Vandenberg LN et al. (2012), Low frequency vibrations induce malformations i...

XB-ART-46397

PLoS One 2012 Jan 01;712:e51473. doi: 10.1371/journal.pone.0051473.

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Low frequency vibrations induce malformations in two aquatic species in a frequency-, waveform-, and direction-specific manner.

Vandenberg LN , Stevenson C , Levin M .

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Environmental toxicants such as industrial wastes, air particulates from machinery and transportation vehicles, and pesticide run-offs, as well as many chemicals, have been widely studied for their effects on human and wildlife populations. Yet other potentially harmful environmental pollutants such as electromagnetic pulses, noise and vibrations have remained incompletely understood. Because developing embryos undergo complex morphological changes that can be affected detrimentally by alterations in physical forces, they may be particularly susceptible to exposure to these types of pollutants. We investigated the effects of low frequency vibrations on early embryonic development of two aquatic species, Xenopus laevis (frogs) and Danio rerio (zebrafish), specifically focusing on the effects of varying frequencies, waveforms, and applied direction. We observed treatment-specific effects on the incidence of neural tube defects, left-right patterning defects and abnormal tail morphogenesis in Xenopus tadpoles. Additionally, we found that low frequency vibrations altered left-right patterning and tail morphogenesis, but did not induce neural tube defects, in zebrafish. The results of this study support the conclusion that low frequency vibrations are toxic to aquatic vertebrates, with detrimental effects observed in two important model species with very different embryonic architectures.

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Species referenced: Xenopus laevis
Genes referenced: fry rpsa

