Acidified water promotes silver-induced toxicity in zebrafish embryos.

Freshwater acidification is a global environmental challenge, yet the effects of acidic water on fish resistance to toxic Ag+ remain an unexplored area. To address this knowledge gap, zebrafish embryos were exposed to different concentrations (0 (control), 0.1, and 0.25 mg/L) of AgNO3 under pH 5 or 7 for 7 days. Notably, AgNO3 at 0.25 mg/L resulted in 100 % mortality in both pH conditions, while AgNO3 at 0.1 mg/L resulted in higher mortality at pH 5 (85 %) compared to pH 7 (20 %), indicating that acidic water enhanced Ag+ toxicity. Several parameters, including body length, inner ear (otic vesicle and otolith) and yolk sac areas, lateral line hair cell number and morphology, the number of ionocytes (H+-ATP-rich cells and Na+/K+-ATP-rich cells), and ion contents (Ag+, Na+, and Ca2+) were assessed at 96 h (day 4) to investigate individual and combined effects of Ag+ and acid on embryos. Acid alone did not significantly alter most parameters, but it decreased the yolk sac area and increased the ionocyte number. Conversely, Ag+ alone caused reductions in most parameters, including body length, the inner ear area, hair cell number, and ionocyte number. Combining acid and Ag+ resulted in greater suppression of the otolith area, hair cell number, and Na+/Ca2+ contents. In conclusion, acidification of freshwater poses a potential risk to fish embryo viability by increasing their susceptibility to silver toxicity, specifically affecting sensory function and ion regulation.

Toxic effects of polystyrene nanoparticles on the development, escape locomotion, and lateral-line sensory function of zebrafish embryos.

Environmental pollution by micro- and nanosized plastic particles is a potential threat to aquatic animals. Polystyrene is one of the most common plastic particles in aquatic environments. Previous studies found that polystyrene nanoparticles (PNs) can penetrate the integument and accumulate in the organs of fish embryos. However, the potential impacts of PNs on fish embryos are not fully understood. To investigate this issue, zebrafish embryos were exposed to different concentrations (10, 25, and 50 mg/L) of PNs (25 nm) for 96 h (4-100 h post-fertilization), and various endpoints were examined, including developmental morphology (body length, sizes of the eyes, otic vesicles, otoliths, pericardial cavity, and yolk sac), locomotion (touch-evoked escape response and spinal motor neurons), and lateral-line function (hair cell number and hair bundle number). Exposure to 50 mg/L of PNs resulted in significant adverse effects across all endpoints studied, indicating that embryonic development was severely disrupted, and both locomotion and sensory function were impaired. However, at 25 mg/L of PNs, only locomotion and sensory function were significantly affected. The effects were insignificant in all examined endpoints at 10 mg/L of PNs. Transcript levels of several marker genes for neuronal function and eye development were suppressed after treatment. Exposure to fluorescent PNs showed that they accumulated in various organs including, the eyes, gills, blood vessels, gallbladder, gut, and lateral line neuromasts. Overall, this study suggests that short-term exposure to a high concentration of PNs can threaten fish survival by impairing embryonic development, locomotion performance, and mechanical sensory function.

Exposure to silver impairs the osmoregulatory capability of euryhaline medaka (Oryzias latipes) subjected to salinity changes.

The widespread use of silver in nanomaterials has led to increases in environmental contamination, which poses a threat to aquatic animals. Euryhaline fish, which live in environments with fluctuating salinity levels, have strong osmotic regulatory abilities to cope with such changes. This study attempted to investigate how silver affects the osmoregulatory capabilities of euryhaline fish, using medaka (Oryzias latipes) embryos as a model. The embryos were exposed to AgNO3 for 7 d in either fresh water (FW) or seawater (SW), and their mortality, heart rate, morphology, and ionocytes were examined. Results showed that the toxicity of AgNO3 was higher in FW than in SW (50% lethal concentrations (LC50) were 0.17 vs. 1.01 ppm). Although AgNO3 (0.05 and 0.1 ppm) did not significantly change the morphology of embryos, it impaired ionocytes and elevated heart rates in FW. While, AgNO3 (0.1 and 0.5 ppm) did not affect the morphology, ionocytes, or heart rate in SW, it impaired the hypo-osmoregulatory capability and elevated the mortality of embryos that were transferred from FW to SW. At 12 h after SW transfer, ionocytes were severely impaired, and water-drinking behavior was suppressed, resulting in body dehydration and sodium overload. In contrast, AgNO3 did not elevate the mortality of embryos that were transferred from SW to FW. To sum up, the presence of silver in FW during the developmental stage of euryhaline fish could potentially endanger their survival during SW adaptation.

