Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

Sardine procalcitonin amino-terminal cleavage peptide has a different action from calcitonin and promotes osteoblastic activity in the scales of goldfish.

The nucleotide sequence of a sardine preprocalcitonin precursor has been determined from their ultimobranchial glands in the present study. From our analysis of this sequence, we found that sardine procalcitonin was composed of procalcitonin amino-terminal cleavage peptide (N-proCT) (53 amino acids), CT (32 amino acids), and procalcitonin carboxyl-terminal cleavage peptide (C-proCT) (18 amino acids). As compared with C-proCT, N-proCT has been highly conserved among teleosts, reptiles, and birds, which suggests that N-proCT has some bioactivities. Therefore, both sardine N-proCT and sardine CT were synthesized, and their bioactivities for osteoblasts and osteoclasts were examined using our assay system with goldfish scales that consisted of osteoblasts and osteoclasts. As a result, sardine N-proCT (10-7M) activated osteoblastic marker enzyme activity, while sardine CT did not change. On the other hand, sardine CT (10-9 to 10-7M) suppressed osteoclastic marker enzyme activity, although sardine N-proCT did not influence enzyme activity. Furthermore, the mRNA expressions of osteoblastic markers such as type 1 collagen and osteocalcin were also promoted by sardine N-proCT (10-7M) treatment; however, sardine CT did not influence their expressions. The osteoblastic effects of N-proCT lack agreement. In the present study, we can evaluate exactly the action for osteoblasts because our scale assay system is very sensitive and it is a co-culture system for osteoblasts and osteoclasts with calcified bone matrix. Both CT and N-proCT seem to influence osteoblasts and osteoclasts and promote bone formation by different actions in teleosts.

Development of bioluminescent enzyme immunoassay for s-equol using firefly luciferase and its application to the assessment of equol-producer status.

In this study, we developed a specific bioluminescent enzyme immunoassay (BLEIA) for S-equol, employing firefly luciferase as a labeling enzyme, as an alternative to HPLC methods. Satisfactory correlation (r=0.992) was shown when this S-equol BLEIA was compared with HPLC. The cross-reactivity with R-equol as its diastereoisomer is <5%, and that with daidzein, which is the substrate of equol, is 0.02%. Frequencies of Japanese equol producers determined using two distinct approaches were compared: a threshold value for urinary S-equol concentration of 232 ng/ml gave frequencies of 32% of men and 19% of women. These values correspond to the results for log(10)-transformed urinary S-equol to daidzein ratio threshold of -1.75, namely, 34% of men and 19% of women. When the changes in concentration of urinary equol and daidzein were measured after ingestion of isoflavone, the maximum concentration (C(max)) of urinary equol appeared after 9.6 h of isoflavone consumption; this C(max) was 2 h later than that for daidzein. The S-equol BLEIA documented in this study is expected to be an important tool for the assessment of equol producer status and demonstration of the bioavailability of isoflavone.

Specificity assessment of immunoassay kits for determination of urinary free cortisol concentrations.

BACKGROUND: In immunoassay kits for determination of urinary free cortisol (UFC) concentrations, the results vary markedly from kit to kit, so we compared in this study the reaction specificity among 4 commercially available immunoassay kits to determine the applicability of these assays in routine determination of UFC concentrations. METHOD: Using 4 commercially available kits, cross-reaction was investigated. In addition, urine samples were fractionated by HPLC to investigate endogenous immunoreactive cortisol responses. HPLC fractions were subjected to gas chromatography-mass spectrometry (GCMS) to identify substances causing inter-kit assay discrepancies. RESULTS: Among the 4 kits, cortisol Kit "TFB" (Immunotech; IOT-RIA method) showed the lowest cross-reaction (2.5%) for prednisolone. Furthermore, on HPLC, 87.8% of the reaction of the entire fraction was seen in the fractions corresponding to the elution position of standard cortisol with the IOT-RIA method; this was the highest percentage among the 4 kits. GCMS revealed that the substance that showed a cross-reaction with the other 3 kits was 5alpha-tetrahydrocortisol (5alpha-THF) glucuronide. CONCLUSIONS: The IOT-RIA method was found to be the most specific for UFC. The other 3 commercially available kits showed cross-reaction with a conjugate of 5alpha-THF, found to be one of the causes of inter-kit assay discrepancies.

Tributyltin inhibits osteoblastic activity and disrupts calcium metabolism through an increase in plasma calcium and calcitonin levels in teleosts.

