Philippe Catroux

Cdc42 downregulates MMP-1 expression by inhibiting the ERK1/2 pathway.

The small GTPases of the Rho family are key intermediates in cellular signalling triggered by activated cell-adhesion receptors. In this study, we took advantage of RNA interference (RNAi) using small interfering RNAs (siRNAs) to define the roles of the best-characterized members of the RhoGTPase family, RhoA, Rac1 and Cdc42, in the control of MMP-1, MMP-2 and type-I-collagen expression in normal human skin fibroblasts (HSFs). A specific and long-lasting repression, up to 7 days after transfection, of the three GTPases was achieved by transient transfection of specific siRNA. The silencing of Cdc42, but not that of RhoA or Rac1, induced a 15-fold increase in MMP-1 secretion. This upregulation was confirmed at the mRNA level and observed with two different siRNAs targeting Cdc42. Such a regulation was also observed in various human cell lines and was rescued by re-expressing wild-type Cdc42 encoded by a construct bearing silent mutations impeding its recognition by the siRNA. By contrast, MMP-2 and type-I-collagen expression was not affected by the individual silencing of each Rho GTPase. Cytokine protein array, enzyme-linked immunosorbent assays and reverse-transcription PCR measurements revealed that ablation of Cdc42 induced an overexpression of interleukin 8 and MCP-1. Although these cytokines are known to induce the expression of MMP-1, we showed that they were not involved in the Cdc42-mediated upregulation of MMP-1. Silencing of Cdc42 also induced an increased phosphorylation of ERK1/2 and p38 MAP kinase. The use of chemical inhibitors on Cdc42-ablated cells revealed that the upregulation of MMP-1 is dependent on the ERK1/2 pathways, whereas the p38 MAP kinase pathway displayed an inhibitory role. Simultaneous knock-down of two or three Rho GTPases allowed us to demonstrate that the RhoA-ROCK pathway was not involved in this regulation but that the silencing of Rac1 reduced the effect of Cdc42 suppression. These data suggest that, in vivo, when cell/extracellular-matrix interactions via integrins induce cytoskeleton organization, MMP-1 expression is maintained at a low level by Cdc42 via a repression of the Rac1 and ERK1/2 pathways. Therefore, Cdc42 contributes to ECM homeostasis and connective tissue integrity.

The silicon microphysiometer for testing ocular toxicity in vitro

The silicon microphysiometer has been used for in vitro evaluation of the ocular irritancy potential of water soluble ingredients and formulations. This light-addressable potentiometric sensor detects changes in cell physiology by monitoring the rate at which cultured cells excrete their acidic products of metabolism. We have mainly determined the metabolic effect of 53 products (21 surfactants and 32 surfactant-based formulations). The related maximal average Draize score (MAS) were available from historical data and varied from 1.7 to 54. All of the Draize categories were represented. Murine fibroblastic cells (L929 clone) were exposed to increasing concentrations of the product for approximately 400 sec per dose. The MRD(50) (dose of product that decreased the metabolic rate of the cells by 50%) was determined by interpolation from a plot of metabolic rate versus test material concentration. Decreases in metabolic rate, as assessed by the MRD(50), occurred over a wide range of concentrations (40 mug/ml-200 mg/ml). The linear (Pearson) and rank (Spearman) correlation between in vivo (MAS) and in vitro (log MRD(50)) data were 0.91 and 0.89, respectively. This study indicates that the silicon microphysiometer method exhibits a high correlation with the Draize test for water-soluble raw materials and formulations and thus can be used as an in vitro screen for ocular irritation.

Evaluation of eye irritation potential: statistical analysis and tier testing strategies☆

Eye irritation testing, specifically the Draize test, has been the centre of controversy for many reasons. Several alternatives, based on the principles of reduction, refinement and replacement, have been proposed and are being used by the industry and government authorities. However, no universally applicable, validated non-animal alternative(s) is currently available. This report presents a statistical analysis and two testing approaches: the partial least squares multivariate statistical analysis of de Silva and colleagues from France, the tier-testing approach for regulatory purposes described by Gerner and colleagues from Germany, and the three-step tier-testing approach of the US Interagency Regulatory Alternatives Group described by Gupta and Hill. These approaches were presented as three separate papers at the November 1993 Interagency Regulatory Alternatives Group (IRAG) Workshop on Eye Irritation Testing; they have been summarized and combined into the following three-part report. The first part (de Silva et al.) presents statistical techniques for establishing test batteries of in vitro alternatives to the eye irritation test. The second (Gerner et al.) and third (Gupta and Hill) parts are similar in that they stage assessment of information by using a combination of screening information and animal testing to effect reductions in animal use and distress.

The use of in vitro methods in the ocular irritation assessment of cosmetic products

The ocular tissue is a complex system consisting of corneal and conjunctival epithelial cells, the underlying corneal stroma and associated endothelial cells. Exposure to chemicals may result in responses ranging from mild, slight redness and itching, to severe injury with loss of corneal epithelium, damage to stroma, inflammatory infiltration and loss of vision. This complexity hinders the development of in vitro methods able to replace animal testing. Various in vitro techniques have been proposed and subsequently developed as potential replacements for ocular toxicity screening on animals. Over the past 2 years, eight methods have been evaluated in these laboratories. The endpoint of these methods could be linked to one or to several clinical events occurring in the in vivo eye irritancy process described above. Using these systems, a battery of four complementary in vitro assays has been developed. For the categories of ingredients and cosmetic products investigated, the promising results obtained suggest that in vitro methods of ocular risk assessment may be used increasingly in the future.

Predictive molecular and genetic toxicology

ConclusionThe search for a link between cellular and molecular events involved in delayed-type CHS reactions and the early molecular activation of xenobiotics is a new field of research. It should largely contribute to the debate on the best way forward for predictive toxicology in general.

Biosensors in pharmacology and toxicology in vitro

The need of disposing of analytical information in real time in many fields of bioanalytical and bioindustries explains the great attention devoted to biosensor development. A biosensor is usually defined as resulting from the combination of a sensitive biological element capable of molecular recognition and a transductor (electrode, optical detector…) which gives a meaningful mostly electrical signal. During the last decade, major development has concerned electrode tipped with enzymes, antibodies or other reagents that interact chemically with analytes -the substances being analysed. Such biosensors are expected to have numerous applications in the field of medicine and biology (Brennan and Krull,1992).