Vincristine exposure impairs skin keratinocytes, ionocytes, and lateral-line hair cells in developing zebrafish embryos.

Environmental contamination by anticancer pharmaceuticals has been widely reported. These drugs are not readily biodegradable, and their parent compounds and/or metabolites have been detected in surface waters and groundwater throughout the world. Adverse effects of anticancer drugs occur frequently in cancer patients, and a large body of clinical knowledge has accumulated. However, the effects of these drugs on aquatic organisms have not been thoroughly studied. This study aimed to investigate the effects of acute exposure to a common anticancer drug, vincristine (VCR), on zebrafish embryonic development and skin function. After 96 h of VCR exposure (0, 1, 10, 15, and 25 mg/L), significant teratogenic effects were observed, including growth retardation, pericardial edema, spine, tail, and yolk sac malformations (VCR ≥ 15 mg/L), a decreased heart rate, and ocular malformations (VCR ≥ 10 mg/L). The value of the half lethal concentration for zebrafish embryos was 20.6 mg/L. At ≥10 mg/L VCR, systemic ion contents and acid secretion in the skin over the yolk-sac decreased, and these findings were associated with decreases in skin ionocytes (H+-ATPase-rich cells and Na+-K+-ATPase-rich cells). Also, the microridge-structure of skin keratinocytes was significantly damaged. The number of lateral line hair cells was reduced when VCR was ≥10 mg/L, and functional impairment was detected when VCR was as low as 1 mg/L. Results of this in vivo study in zebrafish embryos indicate that acute exposure to VCR can lead to developmental defects, impairment of skin functions, and even fish death.

Exposure to colistin impairs skin keratinocytes and lateral-line hair cells in zebrafish embryos.

Environmental contamination by antibiotics has become a global issue. Colistin, a cationic antimicrobial polypeptide, has been widely used in human/veterinary medicine, and growth promotion in aquaculture. However, no study has been conducted to test the toxic effects of colistin on aquatic animals. In this study, we examined the effects of colistin on zebrafish embryos. Zebrafish embryos were incubated in different concentrations (0, 0.01, 0.1, 1, 2, 3, and 10 µM) of colistin for 96 h. Colistin increased the mortality rate in a dose-dependent manner (LC50 was 3.0 µM or 3.5 mg L-1), but it did not change the hatching rate, heart rate, body length, eye size, or yolk size of embryos. However, colistin impaired keratinocytes and lateral-line hair cells in the skin of embryos. Colistin (at concentrations ≥0.1 µM) decreased the number of FM1-43-labeled hair cells and reduced the mechanotransduction-mediated Ca2+ influx at hair bundles, suggesting that sublethal concentrations of colistin can impair lateral line function. To investigate the lethal injury, morphological changes were sequentially observed in post-hatched embryos subjected to lethal concentrations of colistin. We found that skin keratinocytes were severely damaged and detached after exposure, leading to hypotonic swelling of the yolk sac, loss of ion contents, cell lysis, and eventual death. This study revealed that acute colistin exposure can impair skin cells and pose a threat to fish survival.

Exposure to copper nanoparticles impairs ion uptake, and acid and ammonia excretion by ionocytes in zebrafish embryos.

