Elevated arsenic level in fasting serum via ingestion of fish meat increased the risk of hypertension in humans and mice.

Aims: There has been a shortage of human studies to elucidate the association between serum arsenic levels and the prevalence of hypertension. This study multidirectionally investigated associations among arsenic exposure, dietary ingestion, and the risk of hypertension by combined human epidemiological and mouse experimental studies. Methods and results: This study focused on the total arsenic level in fasting serum, a biomarker of arsenic exposure. Associations among ingestion frequencies of 54 diet items of Japanese food separated into six categories, total arsenic level in fasting serum, and the prevalence of hypertension were investigated in 2709 general people in Japan. Logistic regression analysis demonstrated a dose-dependent association between serum arsenic level and hypertension and a positive association between the ingestion of fish meat and hypertension. Further analysis showed that the latter association was fully mediated by increased fasting serum arsenic levels in humans. Similarly, oral exposure to the putative human-equivalent dose of arsenic species mixture with the same ratios in a common fish meat in Japan increased systolic blood pressure and arsenic levels in fasting serum in mice. Conclusion: This interdisciplinary approach suggests that fish-meat ingestion is a potential risk factor for arsenic-mediated hypertension. Because the increased consumption of fish meat is a recent global trend, health risks of the increased ingestion of arsenic via fish meat should be further investigated.

Calcitriol inhibits arsenic-promoted tumorigenesis through regulation of arsenic-uptake in a human keratinocyte cell line.

Chronic arsenic exposure from drinking water causes a variety of diseases and it is now recognized that at least 140 million people in 50 countries have been drinking water containing arsenic at levels above the WHO provisional guideline value of 10 µg/L. Long-term exposure to arsenic is associated with various types of cancers in humans including skin cancers. However, there is limited information on key molecules regulating arsenic-promoted carcinogenesis, and methods for the prevention and therapy of arsenic-promoted carcinogenesis have not yet been fully developed. Our in vitro study in human nontumorigenic HaCaT skin keratinocytes showed that calcitriol (activated vitamin D3, 1,25(OH)2D3) inhibited arsenic-mediated anchorage-independent growth with downregulations of cancer-related activation of MEK, ERK1/2 and AKT and activity of cell cycle. Moreover, calcitriol significantly repressed arsenic uptake in HaCaT cells with inhibition of expressions of aquaporin genes (AQP7, 9 and 10) which were modified by arsenic exposure. VDR, a vitamin D receptor, expression was significantly increased by arsenic exposure whereas calcitriol had no effect on its expression. These results suggest that treatment of calcitriol inhibits arsenic uptake via suppressions of aquaglyceroporin gene expressions resulting in inhibition of arsenic-promoted tumorigenesis in keratinocytes.

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.

Loss-of-function mutation of c-Ret causes cerebellar hypoplasia in mice with Hirschsprung disease and Down's syndrome.

The c-RET proto-oncogene encodes a receptor-tyrosine kinase. Loss-of-function mutations of RET have been shown to be associated with Hirschsprung disease and Down's syndrome (HSCR-DS) in humans. DS is known to involve cerebellar hypoplasia, which is characterized by reduced cerebellar size. Despite the fact that c-Ret has been shown to be associated with HSCR-DS in humans and to be expressed in Purkinje cells (PCs) in experimental animals, there is limited information about the role of activity of c-Ret/c-RET kinase in cerebellar hypoplasia. We found that a loss-of-function mutation of c-Ret Y1062 in PCs causes cerebellar hypoplasia in c-Ret mutant mice. Wild-type mice had increased phosphorylation of c-Ret in PCs during postnatal development, while c-Ret mutant mice had postnatal hypoplasia of the cerebellum with immature neurite outgrowth in PCs and granule cells (GCs). c-Ret mutant mice also showed decreased numbers of glial fibers and mitogenic sonic hedgehog (Shh)-positive vesicles in the external germinal layer of PCs. c-Ret-mediated cerebellar hypoplasia was rescued by subcutaneous injection of a smoothened agonist (SAG) as well as by reduced expression of Patched1, a negative regulator for Shh. Our results suggest that the loss-of-function mutation of c-Ret Y1062 results in the development of cerebellar hypoplasia via impairment of the Shh-mediated development of GCs and glial fibers in mice with HSCR-DS.