To examine the direct effects of tributyltin acetate (TBTA) on osteoclasts and osteoblasts, teleost scale, which has both osteoclasts and osteoblasts and is similar to mammalian membrane bone, was used in the present study. The activities of tartrate-resistant acid phosphatase and alkaline-phosphatase, as respective indicators of activity in both cells, were used. In freshwater teleost (goldfish) and marine teleosts (nibbler and wrasse), the osteoclastic activity in the scales did not change as a result of TBTA treatment (10(-9) to 10(-5) M). However, the osteoblastic activity decreased in the goldfish, nibbler, and wrasse after 6 h of incubation. In goldfish, even 10(-10) M of TBTA significantly inhibited the osteoblastic activity. The inhibitory activity in goldfish was stronger than that in nibbler and wrasse. Therefore, details of the mechanism were examined using goldfish. The mRNA expressions of the estrogen receptor and insulin-like growth factor-I, which participate in osteoblastic growth and differentiation, decreased in the TBTA-treated scales. However, the mRNA expression of metallothionein (MT), a metal-binding protein that protects the organism from heavy metal, increased much less than those of cadmium and methyl-mercury. Furthermore, we showed that the plasma calcium and hypocalcemic hormone (calcitonin) level increased in goldfish kept in water containing TBTA (10(-10) and 10(-8) M). The current data are the first to demonstrate that, in teleosts, TBTA inhibits osteoblastic activity without affecting osteoclastic activity and disrupts the calcium metabolism, including the calcemic hormone, in goldfish.

Both mercury and cadmium directly influence calcium homeostasis resulting from the suppression of scale bone cells: the scale is a good model for the evaluation of heavy metals in bone metabolism.

To examine the effects of heavy metals such as cadmium and mercury on calcium homeostasis, plasma calcium and calcitonin were measured in goldfish. Cadmium induced hypocalcemia both at 4 and at 8 days. In methylmercury-treated goldfish, the plasma calcium level increased at 2 days and then decreased at 8 days. The plasma calcitonin level increased in correspondence with the increased plasma calcium by methylmercury treatment, although cadmium did not cause a significant change. To elucidate the mechanism in detail, fish scales, which have both osteoclasts and osteoblasts and are similar to mammalian membrane bone, were used in the present study. We measured tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) activity as respective indicators of activity in both types of cells. TRAP activity in the scales decreased by treatment of cadmium and methylmercury at 6 h incubation. Particularly, cadmium (even at 10(-13) M) significantly suppressed TRAP activity, suggesting that this system is utilized as an acute biosensor for cadmium. ALP activity decreased after exposures of 64 and 96 h, although the activity did not change after 6, 18, and 36 h. In addition, mRNA expression of the estrogen receptor and insulin-like growth factor 1, which participate in osteoblastic growth and differentiation, was less than the control values by treatment with both metals. This study demonstrates that mercury directly acts on the bone cells and influences calcium homeostasis and indicates that, in a short-term exposure, mercury has a different action from that of cadmium and induces hypercalcemia.

Possible direct induction by estrogen of calcitonin secretion from ultimobranchial cells in the goldfish.

The plasma level of calcitonin (CT), a calcium (Ca)-regulating hormone, is known to increase in female teleosts during the reproductive period. In the present study, a correlation between plasma CT and Ca and one between plasma CT and the gonad somatic index were demonstrated in the female goldfish but not in the male. To clarify the relationship between CT and Ca, we examined the plasma CT and Ca levels after injecting immature goldfish with estrogen. At day 1, the plasma CT level significantly increased, whereas the plasma Ca level was not changed from its initial level. This result suggests that the trigger of CT secretion is estrogen and that estrogen directly acts on the ultimobranchial gland (UBG), a CT-secreting organ. To determine whether the UBG is equipped with estrogen receptor (ER), an ER binding assay and immunohistochemical staining of UBG cells with an antibody against ER were conducted. As a result, estrogen-specific binding (Kd, 18.52 nM; Bmax, 1.35 pmol/mg protein) and ER-immunoreactivity in the UBG were demonstrated. Furthermore, the expression of alpha, beta, and gamma types of ER in the UBG was also detected by use of the reverse-transcription polymerase chain reaction. Thus, we concluded that estrogen acts on the UBG to induce the release of CT, which in turn plays an important role in reproduction directly and/or indirectly through Ca. This is the first report on the existence of ERs in a teleost UBG and the occurrence of CT secretion caused by estrogen.

Enzyme-linked immunosorbent assay for monitoring toxic dioxin congeners in milk based on a newly generated monoclonal anti-dioxin antibody.

To develop an enzyme-linked immunosorbent assay (ELISA) for monitoring the toxicity due to polychlorinated dibenzo-p-dioxins and dibenzofurans contaminated in human breast milk, we have generated novel monoclonal antibodies using some haptenic derivatives linked to bovine serum albumin via the C-1 or C-2 position on the dioxin skeleton. BALB/c or A/J mice were repeatedly immunized with the immunogen, and spleen cells were fused with P3/NS1/1-Ag4-1 myeloma cells. After five fusion experiments, a hybridoma clone was established that secretes an antibody D9-36 group specifically recognizing the major toxic congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 2,3,4,7,8-pentachlorodibenzofran. An ELISA is developed on the basis of the competitive and labeled-antigen format. The toxic congeners extracted from butter or milk specimens by a novel extraction cartridge and a peroxidase-labeled dioxin analogue were sequentially reacted with a fixed amount of D9-36 in the presence of Triton X-100. The bound fraction was captured on a microtiter plate, immobilizing a second antibody, and the enzyme activity was colorimetrically determined. This ELISA afforded a practical sensitivity (measurable range, 1-100 pg/assay; detection limit, 1.0 pg/assay as 2,3,7,8-TCDD equivalent). The assay values for milk and butter samples were in reasonable accordance with the sum of the toxicity-equivalent quantity of each congener, which had been determined by a high-resolution gas chromatography/high-resolution mass spectrometry method.