The potential toxicity of copper nanoparticles (CuNPs) to early stages of fishes is not fully understood, and little is known about their effects on ionocytes and associated functions. This study used zebrafish embryos as a model to investigate the toxic effects of CuNPs on two subtypes of ionocytes. Zebrafish embryos were exposed to 0.1, 1, and 3 mg L-1 CuNPs for 96 h. After exposure, whole-body Na+ and Ca2+ contents were significantly reduced at ≥0.1 mg L-1, while the K+ content had decreased at ≥1 mg L-1. H+ and NH4+ excretion by the skin significantly decreased at ≥1 mg L-1. The number of living ionocytes labeled with rhodamine-123 had significantly decreased with ≥0.1 mg L-1 CuNPs. The ionocyte subtypes of H+-ATPase-rich (HR) and Na+/K+-ATPase-rich (NaR) cells were labeled by immunostaining and had decreased with ≥1 mg L-1. Shrinkage of the apical opening of ionocytes was revealed by scanning electronic microscopy. Functional impairment was also reflected by changes in gene expressions, including ion transporters/channels and Ca2+-regulatory hormones. This study shows that CuNP exposure can impair two subtypes of ionocytes and their associated functions, including Na+/Ca2+ uptake and H+/NH4+ excretion in zebrafish embryos.

Characterization and toxicology evaluation of low molecular weight chitosan on zebrafish.

Chitosan is suggested as no or low toxicity and biocompatible biomaterial. Digestion of chitosan to reduce molecular weight and formulate nanoparticle was generally used to improve efficiency for DNA or protein delivery. However, the toxicity of low-molecular-weight chitosan (LMWCS) towards freshwater fishes has not been well evaluated. Here, we reported the toxic mechanism of LMWCS using zebrafish (Danio rerio) liver (ZFL) cell line, zebrafish larvae, and adult fish. LMWCS rapidly induced cytotoxicity of ZFL cells and death of zebrafish. Cell membrane damaged by LMWCS reduced cell viability. Damaged membrane of epithelial cell in zebrafish larvae induced breakage of the yolk. Adult fish exhibited hypoxia before death due to multiple damages induced by LMWCS. Although the toxicity of LMWCS was revealed in zebrafish model, the toxicity was only present in pH < 7 and easy be neutralized by other negative ions. Collectively, these data improved a new understanding of LMWCS properties.

Acidified water impairs the lateral line system of zebrafish embryos.

Acidification of freshwater ecosystems is recognized as a global environmental problem. However, the influence of acidic water on the early stages of freshwater fish is still unclear. This study focused on the sublethal effects of acidic water on the lateral line system of zebrafish embryos. Zebrafish embryos were exposed to water at different pH values (pH 4, 5, 7, 9, and 10) for 96 (0-96 h post-fertilization (hpf)) and 48 h (48∼96 hpf). The survival rate, body length, and heart rate significantly decreased in pH 4-exposed embryos during the 96-h incubation. The number of lateral-line neuromasts and the size of otic vesicles/otoliths also decreased in pH 4-exposed embryos subjected to 96- and 48-h incubations. The number of neuromasts decreased in pH 5-exposed embryos during the 96-h incubation. Alkaline water (pH 9 and 10) did not influence embryonic development but suppressed the hatching process. The mechanotransducer channel-mediated Ca2+ influx was measured to reveal the function of lateral line hair cells. The Ca2+ influx of hair cells decreased in pH 5-exposed embryos subjected to the 48-h incubation, and both the number and Ca2+ influx of hair cells had decreased in pH 5-exposed embryos after 96 h of incubation. In addition, the number and function of hair cells were suppressed in H+-ATPase- or GCM2-knockdown embryos, which partially lost the ability to secrete acid into the ambient water. In conclusion, this study suggests that lateral line hair cells are sensitive to an acidic environment, and freshwater acidification could be a threat to the early stages of fishes.

Incidence of soft tissue sarcoma in Taiwan: A nationwide population-based study (2007-2013).

