Peptide binding at the GLP-1 receptor.

The receptor for GLP-1 [glucagon-like peptide-1-(7-36)-amide] is a member of the 'Family B' of GPCRs (G-protein-coupled receptors) comprising an extracellular N-terminal domain containing six conserved cysteine residues (the N-domain) and a core domain (or J-domain) comprising the seven transmembrane helices and interconnecting loop regions. According to the two-domain model for peptide binding, the N-domain is primarily responsible for providing most of the peptide binding energy, whereas the core domain is responsible for binding the N-terminal region of the peptide agonists and transmitting the signal to the intracellular G-protein. Two interesting differences between the binding properties of two GLP-1 receptor agonists, GLP-1 and EX-4 (exendin-4), can be observed. First, while GLP-1 requires its full length to maintain high affinity, the eight N-terminal residues of EX-4 can be removed with little reduction in affinity. Secondly, EX-4 (but not GLP-1) can bind to the fully isolated N-domain of the receptor with an affinity matching that of the full-length receptor. In order to better understand these differences, we have studied the interaction between combinations of full-length or truncated ligands with full-length or truncated receptors.

The Use of Human Keratinocytes in the EU/COLIPA International In Vitro Phototoxicity Test Validation Study and the ECVAM/COLIPA Study on UV Filter Chemicals.

The EU/COLIPA in vitro phototoxicity study involved the testing of 30 chemicals in Phase II, and the ECVAM/COLIPA study on UV filter chemicals involved the testing of 20 chemicals, for which in vivo human and/or animal data were available. Primary human keratinocytes, from four separate male donors, were not found to be sensitive to the 5J/cm2 UVA produced by the SOL500 lamp when assayed by using the neutral red uptake endpoint, as employed with the 3T3 cells used in these international interlaboratory validation studies. The primary human keratinocytes tested in one laboratory alongside the 3T3 fibroblasts gave consistent indications of phototoxicity with all the phototoxicants tested in the Phase II and UV filter studies. The one exception was bithionol, which was predicted to be non-phototoxic in both studies. None of the non-phototoxic chemicals resulted in a positive reaction with the Photoirritation Factor (PIF) version of the prediction model. However, when the Mean Photo Effect (MPE) prediction model version was applied (with a cut-off point of 0.1), one sunscreen agent, octyl salicylate, was deemed to have phototoxic potential. The entire set of negative rated chemicals included in Phase II and in the UV filter study were also rated as non-phototoxic by the MPE prediction model.

Nitrite synthesis in a murine macrophage cell line stimulated by products released from irritant-treated normal human keratinocytes.

Nitric oxide is produced in dermal inflammatory reactions; its role as an irritant mediator was therefore examined. Nitrite accumulation (an indicator of nitric oxide synthesis) was measured in human keratinocytes, NCTC 2544 cells and RAW 264 macrophages, following stimulation by lipopolysaccharide (LPS), sodium dodecyl sulfate (SDS) or ultraviolet irradiation (UVA). Nitrite synthesis was low in primary cultured keratinocytes (passage 3) at 0.36 +/- 0.16 nmol/well. This was minimally affected by LPS (0.58 +/- 0.1 nmol/well). SDS and UVA failed to modify keratinocyte nitrite production. No nitrite synthesis was detected in NCTC 2544 cells. RAW 264 macrophages responded to LPS (0-10 mug/ml) by increasing nitrite production from 2 +/- 0.6 to 22 +/- 4.5 nmol/well in keratinocyte serum-free media. Responses of macrophages to LPS were diminished in Dulbecco's media containing serum or dialysed serum. Human interleukin-1 (0-30 pg/ml) increased macrophage nitrite production and l-N-monomethyl arginine (l-NMMA) (0-1 mm) inhibited nitrite synthesis. Media conditioned by keratinocytes for 24 hr following treatment with SDS (0-100 mug/ml for 0-8 hr) or UVA (1.6 mW/cm(2) for 0-4 hr) enhanced macrophage nitrite synthesis. Conditioned media from UVA-treated NCTC 2544 cells failed to stimulate macrophage nitrite synthesis.

The complement activation assay: The test for immediate phototoxicity.

A new in vitro test for phototoxicity has been developed. Nine substances (eight photosensitizers and one non-photosensitizer) were screened for their ability to activate human complement in the presence of UV light, and all photosensitizers were found to be active. Complement activation was quantified by means of ELISA. In all cases activation was mediated by alternative, but not by classical, pathways. The results of a cell culture test performed with 5-methoxypsoralen suggest the existence of two different mechanisms of phototoxicity. This indicates that the battery of in vitro assays for phototoxicity prediction must contain at least two different tests.

In vitro alternatives for the detection of photoirritant chemicals: The EEC/COLIPA trial.

The effects of UV irradiation on the cytotoxicities of selected chemicals were investigated in cultures of Balb/c 3T3 cells, normal human keratinocytes and Jurkat cells. The chemicals were chosen according to their reported photoirritancy, or lack thereof, and the abilities of the in vitro tests to distinguish between these photoirritants and non-photoirritants were assessed. The data obtained suggest that the neutral red uptake assay with Balb/c 3T3 cells can be used to provide some indication of the potential photoirritancy of these particular chemicals. However, similar results were also obtained using the human keratinocytes and these should provide a more relevant in vitro model for investigating mechanisms of chemical-induced photoirritation.

EEC/COLIPA project on in vitro phototoxicity testing: First results obtained with a Balb/c 3T3 cell phototoxicity assay.

In a joint validation project eight laboratories from the European Cosmetic Industry Association (COLIPA) as well as FRAME (England) and ZEBET (Germany) are trying to develop validated in vitro methods to be incorporated into new international guidelines for acute phototoxicity testing. The first stage of the study involved selection of the most promising in vitro phototoxicity tests for further validation. 20 chemicals with known phototoxic properties (12 phototoxins, four UV-absorbing non-phototoxins and four non-UV absorbing non-phototoxins) were tested under identical conditions of UV exposure conditions (sun simulator, UVA 5 J/cm(2)) in a standardized cytotoxicity assay with Balb/c 3T3 fibroblasts (endpoint: neutral red uptake, NRU). 19 of the 20 chemicals were correctly classified by the 3T3 NRU phototoxicity test, and therefore, this simple assay for phototoxicity seems very promising and should be validated further.