Leukoderma induced by rhododendrol is different from leukoderma of vitiligo in pathogenesis: A novel comparative morphological study.

BACKGROUND: Rhododendrol (rhododenol), an inhibitor of tyrosinase activity, is used as a skin-whitening component. Many cases of leukoderma after the application have been reported, termed rhododenol-induced leukoderma (RIL). The aim of this study was to clarify the pathogenesis of RIL morphologically through comparison with vitiligo. METHODS: We examined 14 cases of RIL and 15 cases of vitiligo using routine histopathology and immunohistochemistry. Thirteen cases of RIL, six cases of vitiligo and specimens of the RIL mouse model were evaluated by electron microscopy. RESULTS: There were common findings in RIL and vitiligo at the light-microscopic level: (a) vacuolar changes in the dermo-epidermal junction, (b) melanophages in the papillary dermis, (c) perifollicular lymphocyte infiltration, (d) loss or decrease of basal melanin pigment and (e) decrease of melanocytes in the lesions. The ultrastructural observations showed specific findings of RIL: (a) remaining melanocytes in depigmented lesions, (b) inhomogeneous melanization in melanocytes and (c) degenerated melanosomes in melanocytes. Some of the findings were observed in a RIL mouse model. Furthermore, it is notable that cell organelles of melanocytes were intact in our RIL cases. CONCLUSION: Morphological changes of RIL targeting melanosomes in melanocytes without degeneration of organelles reflect the reversible clinical course of most cases.

The spectrophotometrical analysis of rhododendrol-induced leucoderma using a novel multispectral camera.

BACKGROUND: Many users in Japan of skin brightening/lightening cosmetics containing rhododendrol (RD) have developed leucoderma. Leucoderma appears on skin areas repeatedly treated with RD-containing cosmetics. RD-induced leucoderma (RDIL) presents different degrees of well-defined hypopigmentation. It is crucial to determine the degree of hypopigmentation to differentiate RDIL from vitiligo vulgaris (VV). OBJECTIVES: To quantitatively evaluate hypopigmentation of RDIL lesions and the recovery of pigmentation, and to compare the hypopigmentation with VV and normal skin. MATERIALS AND METHODS: Sixteen cases of RDIL, nine cases of VV and 15 healthy controls were examined using a novel multispectral camera (MSC) that can simultaneously obtain the reflection intensity at 10-nm wavelength intervals from 400 to 760 nm of the photographed area. ∆Absorbance was calculated by subtracting the log of reflection intensity of the target area from that of a white reflection standard. RESULTS: Most RDIL lesions showed lower ∆Absorbance than healthy skin and higher ∆Absorbance than VV lesions between 400 and 550 nm. Statistical comparison of the maximum ∆Absorbance from 420 to 460 nm (Max∆Absorbance) for VV, RDIL and control skin showed that the Max∆Absorbance of RDIL was significantly higher than that of VV and lower than that of control skin. The comparison of ∆Absorbance of the same sites in RDIL lesions between the initial visit and 6 months later showed significant improvement after 6 months. CONCLUSIONS: These studies demonstrated quantitative changes in RDIL and its recovery phase and suggested the utility of a MSC in obtaining objective colour information of skin disorders.

[Leukoderma caused by chemicals: mechanisms underlying 4-alkyl/aryl-substituted phenols- and rhododendrol-induced melanocyte loss].

Chemical leukoderma is a skin depigmentation disorder known to occur in manufactural workplace through contact with chemicals, such as monobenzyl ether of hydroquinone (MBEH) and 4-tert- butylphenol (4-TBP). In the skin depigmented -legions induced by these chemicals, the number of melanocyte was severely decreased. Anti-melanoma agent 4-cysteaminylphenol (4-SCAP) and its derivatives are also known to cause leukoderma. Evidence has accumulated supporting that typical class of chemicals causing leukoderma is "4-alkyl/aryl-substituted phenols/catechols", which are structurally similar to melanin precursor tyrosine. Tyrosinase-mediated oxidation of these chemicals yields toxic ortho-quinones which bind to cellular proteins and produce reactive oxygen species. Accordingly, this tyrosinase-dependent metabolic activation is thought to cause melanocyte-specific damage and subsequent immune reactions toward melanocytes. Recently, rhododendrol, an inhibitor of tyrosinase developed for so-called lightening/whitening cosmetics, was shown to cause leukoderma in the users. In this review, I document the causes of known chemical leukoderma and rhododendrol- induced leukoderma, focusing on their common mechanisms underlying melanocyte loss.