Hair graying with aging in mice carrying oncogenic RET.

Hair graying is a representative sign of aging in animals and humans. However, the mechanism for hair graying with aging remains largely unknown. In this study, we found that the microscopic appearance of hair follicles without melanocyte stem cells (MSCs) and descendant melanocytes as well as macroscopic appearances of hair graying in RET-transgenic mice carrying RET oncogene (RET-mice) are in accordance with previously reported results for hair graying in humans. Therefore, RET-mice could be a novel model mouse line for age-related hair graying. We further showed hair graying with aging in RET-mice associated with RET-mediated acceleration of hair cycles, increase of senescent follicular keratinocyte stem cells (KSCs), and decreased expression levels of endothelin-1 (ET-1) in bulges, decreased endothelin receptor B (Ednrb) expression in MSCs, resulting in a decreased number of follicular MSCs. We then showed that hair graying in RET-mice was accelerated by congenitally decreased Ednrb expression in MSCs in heterozygously Ednrb-deleted RET-mice [Ednrb(+/-);RET-mice]. We finally partially confirmed common mechanisms of hair graying with aging in mice and humans. Taken together, our results suggest that age-related dysfunction between ET-1 in follicular KSCs and endothelin receptor B (Ednrb) in follicular MSCs via cumulative hair cycles is correlated with hair graying with aging.

Increased levels of renal damage biomarkers caused by excess exposure to trivalent chromium in workers in tanneries.

BACKGROUND: The process for leather material production is carried out in developing countries using a large amount of trivalent chromium [Cr(III)]. Assesment of health risks for millions of workers in tanneries worldwide that are highly polluted with Cr(III) is needed. METHODS: Levels of total Cr and its chemical species in wastewater samples from tannery built-up areas of Bangladesh were investigated. Cr-mediated renal damage was assessed in 100 male tannery workers by epidemiological analysis consisting of questionnaires and measurements of levels of urinary Cr and urinary renal damage markers [urinary levels of total protein and kidney injury molecule-1 (KIM-1)]. RESULTS: High levels of total Cr (mean ± standard deviation = 1,908,762 ± 703,450 µg/L) were detected in wastewater samples from 13 sites of tanneries. More than 99.99% of total Cr in the wastewater was Cr(III), indicating that workers in the tanneries were exposed to large concentrations of Cr(III). Cr levels (mean ± standard, 2.89 ± 4.23 µg/g creatinine) in urine samples from the workers in tanneries were >24-fold higher than the levels in a general population previously reported. Multivariate analysis showed significant correlations between urinary levels of Cr and urinary levels of renal damage biomarkers. Nagelkerke Pseudo R2 values also showed that Cr level is the strongest contributor to the levels of renal damage biomarkers in the workers. CONCLUSION: Our results newly suggest that excess exposure to Cr(III) could be a risk for renal damage in humans.

Multidisciplinary approach to assess the toxicities of arsenic and barium in drinking water.

Well water could be a stable source of drinking water. Recently, the use of well water as drinking water has been encouraged in developing countries. However, many kinds of disorders caused by toxic elements in well drinking water have been reported. It is our urgent task to resolve the global issue of element-originating diseases. In this review article, our multidisciplinary approaches focusing on oncogenic toxicities and disturbances of sensory organs (skin and ear) induced by arsenic and barium are introduced. First, our environmental monitoring in developing countries in Asia showed elevated concentrations of arsenic and barium in well drinking water. Then our experimental studies in mice and our epidemiological studies in humans showed arsenic-mediated increased risks of hyperpigmented skin and hearing loss with partial elucidation of their mechanisms. Our experimental studies using cultured cells with focus on the expression and activity levels of intracellular signal transduction molecules such as c-SRC, c-RET, and oncogenic RET showed risks for malignant transformation and/or progression arose from arsenic and barium. Finally, our original hydrotalcite-like compound was proposed as a novel remediation system to effectively remove arsenic and barium from well drinking water. Hopefully, comprehensive studies consisting of (1) environmental monitoring, (2) health risk assessments, and (3) remediation will be expanded in the field of environmental health to prevent various disorders caused by environmental factors including toxic elements in drinking water.