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	Fig 1. Vibration induces heterotaxia in Xenopus embryos, independent of waveform or direction. A) Shown are embryos with different organ situs (position), including three with various forms of heterotaxia, i.e. the inversion in placement of one or more organs. Red arrows indicate the apex of the heart; green arrows indicate the gall bladder; yellow arrows indicate the coiling of the gut. These animals were exposed to one of three vibrations that were previously shown to affect left-right patterning. Here, the waveforms were varied between sine, square and triangle waves. B) Vibrations applied vertically induce heterotaxia, regardless of which frequency and waveform combination was applied. C) Vibrations applied horizontally also induce heterotaxia. Heterotaxia rates in un-vibrated controls were approximately 1%. Numbers on bars indicate frequencies of phenotypes for that treatment. At least 110 embryos were included for each treatment. p<0.01; *p<<0.001 relative to controls; on panel C, # indicates significant differences from vertical treatment (p<0.01).
	Figure S2 Vibration can induce a range of developmental patterning defects in Xenopus embryos. In addition to scored phenotypes, other severe developmental defects were occasionally observed in treated groups. However, these defects were observed infrequently enough that their incidence was not recorded. A Edema (indicated by orange arrowheads) was observed in a small number of embryos. This edema was often observed in tadpoles with tail defects including bent and curly tails (indicated by the dotted yellow lines, kinks indicated by green arrows). However, embryos with edemas were not scored for any phenotype due to their severe malformations. B) Hyperpigmentation was occasionally observed, but not related to any specific treatment. C) Craniofacial defects were observed including animals with narrow jaws and conjoined eyes, as shown here (blue arrows). Other craniofacial defects included missing facial structures and malformations in the eyes, mouth, nostrils and otoliths (not shown). D) Occasionally, we observed tadpoles with normal anterior structures but completely truncated tails (red arrowhead).
	Figure 2. Vibration induces neural tube defects in a frequency-, waveform- and direction-specific manner in Xenopus embryos.Xenopus laevis embryos were vibrated from 1-cell through st. 19 (late neurulation) and then allowed to develop in a vibration-free environment approximately stage 45. A) The majority of embryos develop as tadpoles with properly fused spinal cords. B, B’, B”) Examples of tadpoles with neural tube defects (NTDs). NTDs were defined by shortened axes and un-fused spinal cords. Animals often developed split tails. (These animals were produced with 15 Hz sine vertical vibrations.) C) Applying vibrations vertically to embryos induced significant numbers of NTDs at all frequencies tested, but only for certain waveforms. D) NTDs were also observed following vibration in the horizontal direction, but only for specific combinations of frequencies/waveforms. Numbers on bars indicate frequencies of phenotypes for that treatment. NTDs were observed in <1% of controls. Each group includes at least 100 treated embryos. p<0.01, *p<<0.001 relative to controls; on panel D, # indicates significant differences from vertical treatment (p<0.01).
	Figure 3. Vibrations induce abnormal tail morphology in Xenopus embryos.Xenopus laevis embryos were exposed to vibrations with a range of frequencies, waveforms and directions from 1-cell until approximately st. 19 (late neurulation). These embryos, as well as control embryos raised in a vibration-free environment, were scored at approximately stage 45 for abnormal tail morphologies. Compared to the normal tail appearance (A), a wide variety of bent tail phenotypes were seen, including: B) a single kink in the spine of the tail in the dorsal-ventral plane (kink marked by a green arrow in B’); C) two distinct kinks located within a small distance of each other (both kinks marked by red arrows in C’); D) a more gentle bending of the tail tissue in the dorsal-ventral plane; and E) a distinct kink in the tail in the left-right plane. (Animals shown in panels B-E were all produced with 15 Hz sine vertical vibrations.) For both vertical (F) and horizontal (G) vibrations, several frequencies and waveforms induced significant numbers of tail abnormalities. Numbers on bars indicate frequencies of phenotypes for that treatment. In un-vibrated controls, bent tails were observed in <8%. Each group includes at least 100 tadpoles. p<0.01, *p<<0.001 relative to controls; on panel G, # indicates significant differences from vertical treatment (p<0.01).
	Figure 4. Vibration induces heterotaxia and disrupts tail morphogenesis in Zebrafish embryos.A) Organ situs was scored in zebrafish fry 7 days post fertilization using the auto-fluorescence of the pancreas and gall bladder (circled). In vibrated fish, inversions were often observed (with the gall bladder positioned to the left of the pancreas); isomerisms (with the gall bladder directly above the pancreas) were observed less often. These animals were generated by 20 or 30 Hz vertical sine vibrations. B) The incidence of heterotaxia observed in vibrated zebrafish for a total of 18 treatments. Heterotaxia was observed in 3–4% of un-vibrated controls. C) Isomerisms were induced by a few horizontal vibration treatments only. Isomerisms were never observed in untreated fish. D) Typical tail morphology in zebrafish fry at 7 days post fertilization. E-E”) Examples of abnormal tail morphologies observed in zebrafish that were vibrated from 1-cell overnight. These animals were generated by 20 or 30 Hz horizontal sine vibrations. F) Abnormal tail morphologies were rare, but significant numbers were observed following two horizontal vibration treatments. For all graphs, each group includes at least 110 fish. p<0.01, *p<<0.001 relative to controls; # indicates significant differences between horizontal and vertical treatments (p<0.01).
	Figure 5. Vibration induces LR patterning and tail morphogenesis defects in zebrafish in a frequency-, waveform- and direction-specific manner.Zebrafish embryos were vibrated from 1-cell through the 5 somite stage, and then allowed to develop in a vibration-free environment until 7 days post-fertilization when they were scored for LR patterning defects (heterotaxia+isomerisms) and abnormal tail morphologies. A) Effects of vibrations applied vertically to LR patterning. Only two treatments induced LR defects. B) Vibrations applied horizontally also induce LR defects. When vibrations were applied in this direction, every treatment with the exception of 15 Hz triangle waves induced significant numbers of LR patterning defects. LR defects were observed in approximately 5% of untreated controls. C) Vertical vibrations induce abnormal tail morphologies in two treatments: 15 Hz triangle and 150 Hz square. D) Horizontal vibrations also induced malformed tails in two different treatments: 15 Hz sine and 150 Hz square waves. Numbers on bars indicate frequencies of phenotypes for that treatment. For all treatments, each group includes at least 110 fish. On graphs, p<0.01, *p<<0.001 relative to controls; # indicates significant differences between horizontal and vertical treatments (p<0.01).
	Figure 1. Vibration induces heterotaxia in Xenopus embryos, independent of waveform or direction.A) Shown are embryos with different organ situs (position), including three with various forms of heterotaxia, i.e. the inversion in placement of one or more organs. Red arrows indicate the apex of the heart; green arrows indicate the gall bladder; yellow arrows indicate the coiling of the gut. These animals were exposed to one of three vibrations that were previously shown to affect left-right patterning. Here, the waveforms were varied between sine, square and triangle waves. B) Vibrations applied vertically induce heterotaxia, regardless of which frequency and waveform combination was applied. C) Vibrations applied horizontally also induce heterotaxia. Heterotaxia rates in un-vibrated controls were approximately 1%. Numbers on bars indicate frequencies of phenotypes for that treatment. At least 110 embryos were included for each treatment. p<0.01; *p<<0.001 relative to controls; on panel C, # indicates significant differences from vertical treatment (p<0.01).

