Distribution of cionin, a cholecystokinin/gastrin family peptide, and its receptor in the central nervous system of Ciona intestinalis type A.

The cholecystokinin (CCK)/gastrin family peptides are involved in regulation of feeding and digestion in vertebrates. In the ascidian Ciona intestinalis type A (Ciona robusta), cionin, a CCK/gastrin family peptide, has been identified. Cionin is expressed exclusively in the central nervous system (CNS). In contrast, cionin receptor expression has been detected in the CNS, digestive tract, and ovary. Although cionin has been reported to be involved in ovulation, its physiological function in the CNS remains to be investigated. To elucidate its neural function, in the present study, we analyzed the expression of cionin and cionin receptors in the CNS. Cionin was expressed mainly in neurons residing in the anterior region of the cerebral ganglion. In contrast, the gene expressin of the cionin receptor gene CioR1, was detected in the middle part of the cerebral ganglion and showed a similar expression pattern to that of VACHT, a cholinergic neuron marker gene. Moreover, CioR1 was found to be expressed in cholinergic neurons. Consequently, these results suggest that cionin interacts with cholinergic neurons as a neurotransmitter or neuromodulator via CioR1. This study provides insights into a biological role of a CCK/gastrin family peptide in the CNS of ascidians.

Multi-regional expression of pancreas-related digestive enzyme genes in the intestinal chamber of the ascidian Ciona intestinalis type A.

Bilateria share sequential steps in their digestive systems, and digestion occurs in a pre-absorption step within a chamber-like structure. Previous studies on the ascidian Ciona intestinalis type A, an evolutionary research model of vertebrate organs, revealed that Ciona homologs of pancreas-related exocrine digestive enzymes (XDEs) are exclusively expressed in the chamber-like bulging stomach. In the development of the gastrointestinal tract, genes for the pancreas-related transcription factors, namely Ptf1a, Nr5a2, and Pdx, are expressed near the stomach. Recent organ/tissue RNA-seq studies on two Ciona species reported that transcripts of the XDE homologs exist in the intestinal regions, as well as in the stomach. In the present study, we investigated the spatial gene expression of XDE homologs in the gastrointestinal region of the C. intestinalis type A. Whole-mount in situ hybridization using adult and juvenile specimens revealed apparent expression signals of XDE homologs in a small number of gastrointestinal epithelial cells. Furthermore, two pancreas-related transcription factor genes, Nr5a2 and Pdx, exhibited multi-regional expression along the Ciona juvenile intestines. These results imply that ascidians may form multiple digestive regions corresponding to the vertebrate pancreas.

Repetitive and zonal expression profiles of absorption-related genes in the gastrointestinal tract of ascidian Ciona intestinalis type A.

Intestinal absorption is essential for heterotrophic bilaterians with a tubular gut. Although the fundamental features of the digestive system were shared among chordates with evolution, the gut morphologies of vertebrates diverged and adapted to different food habitats. The ascidian Ciona intestinalis type A, a genome-wide research model of basal chordates, is used to examine the functional morphology of the intestines because of its transparent juvenile body. In the present study, the characteristic gene expression patterns (GEP) of Ciona absorptive proteins, e.g., brush border membrane enzymes for terminal digestion (lactase, maltase, APA, and APN) and transporters (SGLT1, GLUT5, PEPT1, and B0AT1), were investigated in juveniles and young adults, with a special reference to the absorption of other nutrients by pinocytosis- and phagocytosis-related proteins (megalin, cubilin, amnionless, Dab2, Rab7, LAMP, cathepsins, and MRC1). Whole-mount in situ hybridization revealed that these GEP showed multi-regional and repetitive features along the Ciona gastrointestinal tract, mainly in the stomach and several regions of the intestines. In young adults, many absorption-related genes, including pinocytosis-/phagocytosis-related genes, were also expressed between the stomach and mid-intestine. In the gastrointestinal epithelium, absorption-related genes showed zonal GEP along the epithelial structure. Comparisons of GEP, including other intestinal functions, such as nutrient digestion and intestinal protection, indicated the repetitive assignment of a well-coordinated set of intestinal GEP in the Ciona gastrointestinal tract.

Deep ocean water alters the cholesterol and mineral metabolism of squid Todarodes pacificus and suppresses its weight loss.