Peptides containing the MXXCW motif inhibit oncogenic RET kinase activity with a novel mechanism of action.

REarranged during Transition (RET) is a tyrosine kinase associated with the development of several malignancies. Identification of RET kinase inhibitors promises valuable therapeutic tools for the intervention of RET-driven tumors. Most currently available tyrosine kinase inhibitors target the ATP binding site, but there are several drawbacks of these ATP-competitive drugs. Therefore, there is a need to develop new kinase inhibitors with alternative mechanisms of action. We have previously reported that a conserved cysteine in the MXXCW motif of RET is crucial to the disulfide-bonded dimerization-linked activation of RET kinases. Reagents which bind to this cysteine may inhibit the activity of RET kinases through disulfide-bond mediated dimerization. Here, we examine the potential of MXXCW motif-containing peptides as candidate kinase inhibitors. We demonstrate that MXXCW motif-containing peptides bind to RET in a redox-sensitive manner and block enzymatic activity, causing inhibition of the RET-dependent activity of extracellular signal-regulated kinases and effectively reducing the malignant potential of RET-papillary thyroid carcinoma-1 (PTC)-expressing cells. These motif-containing peptides were also found to be effective against the drug resistant mutant of RET. The inhibition of RET kinase activity by these peptides resulted in suppression of RET-PTC-1-mediated cancer growth. The great potency of these cysteine targeted peptides could indicate promising approaches for novel molecular-targeted therapies for RET-associated cancers.

A unique system that can sensitively assess the risk of chemical leukoderma by using murine tail skin.

Chemical leukoderma is a patchy hypopigmentation in the skin. Phenol derivatives such as raspberry ketone have been reported to cause the development of occupationally induced leukoderma. Recently, 2% (w/w) rhododenol, a reduced form of raspberry ketone used in a skin-lightning agent, also caused the development of leukoderma in >16,000 users, about 2% of all users, in Asian countries including Japan. However, a method for assessing the risk of leukoderma caused by 2% rhododenol has not been established despite the fact that the development of leukoderma caused by 30% rhododenol was previously shown in animal experiments. Establishment of a novel technique for risk assessment of leukoderma in humans caused by external treatment with chemicals is needed to prevent a possible future chemical disaster. This study demonstrated that external treatment with 2% rhododenol and the same concentration of raspberry ketone caused the development of leukoderma in murine tail skin without exception with significant decreases in the amount of melanin and number of melanocytes in the epidermis. Thus, a novel in vivo technique that can assess the risk of leukoderma caused by 2% rhododenol was developed. The unique technique using tail skin has the potential to prevent chemical leukoderma in the future.

Hearing loss in humans drinking tube well water with high levels of iron in arsenic-polluted area.

Well water for drinking with increased levels of iron in arsenic-polluted areas has been reported worldwide. Oral exposure to arsenic has been shown to be associated with hearing loss, while there is no evidence for an association between excessive exposure to iron and hearing loss in humans. In this study, we determined iron and arsenic levels in biological samples and hearing levels by pure tone audiometry (PTA) in subjects in a control area and an arsenic-polluted area in Bangladesh. The iron level in well water in the arsenic-polluted area was significantly higher than that in piped supply water in the control area. Subjects in the polluted area (n = 109), who had higher iron and arsenic levels in hair and toenails than those in subjects in the control area (n = 36), had an increased risk of hearing loss at 8 kHz and 12 kHz after adjustments for age, gender, smoking and BMI. Significant associations of the exposure group with hearing loss at 8 kHz and 12 kHz remained after further adjustment for arsenic levels in toenails and hair. Thus, this pilot study showed that excessive exposure to iron via drinking water is a potential risk for hearing loss in humans.

Chromium-mediated hyperpigmentation of skin in male tannery workers in Bangladesh.

