Utilization of a mouse/human chimeric model for long term metabolic testing of human skin.

Until now, ex vivo human skin explant utilization in tissue culture has consisted of limited short-term studies (less than a week). This short timeframe does not allow for the investigation of metabolic responses of complex tissues to specific molecules or compounds. Here, we aim to develop an improved mouse transplantation model that maintains the viability, structure and functionality of the human skin explants for prolonged periods of time. Healthy human skin explants derived from biopsies were grafted onto nude mice and used to perform a toxicological study of the reactivity and functionality of grafted skin explants after one month. Histological observations suggest that the tissue properties and phenotype of the human skin graft are conserved as a result of re-vascularization upon tissue integration. The toxicological test performed shows that the human skin graft reacts to systemic exposure of a xenobiotic metabolic inducer when applied to this mouse model. This mouse/human chimeric model can be effective for the long-term study of human skin reactivity to chemicals as well to study in vivo responses to complex co-exposures.

Mechanisms of pollutant-induced toxicity in skin and detoxification: Anti-pollution strategies and perspectives for cosmetic products.

With the development of industry and increase in road traffic, atmospheric pollution has reached unprecedented levels in many regions of the world. Concentrations of pollutants are often far beyond the recommendations of the World Health Organization. Skin, as the first interface between the human body and its environment, is one of the main organs exposed to pollutants and to other environmental factors such as UV irradiation. As much as the effects of pollution and UV irradiation on human skin have been described, the underlying mechanisms remain to be elucidated. This state of the art study aims at exposing the numerous adverse effects of UV and pollution as well as their mode of action on skin. We summarize how these environmental factors negatively impact skin cells: by upregulating xenobiotic metabolism (and bioactivation) and inducing oxidative stress and inflammation, leading to premature aging and a disrupted barrier function. Consequently, we suggest adapted protective measures for the cosmetic industry to support anti-pollution claims.

Perspectives and strategies of alternative methods used in the risk assessment of personal care products.

Risk assessment for personal care products requires the use of alternative methods since animal testing is now totally banned. Some of these methods are effective and have been validated by the "European Union Reference Laboratory for alternatives to animal testing"; but there is still a need for development and implementation of methods for specific endpoints. In this review, we have focused on dermal risk assessment because it is the prime route of absorption and main target organ for personal care products. Within this field, various areas must be assessed: irritation, sensitisation and toxicokinetic. Personal care product behaviour after use by the consumer and potential effects on the environment are also discussed. The purpose of this review is to show evolution and the prospects of alternative methods for safety dermal assessment. Assessment strategies must be adapted to the different chemical classes of substances studied but also to the way in which they are used. Finally, experimental and theoretical technical parameters that may impact on measured effects have been identified and discussed.

Rhein, the metabolite of diacerhein, reduces the proliferation of osteoarthritic chondrocytes and synoviocytes without inducing apoptosis.

OBJECTIVE: The aim of this study was to determine the effects of pharmacologically relevant concentrations of rhein (1,8-dihydroxy-3-carboxyanthraquinone) on the cell proliferation rate of human chondrocytes and synoviocytes. METHODS: Cultures of human osteoarthritic synoviocytes and chondrocytes were incubated with 10(-6), 10(-5), and 10(-4) M rhein. [3H]thymidine incorporation was used to determine rhein proliferative effects after incubation periods of 24 h, 48 h, and 1 week. The cytotoxicity of the drug was assayed with a nonradioactive assay kit. Nuclear extracts were used to detect variations in cell-cycle proteins (p21, p27, and cyclin D1) by Western blotting. The effect of rhein on apoptosis was investigated by measurement of caspase-3/7 activity and DNA fragmentation. RESULTS: Rhein was found to downregulate the proliferation rate of both chondrocytes and synoviocytes, two-fold for 10(-5) M rhein and five- to six-fold for 10(-4) M rhein. No cytotoxicity of the drug was observed. Rhein (10(-4) M) decreased caspase-3/7 activity and did not induce DNA fragmentation. Western blots showed that 10(-4) M rhein increased the expression of p21 and/or p27, but not that of cyclin D1. CONCLUSIONS: Rhein has previously been shown to reduce the interleukin (IL)-1beta deleterious effects on osteoarthritis (OA) cartilage through inhibition of the expression of degrading enzymes. Here, rhein was also found to inhibit proliferation of both synoviocytes and chondrocytes, suggesting that the drug may decrease the development of the inflammatory synovial tissue that accompanies joint pathologies. Both its anti-catabolic and anti-proliferative effects may explain its beneficial effect in the treatment of joint diseases.

[Photodynamic therapy: principles and therapeutic indications]. / Principes et applications thérapeutiques de la photothérapie dynamique.

