EVALUATION OF THE EFFECT OF THE COMPOUND RIBOXYL ON THE ATP LEVEL AND CELLULAR RESPIRATION FROM HUMAN DERMAL FIBROBLASTS

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1 EVALUATION OF THE EFFECT OF THE COMPOUND RIBOXYL ON THE ATP LEVEL AND CELLULAR RESPIRATION FROM HUMAN DERMAL FIBROBLASTS FINAL REPORT OF THE STUDY GT Study promoter: LUCAS MEYER COSMETICS ZA Les belles Fontaines 99 route de Versailles CHAMPLAN Phone : Fax : info@lmcosmetics.fr Date: February 14th, 2006

2 1 - INTRODUCTION MATERIALS AND METHODS BIOLOGICAL MODEL TEST COMPOUND PRELIMINARY CYTOTOXICITY OXYGEN CONSUMPTION ASSAY ATP ASSAY DATA MANAGEMENT 5 3. RESULTS PRELIMINARY CYTOTOXICITY DETERMINATION OXYGEN CONSUMPTION ASSAY Effects on the basal cell respiration rate Effects on the mitochondrial respiration rate ATP ASSAY Effects on ATP level without hypoxia Effect on ATP level in hypoxia conditions 7 4. TABLES AND FIGURES 8 2

3 1 - INTRODUCTION The laboratories LUCAS MEYER COSMETICS have the compound RIBOXYL for which the effect on cutaneous energy metabolism is researched. This study was performed in order to evaluate the effects of this compound on several parameters in human dermal fibroblasts: - effects of the compound on the basal and mitochondrial cell respiration rate (oxygen consumption). - effects of the compound on the ATP level after hypoxia. 2 - MATERIALS AND METHODS 2.1. Biological model - Type: Normal Human Dermal Fibroblasts (NHDF) pool PF2, 8th passage - Culture medium: DMEM (Invitrogen ) Glutamine 2 mm (Invitrogen ) Penicillin 50 UI/ml-Streptomycin 50 µg/ml (Invitrogen ) Foetal calf serum 10% (FCS, Invitrogen ). - Hypoxia Assay medium: DMEM glucose 4.5 g/l, hepes 25 mm (Invitrogen ) Glutamine 2 mm (Invitrogen ) Penicillin 50 UI/ml-Streptomycin 50 µg/ml (Invitrogen ) Foetal calf serum 10% (FCS, Invitrogen ) - Assay medium : HBSS (Invitrogen ) (mitochondrial respiration) pyruvate 625 µm (Sigma P4562) malate 313 µm (Sigma M6773) - Assay medium : HBSS (Invitrogen ) (basal respiration) glucose 1.25 mm (Sigma G7021) 2.2. Test compound Test compound Stock solution Dilution Final tested concentration RIBOXYL White powder supplied by the study promoter and stored at in assay 0.05 % (respiration) lot n C. Solution prepared at medium (GT050923/1) 10 % (100 mg/ml) in assay medium 0.05 % (ATP assay) 3

4 2.3. Preliminary cytotoxicity The cell viability was assessed using a standard reduction assay (MTT assay). MTT salt 3-(4,5- dimethyl thiazol-2-yl)-2,5 diphenyl-tetrazolium bromide is reduced by mitochondrial succinate dehydrogenase in living cells, this reaction produces formazan crystals, soluble in isopropanol, that are quantified by photometry (OD540nm). Consequently, the mitochondrial succinate dehydrogenase-dependent transformation of MTT in formazan crystal is proportional to the amount of living cells in the tissue. - plates: 96-wells plates - tested concentrations: 10 % (100 mg/ml) and then dilution factor 5 - replicates: 6 - cells/compound contact: 24 h - evaluation parameter: MTT hydrolysis assay and morphological observation 2.4. Oxygen consumption assay This assay was performed in two different conditions: - effect on the basal cell respiration rate realized on non-permeabilized cells in presence of glucose (1.25 mm final). - effect on the mitochondrial rate on saponine-permeabilized fibroblasts in the presence of the respiratory substrate pyruvate/malate (625 µm/313 µm final). A suspension of fibroblasts ( cells/well), saponine-permeabilized (mitochondrial condition) or non-permeabilized (basal condition), were deposited in presence or not (control) of the tested compound and substrate (glucose or pyruvate/malate) in specific plates BD Oxygen Biosensor System (BD ). The mix was recovered with mineral oil (Sigma M-5904) and the fluorescence intensity was measured in kinetic during 6 h (in this system, the fluorescence increasing is proportional to the oxygen consumption by the cells). All the conditions were realized in hexaplicates. The results corresponding to the oxygen consumption rate were expressed in arbitrary units (AU)/minute. The oxygen consumption was calculated in the linear part of the kinetic curve of fluorescence emission, corresponding to the first two hours of contact ATP assay The fibroblasts were seeded in two series of 96-wells plates in DMEM 10 % FCS until confluence (D0). The culture medium of the first series of plates was removed and replaced by hypoxia assay medium (DMEM/ HEPES/glucose) supplemented with 10 % FCS and the cells were incubated 24 h at 37 C and 5 % CO2 (D1). In the second series of plates, the cells were incubated, without medium change, 24 h at 37 C and 5 % CO2. After incubation, the culture medium was removed and replaced by hypoxia assay medium containing or not (control) the indicated concentrations of the test compound and the cells were incubated 24 h at 37 C and 5 % CO2. After treatment (D2), one plate was incubated at 37 C and 5 % CO2 (series without hypoxia) and the second was incubated at 37 C in N2 atmosphere (series with hypoxia). All treatments were realized in hexaplicate. After 6 h for each incubation, the culture medium was removed and the cells monolayers were frozen at -80 C. The ATP was extracted in Tris 100 mm/edta 4mM, ph 7.75 and ATP 4