Since tannery workers in developing countries are chronically exposed to high levels of chromium (Cr), there are serious concerns about health problems. However, there has been limited study in which Cr levels were measured in tannery workers, who are chronically exposed to Cr. Our preliminary inspection showed that there was hyperpigmented skin in tannery workers. We therefore investigated the correlation between skin pigmentation levels digitally evaluated as L* values by using a reflectance spectrophotometer and Cr levels in skin appendages in 100 male tannery workers and in 49 male non-tannery workers in Bangladesh. Digitalized skin pigmentation levels of the face and feet in addition to Cr levels in hair and toenails in tannery workers were significantly higher than those in non-tannery workers in our univariate analysis. Spearman's rank correlation coefficient analysis showed significant correlation between duration of tannery work (years) and Cr levels in hair (r = 0.62) and toenails (r = 0.61). Our multivariate analysis also showed that Cr levels in hair and toenails were significantly correlated with digitalized skin pigmentation levels of the face and feet in addition to duration of tannery work in all participants. Thus, our results showed the development of hyperpigmented skin in tannery workers. Our results also suggested that hyperpigmented skin could be a useful diagnostic marker for chronic exposure to Cr. Furthermore, cutaneous L* value might be a convenient marker for detection of chronic Cr poisoning, since the digitalized values enable objective evaluation of skin pigmented levels by general people as well as dermatologists.

Inhibition of mast cell degranulation by melanin.

Melanin is a dark naturally occurring pigment produced in nature and in many organisms. Although several reports have demonstrated applications for melanins in various therapeutic treatments, to date, no research has examined the anti-allergic effect of melanin. In this study, we for the first time found that solubilized or synthesized soluble melanin acts as a potent inhibitor of the degranulation of mast cells. We found that squid-ink-derived melanin significantly inhibited antigen-IgE-FcεRI-mediated degranulation of the mucosal mast cell line RBL-2H3. A homogenized melanin nanoparticle prepared by laser ablation also clearly suppressed antigen-induced mast cell degranulation. We also successfully solubilized synthetic melanin in a neutral biochemical buffer and found that it also significantly inhibited IgE-sensitized mast cells. The anti-degranulation activity of synthesized melanin was abolished in the melanin fraction below 50-kD molecular weight. All melanins used in this study did not exert significant cell death. Signal transduction analysis revealed that melanin suppressed antigen-triggered phosphorylation of signaling molecules as well as calcium influx. Transmission electron microscopy revealed that homogenized melanin nanoparticles partially attached to the cell surface and some nanoparticles were internalized to the cell. Flow cytometry revealed that the number of FcεRI-bound IgE molecules was decreased by melanin. Fluorescence recovery after photobleaching analysis indicated that melanin attenuated both plasma membrane and cytoplasmic fluidity, implying that melanin increased their viscosities. In vivo experiments using passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) mouse models demonstrated that oral administration of melanin accelerated the recovery of decreased body temperature after antigen infection in PSA, and combination sensitization of IgE with melanin attenuated antigen-induced extravasation in PCA. These findings indicated that melanin exhibits preventative effects against IgE-mast cell-mediated anaphylaxis. This study provides the first evidence that homogenized melanin may be a potential therapeutic agent for diseases involving mast cells.

Label-Free Imaging of Melanoma with Confocal Photothermal Microscopy: Differentiation between Malignant and Benign Tissue.

Label-free confocal photothermal (CPT) microscopy was utilized for the first time to investigate malignancy in mouse skin cells. Laser diodes (LDs) with 405 nm or 488 nm wavelengths were used as pumps, and a 638 nm LD was used as a probe for the CPT microscope. A Grey Level Cooccurrence Matrix (GLCM) for texture analysis was applied to the CPT images. Nine GLCM parameters were calculated with definite definitions for the intracellular super-resolved CPT images, and the parameters Entropy, Contrast, and Variance were found to be most suited among the nine parameters to discriminate clearly between healthy cells and malignant cells when a 405 nm pump was used. Prominence, Variance, and Shade were most suited when a pump wavelength of 488 nm was used.