Photodynamic therapy consists in destroying a tumoral or a non tumoral tissue by the effect of both a photosensitizing molecule and a laser light. This simple concept has needed numerous years in order to be used in routine treatments with both photosensitizers and laser light delivered optimally. Researches in chemistry lead to new porphyrin and bacteriochlorophyl derivatives which alleviate the decrease of light absorption by endogenous molecules and in consequence allow a deeper light penetration. Short half-life of these compounds allows an easier treatment monitoring. In parallel, improvements in both laser technology and fibers allow new indications in various pathologies. First applications took place in treatment of respiratory, digestive and urologic cancers. The biggest success to date is recorded in ophthalmology with the treatment of age related macular degeneration. New approaches are explored and clinical studies are ongoing.

Modulatory effect of rhein on IL-1alpha-induced responses in human chondrocytes: a comparative study between antibody microarrays and specific ELISAs.

The present work aimed to take advantage of the screening capacity of protein arrays to search for additional targets of rhein in interleukin (IL)-1-stimulated chondrocytes. Primary cultures of chondrocytes from osteoarthritic (OA) patients were stimulated for 24 and 48 h with 1 ng/ml of IL-1alpha, in the presence or absence of 10(-5) M of rhein. Culture supernatants were analyzed with arrays membranes consisting of 120 antibodies directed against cytokines, chemokines, and angiogenic or growth factors and were controlled for 8 proteins by specific immuno-enzymatic assays (ELISA). Protein arrays showed that several CC or CXC chemokines, the growth factor GM-CSF, the cytokines IL-6, IL-7 and IL-10 (but unexpectedly not IL-1beta or TNFalpha) and the adhesion molecule ICAM-1 were induced maximally by IL-1alpha. In IL-1-stimulated chondrocytes, rhein reduced slightly the production of MCP-1 and increased those of IL-1Ra, of the cytokine receptors sgp130, IL-6R, sTNFR I and R II, but also of some chemokines or ICAM-1. Specific ELISAs confirmed the effect of rhein on MCP-1, IL-1Ra, sgp130, IL-6R and sTNFR II but was discrepant for GROalpha and were always more sensitive than protein arrays to detect IL-1 effects such as IL-1Ra and TNFalpha release. The present data show that rhein modulated some IL-1-induced responses contributing possibly to its chondroprotective (IL-1Ra, MCP-1) or cytokine modifying (sTNFR II, sgp130) properties, but that protein arrays were poorly sensitive to check for IL-1- and/or rhein-induced changes.

Rhein, a diacerhein-derived metabolite, modulates the expression of matrix degrading enzymes and the cell proliferation of articular chondrocytes by inhibiting ERK and JNK-AP-1 dependent pathways.

OBJECTIVE: To determine the effects of rhein on the expression of matrix metalloproteinases (MMP-1, -3, 13) and ADAMTs 4, 5 (a disintegrin and metalloproteinase with thrombospondin type-I repeat)/aggrecanases-1, -2 in interleukin-1-stimulated bovine articular chondrocytes, and to investigate the signalling pathways involved in the effects of the drug on gene expression and cell proliferation. METHODS: Bovine chondrocytes were treated with 10(-4) M rhein for 18 h, followed by 10 ng/ml IL-1Beta for 30 min (cytoplasmic extracts) or 24 h (RNA extraction and EMSA). mRNA was assessed by RT-PCR for the expression of MMPs and aggrecanases, and the phosphorylation of MAP kinases was studied by Western blotting. NF-kappaB and AP-1 DNA binding were determined by gel retardation assay. The effects of inhibitors of these signalling pathways were compared to those of rhein. The proliferation of human chondrocytes and synoviocytes treated with the drug was also investigated. RESULTS: IL-1Beta-induced stimulation of the MMPs and aggrecanase-1 was markedly inhibited by rhein. The drug reduced IL-1Beta-induced NF-kappaB and AP-1 DNA binding, as well as the phosphorylation of ERK and JNK. Similar effects were produced by the specific inhibitors of these signalling pathways. In addition, rhein reduced the proliferation of both human chondrocytes and synoviocytes. CONCLUSION: Our data indicate that rhein may reduce the deleterious effects of IL-1Beta on osteoarthritic cartilage through its effects on the ERK- and JNK-dependent pathways. Both its anti-catabolic and anti-proliferative properties may explain its value in the treatment of joint diseases.

Inhibition of interleukin-1beta-induced activation of MEK/ERK pathway and DNA binding of NF-kappaB and AP-1: potential mechanism for Diacerein effects in osteoarthritis.

