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Alternatives to Laboratory Animals - ATLA

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In Vitro Alternatives and Phototoxicity Testing. II. Effects of Reactive Oxygen Species in In Vitro Phototoxicity Assays

Yuuko Okamoto, Akemi Ryu and Kenji Ohkoshi

The effects of reactive oxygen species (including singlet oxygen) in two in vitro phototoxicity assays — the 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) assay and the photohaemolysis assay — were assessed by using scavengers. Fifteen test substances, which had previously been shown to be phototoxic in vitro, were assessed. Eleven of these produced singlet oxygen. The major factor in the photodynamic reaction of bithionol was thought to be a Type I reaction, because bithionol did not produce singlet oxygen and did not react to histidine. Acridine was regarded as a Type II substance, because of the evident effect of histidine as a scavenger. 8-Methoxypsoralen and 5-methoxypsoralen produced singlet oxygen, but their actions were not affected by the scavengers. In this study, we confirmed that reactive oxygen species have great effects in in vitro phototoxicity, and that the 3T3 NRU PT assay can be used to detect effects which are thought to be the direct reaction of an excited photosensitiser to biological substrates (Type III reaction), for example, 8-methoxypsoralen. Therefore, we suggest that photohaemolysis and phototoxicity could be used to evaluate the photodynamic mechanisms of photosensitising chemicals.