Butanol fraction of Alstonia boonei De Wild. leaves ameliorate oxidative stress and modulate key hypoglycaemic processes in diabetic rats.

OBJECTIVE: The effect of Alstonia boonei fractions on glucose homeostasis was investigated via in vitro enzyme inhibition activity, ex vivo glucose uptake assay, and in vivo methods in diabetic rats. METHODOLOGY: A. boonei fractions were subjected to in vitro α-glucosidase inhibitory assay and then ex vivo glucose uptake activity. The butanol fraction of the leaves (ABBF) was picked for the in vivo assay since it showed more activity in the initial tests conducted. ABBF was administrated via oral dosing to six-weeks old fructose-fed STZ-induced type 2 diabetic rats over a 5-week experimental period. RESULTS: ABBF treatment at a low dose of 150 mg/kg bw, significantly (p < .05) reduced blood glucose level, enhanced oral glucose tolerance ability, restored insulin secretion and hepatic glycogen synthesis as well as promoted islet regeneration than the high dose (300 mg/kg bw). CONCLUSION: These results suggest that ABBF could be exploited as a therapeutic potential for treating T2D.

Zebrafish as a new model for rhododendrol-induced leukoderma.

Idiopathic leukoderma is a skin disorder characterized by patchy loss of skin pigmentation due to melanocyte dysfunction or deficiency. Rhododendrol (RD) was approved as a cosmetic ingredient in Japan in 2008. However, it was shown to induce leukoderma in approximately 20,000 customers. The prediction of cytotoxicity, especially to melanocytes in vivo, is required to avoid such adverse effects. Since the use of higher vertebrates is prohibited for medicinal and toxicological assays, we used zebrafish, whose melanocytes were regulated by mechanisms similar to mammals. Zebrafish larvae were treated with RD in breeding water for 3 days, which caused body lightening accompanied by a decrease in the number of melanophores. Interestingly, black particles were found at the bottom of culture dishes, suggesting that the melanophores peeled off from the body. In addition, RT-PCR analysis suggested that the mRNA levels of melanophore-specific genes were significantly low. An increase in the production of reactive oxygen species was found in larvae treated with RD. The treatments of the fish with other phenol compounds, which have been reported to cause leukoderma, also induced depigmentation and melanophore loss. These results suggest that zebrafish larvae could be used for the evaluation of leukoderma caused by chemicals, including RD.

Nutrient composition and safety evaluation of simulated isobutanol distillers dried grains with solubles and associated fermentation metabolites when fed to male Ross 708 broiler chickens (Gallus domesticus).

Saccharomyces cerevisiae genetically engineered to enhance butanol production will be used in a manufacturing process similar to that of fuel ethanol production, including co-production of distillers products for animal feed. A poultry feeding trial was conducted with simulated isobutanol-derived dried distillers grains with solubles (bDDGS), comprising non-fermentable corn solids and heat-inactivated Butamax modified yeast (BMY), to determine potential health effects. Simulated dried distillers grains were produced in 2 variants: bDDGS containing 10% (B10) or 50% (B50) BMY. The BMY concentrations were selected based on a conservative estimate from ethanol-derived distillers grains (eDDGS) approximating 2.5 and 12-fold margins of exposure. The B10 and B50 DDGS were evaluated in a 42-day feeding trial using male Ross 708 broiler chickens fed diets containing eDDGS, B50 DDGS, or B10 DDGS without or with isobutanol, 2,3-butanediol, and isobutyric acid metabolites each at target concentrations of 2 (B10-2), 5 (B10-5), or 10 (B10-10) times the anticipated specification limit in the commercial product. Diets were fed (n = 50 broilers/treatment) in 3 phases: starter phase with 8% DDGS and grower and finisher phases each with 15% DDGS. No statistically significant differences or diet-related effects on mortality, clinical pathology, or organ weights, and no microscopic observations associated with consumption of diets containing B10, B50, or B10 supplemented with metabolites at any targeted exposure level were observed. A lower (P < 0.05) mean absolute bursa of Fabricius weight in the B10-10 group compared to the B10 group was considered to be within the range of biological variability. A non-significant trend toward lower weight, gains, and feed intake, and higher feed:gain ratio was observed in the B10-10 group, and was considered a non-adverse palatability effect of consuming high concentrations of metabolites. These results demonstrate that consumption of phase diets containing simulated DDGS from a novel isobutanol production process was well-tolerated.

