Increase of ß2-integrin on adhesion of THP-1 cells to collagen vitrigel membrane.

When human monocyte-derived leukemia (THP-1) cells, which are floating cells, are stimulated with lipid peroxides, or Streptococcus suis, these cells adhere to a plastic plate or endothelial cells. However, it is unclear whether or not non-stimulated THP-1 cells adhere to collagen vitrigel membrane (CVM). In this study, firstly, we investigated the rate of adhesion of THP-1 cells to CVM. When THP-1 cells were not stimulated, the rate of adhesion to CVM was high. Then, to identify adhesion molecules involved in adhesion of THP-1 cells to CVM, expressions of various cell adhesion molecules on the surface of THP-1 cells adhering to CVM were measured. ß-actin, ß-catenin, and ß1-integrin expressions did not change in non-stimulated THP-1 cells cultured on CVM compared with those in cells cultured in a flask, but ß2-integrin expression markedly increased.

Lack of promoting effect of titanium dioxide particles on ultraviolet B-initiated skin carcinogenesis in rats.

Titanium dioxide (TiO(2)) is used in sunscreens and cosmetics as an ultraviolet light screen. TiO(2) has carcinogenic activity in the rat lung, but its effect on the skin has not been reported. We examined the promoting/carcinogenic effect of nano-size TiO(2) particles using a two-stage skin model. c-Ha-ras proto-oncogene transgenic (Hras128) rats, which are sensitive to skin carcinogenesis, and their wild-type siblings were exposed to ultraviolet B radiation on shaved back skin twice weekly for 10 weeks; then the shaved area was painted with a 100 mg/ml TiO(2) suspension twice weekly until sacrifice. All rats were killed at week 52 except for female Hras128 rats which were sacrificed at week 16 because of early mammary tumor development. Skin tumors developed in male Hras128 rats and mammary tumors developed in both sexes of Hras128 rats and in wild-type female rats, but tumor incidence was not different from controls. TiO(2) particles were detected in the upper stratum corneum but not in the underlying skin tissue layers. TiO(2) particles also did not penetrate a human epidermis model in vitro. Our data suggest that TiO(2) does not cause skin carcinogenesis, probably due to its inability to penetrate through the epidermis and reach underlying skin structures.

Effects of coating materials and size of titanium dioxide particles on their cytotoxicity and penetration into the cellular membrane.

In order to estimate the effects of the size and surface treatment (coating or non-coating) of titanium dioxide particles on their cytotoxicity and penetration into the cellular membrane, two types of non-treated titanium dioxide (TiO(2)) particles of 20 nm (LU175) and 250 nm (LU205) were exposed to CHO cells, RBL-2H3 cells, A431 cells, B16 melanoma, NHEK(F), and NHSF, and six types of surface-treated or non-treated TiO(2) particles of 35 nm were exposed to RBL-2H3 cells and NHSF. The order of half-maximal inhibitory concentrations (IC50s) of LU175 was NHSF < CHO, RBL-2H3 < A431 < B16 melanoma, NHEK(F). On the other hand, LU205 showed no cytotoxicity against any cells. Surface-treated TiO(2) showed much less cytotoxicity against RBL-2H3 cells than non-treated TiO(2). Then, between 0.5 and 10 mg of LU175 or LU205 was exposed to CHO cells. After 24 hr, the amount of LU175 in cellular cytosol increased dose-dependently. On the other hand, the amount of LU205 in cellular cytosol was much less than that of LU175. The proportion of surface-treated TiO(2) in the cellular cytosol of RBL-2H3 cells differed for each coating material. These results suggested that TiO(2) has different cytotoxicities among cell lines, and that of surface-treated TiO(2) was weaker than that of non-treated TiO(2). TiO(2) located in cytosol might be the main cause of cytotoxicity.

[Analysis of volcanic-ash-based insoluble ingredients of facial cleansers].

The substance termed "Shirasu balloons", produced by the heat treatment of volcanic silicates, is in the form of hollow glass microspheres. Recently, this substance has gained popularity as an ingredient of facial cleansers currently available in the market, because it lends a refreshing and smooth feeling after use. However, reports of eye injury after use of a facial cleanser containing a substance made from volcanic ashes are on the rise. We presumed that the shape and size of these volcanic-ash-based ingredients would be the cause of such injuries. Therefore, in this study, we first developed a method for extracting water-insoluble ingredients such as "Shirasu balloons" from the facial cleansers, and then, we examined their shapes and sizes. The insoluble ingredients extracted from the cleansers were mainly those derived from volcanic silicates. A part of the ingredients remained in the form of glass microspheres, but for the most part, the ingredients were present in various forms, such as fragments of broken glass. Some of the fragments were larger than 75 microm in length. Foreign objects having a certain hardness, shape, and size (e.g., size greater than 75 microm) can possibly cause eye injury. We further examined insoluble ingredients of facial scrubs, such as artificial mineral complexes, mud, charcoal, and polymers, except for volcanic-silicate-based ingredients. The amounts of insoluble ingredients extracted from these scrubs were small and did not have a sharp edge. Some scrubs had ingredients with particles larger than 75 microm in size, but their specific gravities were small and their hardness values were much lower than those of glass microspheres of ingredients such as "Shirasu balloons". Because the fragments of glass microspheres can possibly cause eye injury, the facial cleansers containing large insoluble ingredients derived from volcanic ashes should be avoided to use around eyes.

[Development of novel cell culture systems utilizing the advantages of collagen vitrigel membrane].

