell viability induced by irradiation.

Adenosine Triphosphate↗
PubMed2001

Modulation of exogenous and endogenous levels of thioredoxin in human skin fibroblasts prevents DNA damaging effect of ultraviolet A radiation.

Thioredoxin (Trx) plays important biological roles both intra- and extracellularly via thiol redox control. We have previously demonstrated that Trx exhibited protective effects against UVA cytotoxicity in human skin fibroblasts. As an extension of the latter investigation, the present work is aimed at assessing ability of Trx to maintain genomic integrity in human skin fibroblasts upon exposure to UVA radiation. Indeed, UVA (320--380 nm) is mutagenic and induces genomic damage to skin cells. The alkaline comet assay was used in association with DNA repair enzyme including formamido pyrimidine glycosylase (Fpg) and endonuclease III (endo III) to estimate the amount of modified bases together with the level of strand breaks and alkali-labile sites. The HPLC-EC assay was applied to assess 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) levels and to permit the calibration of comet assay as previously described. We reported that overexpression of human Trx (transient transfection) as well as exogenous human recombinant Trx added to the culture medium, decreased the level of DNA damage in UVA irradiated cells. Interestingly, transfection appeared to prevent UVA-induced 8-oxodGuo (3.06 au per Joules.cm(-2) compared to 4.94 au per Joules.cm(-2) for nontransfected cells). Moreover, Trx accumulates into nuclei in transfected cells. This finding supports the notion that Trx is important for the maintenance of the integrity of genetic information. This work demonstrated that under conditions of UVA oxidative stress, Trx prevented the UVA-induced DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

[Enhancement of endogenous antioxidant defenses: a promising strategy for prevention of skin cancers].

There is considerable evidence that ultraviolet radiation (UV) from sunlight is implicated in skin carcinogenesis. So the risks of cutaneous cancer have increased during the last decade due to increase of sun exposure. For a long time, ultraviolet B radiation (UVB: 290-320 nm) have been considered to be the more efficient wavelength in eliciting carcinogenesis in human skin. It is today clear that UVA (320-400 nm), especially UVA1 (340-400 nm) also participate to photo carcinogenesis. One of molecular mechanisms in the biological effects of UV is the induction of reactive oxygen species (ROS) directly or through endogenous photosensitization reactions. UVA radiation mainly acts via this production of ROS and the subsequent oxidative stress seems to play a crucial role in the deleterious effects of UVA. Fortunately, the skin possesses a wide range of interlinked antioxidant defence mechanism to protect itself from damage by UV-induced ROS. However, the capacity of these systems is not unlimited; they can be overwhelmed by excessive exposure to UV and then ROS can reach damaging levels. An interesting strategy to provide photoprotection would be to support or enhance one or more of these endogenous systems. In our experiments, we have evaluated the protective effect of glutathion, selenium and zinc, three compounds playing a pivotal role in the cellular defence against oxidative damage. We have irradiated both by UVA1 and UVB culture of human normal skin fibroblasts or of spontaneously immortalised human keratinocyte cell line Hacat. Before irradiation, treated cells were submitted to zinc deprivation by a diffusible zinc chelator, NNN' N'-tetrakis (2-pyridylmethyl) ethylene diamine (TPEN) or supplied with zinc chloride, thiols (N-acetyl-cysteine; N-acetyl-homocysteine-thiolactone, L2oxothiozolidine-4 carboxylate) selenium as sodium selenite. The cell viability was measured using the adhesion-proliferation method or a tetrazolium colorimetric assay. The damages induced to cellular membrane were appreciated by determination of thiobarbituric reactants (Tbars) by a fluorometric micromethod. Cellular DNA damage was examined by strand break determination carried out using the method described by Birnboim and Jevcak and by the Comet assay. Our results show that: UVA1 have a main part in cytotoxic effect of UVA and this effect is linked to ROS; thiols and selenium protect cells against UVA radiation with a synergic interaction and this protection acts though an increase in glutathion peroxidase activity; zinc protects against cytotoxicity of UVA and UVB and against UVB induced DNA damages; above all, DNA damages induced by UVA or UVB are significantly prevented by thiol molecules, selenium and zinc. As DNA damages have a main place in photocarcinogenesis, our results point out the potential interest of a photoprotection based on the support of endogenous antioxidant system. The research of new ways for photoprotection is indeed a necessity because sunscreens did not give convincing evidences of efficacy in preventing skin cancers.

Antioxidants↗
PubMed1999

Impairment of cultured cell proliferation and metallothionein expression by metal chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylene diamine.

