To examine the sensitivity of vitiligo melanocytes to external oxidative stress, we studied enzymatic and non-enzymatic anti-oxidants in cultured melanocytes of normal subjects (n = 20) and melanocytes from apparently normal skin of vitiligo patients (n = 10). The activity of superoxide dismutase and catalase and the intracellular concentrations of vitamin E and ubiquinone were evaluated in cultures at the fourth or fifth passage. In addition, cells were exposed to various concentrations of a peroxidizing agent, cumene hydroperoxide (CUH, 0.66-20 microM), for 1 and 24 h. Compared to normal melanocytes, vitiligo melanocytes showed normal superoxide dismutase and significantly lower catalase activities and higher vitamin E and lower ubiquinone levels. At the concentration used, CUH did not significantly affect cell number or viability of melanocytes after either period of culture. On the contrary, vitiligo melanocytes were susceptible to the toxic effect of CUH after 24 h of continuous treatment at concentrations greater than 6.6 microM. The degree of CUH toxicity correlated strictly with the anti-oxidant pattern, defined as the ratio between vitamin E concentration and catalase activity, suggesting that the alteration in the antioxidants was the basis for sensitivity to the external oxidative stress. Our results demonstrate the presence of an imbalance in the anti-oxidant system in vitiligo melanocytes and provide further support for a free radical-mediated damage as an initial pathogenic event in melanocyte degeneration in vitiligo.

In order to evaluate the free radical defense systems of melanocytes and their possible correlation with melanoma, we have studied in cultured normal human melanocytes (20), normal melanocytes from melanoma patients (15), and melanoma cells (40) the fatty acid pattern of membrane phospholipids as a target of peroxidative damage and the superoxide dismutase and catalase activities, vitamin E, and ubiquinone levels as intracellular antioxidants. Cells were cultured in the same medium and analyzed at III or IV passage. Compared to the values obtained in normal human melanocytes, melanoma cells showed on average: a) higher levels of polyunsaturated fatty acids, b) increased superoxide dismutase and decreased catalase activities, higher vitamin E, and lower ubiquinone levels. Among the normal melanocytes from melanoma patients studied, two groups were differentiated: a) cultures (7) with enzymatic and non-enzymatic antioxidants level similar to those of normal human melanocytes; b) cultures (8) with antioxidant patterns similar to those observed in melanoma cells. Polyunsaturated fatty acids were also increased in the latter group. The results indicate that in melanoma cells and in a percentage of normal melanocytes from melanoma patients, an imbalance in the antioxidant system can be detected that can lead to endogenous generation of reactive oxygen species and to cellular incapability of coping with exogenous peroxidative attacks. These alterations could be correlated with the malignant transformation of cells and with the progression of the disease.

We have previously shown an imbalance of the antioxidant system in some cultures of normal melanocytes from patients with melanoma. In order to evaluate if the alteration of the antioxidants could be the basis of an increased sensitivity to exposure to peroxidative agents, in cultured melanocytes from normal individuals (n = 11) and from patients with melanoma (n = 11), superoxide dismutase and catalase activities were evaluated by spectrophotometer, and the levels of vitamin E and of the polyunsaturated fatty acid of cell membranes were determined by gas chromatography mass spectrometry. In 5 out of the 11 cultures of melanocytes from melanoma patients, with respect to those from normal individuals, a significant decrease of catalase activity (Cat) associated with an increase of vitamin E (Vit E) concentration was found, whereas no significant modification of superoxide dismutase activity (SOD) was observed. A wide range of variability was detected in the percentage of the polyunsaturated fatty acids of the cell membranes and a correlation was found between the ratio SOD/Cat and the percentage of linoleic acid, indicating that the imbalance of the enzymatic antioxidants leads to a lipoperoxidative process. The electron microscopic examination of these cultures revealed many microvilli in the plasma membranes and nuclear infoldings and in the cytoplasm light vacuoles. Moreover some cells contained several dense bodies with a round shape and numerous spherical lamellae possibly representing immature melanosomes. Treatment with cumene hydroperoxide between 0.66 and 20 microM did not produce a significant modification of cell viability in melanocytes from normal individuals. On the contrary in melanocytes from melanoma patients correlated with the ratio Vit E/Cat, considered as a parameter of the antioxidant imbalance, a stimulatory effect was observed at 0.66 microM CUH and a cytotoxic effect at 20 microM. In conclusion our results suggest that a constitutional alteration of the scavenger system could be present in normal melanocytes from melanoma patients and that this could be the basis for an increased sensitivity to pro-oxidant agents.

One of the primary goals in medical genetics is a precise clinical definition of chromosomal diseases. This is now possible because of the increased number of case reports and new techniques. A male patient, without a clear‐cut syndrome, was cytogenetically investigated. Chromosomal analysis showed a small unidentified bisatellited supernumerary marker. In situ hybridization with a biotin‐labeled DNA probe for the chromosome 15 centromere (D15Z1) demonstrated that the marker was derived from chromosome 15. Hybridization with the Prader‐Willi Syndrome Cosmid biotinylated probe (localized to band 15q11‐q13) showed a signal on both ends suggesting a marker with a symmetrical inv dup(15) and a breakpoint localized in q13. It was then possible to define the karyotype as: 47,XY,+ inv dup(15) (pter‐q13::q13‐pter). All cases of inv dup(15) reported in the literature were reviewed, paying particular attention to the different breakpoints involved, in order to provide a better clinical definition of this syndrome.

The GRO1 oncogene (melanoma growth-stimulating activity alpha) has been localized in region 4q21. The involvement of this chromosomal region in clonal aberrations found in primary melanoma cell cultures could have an important role in the etiology and pathogenesis of this tumor. We characterized three primary cell cultures obtained from different patients, each of which showed clonal chromosomal aberrations involving the 4q21 region.