BACKGROUND: Asian studies on soft tissue sarcoma (STS) incidence, irrespective of the primary site, are scant. METHODS: STS data were acquired from the population-based 2007-2013 Taiwan Cancer Registry of the Health and Welfare Data Science Center, Taiwan. Histological subtype-, site-, sex-, and age-specific STS incidence rates were analyzed according to the 2013 classification of the World Health Organization. RESULTS: In total, 11,393 patients with an age-standardized incidence rate (ASIR) of 5.62 (95% confidence interval, 5.51-5.73) per 100,000 person-years were identified. Overall, a male predominance (sex-standardized incidence rate ratio, 1.2) was noted, and the rate increased with age, peaking at >75 years. Approximately 30% of STSs occurred in connective, subcutaneous, and other soft tissues and 70% in other sites. In addition to connective, subcutaneous, and other soft tissues, the three most common primary sites were the stomach (15.9%), skin (14.3%), and small intestines (10.5%). Gastrointestinal stromal tumor was the most common subtype (29.2%; ASIR, 1.55/100,000 person-years), followed by liposarcoma (11.5%; ASIR, 0.63/100,000 person-years) and leiomyosarcoma (9.7%; ASIR, 0.53/100,000 person-years). Compared with relevant data from Western countries, the incidence rate of angiosarcomas was higher than that in other regions, whereas the incidence rates of leiomyosarcoma and Kaposi sarcoma were lower than those in other regions. CONCLUSION: STS incidence varied by histological subtype, sex, age, and primary site in an Asian population. Our results suggested regional and racial discrepancies in the incidence rates of certain STS subtypes.

Systemic Epstein-Barr Virus-positive T-Cell Lymphoma of Childhood Presentation With Hemophagocytosis.

A 2-year-old Asian girl presented to our facility for the evaluation of thrombocytopenia. She was treated with intravenous immunoglobulin under the impression of immune thrombocytopenia. However, her body temperature spiked and progressive pancytopenia, hepatosplenomegaly, abnormal liver function, coagulopathy, and pulmonary infiltration developed. The final diagnosis was systemic Epstein-Barr virus (EBV)-positive T-cell lymphoma of childhood with hemophagocytic syndrome. This type of cancer is extremely rare but occurs more commonly in Asians. Its prognosis is generally poor, and a treatment strategy is yet to be established. Double staining for EBV-encoded RNA and CD3 or CD8 is crucial for diagnosis. This type of lymphoma must be diagnosed differentially from acute EBV-associated hemophagocytic lymphohistiocytosis, which is considered nonmalignant. This case report highlights the importance of awareness of this type of rare cancer, a comprehensive diagnostic approach, and close communication between primary care physicians and pathologists.

Role of Calcium-Sensing Receptor in Mechanotransducer-Channel-Mediated Ca2+ Influx in Hair Cells of Zebrafish Larvae.

The calcium-sensing receptor (CaSR) is an extracellular Ca2+ sensor that plays a critical role in maintaining Ca2+ homeostasis in several organs, including the parathyroid gland and kidneys. In this study, through in situ hybridization, the expression of CaSR mRNA was found in the neuromasts of zebrafish larvae. Immunohistochemistry further demonstrated that the CaSR protein was present in neuromast hair cell stereocilia and basolateral membranes. Based on the expression and subcellular localization of the CaSR in hair cells, we hypothesized that the CaSR is expressed in zebrafish lateral-line hair cells to regulate mechanotransducer (MET)-channel-mediated Ca2+ entry. Using the scanning ion-selective electrode technique, MET-channel-mediated Ca2+ influx at the stereocilia of hair cells was measured in intact larvae. Ca2+ influx was suppressed after larvae were pretreated with a CaSR activator (R-568) or high-Ca2+ (HCa) medium. Gene knockdown by using morpholino oligonucleotides decreased CaSR expression in hair cells and eliminated the effects of R-568 and HCa on Ca2+ influx. In addition, we found that treatment with R-568 attenuated neomycin-induced hair cell death. This study is the first to demonstrate that the CaSR is involved in mechanotransduction in zebrafish hair cells.

Hepatoblastoma incidence in Taiwan: A population-based study.