Metallothioneins (MT) are a family of intracellular, cysteine-rich, zinc-binding proteins. Their expression is constitutive but can also be induced at the transcriptional level by various stimuli. In this study, we exposed HaCaT human keratinocytes to excess zinc (ZnCl2) or to zinc deprivation by the diffusible chelator NNN'N'-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), and to ultraviolet B (UVB) irradiation. We examined both cell proliferation and MT expression. Cell proliferation was maximally stimulated by 100 microM Zn2+ supply and was markedly inhibited by zinc deprivation or UVB irradiation. Zinc and UVB irradiation both increased MTI and/or MTII as detected by immunocytochemistry and enhanced the baseline level of MT-IIA mRNA, whereas TPEN treatment inhibited MT basal expression. Zinc partially prevented the concentration-dependent, UVB-induced decrease in cell proliferation. On the other hand, TPEN partially prevented the UVB-induced increase in MTIIA mRNA. These results suggest that zinc is involved in defense mechanisms of skin keratinocytes and in their stress-induced response.

Blotting, Northern↗
PubMed1999

Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts.

Ultraviolet A1 (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340-450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dose-dependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.

Apoptosis↗
PubMed1999

L-arginine increases UVA cytotoxicity in irradiated human keratinocyte cell line: potential role of nitric oxide.

Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), which metabolize L-arginine (L-Arg) for producing nitric oxide (NO*). This report delineates the relations between NO* and UVA in the human keratinocyte cell line HaCaT. NOS activity was stimulated by exposure of cells to L-Arg just after irradiation. L-Arg (5 mM) supply led to an increase in UVA (25.3 J/cm(2)) cytotoxicity (% of viability 18 +/- 3%) whereas neither L-Arg itself nor UVA irradiation induced cell death at the doses used in this study. Cells were also treated either with L-thiocitrulline (L-Thio), an irreversible inhibitor of NOS, or with exogenous superoxide dismutase (SOD) and catalase. L-Thio and SOD prevented L-Arg-mediated deleterious effects in irradiated cells, whereas catalase was ineffective. Intracellular antioxidant enzyme activities were also determined. UVA/L-Arg stress altered catalase (66% decrease) and glutathione peroxidase (83% decrease). DNA damage was evaluated using the 'comet assay' and quantified using the 'tail moment'. UVA alone was genotoxic (mean tail moment: 25.43 +/- 1.23, P<0.001 compared control cells). The addition of L-Arg potentiated DNA damage (mean tail moment: 41.05+/-3.9) whereas L-Thio prevented them (mean tail moment 9.86 +/- 0.98). We attempted to assess the effect of poly(ADP-ribose) polymerase (PARP) inhibition on cell death. Using the PARP inhibitor 3-aminobenzamide, we established that PARP determines both cell lysis and DNA damage induced by UVA and/or L-Arg. Our findings demonstrated that L-Arg was able to increase UVA-mediated deleterious effects in keratinocytes (both DNA damage and cytotoxicity) and that the ratio NO*/O2*- plays a key role in these processes.

Antioxidants↗
PubMed1998

Metal chelator NNNNN-tetrakis-(2-pyridymethyl)ethylene diamine inhibits the induction of heat shock protein 70 synthesis by heat in cultured keratinocytes.

Heat shock protein (HSP) synthesis results from various types of injury, including heat shock (HS) and some oxidants. The intracellular signals leading to HSP synthesis are not yet fully elucidated. We have studied the influence of NNN'N'-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a metal chelator known to induce cellular zinc and copper deprivation, on resistance to heat and on hsp70 synthesis in HaCaT keratinocytes. TPEN was shown to sensitize HaCaT cells to heat shock. The effect of TPEN was neutralized by equimolar Zn2+. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blotting characterization of hsp70, it was shown that cultured HaCaT cells constitutively express the inducible form of hsp70. The application of TPEN alone slightly increases the level of hsp70 but inhibits its induction by HS. This inhibitory effect is related to metal deprivation, because it is eliminated when Cu2+ or Zn2+ ions are supplied together with TPEN. These results suggest that these metals are involved in the expression by keratinocytes of a stress protein which has a protective action against environmental stress.

Cell Survival↗
PubMed1998

Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture.

Ultraviolet A radiation (UVA, 320-400 nm) is mutagenic and induces genomic damage to skin cells. N-acetyl-cysteine (NAC), selenium and zinc have been shown to have antioxidant properties and to exhibit protective effects against UVA cytotoxicity. The present work attempts to delineate the effect of these compounds on genomic integrity of human skin fibroblasts exposed to UVA radiation using the single cell gel electrophoresis (SCGE) or Comet assay. The cells were incubated with NAC (5 mM), sodium selenite (0.6 microM) or zinc chloride (100 microM). Then cells were embedded in low melting point agarose, and immediately submitted to UVA fluences ranging from 1 to 6J/cm2. In the Comet assay, the tail moment increased by 45% (1 J/cm2) to 89% (6J/cm2) in non-supplemented cells (p)<0.01). DNA damage was significantly prevented by NAC, Se and Zn, with a similar efficiency from 1 to 4J/cm2 (p < 0.05). For the highest UVA dose (6J/cm2), Se and Zn were more effective than NAC (p < 0.01).