5 concentrations were determined using a specific kit (Roche ) according to the manufacturer s procedure. The raw data were expressed in nm of ATP Data management - The raw data were analyzed with PRISM software (Graph Pad Software). - The inter-group comparisons were performed by variance analysis (ANOVA) with multiple comparisons test of Dunnett. 5

6 3. RESULTS 3.1. Preliminary cytotoxicity determination The aim of this first step was to study the cytotoxicity of the compound RIBOXYL on human fibroblasts using a standard MTT assay. The results are shown in table Oxygen consumption assay Effects on the basal cell respiration rate This assay was performed on non-permeabilized whole cells in presence of glucose as substrate. This protocol involves the study of the global energy metabolism, including the glycolyse. The results are shown in table 2 and figure 1. In these experimental conditions, the compound RIBOXYL tested at 0.05 % increased significantly the basal cell respiration rate (137 % compared to the control, p<0.01) Effects on the mitochondrial respiration rate This assay was realized on saponine-permeabilized cells in presence of pyruvate-malate as substrate (direct substrate of Krebs cycle). The results are shown in table 3 and figure 2. In these experimental conditions, the compound RIBOXYL tested at 0.05 % increased significantly the mitochondrial respiration rate (131 % compared to the control, p<0.01). In conclusion, the compound RIBOXYL induced a significant stimulated effect on basal and mitochondrial respiration at 0.05 %. 6

7 3.3. ATP assay Table 4 In these experimental conditions, incubation for additional 24h without hypoxia induced an increase in the ATP production by the fibroblasts. This increase is normal and due to the cell culture proliferation. In hypoxia conditions, the ATP rate noticeably decreased. This result caused by the stress of oxygen deprivation of the cells was expected and validated the assay Effects on ATP level without hypoxia The results are shown in table 5 and figure 3. In absence of hypoxia, the compound RIBOXYL tested at 0.05 % did not show effect on ATP level Effect on ATP level in hypoxia conditions The results are shown in table 6 and figure 4. In these experimental conditions, the compound RIBOXYL tested at 0.05 % increased the ATP level in hypoxia condition. 7

8 4. TABLES AND FIGURES Morphological observations: +, normal population +/ -, growth reduction or morphological modifications -, toxicity 0, cell mortality Table 1: preliminary cytotoxicity on human fribroblasts. Determination of the compound cytotoxicity by the MTT assay. 8

9 Table 2 and figure 1: effects of the compound RIBOXYL on the basal cell respiration rate in normal human dermal fibroblast culture. 9

10 Table 3 and figure 2: effects of the compound RIBOXYL on the mitochondrial respiration rate in normal human dermal fibroblast culture. 10

11 Table 4: measurement of ATP level in untreated fibroblasts. Control without hypoxia and incubated 24h at 37 C and 5 % CO2 (D1) and control incubated 48h + 6h without or with hypoxia (D2). 11

12 Table 5 and figure 3: effects of the compound RIBOXYL on the ATP level in NHDF cultivated in normal conditions (without hypoxia). 12

13 Table 6 and figure 4: effects of the compound RIBOXYL on the ATP level in NHDF cultivated in hypoxia conditions. 13