Shinobu Kato

Effect of the photocatalytic activity of TiO2 on plasmid DNA

We investigated the photodynamic DNA strand-breaking activity of TiO(2). A solution of super-coiled pBR 322 DNA was irradiated with 5 J/cm(2) of UVA in the presence of TiO(2) and the products were analyzed by using gel electrophoresis. The ratio of open-circular DNA to super-coiled circular DNA was calculated from the resulting peak areas as a DNA strand-breaking index (SBI). The SBI of anatase-structure TiO(2) (band gap=3.23 eV) was greater than that of rutile structure (band gap=3.06 eV), and the level of SBI correlated with the photocatalytic activity for degradation of 2-propanol. The inhibitory effects of active oxygen scavengers, including DMSO, glutathione and histidine, on the DNA strand-breaking activity were examined. All of the scavengers except ascorbic acid showed inhibitory effects, as did several polyhydric alcohols including mannitol, a well-known hydroxyl radical scavenger. These results suggest that the photodynamic DNA strand-breaking activity of TiO(2) is due to active oxygen species, especially hydroxyl radicals. Polyhydric alcohols showed an inverse correlation between the inhibitory effect on DNA strand-breaking activity and the octanol/water partition coefficient (logP).

An in vitro alternative to the Draize eye-irritation test: Evaluation of the crystal violet staining method

To evaluate a simple in vitro method as an alternative to the Draize eye-irritation test, 12 surfactants were tested by both of the above methods, and the correlation between the results was examined. The in vitro method used was the crystal violet staining method. HeLa cells or SIRC cells from rabbit cornea were inoculated into each well of 96-well microplates which contained serially diluted test chemicals. They were cultured for 72 hr, and then fixed and stained with crystal violet. After spectrophotometric measurement, the concentrations of test materials that inhibited the absorbance to 50% of the control level (IC(50)) were determined. The in vivo data used for comparison were the maximum corneal score and the maximum total score obtained by the eye test when the substances were tested at a concentration of 10%. The correlation coefficients between the maximum corneal scores of the 12 surfactants and their IC(50)s in HeLa cells and in SIRC cells were -0.821 and -0.816, respectively, while those between the maximum total scores and IC(50)s in both cell lines were -0.855 and -0.863, respectively. All of these values are statistically highly significant, indicating that the crystal violet staining method may have value in predicting the eye irritancy of surfactants. Further interlaboratory validation will, however, be needed.

A modification of the local lymph node assay for contact allergenicity screening : measurement of interleukin-2 as an alternative to radioisotope-dependent proliferation assay

The local lymph node assay is an effective prediction method for contact allergenicity, but employs radioisotopes. We investigated whether measurement of interleukin-2 (IL-2) production by lymph node cells could be used instead to predict contact allergenicity of chemicals. Test chemicals were applied for three consecutive days to both ears of Balb/c mice and the auricular lymph nodes were obtained on either the fourth or fifth day after the first application. Both IL-2 concentration in supernatant of the suspension and proliferative activity of lymph node cells were determined after 24-, 48-, 72-h cell culture in RPMI-1640 medium by ELISA and by measuring [3H]methylthymidine incorporation, respectively. These two methods detected allergenicity similarly except in the case of TNCB and oxazolone, which showed excessive proliferation-inducing capacity as compared to IL-2 release-increasing effect. Flow cytometry showed that these two chemicals also increased the percentage of Iad-positive cells in the lymph nodes, suggesting that these chemicals might induce not only cellular immunity but also humoral immunity. We conclude that interleukin-2 assay is a convenient and dependable method for screening strong contact allergens without using radioisotopes.

Quantitative evaluation to predict the eye irritancy of chemicals: Modification of chorioallantoic membrane test by using trypan blue.

The chorioallantoic membrane (CAM) test using fertile hens eggs has been reported as a promising alternative method to predict the eye irritancy of chemicals. However, the scoring system of the original CAM test is not satisfactory because of its lack of objectivity and quantitativeness. Furthermore, the test requires skill when the effects of test substances are to be graded. To overcome these disadvantages, a more objective means of judging the injurious effects of chemicals was examined as follows: after treatment with the test chemical, the CAM was stained with trypan blue and the amount of pigment adsorbed was measured spectrophotometrically. The amounts of trypan blue adsorbed with the CAM showed a good correlation with the in vivo eye irritation test scores (r = 0.89). The findings suggest that the trypan blue staining method is very simple and reproducible, and thus would be a promising alternative method for predicting the eye irritancy of chemicals. However, further studies will be required to validate this test method with a wide variety of chemicals and formulations.

