Melanin potentiates kanamycin-induced inhibition of collagen biosynthesis in human skin fibroblasts

Ototoxicity is one of the well known side effects of kanamycin. The mechanism underlying the organ specificity of the side effect is not understood. Since many pharmacologic agents are known to form complexes with melanin and melanin is an abundant constituent of the inner ear, we investigated whether kanamycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. We found that kanamycin forms complexes with melanin. The amount of kanamycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the kanamycin-melanin complex formation: strong binding sites with the association constant K-1 similar to 3 x 10(5) M-1, and the weak binding sites with K-2 similar to 4 x 10(3) M-1. The number of total binding sites (n(1) + n(2)) was calculated as about 0.64 mu mol kanamycin per 1 mg melanin. We found that kanamycin induced inhibition of collagen and DNA biosynthesis (IC50 similar to 5 mu M). Melanin at 100 mu g/ml produced about 25% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (1100 mu g/ml) to kanamycin-treated cells (5 mu M) augmented the inhibitory action of kanamycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in cell growth and collagen metabolism, may be one of the targets for kanamycin-induced inhibition of these processes. As shown by Western immunoblot analysis melanin augmented kanamycin-induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2. The obtained results demonstrate that melanin potentiates the inhibitory effect of kanamycin on IGF-I receptor-dependent signaling pathway in cultured fibroblasts. The data suggest a potential mechanism for the organ specificity of kanamycin-induced hearing loss in patients which may result from melanin-induced augmentation of the inhibitory effects of kanamycin on collagen and DNA biosynthesis.