Melanin bleaching in argyrophilic staining of AgNORs in pigmented lesions: a morphometric evaluation

OBJECTIVE: To establish a procedure that can effectively bleach melanin from pigmented lesions without affecting quantification of argyrophilic staining of nucleolar organizer regions (AgNORs). STUDY DESIGN: Twenty banal compound nevi, five from each of nonpigmented, slightly pigmented, moderately pigmented and heavily pigmented groups, were bleached by 10% H202 for periods of 0 (nonbleached controls) and 24 hours. AgNOR size and count parameters of nevomelanocytic nuclei were measured by video image analysis. Melanin bleaching using KMnO4 was also investigated. RESULTS: In all lesions treated with 10% H202 for 24 hours, the melanin was bleached effectively, with no qualitative change in AgNOR appearance. There were no significant differences in mean AgNOR number per nucleus (AgNOR number), mean individual AgNOR size (AgNOR size) or mean percentage of AgNOR area per nucleus (% nuclear area) between nonbleached and bleached sets in both the nonpigmented and slightly pigmented groups. However, disintegration of AgNOR dots was observed in those treated with 1% KMnO4 for 5, 10 and 15 minutes. There were significant decreases in AgNOR size (P = .002) and % nuclear area (P = .003) and increase in AgNOR number (P = .05) in the slightly pigmented group evaluated when treated with 1% KMnO4 for five minutes. CONCLUSION: Melanin in pigmented lesions can be bleached effectively with an H202 procedure without significantly affecting AgNOR staining properties in contrast to bleaching with KMnO4.     

Effect of melanin bleach on Feulgen-DNA microdensitometry in pigmented lesions

OBJECTIVE: To investigate the effect of melanin bleach on Feulgen-DNA microdensitometry in pigmented melanocytic lesions. STUDY DESIGN: Twenty banal compound nevi with various grades of pigmentation were bleached by 0.5% and 1% KMnO4 for 0 to 20 minutes and by 10% H2O2 for 24 hours prior to Feulgen staining. DNA microdensitometry was performed by video image analysis to measure the integrated optical density (IOD) in nuclei from nevomelanocytes, lymphocytes and spinous keratinocytes. The DNA index of nevomelanocytes was calculated using spinous keratinocytes as the diploid controls. RESULTS: There were significant decreases in IOD (P < .05) in the nuclei of nevomelanocytes, lymphocytes and spinous keratinocytes after treatment with 1% KMnO4 for 5 and 10 minutes, but no significant changes were detected after treatment with 0.5% KMnO4 for 5 and 10 minutes. Severe tissue damage was observed in the Feulgen-stained slides treated with 1% KMnO4 for 15 and 20 minutes and with 10% H2O2 for 24 hours. There was no significant change in DNA index in any bleached sets measured. CONCLUSION: KMnO4 can affect Feulgen-DNA content if used in high concentrations or for long periods of incubation. The DNA index, which is derived from internal controls, is not affected by the bleach procedure.     

The stepwise two-photon excited melanin fluorescence is a unique diagnostic tool for the detection of malignant transformation in melanocytes

Malignant transformation of melanocytes is associated with changes in melanogenesis. Therefore, fluorescence of melanin may be an informative indicator of this process. But the conventionally excited autofluorescence of melanin in skin tissue is ultra-weak and its main part in the visible spectral region is hidden by the much stronger fluorescence from other endogenous fluorophores. Here, using a new mode of stepwise two-photon excitation, melanin-dominated fluorescence spectra of pigmented skin lesions are reported. From these, pure melanin fluorescence spectra of normal pigmented skin, melanocytic nevi and malignant pigmented melanoma were analyzed. They show distinctly different spectral shapes: melanoma gave a characteristic fingerprint with a fluorescence band peaking at 640 nm, independent of the melanoma subtype. The melanin fluorescence spectra peaked at 590 nm for all types of common melanocytic nevi. These differences in the fluorescence spectra are probably based on different contents of eumelanin and pheomelanin. In a series of 167 cases with melanocytic nevi and melanomas, the sensitivity of this new method to diagnose melanoma was 93.5%, the specificity 80.0% and the diagnostic accuracy 82.6%. The two-photon excitation fluorescence method is a new diagnostic tool which may in future supplement conventional dermatohistopathology.     

Nucleolar organizer regions in pigmented skin lesions. Value in the differential diagnosis of Spitz nevi.

