Muscular contractions in the zebrafish embryo are necessary to reveal thiuram-induced notochord distortions

Author Posting. © The Authors, 2006. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Toxicology and Applied Pharmacology 212 (2006): 24-34, doi:10.1016/j.taap.2005.06.016.Dithiocarbamates form a large group of chemicals that have numerous uses in agriculture and medicine. It has been reported that dithiocarbamates, including thiuram (tetramethylthiuram disulfide), cause wavy distortions of the notochord in zebrafish and other fish embryos. In the present study, we investigated the mechanism underlying the toxicity of thiuram in zebrafish embryos. When embryos were exposed to thiuram (2-1,000 nM: 0.48-240 µg/L) from 3 hours post fertilization (hpf) (30% epiboly) until 24 hpf (Prim-5), all embryos develop wavy notochords, disorganized somites, and have shortened yolk sac extensions. The thiuram response was specific and did not cause growth retardation or mortality at 24 hpf. The thiuram-dependent responses showed the same concentration dependence with a waterborne EC50 values of approximately 7 nM. Morphometric measurements revealed that thiuram does not affect the rate of notochord lengthening. However, the rate of overall body lengthening was significantly reduced in thiuram exposed animals. Other dithiocarbamates, such as ziram, caused similar malformations to thiuram. While expression of genes involved in somitogenesis was not affected, the levels of notochord specific transcripts were altered before and after the onset of malformations. Distortion of the notochord started precisely at 18 hpf, which is concomitant with onset of spontaneous rhythmic trunk contractions. Abolishment of spontaneous contractions using tricaine, α-bungarotoxin, and a paralytic mutant sofa potato, resulted in normal notochord morphology in the presence of thiuram. These results indicate that muscle activity is necessary to reveal the underlying functional deficit and suggest that the developmental target of dithiocarbamates impairs trunk plasticity through an unknown mechanism.This work was supported by grants-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (T.H. and H.T.), and grant-in-aid for JSPS fellows from the Japanese Ministry of Education, Culture, Sports, Science and Technology (W. D.), a grant-in-aid for High Technological Research Center (Rakuno Gakuen University) from the Ministry of Education, Science, Sports and Culture of Japan (H.T.), Technology, cooperative research from active research in Rakuno Gakuen University 2004-7 (H.T.), and NIH/NIEHS grants ES00210 and ES03850 (RT)

EFFECT OF CYCLOPHOSPHAMIDE ON THE THYMUS AND THE BURSA OF FABRICIUS IN CHICKENS

The effect of cyclophosphamide (Cy) on the thymus and the bursa of Fabricius in chickens, aged one day to 7 weeks, was examined morphologically. Chickens, which were daily injected i.m. with 6 mg Cy 1,2,or 3 times from the hatching day, were compared with saline control chickens. The relative weights of both the thymus and the bursa decreased abruptly just after the injection of Cy. The weight of the thymus had an involutional bottom at the age of 4〜5 days, and thereafter recovered to the normal level at age 3 weeks, while that of the bursa never returned to the control level until age 7 weeks. The histological changes in the thymus appeared to be greater in the cortex than in the medulla. A degeneration of lymphocytes, an increase of macrophages, hypertrophy of the reticular cells, and a disappearance of mitotic cells were observed in the cortex during early involution. Recovery began as soon as the large lymphocytes appeared in the cortex, and the histological structures of the thymuses in the Cy-treated birds recovered completely at age 3 weeks. The effect of Cy in the bursa was similar to that in the thymus, but these changes were seen earlier than they were in the thymus. Restoration of the lymphoid follicles was observed from 5 to 10 days of age mainly in the groups treated with a smaller quantity of Cy. In the chickens treated with 18 mg Cy, however, only a few restorative follicles were found

Growth of Boron Nitride Nanohorn Structures

Boron nitride (BN) nanohorns were fabricated, and their structures were investigated by transmission electron microscopy and molecular mechanics calculation. The multi-walled BN nanohorns would be stabilized by stacking of nanohorn structures. Growth of the BN nanohorn was observed at elevated temperatures, and the activation energy for the nanohorn growth was estimated to be 2.3 eV. [doi:10.2320/matertrans.MB200806

Correlation of ghrelin concentration and ghrelin, ghrelin-O-acetyltransferase (GOAT) and growth hormone secretagogue receptor 1a mRNAs expression in the proventriculus and brain of the growing chicken