This study is the first to demonstrate that deep ocean water (DOW) has physiological significant effects on squid. After 36 h of rearing squids, those reared with DOW had significantly higher total and free cholesterol levels and lower alanine transaminase activity in hemolymph as compared with those reared with surface sea water (SSW). SSW rearing also resulted in 6.95% weight loss, while DOW rearing caused only 2.5% weight loss, which might be due to liver metabolism suppression. Furthermore, both monovalent (sodium, chloride, and potassium ions) and divalent (calcium, inorganic phosphorus, and magnesium ions) ions in hemolymph were elevated when reared with DOW compared to those when reared with SSW. A study of genes expressed in the brain revealed that five genes were specifically remarked in DOW rearing. Most altered genes were neuropeptides, including those from vasopressin superfamily. These neuropeptides are involved in cholesterol and/or mineral metabolisms and physiological significant effects on squid. This study is the first report the effects of DOW on cholesterol and mineral metabolism of squid and will contribute to squid aquaculture using DOW.

Kynurenine promotes Calcitonin secretion and reduces cortisol in the Japanese flounder Paralichthys olivaceus.

Deep ocean water (DOW) exerts positive effects on the growth of marine organisms, suggesting the presence of unknown component(s) that facilitate their aquaculture. We observed that DOW suppressed plasma cortisol (i.e., a stress marker) concentration in Japanese flounder (Paralichthys olivaceus) reared under high-density condition. RNA-sequencing analysis of flounder brains showed that when compared to surface seawater (SSW)-reared fish, DOW-reared fish had lower expression of hypothalamic (i.e., corticotropin-releasing hormone) and pituitary (i.e., proopiomelanocortin, including adrenocorticotropic hormone) hormone-encoding genes. Moreover, DOW-mediated regulation of gene expression was linked to decreased blood cortisol concentration in DOW-reared fish. Our results indicate that DOW activated osteoblasts in fish scales and facilitated the production of Calcitonin, a hypocalcemic hormone that acts as an analgesic. We then provide evidence that the Calcitonin produced is involved in the regulatory network of genes controlling cortisol secretion. In addition, the indole component kynurenine was identified as the component responsible for osteoblast activation in DOW. Furthermore, kynurenine increased plasma Calcitonin concentrations in flounders reared under high-density condition, while it decreased plasma cortisol concentration. Taken together, we propose that kynurenine in DOW exerts a cortisol-reducing effect in flounders by facilitating Calcitonin production by osteoblasts in the scales.

Adaptation to the shallow sea floor environment of a species of marine worms, Oligobrachia mashikoi, generally inhabiting deep-sea water.

Beard worms from the family Siboglinidae, are peculiar animals and are known for their symbiotic relationships with sulfur bacteria. Most Siboglinids inhabit the deep-sea floor, thus making difficult to make any observations in situ. One species, Oligobrachia mashikoi, occurs in the shallow depths (24.5 m) of the Sea of Japan. Taking advantage of its shallow-water habitat, the first ecological survey of O. mashikoi was performed over a course of 7 years, which revealed that its tentacle-expanding behavior was dependent on the temperature and illuminance of the sea water. Furthermore, there were significantly more O. mashikoi with expanding tentacles during the nighttime than during the daytime, and the prevention of light eliminated these differences in the number of expending tentacles. These results confirmed that the tentacle-expanding behavior is controlled by environmental light signals. Consistent with this, we identified a gene encoding a photoreceptor molecule, neuropsin, in O. mashikoi, and the expression thereof is dependent on the time of day. We assume that the described behavioral response of O. mashikoi to light signals represent an adaptation to a shallow-water environment within the predominantly deep-sea taxon.

Expression of Chrna9 is regulated by Tbx3 in undifferentiated pluripotent stem cells.

It was reported that nicotinic acetylcholine receptor (nAChR)-mediated signaling pathways affect the proliferation and differentiation of pluripotent stem cells. However, detail expression profiles of nAChR genes were unrevealed in these cells. In this study, we comprehensively investigated the gene expression of α subunit of nAChRs (Chrna) during differentiation and induction of pluripotent stem cells. Mouse embryonic stem (ES) cells expressed multiple Chrna genes (Chrna3-5, 7 and 9) in undifferentiated status. Among them, Chrna9 was markedly down-regulated upon the differentiation into mesenchymal cell lineage. In mouse tissues and cells, Chrna9 was mainly expressed in testes, ES cells and embryonal F9 teratocarcinoma stem cells. Expression of Chrna9 gene was acutely reduced during differentiation of ES and F9 cells within 24 h. In contrast, Chrna9 expression was increased in induced pluripotent stem cells established from mouse embryonic fibroblast. It was shown by the reporter assays that T element-like sequence in the promoter region of Chrna9 gene is important for its activities in ES cells. Chrna9 was markedly reduced by siRNA-mediated knockdown of Tbx3, a pluripotency-related transcription factor of the T-box gene family. These results indicate that Chrna9 is a nAChR gene that are transcriptionally regulated by Tbx3 in undifferentiated pluripotent cells.