Forty-one cases of typical melanocytic skin lesions (15 intradermal nevi, 14 Spitz nevi and 12 malignant melanomas) were used to investigate the value of staining of nucleolar organizer regions (NORs) in the differential diagnosis of such pigmented lesions. Histologic sections were stained by the silver colloid (Ag) method, with and without the prior use of a melanin blocking agent. There were statistically significant differences in the mean numbers of AgNORs per nucleus between the groups of lesions studied (1.658 for intradermal nevi, 3.0042 for Spitz nevi and 6.669 for malignant melanomas). Sections treated with potassium permanganate (melanin blocking agent) prior to staining showed an obvious increase in the AgNOR scores in all groups; this increase was highest for Spitz nevi. Although AgNOR staining allows a distinction to be made between intradermal nevi and malignant melanomas, the striking overlap between the counts for Spitz nevi and malignant melanomas precludes the use of this technique as the sole method for establishing the diagnosis of malignancy. Other clinical and morphologic data are especially required to make the diagnosis of Spitz nevi.     

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.     

Satellite observation of Keppel Islands (Great Barrier Reef) 2002 coral bleaching using IKONOS data

An examination of IKONOS satellite imagery of the Keppel Islands (Great Barrier Reef) acquired before and during a coral bleaching event indicates that severe bleaching of reefs can be detected as an increase in brightness in the band 1 (blue) and band 2 (green) IKONOS spectral bands (4-m resolution). The bleaching was not detected in band 3 (red), band 4 (near-infrared), or in the 1-m panchromatic band data. A total of 0.74 km² of bleached coral was identified, with detection occurring in waters as deep as 15 m. The procedure requires that one of the scenes be radiometrically normalized to match the reference scene prior to image differencing. A relative radiometric normalization was used in this case because variable cloud cover present in the image acquired during the bleaching event prevented reliable modeling of atmospheric effects. The success at coral bleaching detection at Keppel Islands represents both a best-case and a cloud-challenged scenario. It was a best-case scenario in that coral cover was extensive (70–90% live coral cover, mostly acroporids) and the bleaching level was extreme (92–95% of coral cover white bleached). It was a cloud-challenged scenario in terms of having extensive and highly variable cloud cover present in the image acquired during the bleaching event. Color difference images reveal extensive areas of bleached coral at sites away from our study area, indicating that this platform and methodology may be a valuable tool for mapping high coral cover areas during bleaching events. Additional studies and technique refinements would be required to test the detection limits of bleaching with IKONOS imagery or to develop a spectrally based bleaching detection index.An erratum to this article can be found at     

Towards in vivo melanin radicals detection in melanomas by electron paramagnetic resonance (EPR) spectroscopy: a proof-of-concept study

Melanoma is the most aggressive skin tumour type. Although complete cure can be achieved when the whole tumour is resected, prognostic dramatically drops when melanoma cells reach deeper tissues and lymph nodes. Hence, there is an urgent need to develop accurate tools allowing (i) discriminating benign naevi from malignant tumours and (ii) being able to characterise melanoma infiltration. For that purpose, we exploited the paramagnetic properties of melanin by using electron paramagnetic resonance (EPR) spectroscopy to measure the melanin content in pigmented (B16F10 cancer cells) and non-pigmented melanomas (WM2664 cancer cells) inoculated intradermally in nude mice. Specifically, we took advantage of a new clinical EPR device (1?GHz), which provides sensitive measurements of radical species in vivo. Results showed that the melanin-specific EPR signal increased with tumour growth in pigmented tumours, whereas no EPR signal could be detected in achromic melanomas. These data plead for the development of new EPR spectrometers/imagers with an improved in-depth resolution for the detection of invasive melanomas.     

Ki-67 immunostaining and stereologic estimation of nuclear volume in melanocytic skin tumors

OBJECTIVE: To investigate the proliferative activity and mean nuclear volume (MNV) of melanocytic skin tumors. STUDY DESIGN: Proliferative activity, assessed by immunostaining for the Ki-67 monoclonal antibody (reactive with all actively cycling cells), and MNV, estimated by means of a stereologic method, were determined in 60 cutaneous melanocytic tumors, including 28 primary malignant melanomas (PMM), 13 compound nevi (CN), 11 dysplastic nevi and 8 metastatic malignant melanomas. RESULTS: Both MNV and Ki-67 expression differed significantly between CN and other melanocytic tumors and showed a good correlation with Clark's level (a well-established prognostic parameter in PMM). CONCLUSION: The association of proliferative activity and quantitative nuclear features may be helpful in the interpretation of the degree of malignancy in melanocytic skin tumors.     