To determine mechanisms for age-related decrease of GHS-R1a expression in the chicken proventriculus, changes in mRNA expression of ghrelin and ghrelin-O-acetyltransferase (GOAT) as well as ghrelin concentrations in the proventriculus and plasma were examined in growing chickens. Changes in expression levels of ghrelin, GOAT and GHS-R1a mRNAs were also examined in different brain regions (pituitary, hypothalamus, thalamus, cerebellum, cerebral cortex, olfactory bulb, midbrain and medulla oblongata). Ghrelin concentrations in the proventriculus and plasma increased with aging and reached plateaus at 30–50 days after hatching. High level of ghrelin mRNA decreased at 3 days after hatching, and it became stable at half of the initial level. Expression levels of GHS-R1a and GOAT decreased 3 or 5 days after hatching and became stable at low levels. Significant negative correlations were found between plasma ghrelin and mRNA levels of GOAT and GHS-R1a. Expression levels of ghrelin mRNA were different in the brain regions, but a significant change was not seen with aging. GHS-R1a expression was detected in all brain regions, and age-dependent changes were observed in the pituitary and cerebellum. Different from the proventriculus, the expression of GOAT in the brain increased or did not change with aging. These results suggest that decreased GHS-R1a and GOAT mRNA expression in the proventriculus is due to endogenous ghrelin-induced down-regulation. Expression levels of ghrelin, GOAT and GHS-R1a in the brain were independently regulated from that in the proventriculus, and age-related and region-dependent regulation pattern suggests a local effect of ghrelin system in chicken brain

Molecularidentification of ghrelinreceptor (GHS-R1a) and its functionalrole in the gastrointestinaltract of the guinea-pig

Ghrelin stimulates gastric motility in vivo in the guinea-pig through activation of growth hormone secretagogue receptor (GHS-R). In this study, we identified GHS-R1a in the guinea-pig, and examined its distribution and cellular function and compared them with those in the rat. Effects of ghrelin in different regions of gastrointestinal tract were also examined. GHS-R1a was identified in guinea-pig brain cDNA. Amino acid identities of guinea-pig GHS-R1a were 93% to horses and 85% to dogs. Expression levels of GHS-R1a mRNA were high in the pituitary and hypothalamus, moderate in the thalamus, cerebral cortex, pons, medulla oblongata and olfactory bulb, and low in the cerebellum and peripheral tissues including gastrointestinal tract. Comparison of GHS-R1a expression patterns showed that those in the brain were similar but the expression level in the gastrointestinal tract was higher in rats than in guinea-pigs. Guinea-pig GHS-R1a expressed in HEK 293 cells responded to rat ghrelin and GHS-R agonists. Rat ghrelin was ineffective in inducing mechanical changes in the stomach and colon but caused a slight contraction in the small intestine. 1,1-Dimethyl-4-phenylpiperazinium and electrical field stimulation (EFS) caused cholinergic contraction in the intestine, and these contractions were not affected by ghrelin. Ghrelin did not change spontaneous and EFS-evoked [^3H]-efflux from [^3H]-choline-loaded ileal strips. In summary, guinea-pig GHS-R1a was identified and its functions in isolated gastrointestinal strips were characterized. The distribution of GHS-R1a in peripheral tissues was different from that in rats, suggesting that the functional role of ghrelin in the guinea-pig is different from that in other animal species

Malformation of certain brain blood vessels caused by TCDD activation of Ahr2/Arnt1 signaling in developing zebrafish

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). The AHR also plays important roles in normal development in mice, and AHR^ mice show abnormal development of vascular structures in various blood vessels. Our previous studies revealed that Ahr type 2 (Ahr2) activation by TCDD and β-naphthoflavone (BNF) caused a significant decrease in blood flow in the dorsal midbrain of zebrafish embryos. Here we report effects of TCDD exposure on the morphology of some blood vessels in the head of developing zebrafish. TCDD caused concentration-dependent anatomical rearrangements in the shape of the prosencephalic artery in zebrafish larvae. In contrast, no major vascular defects were recognized in the trunk and tail regions following exposure to TCDD at least at the concentrations used. Essentially, the same observations were also confirmed in BNF-exposed larvae. Knock-down of either Ahr2 or Ahr nuclear translocator type 1 (Arnt1) by morpholino oligonucleotides (MOs) protected larvae against abnormal shape of the prosencephalic artery caused by TCDD and BNF. On the other hand, knock-down of Ahr2 or Arnt1 in vehicle-exposed zebrafish larvae had no clear effect on morphology of the prosencephalic artery or trunk vessels. Ascorbic acid, an antioxidant, protected against the TCDD-induced decrease in blood flow through the prosencephalic artery, but not the abnormal morphological changes in the shape of this artery. These results indicate that activation of Ahr2/Arnt1 pathway by TCDD and BNF affects the shape of certain blood vessels in the brain of developing zebrafish