No abstract

No abstract

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.

A hitherto unknown glutathione-S-transferase in human erythrocytes displays polymorphism: three quarters of the population ("conjugators") possess, whereas one quarter ("non-conjugators") lack this specific activity. A standard method for the identification of conjugators and non-conjugators with the use of methyl bromide and gas chromatography (head space technique) is described. Three substrates of the polymorphic enzyme, methyl bromide, ethylene oxide and dichloromethane (methylene chloride), were incubated in vitro with individual whole blood samples of conjugators and non-conjugators. All three substances led to a marked increase of sister chromatid exchanges (SCE) in the lymphocytes of the non-conjugators but not in those of conjugators. A protective effect of the glutathione-S-transferase activity in human erythrocytes for the cytogenetic toxicity of these chemicals in vitro is thus confirmed. Since the enzyme activity is not found in erythrocytes of laboratory animals, species extrapolations for risk assessment of methyl bromide, ethylene oxide and dichloromethane should be reconsidered.

Background and PurposeInsulin stimulates the transport of glucose in target tissues by triggering the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Resistance to insulin, the major abnormality in type 2 diabetes, results in a decreased GLUT4 translocation efficiency. Thus, special attention is being paid to search for compounds that are able to enhance this translocation process in the absence of insulin.Experimental ApproachTotal internal reflection fluorescence (TIRF) microscopy was applied to quantify GLUT4 translocation in highly insulin-sensitive CHO-K1 cells expressing a GLUT4-myc-GFP fusion protein.Key ResultsUsing our approach, we demonstrated GLUT4 translocation modulatory properties of selected substances and identified novel potential insulin mimetics. An increase in the TIRF signal was found to correlate with an elevated glucose uptake. Variations in the expression level of the human insulin receptor (hInsR) showed that the insulin mimetics identified stimulate GLUT4 translocation by a mechanism that is independent of the presence of the hInsR.Conclusions and ImplicationsTaken together, the results indicate that TIRF microscopy is an excellent tool for the quantification of GLUT4 translocation and for identifying insulin mimetic drugs.