Stabilization of ß-catenin promotes melanocyte specification at the expense of the Schwann cell lineage.

The canonical Wnt/ß-catenin pathway governs a multitude of developmental processes in various cell lineages, including the melanocyte lineage. Indeed, ß-catenin regulates transcription of Mitf-M, the master regulator of this lineage. The first wave of melanocytes to colonize the skin is directly derived from neural crest cells, whereas the second wave of melanocytes is derived from Schwann cell precursors (SCPs). We investigated the influence of ß-catenin in the development of melanocytes of the first and second waves by generating mice expressing a constitutively active form of ß-catenin in cells expressing tyrosinase. Constitutive activation of ß-catenin did not affect the development of truncal melanoblasts but led to marked hyperpigmentation of the paws. By activating ß-catenin at various stages of development (E8.5-E11.5), we showed that the activation of ß-catenin in bipotent SCPs favored melanoblast specification at the expense of Schwann cells in the limbs within a specific temporal window. Furthermore, in vitro hyperactivation of the Wnt/ß-catenin pathway, which is required for melanocyte development, induces activation of Mitf-M, in turn repressing FoxD3 expression. In conclusion, ß-catenin overexpression promotes SCP cell fate decisions towards the melanocyte lineage.

Assessment of Betulinic Acid Cytotoxicity and Mitochondrial Metabolism Impairment in a Human Melanoma Cell Line.

Melanoma represents one of the most aggressive and drug resistant skin cancers with poor prognosis in its advanced stages. Despite the increasing number of targeted therapies, novel approaches are needed to counteract both therapeutic resistance and the side effects of classic therapy. Betulinic acid (BA) is a bioactive phytocompound that has been reported to induce apoptosis in several types of cancers including melanomas; however, its effects on mitochondrial bioenergetics are less investigated. The present study performed in A375 human melanoma cells was aimed to characterize the effects of BA on mitochondrial bioenergetics and cellular behavior. BA demonstrated a dose-dependent inhibitory effect in both mitochondrial respiration and glycolysis in A375 melanoma cells and at sub-toxic concentrations (10 µM) induced mitochondrial dysfunction by eliciting a decrease in the mitochondrial membrane potential and changes in mitochondria morphology and localization. In addition, BA triggered a dose-dependent cytotoxic effect characterized by apoptotic features: morphological alterations (nuclear fragmentation, apoptotic bodies) and the upregulation of pro-apoptotic markers mRNA expression (Bax, Bad and Bak). BA represents a viable therapeutic option via a complex modulatory effect on mitochondrial metabolism that might be useful in advanced melanoma or as reliable strategy to counteract resistance to standard therapy.

Whole genome landscapes of uveal melanoma show an ultraviolet radiation signature in iris tumours.

Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline MBD4 mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX). We identify three other significantly mutated genes (TP53, RPL5 and CENPE).

Quantitative assessment of neuropilin-2 as a simple and sensitive diagnostic assay for spitzoid melanocytic lesions.

There is a significant need for the development of diagnostic tools that can precisely distinguish Spitz nevi and spitzoid melanomas. Here, we report the development of a PCR-based quantitative diagnostic assay for spitzoid melanocytic lesions utilizing the expression ratio of neuropilin-2 and melan-A genes in primary tumor specimens. We find that the expression ratio of neuropilin-2/melan-A is significantly increased in spitzoid melanomas compared with Spitz nevi. The diagnostic potential of this quantitative assay was validated in two independent sets of patient samples as demonstrated in a receiver operating characteristic curve analysis showing an area under the curve value of 91.8%. Furthermore, the assay was found to quantitatively distinguish the clinical nature of atypical spitzoid melanocytic lesions that were diagnostically undetermined using histopathologic criteria alone. Our data indicate that this quantitative assay may be used as a tool in determining the diagnostic classification of histologically challenging spitzoid tumors.

Point of care assessment of melanoma tumor signaling and metastatic burden from µNMR analysis of tumor fine needle aspirates and peripheral blood.

This study evaluates µNMR technology for molecular profiling of tumor fine needle aspirates and peripheral blood of melanoma patients. In vitro assessment of melanocyte (MART-1, HMB45) and MAP kinase signaling (pERK, pS6K) molecule expression was performed in human cell lines, while clinical validation was performed in an IRB-approved study of melanoma patients undergoing biopsy and blood sampling. Tumor FNA and blood specimens were compared with BRAF genetic analysis and cross-sectional imaging. µNMR in vitro analysis showed increased expression of melanocyte markers in melanoma cells as well as increased expression of phosphorylated MAP kinase targets in BRAF-mutant melanoma cells. Melanoma patient FNA samples showed increased pERK and pS6K levels in BRAF mutant compared with BRAF WT melanomas, with µNMR blood circulating tumor cell level increased with higher metastatic burden visible on imaging. These results indicate that µNMR technology provides minimally invasive point-of-care evaluation of tumor signaling and metastatic burden in melanoma patients.