Expression of Metazoan Annexins in Yeast Provides Protection Against Deleterious Effects of the Biofuel Isobutanol.

The ability of microorganisms to produce biofuels by fermentation is adversely affected by the perturbing effects of the hydrophobic biofuel on plasma membrane structure. It is demonstrated here that heterologous expression of metazoan, calcium-dependent, membrane-binding proteins of the annexin class can reduce deleterious effects of isobutanol on Saccharomyces cerevisiae viability and complex membrane functions. Therefore, expression of annexins in industrial strains of yeast or bacteria may prove beneficial in biofuel production.

Efficacy of oral cholecalciferol on rhododendrol-induced vitiligo: A blinded randomized clinical trial.

Rhododendrol (RD), 4-(4-hydroxyphenyl)-2-butanol, inhibits melanin synthesis and has been used for skin-whitening cosmetic products. RD has been very effective in lightening skin pigmentation, but some persons have developed so-called RD vitiligo, in which vitiligo starts on the face, neck and hands where topical RD has been applied and even extended over skin areas where RD has not been applied. RD vitiligo lesions in some patients have lasted for years and have been resistant to conventional vitiligo treatments. We examined the effects of cholecalciferol on RD vitiligo in a blinded randomized clinical trial. Forty-eight female RD vitiligo patients were recruited for the trial and were randomized into two groups: the vitamin D (VD)-intervention group that received daily 5000 IU cholecalciferol for 5 months and the control group. Three blinded investigators scored vitiligo improvement by comparing photographic images of baseline and at 5-month observation. Serum 25(OH)D3 of RD vitiligo patients was not significantly different from age-matched healthy volunteers. Twenty-two in the VD-intervention group and 23 in the control group completed the 5-month observation. Serum 25(OH)D3 levels were significantly increased after the 5-month VD intervention, while the control group did not change. The improvement scores were significantly higher in the VD-intervention group than the control group. The improvement scores were positively correlated with the serum 25(OH)D3 levels after the 5-month intervention period but not before the treatment. This blinded randomized clinical trial showed favor in administrating 5000 IU cholecalciferol daily to RD vitiligo patients.

Open-label pilot study to evaluate the effectiveness of topical bimatoprost on rhododendrol-induced refractory leukoderma.

Rhododendrol (RD), 4-(4-hydroxyphenyl)-2-butanol, inhibits melanin synthesis and had been used in skin-whitening cosmetic products until 2013. However, some individuals developed leukoderma on the skin where RD had been applied and have suffered from refractory depigmentation even after discontinuing RD application. Bimatoprost is a prostaglandin F2α analog and is often used for eyelash growth for cosmetic reasons as well as in the treatment of glaucoma. It was reported that bimatoprost induced skin pigmentation in addition to iris pigmentation as adverse effects. Therefore, we conducted an open-label single-center pilot study to evaluate the effectiveness of bimatoprost on refractory RD-induced leukoderma. Eleven Japanese female patients with skin type III who developed leukoderma on the exact or slightly extended area of skin where RD had been applied and gained a halt of enlargement of leukoderma or repigmentation on a part of the affected skin after discontinuation of RD were enrolled. Bimatoprost 0.03% solution was applied on the leukoderma once daily for 3 months, and then the frequency of application was increased to twice daily for the subsequent 3 months. Ten patients completed the 6-month course of bimatoprost application. In four patients, bimatoprost application brought slight improvement in RD-induced refractory leukoderma by dermatologists' evaluation. Because the number of enrolled patients was limited, further larger studies are necessary to better assess the effectiveness of bimatoprost in inducing repigmentation in patients with RD-induced refractory leukoderma.