Physiological consequences of space flight, including abnormal bone metabolism, space radiation injury, and circadian clock dysregulation: Implications of melatonin use and regulation as a countermeasure.

Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-h light-dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight.

Evolution of calcitonin/calcitonin gene-related peptide family in chordates: Identification of CT/CGRP family peptides in cartilaginous fish genome.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is a peptide gene family that is widely found in bilaterians. CT, CGRP, adrenomedullin (AM), amylin (AMY), and CT receptor-stimulating peptide (CRSP) are members of the CT/CGRP family. In mammals, CT is involved in calcium homeostasis, while CGRP and AM primarily function in vasodilation. AMY and CRSP are associated with anorectic effects. Diversification of the molecular features and physiological functions of the CT/CGRP family in vertebrate lineages have been extensively reported. However, the origin and diversification mechanisms of the vertebrate CT/CGRP family of peptides remain unclear. In this review, the molecular characteristics of CT/CGRP family peptides and their receptors, along with their major physiological functions in mammals and teleosts, are introduced. Furthermore, novel candidates of the CT/CGRP family in cartilaginous fish are presented based on genomic information. The CT/CGRP family peptides and receptors in urochordates and cephalochordates, which are closely related to vertebrates, are also described. Finally, a putative evolutionary scenario of the CT/CGRP family peptides and receptors in chordates is discussed.

Osteoclastic and Osteoblastic Responses to Hypergravity and Microgravity: Analysis Using Goldfish Scales as a Bone Model.

It is known that the bone matrix plays an important role in the response to physical stresses such as hypergravity and microgravity. In order to accurately analyze the response of bone to hypergravity and microgravity, a culture system under the conditions of coexistence of osteoclasts, osteoblasts, and bone matrix was earnestly desired. The teleost scale is a unique calcified organ in which osteoclasts, osteoblasts, and the two layers of bone matrix, i.e., a bony layer and a fibrillary layer, coexist. Therefore, we have developed in vitro organ culture systems of osteoclasts and osteoblasts with the intact bone matrix using goldfish scales. Using the scale culture system, we examined the effects of hypergravity with a centrifuge and simulated ground microgravity (g-µG) with a three-dimensional clinostat on osteoclasts and osteoblasts. Under 3-gravity (3G) loading for 1 day, osteoclastic marker mRNA expression levels decreased, while the mRNA expression of the osteoblastic marker increased. Upon 1 day of exposure, the simulated g-µG induced remarkable enhancement of osteoclastic marker mRNA expression, whereas the osteoblastic marker mRNA expression decreased. In response to these gravitational stimuli, osteoclasts underwent major morphological changes. By simulated g-µG treatments, morphological osteoclastic activation was induced, while osteoclastic deactivation was observed in the 3G-treated scales. In space experiments, the results that had been obtained with simulated g-µG were reproduced. RNA-sequencing analysis showed that osteoclastic activation was induced by the down-regulation of Wnt signaling under flight-microgravity. Thus, goldfish scales can be utilized as a bone model to analyze the responses of osteoclasts and osteoblasts to gravity.

Vasopressin-oxytocin-type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians.

Vasopressin/oxytocin (VP/OT)-related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the "platytocin" system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an "antidiuretic hormone" and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses.

Hydroxylated benzo[c]phenanthrene metabolites cause osteoblast apoptosis and skeletal abnormalities in fish.