Liposomes as an in vitro model for predicting the eye irritancy of chemicals

Liposomes containing 4-methylumbelliferyl phosphate (Um-P) were prepared using the lipid extracts from bovine eyes and were incubated with seven surface-active agents. The Um-P released from the liposomes by each test agent was hydrolysed with alkaline phosphatase and the resultant 4-methylumbelliferone was assayed spectrofluorometrically. The values for Um-P(50) (the concentration of test material at which 50% of Um-P is released) showed a good inverse correlation with the irritation scores obtained by the Draize eye test. The results suggest that the liposomal assay reported here may be useful as an in vitro model for predicting the eye irritancy of chemicals.

Mechanism for 6-methylcoumarin photoallergenicity☆

6-Methylcoumarin (6-MC), a synthetic fragrance material, has been reported to be photoallergenic both in man and in guinea pigs. To elucidate the possible mechanism of photoallergenicity, 6-MC in ethanolic solution was exposed to 200 joules/cm2 of long-wavelength ultraviolet (UV) ranging from 320 to 400 nm for 16 hr, and was examined for its contact allergenicity by means of a modified guinea pig maximization test. Strong allergic responses were observed, indicating that UV plays a catalytic role in forming contact allergens during irradiation. The solution was fractionated to isolate and purify contact sensitizers by gel-permeation chromatography and high-performance liquid chromatography. By employing mass spectrometry and proton and carbon-13 nuclear magnetic resonance, mono- and diethyl esters of 6-MC dimer were compounds identified which showed allergic reactions in guinea pigs sensitized with UV-irradiated 6-MC.

Multivariate factorial analysis of data obtained in seven in vitro test systems for predicting eye irritancy

Seven in vitro test systems used to predict eye irritancy (EYTEX, SIRC cytotoxicity, HeLa cytotoxicity, chorioallantoic membrane (CAM), liposome, red blood cell and haemoglobin denaturation test system) were applied to 12 surfactants, and the results were subjected to multivariate analysis to evaluate the relative contributions of five factors. These factors were: (1) cellular plasma membrane destruction factor, (2) haemoglobin type protein denaturation factor, (3) EYTEX-type protein denaturation factor, (4) cytotoxicity factor and (5) an unknown (unidentified) factor. The results clarified the basis on which the findings of each test system were related to the Draize results. According to the analysis, the Draize eye irritation test could be explained by the contribution of the protein denaturation factor and cellular plasma membrane destruction factor. This provides support for a previous hypothesis that the major mechanisms of eye irritation are cellular plasma membrane destruction and protein denaturation. The CAM test value showed a higher correlation coefficient (0.906) with the Draize score than did the results of any of the other test systems; this was due to the fact that these two tests showed similar patterns of dependence on the five factors as indicated by the factorial analysis. The haemoglobin denaturation test system had the next highest correlation coefficient at about 0.75. Furthermore, by using further tests to make up the deficiency of the other necessary factors, a desirable battery system could be predicted; for example, the combination of the haemoglobin-denaturation and rat red blood cell tests would provide a result similar to that of the Draize test. This study should contribute to the development of a rational basis for prediction of eye irritancy of chemicals using in vitro test systems.

Role of a metabolite in allergenicity of Sudan I

A metabolite of Sudan I (l‐phenylazo‐2—naphthol) was isolated alter in vitro incubation with the supernatant of guinea pig liver homogenate (S·9). The metabolite was found to be 4′‐hydroxy‐l‐phenylazo‐2—naplithol by gas chromatography‐mass spectrormetry (GC‐MS), and proton and carbon‐13 nuclear magnetic resonance (NMR) analyses. It elicited positive reactions in guinea pigs sensitized ot Sudan I. It was also shown to be allergenic. The results suggest that para‐hydroxylation of the phenyl group of Sudan I may play an important rôle in its allergenicity.

Evaluation of stinging-inducing chemicals using cultured neuronal cells: an electrophysiological approach.

The effects of various cosmetic ingredients, including preservatives, humectants, ultraviolet absorbents and solvents, on the electrical properties of neuronal cells were investigated using rat phaeochromocytoma PC12 cells and cultured rat dorsal root ganglion (DRG) neurons. When membrane current was measured under whole-cell voltage-clamp, all nine test compounds inhibited voltage-activated K(+) current in PC12 cells. With the five compounds selected for further experiments with DRG neurons, two types of current responses were observed, namely inhibition of K(+) current by methyl or butyl p-hydroxybenzoate (MPHB or BPHB) and resorcinol, and induction of a cationic inward current by MPHB, BPHB, ethanol and dipropylene glycol. The order of potency of these chemicals to inhibit the K(+) current in PC12 cells was similar to that of their cytotoxicity, which was determined by MTT assay. Capsaicin, BPHB, MPHB and resorcinol, however, inhibited the K(+) current at concentrations lower than those required for cytotoxicity. These results suggest that these cosmetic ingredients exert significant effects on the electrical properties of cultured neuronal cells. This electrophysiological method may be useful in prescreening test systems to detect stinging, and also to clarify mechanisms underlying the irritation induced by a variety of compounds.