Ki-67 (MIB 5) immunostaining of mouse lung tumors induced by 4-nitroquinoline 1-oxide

 We immunostained mouse lung tumors using a mouse monoclonal antibody against recombinant Ki-67 antigen (clone; MIB 5) to establish an MIB 5 immunostaining method and to determine the extent of MIB 5 labeling to monitor cell proliferation activity in mouse lung tumors. A/J mice, treated with 4-nitroquinoline 1-oxide, were killed after 18 months. One hour before killing, bromodeoxyuridine (BrdU) was injected intraperitoneally. Lung tissues including tumors were fixed with phosphate-buffered 4% paraformaldehyde and embedded in paraffin. For MIB 5 immunostaining, two antigen-retrieval buffers, citrate buffer pH 6 and TRIS-HCl buffer pH 9.5 containing 5% urea, were tested, and constant and reproducible staining was obtained only with the TRIS-HCl buffer. The mean values of the MIB 5-positive cell index (PCI), the BrdU labeling index (LI), and the mitotic cell count for adenocarcinomas were 4.6%, 2.3%, and 7/mm², and those for adenomas were 1.2%, 0.7%, and 1.3/mm², respectively. Each of these values was significantly higher for adenocarcinomas than for adenomas. A close correlation was seen between the MIB 5 PCI and the BrdU LI for adenocarcinomas and adenomas and between the MIB 5 PCI and the mitotic cell count in adenocarcinomas. Thus, MIB 5 immunostaining is a useful method for assessing the proliferative activity of mouse tumor tissues. Accepted: 23 June 1998     

Skeletal isotope microprofiles of growth perturbations in<Emphasis Type="Italic"> Porites</Emphasis> corals during the 1997–1998 mass bleaching event

Severe coral bleaching occurred throughout the tropics in 1997/98. We report high-resolution skeletal oxygen isotope (¹⁸O) and carbon isotope (¹³C) microprofiles for bleached corals from Pandora Reef, Great Barrier Reef, and Ishigaki Island, Japan, in order to examine the ability of Porites corals to record clear signals of bleaching. Analysis of the annual cycle in ¹⁸O revealed abrupt reductions in skeletal extension immediately after the 1997–98 summer temperature maximum, indicating that bleaching inhibits coral calcification. Skeletal ¹³C in the Ishigaki corals showed lower values during bleaching, indicating depressed coral metabolism associated with a reduction in calcification. In contrast, microprofiles of skeletal ¹³C from the shaded sides of Pandora Reef corals exhibited little change, possibly because algal photosynthesis was already slow prior to bleaching, thus subduing the ¹³C-response to bleaching. Comparison of ¹⁸O microprofiles from bleached corals with instrumental temperature records showed that Porites corals can recover following 5 months with little skeletogenesis. The results indicate that isotopic microprofiling may be the key to identifying gaps in coral growth that are diagnostic of past bleaching events. We have tested this hypothesis using blue UV fluorescent bands to guide us to coral skeleton where isotope microprofiling identifies bleaching events in 1986, 1989, and 1990. These events, detected by proxy, suggest that coral bleaching may have occurred more commonly on Ishigaki Island than previously recorded.     

Calcium-induced modification of protein conformation demonstrated by immunohistochemistry: What is the signal?

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463-469, 1999)     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

Separation of pigmented and albino melanocytes and the concomitant evaluation of endogenous peroxide content using flow cytometry

Flow cytometry (FCM) has been used extensively to analyze various biological properties of the cell. In this report, we describe a method by which FCM was used to determine the light scattering profile of a mixed population of pigmented and non-pigmented melanocytes, plus its subsequent use for the sorting and separation of the two cell types. In addition, the relative peroxide content in pigmented and non-pigmented melanocytes was compared by flow cytometry. Cultured avian melanocytes from a pigmented control and from three genetically distinct albino sources were studied. FCM analysis of forward versus side light scatter within a mixed suspension of pigmented and amelanotic melanocytes distinguished two overlapping populations of cells. Sorting of these two populations demonstrated that the population exhibiting much side and minimal forward light scatter was primarily pigmented melanocytes, while conversely the population exhibiting less side and more forward scatter was principally non-pigmented cells. These two melanocyte types also demonstrated differences in levels of endogenous peroxides. The intracellular content of peroxide in the two subpopulations of cells was measured utilizing the nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (DCFH-DA), which within the cell is oxidized by intracellular peroxides to a fluorescent dichlorofluorescein (DCF). Non-pigmented albino melanocytes had the highest quantity of endogenous peroxides, while heavily pigmented cells had considerably less peroxide-related fluorescence. The amount of this DCF fluorescence could be enhanced by increasing concentrations of DCF used in the assay. These flow cytometric methods are useful for isolating and culturing subpopulations of melanocytes expressing various pigment levels and to investigate the relationship between melanin and its precursors with hydrogen and lipid peroxides in melanocytes.     