Acetylcysteine↗
PubMed1997

Involvement of zinc in intracellular oxidant/antioxidant balance.

The effect of zinc (Zn) on cellular oxidative metabolism is complex and could be explained by multiple complementary interactions. In this study, we evaluated the impact of Zn on the pro-oxidant/antioxidant balance of HaCaT keratinocytes. Cells were submitted to a diffusible metal chelator able to induce intracellular Zn deprivation, TPEN, in combination or not with Zn chloride (ZnCl2), in the culture medium. The intracellular amount of Zn, copper (Cu), and iron (Fe) was determined, as well as CuZnSOD and MnSOD activities and glutathione reserves. The consequence of the modulation of Zn concentration on lipid peroxidation was also evaluated. TPEN induced a significant dose-dependent decrease in intracellular Zn and Cu (from 394-181 and 43-21 microg/g protein, respectively, after 6 h of TPEN 50 microM). No significant change in intracellular Fe concentration was found following TPEN exposure. The SOD activities were unchanged after 6 h of TPEN 50 microM application, either CuZnSOD or MnSOD. Cells exposure to TPEN induced a deep time- and dose-dependent decrease in their glutathione content (from 65-8 microM/g protein after 6 h of TPEN 50 microM), and a concomitant increase in glutathione in the cell-culture supernatants. No significant change in lipid peroxidation products was detected.

Antioxidants↗
PubMed1997

Zinc and DNA fragmentation in keratinocyte apoptosis: its inhibitory effect in UVB irradiated cells.

Zinc has been shown to have antioxidant properties and to exhibit inhibitory effects on apoptosis. In this work we investigated the effect of zinc on DNA integrity and on apoptosis of HaCaT keratinocytes. Cells were submitted to zinc deprivation by a diffusible zinc chelator, (N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine) (TPEN) or supplied with zinc chloride and submitted to UVB radiation. After cell exposure to TPEN for 2 h, strand breaks significantly impaired DNA resistance to alkaline denaturation. DNA strand breaks induced by a 6 h TPEN application were significantly prevented if zinc chloride was supplied together with the chelator. TPEN also generated, after 4-6 h of application, cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes), features of cell death by apoptosis. Moreover, UVB irradiation led to early DNA strand breaks and to an increase in cytoplasmic nucleosomes which was maximum 10 h after irradiation. These effects were prevented by the supply of zinc chloride (0.1 mM) in the culture medium. These results suggest that zinc ions interfere with the apoptosis process at an early stage, by decreasing DNA damage able to trigger apoptosis.

Apoptosis↗
PubMed1995

[Candida vasculitis].

Apart from direct skin attacks, yeasts of the Candida group hold a place that is much discussed in the occurrence of immuno-allergic reactions of the skin, because of their usual saprophytic characteristics. We report here on observation of a patient of 62 years who presented with a secondary purpuric maculo-papular eruption making a tri-symptom of Gougerot, for which we have incriminated the role of the immunogenicity of Candida. Skin histology confirmed the presence of an angiitis and necrotising capillarity. An intradermal test with candidine reproduced the skin symptoms and histology, whilst other microbial and fungal tests remained within normal limits. Other examinations showed the transitory presence of circulating immune complexes, although the remainder of the immunity factors were negative. A digestive focus of Candida was present. Recovery occurred after systemic mycological treatment was started, though there was a return of symptoms after a temporary initial check.

Amphotericin B↗
PubMed1994

Evaluation of cutaneous photodamage using a photographic scale.

Clinical assessments of photodamage are based upon a subjective evaluation of characteristic features such as wrinkling and pigmentary change, and are influenced by inter-observer differences in grading criteria. In an effort to standardize the grading of photodamage severity, we have developed a six-point photographic scale in which each of the six grades of overall photodamage severity is depicted by three photographs. The use of three photographs to portray each grade illustrates the diversity and range of manifestations within each grade. This photographic scale was tested by two groups of dermatologists, who used it on two occasions to grade the overall photodamage severity of a single group of female Caucasian subjects. Results indicate high inter-observer agreement, with chance-corrected agreement ranging from 0.44 to 0.63 and from 0.54 to 0.76 on the first and second occasions, respectively. Intra-observer repeatability was high, with chance-corrected agreement ranging from 0.56 to 0.78. Inter- and intra-observer differences were within one category in nearly all cases. Similar grades were assigned by dermatologists with and without experience in treating photodamaged patients. We conclude that application of this scale results in consistent and reproducible clinical evaluations of overall photodamage severity in Caucasian subjects. The scale may be useful in categorizing subjects for epidemiological studies, or in selecting patients for clinical trials.

Dermatology↗