Clinical and epidemiological analysis in 149 cases of rhododendrol-induced leukoderma.

Rhododendrol-induced leukoderma is an acquired depigmentation that develops mainly at the contact site after repeated use of skin-whitening cosmetics containing rhododendrol. In most cases, cessation of further depigmentation or occurrence of repigmentation is observed after discontinuing the use of cosmetics. However, some patients develop vitiligo vulgaris through the spread of depigmentation into the non-exposed areas. Our study aims to investigate the patient-specific factors that may affect the extent of depigmentation or repigmentation, as well as development of vitiligo vulgaris. The degree of depigmentation of the face, neck and hands where exposed to rhododendrol was scored using photographs over time. The relationships between depigmentation score at first visit/improvement rate of depigmentation score and patient demographics were evaluated and three important clinical observations were made. First, repigmentation of the face was superior compared with that of the hands and neck, suggesting a possible role for the migration and differentiation of melanocyte stem cells from hair follicles, as a mechanism of repigmentation. Second, the intensity of rhododendrol exposure did not contribute to differences in the severity of depigmentation. This suggested a possibility of underlying genetic susceptibility to melanocyte cytotoxicity or immune reaction. Third, depigmentation score at first visit and past history of atopic dermatitis were significantly high in patients who developed vitiligo vulgaris. This suggested that severe chemical damage of melanocytes by rhododendrol leads to a higher risk of developing vitiligo vulgaris through the possible involvement of an immune reaction. These clinical observations may help to further understand the pathogenesis of rhododendrol-induced leukoderma.

Glutathione maintenance is crucial for survival of melanocytes after exposure to rhododendrol.

Rhododendrol is a phenolic compound that shows a tyrosinase-dependent toxicity for melanocytes and occasionally induces a vitiligo-like skin depigmentation. The post-tyrosinase mechanisms determining melanocyte death or survival, however, are far from clear. Here, we find that rhododendrol treatment leads to a reduction in the levels of cellular glutathione but also induces a cellular antioxidant response that eventually increases glutathione levels. We further find that rhododendrol toxicity is enhanced when glutathione levels are experimentally reduced and alleviated when glutathione levels are increased. Hence, it appears that the size of the preexisting glutathione pool along with the capacity to supply glutathione via the antioxidant response determines whether melanocytes survive or die after rhododendrol exposure. It is conceivable, therefore, that rhododendrol-induced leukoderma depends on the capacity to maintain appropriate glutathione levels and that enhancement of glutathione levels may preserve a patient's melanocytes and potentially help in repigmentation.

Biochemical, cytological, and immunological mechanisms of rhododendrol-induced leukoderma.

Recently, an unexpected outbreak of patients with leukoderma occurred in Japan with the use of brightening/lightening cosmetics containing rhododendrol (RD). Patients developed leukoderma mostly on the skin sites repeatedly applied with RD, but some patients also had vitiligo-like lesions on the non-applied sites. RD is a tyrosinase-competitive inhibiting substance, thereby serving as an inhibitor of melanin synthesis. Upon inhibition of tyrosinase, RD is converted to new products such as tyrosinase-catalyzed hydroxyl-metabolite, which damage melanocytes. The melanocyte cell lysates seem to induce T-cell response. The frequencies of CD8+ T cells in both lesional skin and peripheral blood are significantly higher in the RD leukoderma as well as non-segmental vitiligo patients than in normal controls. In HLA-A*02:01 positive cases, circulating Melan-A-specific cytotoxic T cells can be detected at a high frequency. It is thus suggested that RD-induced leukoderma is induced by not only cytolysis of melanocytes but also subsequent immune reactions toward melanocytes.

An immune pathological and ultrastructural skin analysis for rhododenol-induced leukoderma patients.