Immunohistochemical study of pepsinogen C expression in cutaneous malignant melanoma: association with clinicopathological parameters

BACKGROUND: The aim of this study was to evaluate the pepsinogen C expression in malignant cutaneous melanomas and analyze its possible relationship to clinical and pathological parameters. Pepsinogen C is an aspartyl proteinase primarily involved in the digestion of proteins in the stomach and represents one of the main androgen-inducible proteins in breast cancer cells. METHOD: Tumoral pepsinogen C expression was retrospectively analyzed in 35 paraffin-embedded tissues from patients with primary malignant cutaneous melanoma and in 10 samples from 10 benign lesions (4 dermal melanocytic nevi, 4 compound melanocytic nevi and 2 dysplastic melanocytic nevi), using immunohistochemical methods. RESULTS: The benign lesions were consistently negative for pepsinogen C, whereas 20 of the 35 malignant melanomas (57%) showed positive immunostaining for pepsinogen C. The percentage of pepsinogen C-positive tumors was significantly higher in men than in women (p=0.01) and in epithelioid melanomas than in fusocellular or mixed type melanomas (p=0.003). In addition, the percentage of pepsinogen-C positive tumors was positively and significantly correlated with lesion thickness (p=0.003), Clark's level of invasion (p=0.028) and tumor stage (p<0.001). CONCLUSION: Pepsinogen C could be a new prognosticator of unfavorable outcome in cutaneous malignant melanoma.     

Angiomatoid giant cellular blue nevus of vaginal wall associated with pregnancy

Background

Blue nevi that arise from the Müllerian tract are rare melanocytic lesions. Several histopathologic variants of cellular blue nevi have been described. The angiomatoid variant is characterized by a vascular component, and is considered to be a rare variant. Few studies have explored the influence of pregnancy on melanocytic lesions.

Case

A 29-year-old woman was presented with a pigmented vaginal lesion that increased gradually during pregnancy. A full term gynecologic examination showed a tumor mass protruding into the vaginal canal. The mass was resected during cesarean-section under the clinical impression of vaginal hemangioma.

Result

Gross examination revealed a cystic mass measuring 6.0 × 4.3 × 3.5 cm, which was filled with dark friable material. Histologically, the mass showed a subepithelial cellular proliferation of heavily pigmented dendritic melanocytes with prominent vascular stroma. Cytologic pleomorphism, junctional activity, atypical mitosis, and necrosis were not found. The proliferation was immunoreactive for HMB-45, S-100 and melan-A, and non-immunoreactive for CD34, smooth muscle actin, and AE1/AE3. The MIB-1 proliferative index was less than 1%. The patient had a postoperative course without complication.

Conclusions

Angiomatoid giant cellular blue nevus arising from the vagina during pregnancy is extremely rare. The low proliferative index and absence of cytologic pleomorphism, or necrosis, supports a benign biological behavior. Clinical follow-up showed no evidence of recurrence at one year after the resection of the mass.

     

Immunohistochemical and autoradiographic findings suggest that minoxidil is not localized in specific cells of vibrissa,pelage, or scalp follicles

Summary Immunohistochemistry with a minoxidil antibody suggested that minoxidil-immunoreactivity is associated with the root sheaths, laterally orientated differentiating matrix cells, and dividing epithelial cells of cultured vibrissa follicles of pigmented and albino neonatal mice. The dermal papilla and connective tissue sheath were devoid of minoxidil-immunoreactivity. To verify that minoxodil-immunoreactivity in the follicles was specific, immunostaining was conducted with dissected whisker pads, formalin-fixed dead follicles, and sections of spleen, liver and kidney (non-haired organs) cultured with minoxidil. Microscopic examination revealed minoxidil-immunoreactivity in all of these tissues. Follicles and whisker pads cultured with minoxidil, then washed for one h in media were devoid of minoxidil-immunoreactivity. These data suggest that minoxidil-immunoreactivity in cultured vibrissa follicles is probably non-specific. Sections of skin from C3H and CF1 mice which were topically dosed with minoxidil (in vivo) phy demonstrated that tritiated minoxidil was bound in vivo and in vitro only to melanin granules in pigmented follicles of rodent and human tissue. This is probably non-specific binding since melanin is known to accumulate several chemically and pharmacologically unrelated drugs. It is reasonable to conclude that, under the conditions of these experiments, minoxidil is not specifically localized in any cells of whisker, pelage or, scalp follicles.     