BACKGROUND: The incidence of hepatoblastoma is not well known in Taiwan. The goal of this study was to investigate the incidence rates of hepatoblastoma by age and sex. METHODS: The data of patients with hepatoblastoma diagnosed from 1995 to 2012 were obtained from the population-based Taiwan Cancer Registry. Incidence rates of hepatoblastoma according to sex and age were analyzed. This study employed the published methods of International Agency for Research on Cancer to calculate the age-standardized incidence rates (ASIRs), standard errors, 95% confidence intervals (CIs), and standardized incidence rate ratios (SIRRs). RESULTS: In total, 211 patients were diagnosed with hepatoblastoma during the 18-year study period. The ASIR was 0.76 per million person-years. Hepatoblastoma was predominantly diagnosed in children (n = 184, 87.2%). By contrast, adolescents/adults (n = 10, 4.7%) and elderly people (n = 17, 8.1%) were rarely affected. The incidence peaked at ages 0-4 years with corresponding ASIR of 7.3 per million person-years. A significant male predilection was only found in children and elderly people, with male-to-female SIRRs of 1.23 and 1.89, respectively. During 1995-2012, the overall incidence of hepatoblastoma significantly increased only in children (annual percent change: 7.4%, 95% CI 3.9%-11.1%, p < 0.05) and specifically in boys (annual percent change: 6.5%, 95% CI 1.9%-11.2%, p < 0.05). CONCLUSION: Only 27 patients aged ≥ 15 years with hepatoblastoma were identified in this study, the existence of adult hepatoblastoma still requires novel molecular tools to elucidate. The association between the upward trend of hepatoblastoma incidence in boys and increased survival of prematurity in Taiwan warrants further investigations.

Cancer in adolescents: Incidences and trends during 1995-2009 in Taiwan.

This study aimed to describe cancer incidence rates and trends specifically for adolescents aged 15-19 years during 1995-2009 in Taiwan. The incidence counts and census data were obtained from the population-based Taiwan Cancer Registry. During the 15-year study period, 4122 adolescents were diagnosed with cancer. The overall incidence rate was 155.2 per million person-years. Other epithelial tumors were the most frequently diagnosed cancer group (23.7%), followed by leukemias (18.0%) and lymphomas (13.9%). When compared to rates in Western countries, a significantly low rate of lymphomas was found. Moreover, rates of the subtypes of melanomas and nasopharyngeal carcinomas being 1/10- and 4-times rates in Western countries were the most striking variations. During 1995-2009, the overall rate of adolescent cancer did not significantly change. However, the most significant upward and declining trends in incidence rates were found for male germ cell neoplasms (annual percent change, APC, 6.4%) and hepatic tumors (APC, -11.1%), respectively. Further investigation and enhancement of the public discourse of possible lifestyle and environmental risk factors associated with increasing trends of certain adolescent cancers should be carried out in Taiwan.

Changing incidence patterns of hepatocellular carcinoma among age groups in Taiwan.

BACKGROUND & AIMS: This study examined and compared the incidence patterns of hepatocellular carcinoma among age groups in Taiwan, 30 years after a universal hepatitis B virus immunization program was launched. METHODS: Data for hepatocellular carcinoma diagnosed in 2003-2011 were collected from the population-based Taiwan Cancer Registry. Age-standardized incidence rates were calculated to analyze and compare the changes in incidence rates and trends. More specific analyses were performed on four age groups separated by sex. RESULTS: A total of 82,856 patients were diagnosed with hepatocellular carcinoma in 2003-2011 in Taiwan, yielding an age-standardized incidence rate of 32.97 per 100,000 person-years. Hepatocellular carcinoma was predominantly diagnosed in middle-aged adults (50.1%) and elderly people (49.1%), in contrast to the low incidences in children (0.04%) and adolescents and young adults (0.8%). Striking variations in trends were found for children (annual percent change: -16.6%, 2003-2010) and adolescents and young adults (annual percent change: -7.9%, 2003-2011). The incidence rate of hepatocellular carcinoma in children decreased to zero in 2011; only a slight decline in trends occurred for the middle-aged group (annual percent change: -2%, 2003-2011), and a slight upward trend was observed for elderly people (1.3%), specifically in women (1.7%). CONCLUSIONS: In Taiwan, hepatitis B virus-related hepatocellular carcinoma was nearly eradicated in children in 2011. The findings on age-specific incidence patterns and trends of hepatocellular carcinoma suggest that different control strategies for treating this devastating disease in the future be made according to age.