To study the toxicity of 3-hydroxybenzo[c]phenanthrene (3-OHBcP), a metabolite of benzo[c]phenanthrene (BcP), first we compared it with its parent compound, BcP, using an in ovo-nanoinjection method in Japanese medaka. Second, we examined the influence of 3-OHBcP on bone metabolism using goldfish. Third, the detailed mechanism of 3-OHBcP on bone metabolism was investigated using zebrafish and goldfish. The LC50s of BcP and 3-OHBcP in Japanese medaka were 5.7 nM and 0.003 nM, respectively, indicating that the metabolite was more than 1900 times as toxic as the parent compound. In addition, nanoinjected 3-OHBcP (0.001 nM) induced skeletal abnormalities. Therefore, fish scales with both osteoblasts and osteoclasts on the calcified bone matrix were examined to investigate the mechanisms of 3-OHBcP toxicity on bone metabolism. We found that scale regeneration in the BcP-injected goldfish was significantly inhibited as compared with that in control goldfish. Furthermore, 3-OHBcP was detected in the bile of BcP-injected goldfish, indicating that 3-OHBcP metabolized from BcP inhibited scale regeneration. Subsequently, the toxicity of BcP and 3-OHBcP to osteoblasts was examined using an in vitro assay with regenerating scales. The osteoblastic activity in the 3-OHBcP (10-10 to 10-7 M)-treated scales was significantly suppressed, while BcP (10-11 to 10-7 M)-treated scales did not affect osteoblastic activity. Osteoclastic activity was unchanged by either BcP or 3-OHBcP treatment at each concentration (10-11 to 10-7 M). The detailed toxicity of 3-OHBcP (10-9 M) in osteoblasts was then examined using gene expression analysis on a global scale with fish scales. Eight genes, including APAF1, CHEK2, and FOS, which are associated with apoptosis, were identified from the upregulated genes. This indicated that 3-OHBcP treatment induced apoptosis in fish scales. In situ detection of cell death by TUNEL methods was supported by gene expression analysis. This study is the first to demonstrate that 3-OHBcP, a metabolite of BcP, has greater toxicity than the parent compound, BcP.

Effect of Omeprazole on Osteoblasts and Osteoclasts in vivo and in the in vitro Model Using Fish Scales.

Omeprazole suppresses excessive secretion of gastric acid via irreversible inhibition of H+/K+-ATPase in the gastric parietal cells. Recent meta-analysis of data revealed an association between the use of proton pump inhibitors (PPIs) and increased risk of bone fractures, but the underlying molecular mechanism of PPI action remains unclear. In this study, we demonstrated that omeprazole directly influences bone metabolism using a unique in vitro bioassay system with teleost scales, as well as the in vivo model. The in vitro study showed that omeprazole significantly increased the activities of alkaline phosphatase and tartrate-resistant acid phosphatase after 6 h of incubation with this PPI. Expression of mRNAs for several osteoclastic markers was upregulated after 3-h incubation of fish scales with 10-7 M omeprazole. The in vivo experiments revealed that the plasma calcium levels significantly increased in the omeprazole-treated group. The results of in vitro and in vivo studies suggest that omeprazole affects bone cells by increasing bone resorption by upregulating expression of osteoclastic genes and promoting calcium release to the circulation. The suggested in vitro bioassay in fish scales is a practical model that can be used to study the effects of drugs on bone metabolism.

Analyses of Molecular Characteristics and Enzymatic Activities of Ovine HSD17B3.

17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3) converts androstenedione (A4) into testosterone (T), which regulates sex steroid production. Because various mutations of the HSD17B3 gene cause disorder of sex differentiation (DSD) in multiple mammalian species, it is very important to reveal the molecular characteristics of this gene in various species. Here, we revealed the open reading frame of the ovine HSD17B3 gene. Enzymatic activities of ovine HSD17B3 and HSD17B1 for converting A4 to T were detected using ovine androgen receptor-mediated transactivation in reporter assays. Although HSD17B3 also converted estrone to estradiol, this activity was much weaker than those of HSD17B1. Although ovine HSD17B3 has an amino acid sequence that is conserved compared with other mammalian species, it possesses two amino acid substitutions that are consistent with the reported variants of human HSD17B3. Substitutions of these amino acids in ovine HSD17B3 for those in human did not affect the enzymatic activities. However, enzymatic activities declined upon missense mutations of the HSD17B3 gene associated with 46,XY DSD, affecting amino acids that are conserved between these two species. The present study provides basic information and tools to investigate the molecular mechanisms behind DSD not only in ovine, but also in various mammalian species.

Profiles of 5α-Reduced Androgens in Humans and Eels: 5α-Dihydrotestosterone and 11-Ketodihydrotestosterone Are Active Androgens Produced in Eel Gonads.