Effective Melanin Depigmentation of Human and Murine Ocular Tissues: An Improved Method for Paraffin and Frozen Sections

Purpose

The removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues.

Methods

Paraffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO₄) with oxalic acid, and the second 10% hydrogen peroxide (H₂O₂). To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H₂O₂ was assessed using phosphate-buffered saline (PBS), and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA) were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation.

Results

Optimal bleaching was achieved using warm 10% H₂O₂ diluted in PBS at 65°C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO₄/oxalic acid demonstrated poor depigmentation with extensive tissue damage.

Conclusions

Warm dilute H₂O₂ at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues.     

Melanin protects melanocytes and keratinocytes against H2O2-induced DNA strand breaks through its ability to bind Ca2+

Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H(2)O(2) in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 microM H(2)O(2) increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH(4)Cl and elevated l-tyrosine, H(2)O(2)-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H(2)O(2)-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca(2+)-chelator BAPTA. Thus, BAPTA reduced the level of H(2)O(2)-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca(2+) and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca(2+) binding capacity and, in addition, correlated inversely with H(2)O(2)-induced increases in intracellular Ca(2+). Our results show that melanin may have an important role in regulating intracellular Ca(2+) homeostasis and it is suggested that melanin protects against H(2)O(2)-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca(2+).     

The effect of prolonged “bleaching” on skeletal banding and stable isotopic composition in Montastrea annularis

X-radiography and carbon and oxygen stable isotope analysis have been used to examine the effects of prolonged bleaching on the growth rate and chemical composition of the skeleton of the massive reef coral, Montastrea annularis. The post-bleaching linear growth of one colony that remained bleached for 10 to 12 months following the 1987 Caribbean-wide bleaching event was only 37% of mean annual growth from pre-bleaching years, and was manifest as a loss of the following year's low density band. Two colonies that did not bleach (normal) and two that bleached and regained their coloration (recovered) had linear growth rates over the same period that were 81 to 98% of mean pre-bleaching annual growth. Linear growth by a third recovered coral was 66% of pre-bleaching growth. No sub-annual stress bands were associated with the bleaching. The skeleton of the bleached colony had carbon and oxygen isotopic compositions that were reduced in range and enriched (increased) in both ¹³C and ¹⁸O in the post-bleaching year. The skeletons of two of the nine colonies, one bleached and one recovered, had depleted (reduced) ¹⁸O values (-5.3 and -4.8%., respectively) during the bleaching episode that agree with the suggestion that positive temperature anomalies occurred during, and may have caused, the bleaching event. The range and values for all other normal and recovered corals, however, were not different between the post-bleaching year and previous years. Our data suggest that stress bands and isotopic analysis of coral skeletons may not always be reliable tools for examining the occurrence, cause or effects of certain discrete stress events that may interrupt skeletal growth.     

A Novel Histochemical Method for a Simultaneous Staining of Melanin and Collagen Fibers

For the histopathologic diagnosis of melanocytic lesions, it could be necessary to identify the melanin pigment because its visualization is unspecific with hematoxylin-eosin (HE). The Fontana-Masson (FM) technique is used in histopathology in this type of lesion, which allows the identification of the pigment, but it loses all the morphologic parameters. The authors describe a modification to the FM method, for the evaluation of the morphology, the argentaffin reaction of the melanin, and collagens fibers of the extracellular matrix simultaneously, for which they have developed the Fontana-Masson picrosirius (FMPS) method. Biopsies of different melanocytic lesions were used for the performance of the HE, FM, and FMPS methods. The pixel intensity of the reaction for melanin, collagen, and epithelium was determined with ImageJ software. The FMPS method allows the evaluation of morphological characteristics, identifying the melanin pigment and collagen fibers with high intensity simultaneously. This method does not differ significantly from FM in the identification of melanin, maintaining its sensitivity and specificity. In addition, it does not differ in the demonstration of the morphology with HE. However, FMPS is significantly superior in the identification of collagen fibers. The FMPS method combines morphological and histochemical parameters that could be useful in the study of pigmented lesions of melanocytic origin.