Aquaporin 1 Is Involved in Acid Secretion by Ionocytes of Zebrafish Embryos through Facilitating CO2 Transport.

Mammalian aquaporin 1 (AQP1) is well known to function as a membrane channel for H2O and CO2 transport. Zebrafish AQP1a.1 (the homologue of mammalian AQP1) was recently identified in ionocytes of embryos; however its role in ionocytes is still unclear. In this study, we hypothesized that zebrafish AQP1a.1 is involved in the acid secretion by ionocytes through facilitating H2O and CO2 diffusion. A real-time PCR showed that mRNA levels of AQP1a.1 in embryos were induced by exposure to 1% CO2 hypercapnia for 3 days. In situ hybridization and immunohistochemistry showed that the AQP1a.1 transcript was highly expressed by acid-secreting ionocytes, i.e., H+-ATPase-rich (HR) cells. A scanning ion-selective electrode technique (SIET) was applied to analyze CO2-induced H+ secretion by individual ionocytes in embryos. H+ secretion by HR cells remarkably increased after a transient loading of CO2 (1% for 10 min). AQP1a.1 knockdown with morpholino oligonucleotides decreased the H+ secretion of HR cells by about half and limited the CO2 stimulated increase. In addition, exposure to an AQP inhibitor (PCMB) for 10 min also suppressed CO2-induced H+ secretion. Results from this study support our hypothesis and provide in vivo evidence of the physiological role of AQP1 in CO2 transport.

A new model for fish ion regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes).

The ion regulation mechanisms of fishes have been recently studied in zebrafish (Danio rerio), a stenohaline species. However, recent advances using this organism are not necessarily applicable to euryhaline fishes. The euryhaline species medaka (Oryzias latipes), which, like zebrafish, is genetically well categorized and amenable to molecular manipulation, was proposed as an alternative model for studying osmoregulation during acclimation to different salinities. To establish its suitability as an alternative, the present study was conducted to (1) identify different types of ionocytes in the embryonic skin and (2) analyze gene expressions of the transporters during seawater acclimation. Double/triple in situ hybridization and/or immunocytochemistry revealed that freshwater (FW) medaka contain three types of ionocyte: (1) Na(+)/H(+) exchanger 3 (NHE3) cells with apical NHE3 and basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC), Na(+)-K(+)-ATPase (NKA) and anion exchanger (AE); (2) Na(+)-Cl(-) cotransporter (NCC) cells with apical NCC and basolateral H(+)-ATPase; and (3) epithelial Ca(2+) channel (ECaC) cells [presumed accessory (AC) cells] with apical ECaC. On the other hand, seawater (SW) medaka has a single predominant ionocyte type, which possesses apical cystic fibrosis transmembrane conductance regulator (CFTR) and NHE3 and basolateral NKCC and NKA and is accompanied by smaller AC cells that express lower levels of basolateral NKA. Reciprocal gene expressions of decreased NHE3, AE, NCC and ECaC and increased CFTR and NKCC in medaka gills during SW were revealed by quantative PCR analysis.

Extracellular Ca(2+) and Mg(2+) modulate aminoglycoside blockade of mechanotransducer channel-mediated Ca(2+) entry in zebrafish hair cells: an in vivo study with the SIET.

