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)

Structures of B type procyanidin.

<p>(a) (-)-epicatechin (EC; monomer), (b) procyanidin B2 (B2; dimer), (c) procyanidin C1 (C1; trimer), (d) cinnamtannin A2 (A2; tetramer).</p

Comparison of the sympathetic stimulatory abilities of B-type procyanidins based on induction of uncoupling protein-1 in brown adipose tissue (BAT) and increased plasma catecholamine (CA) in mice

<div><p>Objectives</p><p>We previously found that elevated energy expenditure following a single oral dose of flavan 3-ols (FL), a mixture of catechins and B type procyanidins, is caused by sympathetic nerve activation. In the present study, we compared the activity of the FL components (-)-epicatechin (EC; monomer), procyanidin B2 (B2; dimer), procyanidin C1 (C1; trimer), cinnamtannin A2 (A2; tetramer), and more than pentamer fraction (P5).</p><p>Methods</p><p>Male ICR mice were treated with a single oral dose of FL, EC, B2, C1, A2, or P5. The animals were sacrificed and blood and brown adipose tissue (BAT) sampled. The plasma catecholamine (CA) levels and BAT uncoupling protein (UCP)-1 mRNA expression were determined.</p><p>Results</p><p>A single dose of 10 mg/kg FL significantly increased plasma CA and UCP-1 mRNA levels. B2, C1, and A2, but not EC and P5 (all at 1 mg/kg), significantly increased plasma adrenaline levels. Plasma noradrenaline was significantly elevated by B2 and A2, but not by EC, C1, or P5. UCP-1 mRNA levels were significantly increased by C1 and P5. In the dose response study of A2, 10⁻³ mg/kg A2 increased UCP-1 mRNA levels significantly, but not 10⁻² and 10⁻¹ mg/kg A2. In addition, combination treatment with 10⁻¹ mg/kg A2 and yohimbine, an α2 adrenalin blocker, remarkably increased UCP-1 mRNA levels.</p><p>Conclusion</p><p>These results suggest that FL and its components, except EC, increase UCP-1 mRNA and plasma CA with varying efficacy.</p></div

Alterations in plasma noradrenaline (a), adrenaline (b), and UCP-1 mRNA in BAT (c) 2 h after ingestion of 1 mg/kg EC, B2, C1, A2, P5, or vehicle.

<p>The values represent mean ± standard deviation (each group, n = 8). **p<0.01. *p<0.05, **p<0.01 (Tukey-Kramer test vs. vehicle)</p

Dose-reactive response of UCP-1 mRNA levels to A2 and A2 plus yohimbine in BAT.

<p>The values represent mean ± standard deviation (each group, n = 8). *p<0.05, **p<0.01 (Tukey-Kramer test between experimental group).</p

Alterations in plasma noradrenaline (a), adrenaline (b), and UCP-1 mRNA levels in BAT (c) after ingestion of 10 mg/kg flavan 3-ols or vehicle.

<p>The values represent mean ± standard deviation (each group, n = 8). *p<0.05, **p<0.01 (Tukey-Kramer test vs. vehicle).</p