Fig. 4

The melanocytotoxicity of MoTP is mediated by tyrosinase activity. (A) The chemical structure of (2-morpholinobutyl)-4-thiophenol (MoTP). (B) Dendritic (healthy) melanocytes (black arrowheads) in an untreated larva at 3 dpf. (C) When larvae were incubated in MoTP solution from 14 to 72 hpf, no neural crest-derived melanocytes were observed, but RPE is lightly pigmented (black arrow). (D,E) When 48 hpf larvae with pigmented melanocytes were shifted to MoTP, within 24 hours, larval melanocytes had become punctate (white arrowheads in D) and began to extrude from the epidermis (white arrow in E), a hallmark of melanocyte cell death. (F) The melanocytotoxicity of MoTP was blocked by PTU, as indicated by the dendritic appearance of lightly pigmented melanocytes (black arrowheads in F) following co-incubation of MoTP and PTU from 48 to 72 hpf. (G) The chemical structure of 4-hydroxyanisole (4-HA). (H) When larvae were incubated with 4-HA from 14 to 72 hpf, the same punctate melanocyte pattern of cell death appeared (white arrowheads). (I) Illustration of the mechanism of 4-HA melanocytotoxicity (see Riley, 1985). Note that tyrosinase converts the prodrug 4-HA to a cytotoxic o-quinone. Because copper is an essential cofactor for tyrosinase, its activity is blocked by co-incubation with PTU, a copper chelator. Timelines (gray) below the panels indicate the period of drug treatments (red) and analysis time (vertical line above timelines). Scale bars: in H, 500 μm for B-D,F,H; in E, 50 μm.