In order to define safety profiles and proper handling procedures for new industrial products, it is essential to determine their potential for ocular irritation. The Draize test is normally employed but it involves using rabbits. There is today a great need for all researchers to limit the use of animals for laboratory experiments and to encourage the development and adoption of alternative in vitro methods to evaluate the potential toxicity of new products. This study proposes a three-dimensional model of bovine corneal stroma and epithelium that is not only easy to reproduce but may also be used in the toxicological field as an alternative to animal experimentation. The data presented here show that this model allows the growth of epithelium similar in features to in vivo epithelium. Basal cells are cube-shaped, whereas superficial areas are horizontally longer; desmosomes and 64 kDa keratin, as a marker for differentiation of corneal epithelial cells, are both expressed; the basal lamina is synthesised also. The 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay was carried out on the model to evaluate the toxicity of some surfactants: benzalkonium chloride, Triton X-100, sodium dodecylsulphate and Tween 20. Since the in vitro data fit very well the results of the Draize test in vivo as reported in the literature, the three-dimensional culture may be used to predict the potential cytotoxicity of surfactants.

The effect of 4-hydroxymethyl-6,7,8,9-tetrahydro-2H-benzofuro-[3,2-g]-1-benzo piran-2-one (compound 1) and 4-hydroxymethyl-2H-benzofuro-[3,2g]-1-benzopiran-2-one (compound 2), two new benzopsoralen derivatives, was tested on HL60 and HeLa cell lines in the dark and by UVA irradiation; 8-methoxypsoralen was used as a reference compound. The action of the compounds was evaluated by means of the neutral red uptake assay, by means of ultrastructure, morphometry and interaction with human erythrocytes membrane. In both HL60 and HeLa cell lines benzopsoralen derivatives showed more antiproliferative activity after UVA irradiation, however less than 8-methoxypsoralen. Compound 1 was more effective than compound 2 both in the dark and after UVA irradiation. The ultrastructure showed a morphological rank damage caused by these compounds: compound 2 induced slight modifications in the cytoplasm organization, compound 1 induced some vacuolizations and 8-methoxypsoralen generated plenty of vacuoles and an empty space around the nucleus. Morphometrical data in HL60 cells turned out to be in accordance with the different action mechanisms existing between 8-methoxypsoralen and the two benzopsoralen derivatives; in HeLa cells we noted an increase in the nuclear area induced by all the three compounds. Only compound 1 caused the formation of echinocytes both in the dark and after UVA irradiation, suggesting the involvement of a mechanism not strictly related to DNA interaction and singlet oxygen production.

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