Zebrafish lateral-line hair cells are an in vivo model for studying hair cell development, function, and ototoxicity. However, the molecular identification and properties of the mechanotransducer (MET) channel in hair cells are still controversial. In this study, a noninvasive electrophysiological method, the scanning ion-electrode technique (SIET), was applied for the first time to investigate properties of MET channels in intact zebrafish embryos. With the use of a Ca(2+)-selective microelectrode to deflect hair bundles and simultaneously record the Ca(2+) flux, the inward Ca(2+) flux was detected at stereocilia of hair cells in 2- to ~4-day postfertilization embryos. Ca(2+) influx was blocked by MET channel blockers (BAPTA, La(3+), Gd(3+), and curare). In addition, 10 µM aminoglycoside antibiotics (neomycin and gentamicin) were found to effectively block Ca(2+) influx within 10 min. Elevating the external Ca(2+) level (0.2-2 mM) neutralized the effects of neomycin and gentamicin. However, elevating the Mg(2+) level up to 5 mM neutralized blockade by gentamicin but not by neomycin. This study demonstrated MET channel-mediated Ca(2+) entry at hair cells and showed that the SIET to be a sensitive approach for functionally assaying MET channels in zebrafish.

Rhcg1 and Rhbg mediate ammonia excretion by ionocytes and keratinocytes in the skin of zebrafish larvae: H+-ATPase-linked active ammonia excretion by ionocytes.

In zebrafish, Rhcg1 was found in apical membranes of skin ionocytes [H⁺-ATPase-rich (HR) cells], which are similar to α-type intercalated cells in mammalian collecting ducts. However, the cellular distribution and role of Rhbg in zebrafish larvae have not been well investigated. In addition, HR cells were hypothesized to excrete ammonia against concentration gradients. In this study, we attempted to compare the roles of Rhbg and Rhcg1 in ammonia excretion by larval skin and compare the capability of skin cells to excrete ammonia against concentration gradients. Using in situ hybridization and immunohistochemistry, Rhbg was localized to both apical and basolateral membranes of skin keratinocytes. A scanning ion-selective electrode technique (SIET) was applied to measure the NH4⁺ flux at the apical surface of keratinocytes and HR cells. Knockdown of Rhbg with morpholino oligonucleotides suppressed ammonia excretion by keratinocytes and induced compensatory ammonia excretion by HR cells. To compare the capability of cells to excrete ammonia against gradients, NH4⁺ flux of cells was determined in larvae exposed to serial concentrations of external NH4⁺. Results showed that HR cells excreted NH4⁺ against higher NH4⁺ concentration than did keratinocytes. Knockdown of the expression of either Rhcg1 or H⁺ -ATPase in HR cells suppressed the capability of HR cells.

Acid secretion by mitochondrion-rich cells of medaka (Oryzias latipes) acclimated to acidic freshwater.

In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/immunocytochemistry, the Na(+)/H(+) exchanger 3 (NHE3) and H(+)-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H(+)-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H(+)-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation; however, the mRNA level of NHE3 was remarkably higher than that of H(+)-ATPase. A scanning ion-selective electrode technique was used to measure H(+), Na(+), and NH(4)(+) transport by individual MR cells in larval skin. Results showed that Na(+) uptake and NH(4)(+) excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na(+) uptake/NH(4)(+) excretion mechanism plays a critical role in acidic equivalent (H(+)/NH(4)(+)) excretion by MR cells of the freshwater medaka.

Functional regulation of H+-ATPase-rich cells in zebrafish embryos acclimated to an acidic environment.

It is important to maintain internal pH homeostasis in biological systems. In our previous studies, H(+)-ATPase-rich (HR) cells were found to be responsible for proton secretion in the skin of zebrafish embryos during development. In this study, zebrafish embryos were exposed to acidic and basic waters to investigate the regulation of HR cell acid secretion during pH disturbances. Our results showed that the function of HR cells on the skin of zebrafish embryos can be upregulated in pH 4 water not only by increasing the cell number but also by enlarging the acid-secreting function of single cells. We also identified an "alveolar-type" apical opening under scanning electron microscopy observations of the apical membrane of HR cells, and the density and size of the alveolar type of apical openings were also increased in pH 4 water. p63 and PCNA immunostaining results also showed that additional HR cells in pH 4 water may be differentiated not only from ionocyte precursor cells but also newly proliferating epithelial stem cells.