The white of an egg, rendered opaque by boiling, can be converted into a thin, transparent and rigid material like glass by evaporating the moisture. This phenomenon is known as the vitrification of heat-denatured proteins. We applied vitrification technology to a collagen gel and converted it into a rigid glass-like material. We attempted to rehydrate the glass-like material and succeeded in preparing a novel stable state of collagen gel that was a thin and transparent membrane with excellent gel strength and protein permeability. We called it "collagen vitrigel" because it was produced from the vitrification process of a traditional hydrogel. Further, a framework-embedded collagen vitrigel membrane that can be easily turned inside out with tweezers was prepared by inserting a nylon membrane ring in the collagen sol prior to the gelation, thereby allowing the membrane to function as a removable cell culture substratum. Different types of anchorage-dependent cells could be cultured on both surfaces of the substratum by the manipulation of two-dimensional cultures, and consequently a three-dimensional crosstalk model with paracrine effects from each cell type was reconstructed. Also, the collagen vitrigel membrane containing a bioactive molecule provided a drug delivery system (DDS) with sustainable release. In this review, we summarize the recent progress of applied studies using the collagen vitrigel membrane as follows: a corneal model for eye irritant and permeability tests, a skin model for sensitization test, a renal glomerular model for evaluating blood filtration, an endometrial model for developing a new treatment and a DDS of hepatocyte growth factor for improving liver disorder.

Involvement of macrophage inflammatory protein 1alpha (MIP1alpha) in promotion of rat lung and mammary carcinogenic activity of nanoscale titanium dioxide particles administered by intra-pulmonary spraying.

Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.

[Analysis of preservatives used in cosmetic products: salicylic acid, sodium benzoate, sodium dehydroacetate, potassium sorbate, phenoxyethanol, and parabens].

Preservatives are used to inhibit the growth of microorganisms in cosmetic products. The Japanese standards for cosmetics set restrictions on the maximum amount of each preservative added to cosmetics as per the purpose of use of cosmetics. For the investigation into the actual conditions of commonly used preservatives in commercial cosmetics, we analyzed parabens, phenoxyethanol, sodium benzoate, sodium dehydroacetate, salicylic acid, and potassium sorbate by high-performance liquid chromatography (HPLC). Twenty-one samples were obtained from cosmetic product manufacturers located in 14 prefectures in Japan. Among different acid- and salt-based preservatives, sodium benzoate was observed to have been used in many products. These acid- and salt-based preservatives were used with parabens in personal washing products, such as shampoo and soap. The labels of two of the cosmetic product samples displayed inaccurate ingredient information, that is, a preservative other than the one used in the corresponding product was listed on them. The amount of preservatives used did not exceed regulatory limits in any of the analyzed samples.

[Detection of clobetasol propionate in a cream advertised to be effective against atopic dermatitis].

Addition of medical ingredients to cosmetics is prohibited. However, last year some cases of illegal cosmetics containing steroids were successfully identified. We have already reported an analytical method to detect steroids in cosmetics [Bull. Natl. Inst. Health Sci, 126, 51-56 (2008)]. In this study, we initially examined whether this method could be applied for the detection of some new steroids as target chemicals. We then used this developed method to detect steroids in cosmetics obtained from manufacturers by spot checks. These manufacturers have been advertising the effectiveness of a steroid-free cream against atopic dermatitis. The results revealed that clobetasol propionate (CP) was present in this facial moisturizing cream, which was available in the market. The steroid was extracted with methanol. After ultrasonication and centrifugation, the resulting supernatant was injected into the high-performance liquid chromatography system equipped with an ODS column. The separation was achieved using a mixture of acetonitrile and water as the mobile phase. The retention times of the observed peaks were in accordance with those of some preservatives and CP. The presence of CP was also confirmed by thin-layer chromatography. The concentration of CP in the cream was approximately 0.039%. CP is a steroid that has the strongest effect as compared to those of other steroids. The cream was therefore recalled for safety reasons.

[Studies on an analytic method for detection of prohibited ingredient, strontium dichloride in cosmetics].

Strontium dichloride is one of the prohibited ingredients in cosmetics due to the Japanese Pharmaceutical Affairs Act. We established the analytical method for strontium dichloride in cosmetics by capillary electrophoresis (CE). The toothpaste was dispersed into water. After ultrasonication for 10 min, the solution was centrifuged at 3000 rpm for 10 min. The supernatant was filtrated through a membrane (0.45 microm), diluted 100-times with water, and injected into CE. The calibration curve showed linear between the concentrations of strontium dichloride (from 2 to 50 microg/ml) and the peak area of strontium ion. Detection limit of strontium dichloride is 2 microg/ml. There was no interference of the ingredients in the toothpaste.

[Construction of three-dimensional human skin model involving dendritic cells and its application to skin sensitization test].

Recently, to study an in vitro evaluation method of skin irritation and acute toxicity, many three-dimensional human skin models consisting of normal human keratinocytes and fibroblasts have been used. However, these skin models did not have any dendritic cells so were difficult to apply to an in vitro skin sensitization test. On the other hand, a single cell-culture model using normal human dendritic cells was recently studied for an in vitro evaluation method of immune-sensitizing compounds. However, these models have various problems: 1) the life span of dendritic cells is short(within 1 week) and 2) it is difficult to apply water-insoluble samples to these models. To study an alternative to animal testing using immune-sensitizing compounds, we therefore constructed a three-dimensional human skin model consisting of three different cells, dendritic cells (keratinocytes, and fibroblasts) then exposed immune-sensitizing compounds and non-sensitizers to the new skin model for 1 h and investigated the effect of these compounds on cytokine release and expression of CD86. Due to immune-sensitizing compounds, the new skin model significantly released cytokine and significantly expressed CD86. On the other hand, non-sensitizers did not induce IL-1alpha, IL-2, and IL- 4 release and expression of CD86. These results suggest that the new skin model is suitable for study as an alternative to animal testing using immune-sensitizing compounds.