A disadvantageous effect of adsorption of barium by melanin on transforming activity.

At present, beneficial effects of melanin and harmful effects of barium have been reported. However, little is known about the adsorption of barium, and even less is known about the biological significance of adsorption of barium by melanin. In this study, we showed that there was a strong correlation between the digitalized level of skin pigmentation and barium level in murine skin compared to the correlations between skin pigmentation level and levels of homologous elements of barium (magnesium, calcium and strontium). The concentration of subcutaneously injected barium in skin with a high level of pigmentation was higher than that in skin with a low level of pigmentation. Our cell-free experiment using the Langmuir isotherm for adsorption of barium in synthetic melanin also provided direct evidence of adsorption of barium by melanin. We then investigated the biological significance of melanin-mediated barium adsorption. We found barium-mediated increase in transforming activity in pigmented melanocytes (melan-a) but not in unpigmented melanocytes (melan-c) after confirming that the barium level in melan-a melanocytes was 3.4-fold higher than that in melan-c melanocytes after culture of 5 µM barium for 24 h. Taken together, our results not only indicate adsorption of barium by melanin in mice, cells and cell-free systems but also suggest a disadvantageous effect of adsorption of barium by melanin on transforming activity in cultured cells.

Arsenic level in toenails is associated with hearing loss in humans.

Arsenic (As) pollution in drinking water is a worldwide health risk for humans. We previously showed hearing loss in young people who live in areas of As-polluted drinking water and in young mice orally treated with As. In this study, we epidemiologically examined associations between As levels in toenails and hearing in 145 Bangladeshi aged 12-55 years in 2014. Levels of As in toenails, but not those in urine, were shown to be significantly correlated with hearing loss at 4 kHz [odds ratio (OR) = 4.27; 95% confidence interval (CI): 1.51, 12.05], 8 kHz (OR = 3.91; 95% CI: 1.47, 10.38) and 12 kHz (OR = 4.15; 95% CI: 1.55, 11.09) by multivariate analysis with adjustments for age, sex, smoking and BMI. Our experimental study further showed a significant association between As levels in inner ears and nails (r = 0.8113, p = 0.0014) in mice orally exposed to As, suggesting that As level in nails is a suitable index to assess As level in inner ears. Taken together, the results of our study suggest that As level in nails could be a convenient and non-invasive biomarker for As-mediated hearing loss in humans.

Manganese in toenails is associated with hearing loss at high frequencies in humans.

PURPOSE: Elevated hearing thresholds from high frequencies are known to be one of the hallmarks of age-related hearing loss. Our recent study showed accumulation of manganese (Mn) in inner ears resulting in acceleration of age-related hearing loss in mice orally exposed to Mn. However, there is no evidence showing an association between Mn in non-invasive biological samples and hearing loss in humans evaluated by pure tone audiometry (PTA). In this study, we evaluated Mn in non-invasive biological samples as a possible biomarker for hearing loss in humans. MATERIALS AND METHODS: We determined hearing levels by PTA and Mn levels in toenails, hair and urine with an inductively coupled plasma mass spectrometer (ICP-MS) in 145 healthy subjects in Bangladesh. RESULTS: Multivariable analyses showed that Mn levels in toenails, but not in hair and urine samples, were significantly associated with hearing loss at 8 kHz and 12 kHz. Moreover, our experimental study showed a significant correlation between Mn levels in inner ears and nails, but not hair, in mice orally exposed to Mn. CONCLUSIONS: The results provide novel evidence that Mn in toenails is a possible biomarker for hearing loss at high frequencies in humans.

A comprehensive study including monitoring, assessment of health effects and development of a remediation method for chromium pollution.