Bisphenol A influences the plasma calcium level and inhibits calcitonin secretion in goldfish.

In teleosts, it is well known that plasma calcium levels increase as a result of treatment with estrogen for at least during 2 weeks and that calcitonin secretion is induced by estrogen. The present study examined the influence of bisphenol A on calcium homeostasis in goldfish and compared the above known estrogenic action. In goldfish kept in water containing bisphenol A (10(-6) M), the plasma calcium concentration increased significantly (P<0.001) at 4 days but decreased significantly (P<0.05) at 8 days. By the treatment of bisphenol A, calcitonin secretion was not induced until 4 days. At 8 days, however, plasma calcitonin, as well as calcium, decreased significantly (P<0.05), although vitellogenin was detected in the plasma. Therefore, bisphenol A influences plasma calcium levels, but its action is different from that of estrogen, which indicates that bisphenol A affects the calcium homeostasis and might bring about abnormal conditions in teleosts.

Circadian changes in serum concentrations of steroids in Japanese char Salvelinus leucomaenis at the stage of final maturation.

Circadian changes in serum concentrations of testosterone (T), 11-ketotestosterone (11KT), estradiol-17beta (E2), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), 17alpha-hydroxyprogesterone (OHP), cortisol (F) and progesterone (P) were investigated in the spermiated/ovulated Japanese char Salvelinus leucomaenis for over three days using newly developed time-resolved fluoroimmunoassays. Testosterone and DHP in both sex and 11KT in male showed significantly (P<0.05) higher serum levels just before/after onset of darkness (15:00 or 18:00), and the levels during night and daytime were significantly (P<0.05) lower than those of the peak levels. Serum F levels in both sex during dark phase were significantly (P<0.05) higher than those levels during daytime. A surge of serum OHP concentrations in both sexes was observed at the time of twilight (03:00). The peak time of serum T, 11KT and DHP levels were approximately 6 hours prior to those of serum F and OHP levels. Serum E2 in female and P in both sex fluctuated intensely during sampling period, and did not show remarkable changes. These results strongly suggest the existence of circadian-like diel changes in serum T, DHP, F and OHP levels in both sex and 11KT in male, and no variations in serum E2 in female and P in both sex in spermiated/ovulated Japanese char under the stage of final maturation. Furthermore, relationship between circadian rhythms of steroid hormones and spawning behaviors are discussed in the present study.

A highly specific heterologous enzyme immunoassay for 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide (androstanediol-17G) and developmental patterns of urinary androstanediol-17G excretions.

We established a highly specific enzyme immunoassay (EIA) for 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide (androstanediol-17G). Rabbit antisera raised against 5 alpha-androstane-3 alpha, 11 alpha, 17 beta-triol 17-glucuronide 11-glutaryl bovine serum albumin and a heterologous tracer of androstanediol-17G conjugated with horseradish peroxidase at the glucuronic acid group were used. The EIA showed excellent specificity: there were no remarkable cross-reactivities with related androgens. The assay range for urine samples was 0.3-30 ng/ml. Recoveries of standards added to samples were 100-108%. Intra-assay and inter-assay coefficients of variation were 2.9-4.4% and 5.7-7.9%, respectively. The EIA was applied to urine samples of 407 males and 322 females to determine developmental patterns and normal ranges of androstanediol-17G excretions in 11 age groups (0 y, 1 y, 2-3 y, 4-5 y, 6-7 y, 8-9 y, 10-11 y, 12-13 y, 14-15 y, 16-17 y, and over 18 y). Urinary androstanediol-17G/creatinine (androstanediol-17G/Cre) ratios in both sexes were high in infancy, tended to decrease during childhood, and began to increase near adolescence. While androstanediol-17G/Cre ratio in girls increased at 8-9 y and reached a plateau during adolescence, that in boys increased at 10-11 y and continued to increase throughout adolescence. Androstanediol-17G/Cre ratios in girls were higher than those in boys at 6-7 y (P < 0.05) and at 8-9 y (P < 0.01). Androstanediol-17G/Cre ratios in boys were higher than those in girls at 12-13 y and at older ages (P < 0.01). These developmental patterns are parallel to age-related changes in androgenicity and serum androstanediol-17G, suggesting that urinary androstanediol-17G/Cre ratio could be a good marker for androgenicity in childhood.