Takuya Ishibashi

Kinetic analysis on the in vitro cytotoxicity using Living Skin Equivalent for ranking the toxic potential of dermal irritants

The extent of cytotoxicity injured by several skin irritants was kinetically measured and analyzed in a three-dimensional cultured human skin model, Living Skin Equivalent-002 (LSE). Colorimetric thiazoyl blue (MTT) conversion assay was selected as a cytotoxicity assay, and olive oil (OO), lactic acid (LA), Triton X-100 (TX) and sodium lauryl sulfate (SLS) were evaluated as model irritants. OO had almost no effect on the viability of LSE. When the other irritants were applied on the full-thickness LSE, two first-ordered decreasing phases, initial slow and following rapid phases, were found in the viability of LSE. LA and TX showed a bigger difference between the slow and rapid rates than SLS to show an inflection. The inflection time point from the slow to rapid rate was dependent on the kind and concentration of irritants applied. The higher the concentration of irritants applied, the more rapid the inflection point was observed. When LA and SLS were applied on the stratum corneum-stripped LSE, on the other hand, viability was mono-exponentially decreased with time. LA, TX and SLS probably decrease the barrier function of the stratum corneum to increase the rate of cytotoxicity during the irritant application. Interestingly, the rate of cytotoxicity on the stripped skin was similar to the late rapid rate on the full-thickness skin in LA not in SLS. These results suggest that cytotoxicity of skin irritants on the full-thickness LSE can be represented by two first-order kinetics, and that the skin irritation rate is closely related by the barrier function of skin as well as the application concentration and intrinsic toxicity of irritants.

Utility of MTT Assay in Three-Dimensional Cultured Human Skin Model as an Alternative for Draize Skin Irritation Test: Approach Using Diffusion Law of Irritant in Skin and Toxicokinetics-Toxicodynamics Correlation

AbstractPurpose. A cytotoxicity assay using a three-dimensional cultured human skin model, Living Skin Equivalent-high (LSE-high) was evaluated as an alternative to the Draize skin irritation tests using animals. A relation between the cytotoxicity and calculated concentration of an irritant in skin was also evaluated. Methods. Colorimetric thiazoyl blue (MTT) conversion assay and a surfactant, cetylpyridinium chloride (CPC), were selected as a cytotoxicity assay and a model irritant. The fraction of dead cell number in the MTT assay or the Draize irritation score (in vitro and in vivo irritation data, respectively) was treated as a function of CPC concentration in the viable skin of LSE-high and guinea pig. Separately, in vitro permeations of CPC through the LSE-high or excised guinea pig skin were determined to calculate the average concentration of CPC in the viable skin using the Fickian diffusion theory. The obtained relations between the irritation scores and CPC concentration were evaluated by the Emax model (Hill equation). Results. CPC concentration showing 50% irritation (IC50) was similar for the MTT assay (18.9%) and Draize test (12.3%), and a good relationship (r = 0.981) was observed between the fraction of dead cell number and the Draize score. In contrast, IC50, 1.32%, for the MTT assay in LSE-high was much lower than that using guinea pig skin. We then corrected the results for the MTT assay using a ratio of IC50 in guinea pig skin against LSE-high, resulting in a good relation between both MTT results in guinea pig skin and LSE-high. Conclusion. The present results suggest that the MTT assay using LSE-high may be utilized as an alternative for the Draize test in animals for evaluating skin irritation.

Double evaluations of chemicals using a cocktail of fused recombinant receptors

Some chemicals have multipotential as endocrine-disrupting chemicals (EDCs). For example, some chemicals act as both estrogens and antiandrogens. Numbers of such chemicals should be evaluated from many aspects; however, labor and expenses are generally limited. We have developed two expression systems for the wild type of human estrogen receptor alpha and the wild type of human androgen receptor fused with a maltose binding protein. They are soluble and have binding activities. They showed dose-responses to natural hormones and well-known potential EDCs. After we established each assay condition for a competitive binding assay using each receptor, we found that two assay systems can be carried out simultaneously under limited and harmonized conditions. Under harmonized conditions using a cocktail of two types of receptors, we could estimate natural hormones and potential EDCs. Interference between two assay systems was not observed under these conditions. We believe that some competitive binding assays can be carried out using a cocktail of receptors at the same time if interference among different assay systems can be avoided by choosing ideal conditions.