Chromium (Cr) pollution caused by wastewater from tanneries is a worldwide environmental problem. To develop a countermeasure, we performed a comprehensive study using Hazaribagh, the tannery area in Dhaka City, Bangladesh, as a model. Our environmental monitoring indicated that the soluble form of Cr, but not barium or arsenic, in Buriganga River is derived from Hazaribagh. Our chemical analysis next showed that Cr, the primary pollutant in canal water at Hazaribagh, consisted of ≤0.7 µM hexavalent Cr [Cr(VI)] and ≤1705 µM trivalent Cr [Cr(III)]. Our biological study then showed that coexposure to Cr(VI) and Cr(III) at possible ratios in canal water at Hazaribagh synergistically promotes transforming activity of human non-tumorigenic HaCaT keratinocytes with activated MEK/ERK and AKT. Our environmental engineering study finally indicated that a magnesium and iron-based hydrotalcite-like compound (MF-HT), our original depurative, can maximally adsorb 9.0 mg/g Cr(VI) and 1041 mg/g Cr(III). Our results suggested the importance of removal of Cr(III) as well as Cr(VI) by showing that Cr(III), which is generally recognized as a chemical with low toxicity, synergistically promoted carcinogenicity of a low level of Cr(VI). Therefore, we propose the use of our original high-efficient and low-cost depurative as a countermeasure to address the worldwide problem of environmental Cr pollution.

Increased expression level of Hsp70 in the inner ears of mice by exposure to low frequency noise.

Previous studies showed that people in urban areas are possibly exposed to 60-110 dB of low frequency noise (LFN) defined as noise of ≤100 Hz in their daily life. Previous studies also showed increased health risks by exposure to high levels (130-140 dB) of LFN in animals. However, little is known about the health effects of exposure to an ordinary level of LFN. We biochemically and immunohistochemically assessed the effects of exposure to inaudible LFN for mice (12 h/day of 100 Hz LFN at 95 dB for 5 days), at a level to which people are possibly exposed in daily life, on a murine inner ear by targeting 9 stress-reactive molecules. There was more than a 5-fold increased transcript level of heat shock protein 70 (Hsp70) in the whole inner ear exposed to LFN. However, the transcript levels of the other 8 stress-reactive molecules including Hsp27 and Hsp90 were comparable in LFN-exposed and unexposed murine inner ears. Only the transcript level of Cebpß among the previously reported 4 transcriptional activators for Hsp70 expression was more than 3-fold increased by LFN exposure. Hsp70 transcript expression levels in the inner ears 3 days after LFN exposure were comparable to those in unexposed inner ears. The protein level of Hsp70, but not the levels of Hsp27 and Hsp90, was also increased in the vestibule by LFN exposure. However, hearing levels as well as expression levels of Hsp70 protein in the cochleae were comparable in LFN-exposed mice and unexposed mice. Our results demonstrated that the inner ear might be one of the organs that is negatively affected by stress from inaudible LFN exposure. Moreover, LFN exposure might increase Hsp70 expression level via Cebpß in the inner ear. Thus, Hsp70 and Cebpß levels could be candidates of biomarkers for response to LFN exposure.

Arsenic levels in cutaneous appendicular organs are correlated with digitally evaluated hyperpigmented skin of the forehead but not the sole in Bangladesh residents.

There has been no report showing the effect of arsenic level on digitized skin pigmentation level, a typical diagnostic marker for arsenicosis. Correlations among history of drinking well water, arsenic levels in hair and toenails, and digitalized skin pigmentation levels (L*-value) in sunlight-exposed (forehead) and unexposed (sole) skin areas digitally evaluated by using a reflectance spectrophotometer were examined in 150 residents of Bangladesh. Univariate analysis showed that arsenic levels in hair and toenails of subjects with a history of drinking well water were 10.6-fold and 7.1-fold higher, respectively, than those in subjects without a history of drinking well water. The mean L*-value of foreheads, but not that of soles, in subjects with a history of drinking well water was 1.15-fold lower (more pigmented) than that in subjects without a history of drinking well water. Significant correlations were found between duration of drinking well water and arsenic concentrations in hair (r=0.63; P<0.01) and toenails (r=0.60; P<0.01). Multivariate analysis showed that the arsenic levels in hair and toenails and the duration of drinking well water were strongly correlated with the digitized pigmented level of the forehead but not that of the sole. An increase in the duration of drinking well water may increase hyperpigmentation in the forehead, but not that in the sole, through an increased arsenic level in the human body as shown in cutaneous appendicular organs (hair and toenails).