As reported in the mass media on July 2013, numerous consumers who had used the cosmetic ingredient containing rhododendrol (4-(4-hydroxyphenyl)-2-butanol, Trade name; rhododenol), which is a melanin inhibitor isolated from Acer nikoense Maxim, released from Kanebo Cosmetics Inc. (Tokyo, Japan) noticed leukoderma patches on their face, neck and hands. We have experienced 32 cases that developed leukoderma after using such cosmetics so far and skin biopsy samples in some cases were obtained from both leukoderma and pigmented lesions. A histopathological analysis for skin lesions obtained from such patients notably showed basal hypo-pigmentation, melanin incontinence, and remaining melanocytes in most patients which is not relevant in vitiligo vulgaris. Subsequently, we comprehensively carried out immunohistochemical analyses of immune-competent cells infiltration to assess the effect of the cellular immune response to inducible hypopigmentation. Furthermore, detailed morphological observations performed by electron-microscopy notably showed the presence of melanocytes with only a small number of melanosomes, dermal fibroblasts containing melanosome globules and melanophages whereas no damage associated with melanosome transfer and the basal layer apparatus. These findings provide a cue to diagnose as rhododenol-induced leukoderma differentiate from vitiligo vulgaris and for rhododendrol to induce local immunity in addition to melanocyte damage.

Guide for medical professionals (i.e., dermatologists) for the management of Rhododenol-induced leukoderma.

Because some users develop depigmentation after the use of melanogenesis-inhibiting products containing the quasi-drug ingredient Rhododenol, Japanese Dermatological Association (JDA) established a Special Committee on the Safety of Cosmetics Containing Rhododenol on July 17, 2013 and management guide for dermatologists has been updated on the website in order to delineate the diagnostic criteria for Rhododenol-induced leukoderma and provides a broad guide for standard treatment based on current knowledge. This guide is produced on the basis of the guide (version 7) updated on June 20, 2014 in the website. Rhododenol-induced leukoderma refers to depigmentation of varying severity that develops after the use of cosmetics containing Rhododenol, mainly at the site of use. In most cases, repigmentation of part or all the affected area is evident after discontinuation. Histopathologically cellular infiltration around the hair follicles and melanophages are present in most cases. The number of melanocytes in the lesion is declined but not totally absent in most cases. Rhododenol itself is a good substrate for tyrosinase, resulting in the formation of Rhododenol metabolites (e.g., Rhododenol quinone). Melanocytes are damaged by Rhododenol metabolites during the subsequent metabolic process. The continued use of cosmetics containing Rhododenol thus induces tyrosinase activity-dependent cytotoxicity in melanocytes in the epidermis at application sites, resulting in decreasing the amount of melanin produced by melanocytes; the addition of some other factor to this process is believed to subsequently cause the decrease or disappearance of melanocytes themselves from the epidermis.

Diphenidol treatment of arrhythmias.

The antiarrhythmic activity of diphenidol, an antiemetic, has been demonstrated both in electrophysiologic studies of patiens and in experimental arrhythmias in animals. Accordingly, 18 patients with tachyarrhythmias were treated with intravenous diphenidol in doses of 0.5 to 1.5 mg/kg. In six patients with atrial arrhythmias, there was no notable effect. Similarly, 12 patients with premature ventricular contractions were treated and studied. In six of them, ectopic beats were abolished, at least transiently; in three the number of ventricular premature contractions decreased; in two there was no effect; and in one, the number of premature beats was increased. Of the total number of 18 patients, 14 suffered adverse effects related to the central nervous system. These adverse effects were of such severity as to suggest that further studies with diphenidol as an antiarrhythmic are not warranted.

A review of the environmental fate and aquatic effects of a series of C4 and C8 oxo-process chemicals.

Environmental fate and aquatic effects data were examined for a series of C4 (butyl acetate, 1-butanol, isobutyl alcohol) and C8 (2-ethylhexanol and 2-ethylhexanoic acid) oxo-process chemicals. Manufacturing of these chemicals requires enclosed equipment, so environmental releases are generally limited to volatilization during their use, handling or transport. C4 compounds are more soluble and volatile, and would bind to soil and sediment to a lesser extent than C8 compounds. All five compounds were readily biodegradable based on OECD and APHA tests conducted up to 28 days. Atmospheric photo-oxidation half-lives range from 0.43 to 3.8 days. Toxicity data show that all five compounds pose generally low concern to fish, invertebrates, algae, and microorganisms. Overall, the data show that inadvertent releases of these compounds into the environment would be rapidly biodegraded in soil and water, volatilize to the atmosphere subject to photo-oxidation, while any residues remaining in water would pose a negligible threat to aquatic life.