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)