A diagnostic algorithm for atypical spitzoid tumors: guidelines for immunohistochemical and molecular assessment.

Atypical spitzoid tumors are a morphologically diverse group of rare melanocytic lesions most frequently seen in children and young adults. As atypical spitzoid tumors bear striking resemblance to Spitz nevus and spitzoid melanomas clinically and histopathologically, it is crucial to determine its malignant potential and predict its clinical behavior. To date, many researchers have attempted to differentiate atypical spitzoid tumors from unequivocal melanomas based on morphological, immonohistochemical, and molecular diagnostic differences. A diagnostic algorithm is proposed here to assess the malignant potential of atypical spitzoid tumors by using a combination of immunohistochemical and cytogenetic/molecular tests. Together with classical morphological evaluation, this algorithm includes a set of immunohistochemistry assays (p16(Ink4a), a dual-color Ki67/MART-1, and HMB45), fluorescence in situ hybridization (FISH) with five probes (6p25, 8q24, 11q13, CEN9, and 9p21), and an array-based comparative genomic hybridization. This review discusses details of the algorithm, the rationale of each test used in the algorithm, and utility of this algorithm in routine dermatopathology practice. This algorithmic approach will provide a comprehensive diagnostic tool that complements conventional histological criteria and will significantly contribute to improve the diagnosis and prediction of the clinical behavior of atypical spitzoid tumors.

Assessment of Cuscuta chinensis seeds׳ effect on melanogenesis: comparison of water and ethanol fractions in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Cuscuta chinensis seeds have traditionally been used to treat freckles and melasma in Asia, although recent reports have revealed that Semen cuscutae is a promoter of melanogenesis. The present study aims to investigate the mechanism of this opposite effect of Semen cuscutae on melanogenesis. MATERIALS AND METHODS: In accordance with traditional usage, the water fraction and the ethanol fraction from Semen cuscutae (WFSC/EFSC) were extracted to determine the herbal effects by examining the activity of mushroom tyrosinase, cellular melanin contents, tyrosinase activity assay, quantitative-reverse transcription polymerase chain reaction (qRT-PCR), and Western blot analysis for tyrosinase in B16F10 mouse melanoma cells. The melanocyte phenotypes of zebrafish larvae were observed while the in vivo melanin contents and tyrosinase activity were determined. RESULTS: The activity of mushroom tyrosinase assay shown that WFSC was an uncompetitive inhibitor of mushroom tyrosinase, while EFSC indicated dose-dependent activation of the mushroom tyrosinase activity. The WFSC markedly inhibited 3-isobutyl-1-methylxanthine (IBMX)-stimulated melanin synthesis and tyrosinase activity in vitro. Howeveran accelerant role in melanin synthesis and tyosinase activity. Neither fraction had any effect on the IBMX-induced expression of tyrosinase protein or mRNA. The WFSC strongly inhibited melanin synthesis and cellular tyrosinase activity in vivo. Furthermore, with the function of WFSC at a higher concentration, a punctate melanocyte pattern appeared that was similar to the pattern induced by arbutin or Mequinol (MQ). The EFSC had no effect on the melanocytes of zebrafish larvae. It was discovered that WFSC did not show a stable inhibitory effect until it was extracted 1 month later. CONCLUSIONS: These results suggest that the opposite effects of Cuscuta chinensis seeds were caused by the extraction methods and that time has an important role on the effect of WFSC. Both WFSC and EFSC significantly influence melanogenesis by regulating enzymatic activity of tyrosinase. In addition, the data indicate that wildtype (WT) zebrafish may be an ideal model for testing inhibitors of melanogenesis from clinically active herbs.

Expression of soluble adenylyl cyclase in lentigo maligna: use of immunohistochemistry with anti-soluble adenylyl cyclase antibody (R21) in diagnosis of lentigo maligna and assessment of margins.