Although 11-ketotestosterone (11KT) and testosterone (T) are major androgens in both teleosts and humans, their 5α-reduced derivatives produced by steroid 5α-reductase (SRD5A/srd5a), i.e., 11-ketodihydrotestosterone (11KDHT) and 5α-dihydrotestosterone (DHT), remains poorly characterized, especially in teleosts. In this study, we compared the presence and production of DHT and 11KDHT in Japanese eels and humans. Plasma 11KT concentrations were similar in both male and female eels, whereas T levels were much higher in females. In accordance with the levels of their precursors, 11KDHT levels did not show sexual dimorphism, whereas DHT levels were much higher in females. It is noteworthy that plasma DHT levels in female eels were higher than those in men. In addition, plasma 11KDHT was undetectable in both sexes in humans, despite the presence of 11KT. Three srd5a genes (srd5a1, srd5a2a and srd5a2b) were cloned from eel gonads. All three srd5a genes were expressed in the ovary, whereas only both srd5a2 genes were expressed in the testis. Human SRD5A1 was expressed in testis, ovary and adrenal, whereas SRD5A2 was expressed only in testis. Human SRD5A1, SRD5A2 and both eel srd5a2 isoforms catalyzed the conversion of T and 11KT into DHT and 11KDHT, respectively, whereas only eel srd5a1 converted T into DHT. DHT and 11KDHT activated eel androgen receptor (ar)α-mediated transactivation as similar fashion to T and 11KT. In contrast, human AR and eel arß were activated by DHT and11KDHT more strongly than T and 11KT. These results indicate that in teleosts, DHT and 11KDHT may be important 5α-reduced androgens produced in the gonads. In contrast, DHT is the only major 5α-reduced androgens in healthy humans.

11-Ketotestosterone is a major androgen produced in porcine adrenal glands and testes.

Porcine steroid hormone profiles have some unique characteristics. We previously studied human and murine steroidogenesis using steroidogenic cells-derived from mesenchymal stem cells (MSCs). To investigate porcine steroidogenesis, we induced steroidogenic cells from porcine subcutaneous preadipocytes (PSPA cells), which originate from MSCs. Using cAMP, adenovirus-mediated introduction of steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP) induced the differentiation of PSPA cells into sex steroid-producing cells. Introducing SF-1/Ad4BP also induced the aldo-keto reductase 1C1 (AKR1C1) gene. Porcine AKR1C1 had 17ß-hydroxysteroid dehydrogenase activity, which converts androstenedione and 11-ketoandrostenedione into testosterone (T) and 11-ketotestosteorne (11KT). Furthermore, differentiated cells expressed hydroxysteroid 11ß-dehydrogenase 2 (HSD11B2) and produced 11KT. HSD11B2 was expressed in testicular Leydig cells and the adrenal cortex. 11KT was present in the plasma of both immature male and female pigs, with slightly higher levels in the male pigs. T levels were much higher in the male pigs. It is noteworthy that in the female pigs, the 11KT levels were >10-fold higher than the T levels. However, castration altered the 11KT and T plasma profiles in the male pigs to near those of the females. 11KT induced endothelial nitric oxide synthase (eNOS) in porcine vascular endothelial cells. These results indicate that 11KT is produced in porcine adrenal glands and testes, and may regulate cardiovascular functions through eNOS expression.

Expression of sclerostin in the regenerating scales of goldfish and its increase under microgravity during space flight.

Osteocytes, osteoblasts (bone-forming cells), and osteoclasts (bone-resorbing cells) are the primary types of cells that regulate bone metabolism in mammals. Sclerostin produced in bone cells activates osteoclasts, inhibiting bone formation; excess production of sclerostin, therefore, leads to the loss of bone mass. Fish scales have been reported to have morphological and functional similarities to mammalian bones, making them a useful experimental system for analyzing vertebrate bone metabolism in vitro. However, whether fish scales contain cells producing sclerostin and/or osteocytes has not been determined. The current study demonstrated, for the first time, that sclerostin-containing cells exist in goldfish scales. Analysis of the distribution and shape of sclerostin-expressing cells provided evidence that osteoblasts produce sclerostin in goldfish scales. Furthermore, our results found that osteocyte-like cells exist in goldfish scales, which also produce sclerostin. Finally, we demonstrated that microgravity in outer space increased the level of sclerostin in the scales of goldfish, a finding suggesting that the induction of sclerostin is the mechanism underlying the activation of osteoclasts under microgravity.

De novo transcriptome analysis and gene expression profiling of fish scales isolated from Carassius auratus during space flight: Impact of melatonin on gene expression in response to space radiation.