References [+] :

Abbate, Affective correlates of occupational exposure to whole-body vibration. A case-control study. 2004, Pubmed

	Fig 1. Vibration induces heterotaxia in Xenopus embryos, independent of waveform or direction. A) Shown are embryos with different organ situs (position), including three with various forms of heterotaxia, i.e. the inversion in placement of one or more organs. Red arrows indicate the apex of the heart; green arrows indicate the gall bladder; yellow arrows indicate the coiling of the gut. These animals were exposed to one of three vibrations that were previously shown to affect left-right patterning. Here, the waveforms were varied between sine, square and triangle waves. B) Vibrations applied vertically induce heterotaxia, regardless of which frequency and waveform combination was applied. C) Vibrations applied horizontally also induce heterotaxia. Heterotaxia rates in un-vibrated controls were approximately 1%. Numbers on bars indicate frequencies of phenotypes for that treatment. At least 110 embryos were included for each treatment. p<0.01; *p<<0.001 relative to controls; on panel C, # indicates significant differences from vertical treatment (p<0.01).
	Figure S2 Vibration can induce a range of developmental patterning defects in Xenopus embryos. In addition to scored phenotypes, other severe developmental defects were occasionally observed in treated groups. However, these defects were observed infrequently enough that their incidence was not recorded. A Edema (indicated by orange arrowheads) was observed in a small number of embryos. This edema was often observed in tadpoles with tail defects including bent and curly tails (indicated by the dotted yellow lines, kinks indicated by green arrows). However, embryos with edemas were not scored for any phenotype due to their severe malformations. B) Hyperpigmentation was occasionally observed, but not related to any specific treatment. C) Craniofacial defects were observed including animals with narrow jaws and conjoined eyes, as shown here (blue arrows). Other craniofacial defects included missing facial structures and malformations in the eyes, mouth, nostrils and otoliths (not shown). D) Occasionally, we observed tadpoles with normal anterior structures but completely truncated tails (red arrowhead).
	Figure 2. Vibration induces neural tube defects in a frequency-, waveform- and direction-specific manner in Xenopus embryos.Xenopus laevis embryos were vibrated from 1-cell through st. 19 (late neurulation) and then allowed to develop in a vibration-free environment approximately stage 45. A) The majority of embryos develop as tadpoles with properly fused spinal cords. B, B’, B”) Examples of tadpoles with neural tube defects (NTDs). NTDs were defined by shortened axes and un-fused spinal cords. Animals often developed split tails. (These animals were produced with 15 Hz sine vertical vibrations.) C) Applying vibrations vertically to embryos induced significant numbers of NTDs at all frequencies tested, but only for certain waveforms. D) NTDs were also observed following vibration in the horizontal direction, but only for specific combinations of frequencies/waveforms. Numbers on bars indicate frequencies of phenotypes for that treatment. NTDs were observed in <1% of controls. Each group includes at least 100 treated embryos. p<0.01, *p<<0.001 relative to controls; on panel D, # indicates significant differences from vertical treatment (p<0.01).
	Figure 3. Vibrations induce abnormal tail morphology in Xenopus embryos.Xenopus laevis embryos were exposed to vibrations with a range of frequencies, waveforms and directions from 1-cell until approximately st. 19 (late neurulation). These embryos, as well as control embryos raised in a vibration-free environment, were scored at approximately stage 45 for abnormal tail morphologies. Compared to the normal tail appearance (A), a wide variety of bent tail phenotypes were seen, including: B) a single kink in the spine of the tail in the dorsal-ventral plane (kink marked by a green arrow in B’); C) two distinct kinks located within a small distance of each other (both kinks marked by red arrows in C’); D) a more gentle bending of the tail tissue in the dorsal-ventral plane; and E) a distinct kink in the tail in the left-right plane. (Animals shown in panels B-E were all produced with 15 Hz sine vertical vibrations.) For both vertical (F) and horizontal (G) vibrations, several frequencies and waveforms induced significant numbers of tail abnormalities. Numbers on bars indicate frequencies of phenotypes for that treatment. In un-vibrated controls, bent tails were observed in <8%. Each group includes at least 100 tadpoles. p<0.01, *p<<0.001 relative to controls; on panel G, # indicates significant differences from vertical treatment (p<0.01).