CONTEXT: Soluble adenylyl cyclase (sAC) is an enzyme that generates cyclic adenosine monophosphate, a signaling molecule involved in regulating melanocyte functions. R21, a mouse monoclonal antibody against sAC, shows a striking pan-nuclear staining in lentigo maligna, indicating possible utility for diagnosis and margin assessment. OBJECTIVE: To evaluate R21 in the diagnosis and evaluation of margins in lentigo maligna. DESIGN: Thirty one re-excision specimens for lentigo maligna were evaluated for R21 expression using previously published protocol. In addition, 153 cases including 41 lentigo malignas, 30 non-lentigo maligna-type melanomas, 38 lentigos, and 44 nevi were evaluated using a modified stringent protocol to eliminate all nonmelanocyte staining. RESULTS: The sensitivity of nuclear staining with R21 in lentigo maligna was 87.8%. Nuclear expression of sAC was observed in 40% of other melanomas and 2.3% of benign nevi. R21 did not stain nuclei of resting melanocytes but was observed in 28.9% of melanocytic hyperplasias. These cases were easily distinguished from lentigo maligna in routine sections. R21 staining facilitated extent of the lesion in resection margins. In cases examined under the less stringent conditions, interpretation was facilitated by comparing R21 and Mart1/Melan A staining. Greater than 9 pan-nuclear staining melanocytes within one high-power field along with a pan-nuclear sAC/Melan A ratio greater than 0.5 was consistent with a positive margin whereas 5 or less pan-nuclear staining melanocytes along with a sAC/Melan A ratio of less than 0.3 constituted a negative margin. CONCLUSION: R21 is a useful diagnostic adjunct in the diagnosis and evaluation of margins in re-excision specimens in lentigo maligna.

Correlation between histologic assessment and fluorescence in situ hybridization using MelanoSITE in evaluation of histologically ambiguous melanocytic lesions.

CONTEXT: The 4-probe, multicolor, fluorescence in situ hybridization (FISH) panel targeting chromosomes 6 and 11 has shown promising sensitivity and specificity in distinguishing between benign nevi and malignant melanoma. Only a few studies have assessed the potential utility of FISH in classification of histologically ambiguous melanocytic lesions. In the United States, this assay is exclusively licensed to NeoGenomics Laboratories (Irvine, California), which provides the technical component and has developed an innovative service (MelanoSITE) allowing pathologists to interpret FISH results using a dedicated Web portal. Thus far, use of MelanoSITE as a diagnostic adjunct in the diagnosis of melanocytic lesions has not, to our knowledge, been reported in the literature. OBJECTIVE: To analyze 1.5 years of experience with the MelanoSITE melanoma FISH assay in the evaluation of histologically ambiguous lesions in the context of second opinion and routine dermatopathology practice. DESIGN: A prospective histologic/FISH correlation study of 140 cases. RESULTS: Twenty-seven percent of abnormal FISH results were false-positive results because of tetraploidy. After correcting for known false-positive results, all lesions considered atypical nevi showed normal FISH signals. Abnormal FISH signals were reported in 30% of lesions considered histologically borderline and in 48% of lesions in which a diagnosis of melanoma was favored. CONCLUSIONS: Four-probe, multicolor FISH results for melanoma correlate with the microscopic assessments of histologically ambiguous lesions. Pathologists using MelanoSITE must be aware of the high rate of false-positive results from tetraploidy.

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions.

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intradermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient's age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma.

In vivo assessment of pigmentary and vascular compartments changes in UVA exposed skin by reflectance-mode confocal microscopy.

Exposure of the skin to ultraviolet A (UVA) results in various biological responses, skin-colour changes being among the major ones. Although intense research has been performed on UVA-induced pigmentation and vascular changes, the process of skin-colour changes after UVA irradiation remains unclear. For a better understanding of the UVA tanning mechanism, we here performed a human study in 27 healthy volunteers with skin phototype (SPT) II to VI. After a single UVA exposure to inner forearm, the skin sites were imaged using reflectance-mode confocal microscopy (RCM), for analysis of melanin and vascular changes. Punch biopsies were also taken from the UVA-exposed or non-exposed sites for histological examination. Skin sections were stained with Fontana-Masson and evaluated by a sensitive tyrosinase assay for comparison with RCM images. Furthermore, the effect of blood flow on skin-colour changes was evaluated visually after administration of an intradermal anesthesia of lidocaine with or without epinephrine. Our RCM analysis showed dendritic melanocytes and a different melanin distribution in the epidermal layer, clearly visible 1 week after the UVA exposure in subjects of SPT V which were supported by histological examination. However, no melanin distribution pattern changes were apparent immediately after the exposure, while RCM images showed accelerated capillary flow patterns. The restriction of this UVA induced-accelerated blood flow by epinephrine inhibited partially or completely the immediate pigment darkening and delayed tanning. These in vivo studies confirmed that vascular change is an important factor for the development of the immediate pigment darkening and delayed tanning.