In the present report we have shown that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/ml) by an increased DNA binding activity of NF-kappaB and AP-l transcription factors. Incubation of the cells with 10(-5) M Rhein, the active metabolite of Diacerhein, for 24 h was found to reduce this activity particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to a 1 h treatment with IL-1beta. This effect was greater in hypoxia (3% O2) than in normoxia (21% O2). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M Rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extra cellular matrix synthesis. IL-1-induced collagenase (MMP1) expression was significantly decreased by Rhein under the same conditions. In conclusion, Rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several pro-inflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMP1 synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the anti-osteoarthritic properties of Rhein and its disease-modifying effects on OA cartilage, in spite of the absence of inhibition at prostaglandin level.

Comparison of the effects of fasting morning, fasting evening and fed bedtime administration of tenatoprazole on intragastric pH in healthy volunteers: a randomized three-way crossover study.

BACKGROUND: The effectiveness of proton pump inhibitors is influenced by meals and administration time. AIM: To compare the effects on intragastric acidity of times of dosing of tenatoprazole, a novel imidazopyridine-based proton pump inhibitor with a prolonged plasma half-life. METHODS: This randomized three-period crossover study included 12 Helicobacter pylori-negative healthy subjects, who received tenatoprazole 40 mg either fasting at 7.00 AM, fasting at 7.00 PM or fed at 9.30 PM for 7 days, with a 2-week washout between periods. Twenty-four hour intragastric pH was monitored on day 7 of each period. RESULTS: On day 7, median 24-h pH was 4.7, 5.1 and 4.7 after breakfast, dinner and bedtime dosing, respectively (P = 0.11), whereas night-time pH was 4.2, 5.0 and 4.4 (P = 0.13). The mean 24-h percentage of time over pH 4 was 62, 72 and 64 after breakfast, dinner and bedtime dosing, respectively (N.S.), and 54, 68 and 56 during night-time (P = 0.06). Nocturnal acid breakthrough incidence decreased from 100% at baseline to 83%, 55% and 75% after 7.00 AM, 7.00 PM and 9.30 PM dosing, respectively (P = 0.18), and its mean duration dropped from 6.2 to 2.8, 1.0 and 2.2 h, respectively (P < 0.05). CONCLUSION: Seven-day administration of tenatoprazole provides a prolonged duration of acid suppression, especially during the night-time, with little effect of food or time of dosing.

Articular chondrocytes cultured in hypoxia: their response to interleukin-1beta and rhein, the active metabolite of diacerhein.

In the present report, we show that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/ml) by an increased DNA binding activity of NF-kappaB and AP-1 transcription factors. Incubation of the cells with 10(-5) M rhein for 24 h was found to reduce this activity, particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to 1-h treatment with IL-1beta. This effect was greater in hypoxia (3% O(2)) than in normoxia (21% O(2)). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extracellular matrix synthesis. IL-1-induced collagenase (MMP1) expression was significantly decreased by rhein in the same conditions. In conclusion, rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several pro-inflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMP1 synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the anti-osteoarthritic properties of rhein and its disease-modifying effects on OA cartilage, in spite of absence of inhibition at prostaglandin level.

Simultaneous determination of hemoglobin and coproporphyrin by second derivative differential spectrophotometry: application to the diagnosis of meconium aspiration.

We describe a simple, fast method for simultaneous measurement of hemoglobin and coproporphyrin in urine and amniotic fluid, by second-derivative differential spectrophotometry. Both pigments were determined in biological samples without prior extraction. Despite the slightly overlapping spectra, the method permitted satisfactory resolution and avoided interspectral interference. Its sensitivity, sufficiently good, allowed to detect hemoglobin and coproporphyrin in slightly pathological urine and amniotic fluid as an aid to the diagnosis of meconium aspiration.

[Direct spectrophotometric determination of urinary uroporphyrin and coproporphyrin using zerocrossing second derivative spectrophotometry]. / Dosage spectrophotométrique direct de l'uroporphyrine et de la coproporphyrine urinaires par la méthode du point d'annulation en dérivée seconde.

The use of zero-crossing second-derivative spectrophotometry allows simultaneous determination of uroporphyrin and coproporphyrin directly in urines. Linearity and repeatability are excellent. Detection limit is about 8 nmol/l. Specificity is excellent too, except in unusual porphyrias where porphyrins with 5, 6 or 7 carboxylic groups are encountered. In such a case, this method allows the detection of increased porphyrins contents. The main advantage of this technique is its simplicity: it needs just to acidify urines and to plot a spectrum from 380 to 440 nm to determine the uroporphyrin and coproporphyrin concentrations. So it may be a very useful technic for clinical biochemistry laboratories in charge of monitoring patients suffering from porphyria.