	Figure 4. Vibration induces heterotaxia and disrupts tail morphogenesis in Zebrafish embryos.A) Organ situs was scored in zebrafish fry 7 days post fertilization using the auto-fluorescence of the pancreas and gall bladder (circled). In vibrated fish, inversions were often observed (with the gall bladder positioned to the left of the pancreas); isomerisms (with the gall bladder directly above the pancreas) were observed less often. These animals were generated by 20 or 30 Hz vertical sine vibrations. B) The incidence of heterotaxia observed in vibrated zebrafish for a total of 18 treatments. Heterotaxia was observed in 3–4% of un-vibrated controls. C) Isomerisms were induced by a few horizontal vibration treatments only. Isomerisms were never observed in untreated fish. D) Typical tail morphology in zebrafish fry at 7 days post fertilization. E-E”) Examples of abnormal tail morphologies observed in zebrafish that were vibrated from 1-cell overnight. These animals were generated by 20 or 30 Hz horizontal sine vibrations. F) Abnormal tail morphologies were rare, but significant numbers were observed following two horizontal vibration treatments. For all graphs, each group includes at least 110 fish. p<0.01, *p<<0.001 relative to controls; # indicates significant differences between horizontal and vertical treatments (p<0.01).
	Figure 5. Vibration induces LR patterning and tail morphogenesis defects in zebrafish in a frequency-, waveform- and direction-specific manner.Zebrafish embryos were vibrated from 1-cell through the 5 somite stage, and then allowed to develop in a vibration-free environment until 7 days post-fertilization when they were scored for LR patterning defects (heterotaxia+isomerisms) and abnormal tail morphologies. A) Effects of vibrations applied vertically to LR patterning. Only two treatments induced LR defects. B) Vibrations applied horizontally also induce LR defects. When vibrations were applied in this direction, every treatment with the exception of 15 Hz triangle waves induced significant numbers of LR patterning defects. LR defects were observed in approximately 5% of untreated controls. C) Vertical vibrations induce abnormal tail morphologies in two treatments: 15 Hz triangle and 150 Hz square. D) Horizontal vibrations also induced malformed tails in two different treatments: 15 Hz sine and 150 Hz square waves. Numbers on bars indicate frequencies of phenotypes for that treatment. For all treatments, each group includes at least 110 fish. On graphs, p<0.01, *p<<0.001 relative to controls; # indicates significant differences between horizontal and vertical treatments (p<0.01).
	Figure 1. Vibration induces heterotaxia in Xenopus embryos, independent of waveform or direction.A) Shown are embryos with different organ situs (position), including three with various forms of heterotaxia, i.e. the inversion in placement of one or more organs. Red arrows indicate the apex of the heart; green arrows indicate the gall bladder; yellow arrows indicate the coiling of the gut. These animals were exposed to one of three vibrations that were previously shown to affect left-right patterning. Here, the waveforms were varied between sine, square and triangle waves. B) Vibrations applied vertically induce heterotaxia, regardless of which frequency and waveform combination was applied. C) Vibrations applied horizontally also induce heterotaxia. Heterotaxia rates in un-vibrated controls were approximately 1%. Numbers on bars indicate frequencies of phenotypes for that treatment. At least 110 embryos were included for each treatment. p<0.01; *p<<0.001 relative to controls; on panel C, # indicates significant differences from vertical treatment (p<0.01).