Assessment of polymorphic variants in the melanocortin-1 receptor gene with cutaneous pigmentation using an evolutionary approach.

The melanocortin-1 receptor gene (MC1R) encodes a membrane-bound receptor protein that is central to melanin synthesis. The coding region of MC1R is highly polymorphic and associations of variants with pigmentation phenotypes and risk for cutaneous neoplasms have been reported. We sought to determine the distribution and frequency of MC1R variants and their relationship to pigmentation characteristics in 179 Caucasian controls from the United States. One hundred thirty-five (75.4%) subjects carried one or more variants, and we determined that carriage of the previously designated "red hair color" (RHC) alleles, R151C, R160W, and D294H was strongly associated with fair pigmentation phenotypes including light hair and eye color, tendency to burn, decreased tendency to tan, and freckling. We used SIFT software to define MC1R protein positions that were predicted intolerant to amino acid substitutions; detected variants that corresponded to intolerant substitutions were D84E, R142H, R151C, I155T, R160W, and D294H. Carriage of one or more of these putative functionally important variants or the frameshift variant ins86A was significantly associated with fair pigmentation phenotypes. Analyses limited to carriage of ins86A and the three non-RHC alleles identified by SIFT were attenuated and no longer reached statistical significance. This is the first study to describe MC1R variants among control subjects from the U.S. Our results indicate that the frequency of variants is similar to that previously observed among non-U.S. Caucasians. Risk variants defined by either the published literature or by evolutionary criteria are strongly and significantly associated with all fair pigmentation phenotypes that were measured.

Assessment of genetic instability in melanocytic skin lesions through microsatellite analysis of benign naevi, dysplastic naevi, and primary melanomas and their metastases.

Microsatellite instability (MSI) is caused by replication errors due to deficient DNA mismatch repair and has been associated with tumour progression in various types of cancer. Controversial results have been reported concerning the frequency and significance of MSI in malignant melanoma. In this study, the time of onset and relative incidence of MSI were determined during the progression of melanocytic tumours, starting with benign melanocytic naevi. MSI was studied at 13 loci containing single, di- or trinucleotide repeat sequences and mapping to five different chromosomal locations. Tumours were classified as being low frequency MSI (L-MSI+) or high frequency MSI (H-MSI+) when either one or at least two marker loci, respectively, displayed mutant alleles in tumour DNA compared with the corresponding normal tissue DNA. None of the eight melanocytic naevi studied showed MSI, whereas a moderate frequency of H-MSI was detected in dysplastic naevi (one out of 11; 9%) and primary melanomas (six out of 56; 11%). The incidence of H-MSI was increased in melanoma metastases from the same patients (nine out of 42; 21%). In contrast to previously reported data showing higher rates of MSI in melanoma, genetic instability seems to be present in a minority of malignant melanoma lesions. However, our findings are consistent with the hypothesis that MSI may be sequentially induced during malignant evolution, contributing to the progression of a subset of melanocytic tumours.

Accurate molecular detection of melanoma nodal metastases: an assessment of multimarker assay specificity, sensitivity, and detection rate.

BACKGROUND: The application of lymphoscintigraphy followed by sentinel lymph node (SN) biopsy to patients with primary melanoma has revolutionised the ability to identify accurately, yet conservatively, those patients who harbour occult nodal metastases. The molecular detection of SN micrometastases facilitates the cost effective analysis of the entire SN using multiple markers. Currently, a lack of marker specificity is the main barrier preventing the molecular evaluation of SN tissue from becoming clinically applicable. AIMS: To develop a reproducible multimarker reverse transcription-polymerase chain reaction (RT-PCR) assay, with the emphasis on achieving high specificity for the accurate detection of melanoma metastases in nodal tissue. METHODS: Three pigment cell specific (PCS) markers-tyrosinase, Pmel-17, and MART-1-and one cancer testis antigen (CTA)-MAGE-3-were selected for use in a multimarker RT-PCR assay. The conditions for this assay were optimised. RESULTS: High specificity was achievable for each marker by optimising the PCR cycle number such that unwanted transcripts (that is, illegitimate transcripts and/or specific transcripts from other low abundance nodal cell types) remained undetectable in appropriate controls (normal visceral nodes). Tyrosinase was 100% specific at 40 PCR cycles, MAGE-3 and MART-1 at 35 PCR cycles, and Pmel-17 at 30 PCR cycles. Tyrosinase proved to be the most sensitive marker, detecting 10 melanoma cells in 0.1 g of nodal tissue. CONCLUSIONS: Excellent reproducibility of the entire nodal processing and RT-PCR protocol for the detection of very low numbers of melanoma cells in nodal tissue was shown, although there is a risk of false positives using the PCS markers alone, because of an approximate 4-8.5% incidence rate of nodal nevi in melanoma draining SNs (these nevi being absent in all other normal nodes). MAGE-3 was shown to be the only marker that is not expressed by melanocytes. However, because not all melanomas express MAGE-3, it is recommended that more emphasis should be placed on the development of a panel of CTA markers to ensure a zero false positive rate and to provide optimum detection.

