Azelaic acid has been shown clinically to have a cytotoxic effect on the abnormally active and malignant human melanocyte, but it has no apparent effect upon normal melanocytes. This difference in reactivity between normal and abnormal cells in vivo is further examined here in vitro. The disodium salt of azelaic acid (C(9)2Na) was added to pure and mixed cultures of normal human melanocytes and to cultured human melanoma cells, at 10(-3) M, 10(-2) M, 5 X 10(-2) M, and 10(-1) M for 1 and 6 h. Control cultures and cultures exposed to the same concentrations of the disodium salt of adipic acid (C(6)2Na) were also examined. No damage to cells of any line was observed with diacids at 10(-3) M or 10(-2) M up to 6 h. At 5 X 10(-2) M some mitochondria of melanoma cells appeared swollen. With C(6)2Na at 10(-1) M for I and 6 h, minimal swelling of mitochondria was observed in some cells of all lines. Pure normal melanocytes and melanocytes of mixed cultures exhibited greater swelling of mitochondria with 10(-1) M C(9)2Na at 1 and 6 h, but the mitochondria of the malignant melanocytes were massively swollen with destruction of cristae. Plasma and nuclear membranes and membranes of rough endoplasmic reticulum were intact, but Golgi membranes exhibited vesiculation. These results provide further evidence that azelaic acid damages the human malignant melanocyte and that one of its targets is the mitochondrion. Damage to normal melanocytes, found here, may be due to the fact that, in culture, they are more active than in intact epidermis.

Clinically, dicarboxylic acids have a cytotoxic effect on the abnormally hyperactive and malignant epidermal melanocyte, and diacids from C8 to C13 have been shown to inhibit mitochondrial oxidoreductases. Here, their effect on the growth kinetics and ultrastructure of murine melanoma cells in culture is examined. Cultures of Harding-Passey and Cloudman S91 melanoma cells were exposed to single doses of the disodium salts of C12, C9, and C6 (which does not significantly inhibit mitochondrial enzymes) dicarboxylic acids at concentrations of 10(-3) M to 10(-1) M. With C12 and C9, viability and cell proliferation over 3 days were significantly affected by concentrations greater than 10(-2) M. With exposure to C6 at 10(-1) M and to medium to which NaCl was added to produce equal osmolarity, the effect was much less. Electron microscopy of cells exposed to C9 at 10(-1) M for 1 h and 6 h revealed massive swelling of mitochondria with destruction of cristae, but plasma and nuclear membranes and membranes of endoplasmic reticulum were intact. Similar damage was not seen with C6 at 10(-1) M nor with equiosomolar NaCl. The results confirm (1) the cytotoxicity of dicarboxylic acids for malignant melanocytes, and (2) that the mitochondrion is a prime target for their action.

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Cultures of melanocytes and keratocytes were exposed to 15 micrograms ml-1 tyrosinase and 4-hydroxyanisole (4-OHA) 5 X 10(-4) M to 5 X 10(-2) M for 1 to 24 h. No damage was suffered by either cell below 5 X 10(-3) M 4-OHA for 6 h, but higher concentrations and longer exposures extensively damaged both cells. Exposure of cells washed free of culture medium to tyrosinase and 4-OHA 1 X 10(-3) M for 1 h resulted also in extensive damage. This indicates that an early-formed toxic product of the reaction between tyrosinase and 4-OHA is inactivated by constituents of the medium. This was confirmed by Liquid Chromatography and Scanning Spectrophotometry which showed that a toxic 4-OHA quinone immediately reacted with nucleophilic substances in the medium resulting in products which, on accumulation, are probably responsible for the later (6 h plus) damage to melanocytes and keratocytes. A possible effect of allegedly specific melanocytotoxic drugs on keratocytes should always be borne in mind with tissue culture experiments. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9

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