Astronauts are inevitably exposed to two major risks during space flight, microgravity and radiation. Exposure to microgravity has been discovered to lead to rapid and vigorous bone loss due to elevated osteoclastic activity. In addition, long­term exposure to low­dose­rate space radiation was identified to promote DNA damage accumulation that triggered chronic inflammation, resulting in an increased risk for bone marrow suppression and carcinogenesis. In our previous study, melatonin, a hormone known to regulate the sleep­wake cycle, upregulated calcitonin expression levels and downregulated receptor activator of nuclear factor­κB ligand expression levels, leading to improved osteoclastic activity in a fish scale model. These results indicated that melatonin may represent a potential drug or lead compound for the prevention of bone loss under microgravity conditions. However, it is unclear whether melatonin affects the biological response induced by space radiation. The aim of the present study was to evaluate the effect of melatonin on the expression levels of genes responsive to space radiation. In the present study, to support the previous data regarding de novo transcriptome analysis of goldfish scales, a detailed and improved experimental method (e.g., PCR duplicate removal followed by de novo assembly, global normalization and calculation of statistical significance) was applied for the analysis. In addition, the transcriptome data were analyzed via global normalization, functional categorization and gene network construction to determine the impact of melatonin on gene expression levels in irradiated fish scales cultured in space. The results of the present study demonstrated that melatonin treatment counteracted microgravity­ and radiation­induced alterations in the expression levels of genes associated with DNA replication, DNA repair, proliferation, cell death and survival. Thus, it was concluded that melatonin may promote cell survival and ensure normal cell proliferation in cells exposed to space radiation.

Oral administration of melatonin contained in drinking water increased bone strength in naturally aged mice.

Melatonin has recently been found to be a possible new regulator of bone metabolism. However, the influence of melatonin in natural age-related osteoporosis has not been fully elucidated yet, although there have been some reports regarding postmenopausal osteoporosis with melatonin treatments. The present study investigated the effects of long-term melatonin administration during the aging process on bone metabolism. Using quantitative computed tomography methods, we found that the total bone density of both the femur metaphysis and diaphysis decreased significantly in 20-month-old male mice. In the metaphysis, both trabecular bone mass and Polar-Strength Strain Index (SSI), which is an index of bone strength, decreased significantly. Judging from bone histomorphometry analysis, trabecular bone in 20-month-old male mice decreases significantly with age and is small and sparse, as compared to that of 4-month-old male mice. Loss of trabecular bone is one possible cause of loss of bone strength in the femoral bone. In the metaphysis, the melatonin administration group had significantly higher trabecular bone density than the non-administration group. The Polar-SSI, cortical area, and periosteal circumference in the diaphysis was also significantly higher with melatonin treatments. Since the melatonin receptor, MT2, was detected in both osteoblasts and osteoclasts of the femoral bone of male mice, we expect that melatonin acts on osteoblasts and osteoclasts to maintain the bone strength of the diaphysis and metaphysis. Thus, melatonin is a potential drug for natural age-related osteoporosis.

Influence of Benz[a]anthracene on Bone Metabolism and on Liver Metabolism in Nibbler Fish, Girella punctata.

It has been reported that spinal deformity was induced in developing fish by the addition of polycyclic aromatic hydrocarbons (PAHs). To examine the mechanism of the disruption of fish bone metabolism, the effect of benz[a]anthracene (BaA), a kind of PAH, on plasma calcium, inorganic phosphorus, osteoblasts, and osteoclasts was investigated in this study. We also measured several plasma components to analyze the toxicity of BaA on other metabolisms. BaA (1 or 10 ng/g body weight) was intraperitoneally injected (four times) into nibbler fish during breeding, for 10 days, and it was indicated, for the first time, that injecting high doses of BaA to nibbler fish induced both hypocalcemia and hypophosphatemia. Furthermore, in the scales of nibbler fish treated with high doses of BaA, both osteoclastic and osteoblastic marker messengerRNA (mRNA) expressions decreased. These results are a cause of disruption of bone metabolism and, perhaps, the induction of spinal deformities. In addition, we found that total protein, metabolic enzymes in the liver, total cholesterol, free cholesterol, and high-density lipoprotein cholesterol levels significantly decreased in BaA-injected fish. These results indicate that BaA may affect liver diseases and emphasize the importance of prevention of aquatic PAH pollution.