The human melanocyte as a particular target for UVA radiation and an endpoint for photoprotection assessment.

The induction of DNA breaks by UVA (320-400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin-related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.

Molecular control of melanogenesis in malignant melanoma: functional assessment of tyrosinase and lamp gene families by UV exposure and gene co-transfection, and cloning of a cDNA encoding calnexin, a possible melanogenesis "chaperone".

Melanogenesis is a cascade of events significantly controlled by regulatory genes which are associated with the melanosomal membrane. This report introduces our current research efforts dealing with (a) the gene and protein expressions of tyrosinase and Lamp (lysosome-associated membrane protein) families by human melanoma cells after repeated exposures to UV light, (b) the coordinated alterations in the expression of the Lamp family gene and its encoding product after transfection of two genes of the tyrosinase family in human melanoma cells and (c) cloning and sequencing of a Ca(2+)-binding phosphoprotein, calnexin, which could be a candidate as a chaperone for sorting and maturation of tyrosinase and Lamp family glycoproteins in melanogenesis cascade. Our UV exposure study, as well as gene transfection and antisense hybridization experiments, has clearly indicated a marked and coordinated interaction of the Lamp-1 gene with the tyrosinase and TRP-1 genes in this process. We propose that melanogenesis is controlled at least by two major gene family products, i.e., (a) the tyrosinase family of tyrosinase, TRP-1 and TRP-2, and the Lamp family of Lamp-1, Lamp-2 and Lamp-3. These two gene families probably derived from primordial melanogenesis-associated genes which are common or closely related to each other.

Quantitative assessment of epidermal melanogenesis in C3H/Tif hr/hr mice treated with topical furocoumarins and UVA radiation.

We report quantitative data on epidermal melanogenesis by established and new furocoumarins. The ears and dorsal skin of pigmented hairless mice were treated for 12 d with compounds in ethanol, at equi-optical concentrations, and exposed to subphototoxic doses of ultraviolet A. Increased pigmentation was observed with 6,4,4'-trimethylangelicin > psoralen > 8-methoxypsoralen > 5-methoxypsoralen > 4,4',5'-trimethylazapsoralen = bergamot oil. Assessment of melanocyte numbers and morphology in epidermal sheet dihydroxyphenylalanine preparations showed that 6,4,4'-trimethylangelicin was the best compound with 536 ear melanocytes/mm2 +/- 15 SEM compared with 46 +/- 4 in controls. Psoralen induced 297/mm2 +/- 33, compared with its methoxy derivatives with ranges between 200 and 240/mm2.6,4,4'-trimethylangelicin had a striking effect on dorsal skin with 462 +/- 18 melanocytes/mm2 compared to less than 80/mm2 in all other ultraviolet A treatment groups. Khellin, 5-GOP and ultraviolet A only and all non-ultraviolet A controls had no effect. Melanogenesis was associated with increased dendricity, melanocyte size, especially with 5-methoxypsoralen, and giant melanocytes were noted with some treatments. The potency of 6,4,4'-trimethylangelicin, which does not form DNA interstrand crosslinks, may be related to its high DNA binding constant. Our data may be useful in the selection of compounds to treat vitiligo.

Further assessment of a pharmacological effect of Tris buffer.

1. Data are presented confirming that Tris buffer can potentiate teleost melanophore responses to noradrenalin, as recently reported for the first time in Poecilia reticulata (Visconti and Castrucci, 1985). 2. In vitro melanophore responses of Pseudopleuronectes americanus were compared in Tris- and bicarbonate-buffered physiological saline solutions. 3. Dose-response curves demonstrate that Tris significantly potentiates melanophore responses to noradrenalin doses between 10(-6) and 10(-5)M in this species. 4. Time-response curves further characterized this effect of Tris on melanophore activity, quantifying the enhancement of the melanosome aggregation rate as well as the dose-related equilibrium levels.