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Zebrafish biosensor for toxicant induced muscle hyperactivity.

Shahid M, Takamiya M, Stegmaier J, Middel V, Gradl M, Klüver N, Mikut R, Dickmeis T, Scholz S, Rastegar S, Yang L, Strähle U - Sci Rep (2016)

Bottom Line: Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression.Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency.TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Postfach 3640, D76021 Karlsruhe.

ABSTRACT
Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

No MeSH data available.


Related in: MedlinePlus

The hspb11 transgene responses to pesticides.Embryos from TgBAC(hspb11:GFP) were treated with either DMSO (solvent control; A–C and M), or the pesticides azinphosmethyl (APM; D–F), propoxur (PPX; G–I) or galanthamine (GAL; J–L). The embryos were treated from 90%-epiboly (9 hpf) to 48 hpf and examined for birefringence as a readout of muscle integrity (B,E,H,K; lateral view of the trunk region) and hspb11 transgene expression (C,F,I,L; dorsal view of the trunk region). Corresponding bright field images are given in (A,D,G,J; lateral view). The stippled lines (in C,F,I,L) delineate the notochord (n). sm: slow muscle. Anterior left. (M–S) Embryos from TgBAC(hspb11:GFP) were treated with either azinphosmethyl (N,O), propoxur (P,Q) or galanthamine (R,S). The hspb11 transgene level was compared between two groups: one group treated with chemicals from 9–48 hpf (“not washed”; M,N,P,R), another treated from 9–24 hpf, followed by several washes in fresh fish water until 48 hpf (“washed”; O,Q,S). The intensity of hspb11 transgene is expressed in fold increase over control. Scale bars: (A,B,D,E,G,H,J,K,M–S) 100 μm; (C,F,I,L) 50 μm. (T) Quantification of the experiments exemplified in (M–S). The intensity of hspb11 transgene expression is shown as fold induction over DMSO control. There were significant effects of the washout of the compounds on the hspb11 transgene level (one-way ANOVA, F[1, 118] = 77.762, p = 1.228 × 10−14), with azinphosmethyl (TukeyHSD test from here onward, p = 0.0000000), propoxur (p = 0.0000429), and galanthamine (p = 0.0000087) treated embryos now showing lower hspb11 transgene expression. However, all washed groups showed still higher hspb11 transgene levels in comparison to DMSO solvent control (one-way ANOVA, F[3, 118] = 29.316, p = 3.09 × 10−14; TukeyHSD test, ***p = 0.0000000 for azinphosmethyl vs. control, p = 0.0507833+ with a substantial difference for propoxur vs. control, and *p = 0.0124261 for galanthamine vs. control).
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f2: The hspb11 transgene responses to pesticides.Embryos from TgBAC(hspb11:GFP) were treated with either DMSO (solvent control; A–C and M), or the pesticides azinphosmethyl (APM; D–F), propoxur (PPX; G–I) or galanthamine (GAL; J–L). The embryos were treated from 90%-epiboly (9 hpf) to 48 hpf and examined for birefringence as a readout of muscle integrity (B,E,H,K; lateral view of the trunk region) and hspb11 transgene expression (C,F,I,L; dorsal view of the trunk region). Corresponding bright field images are given in (A,D,G,J; lateral view). The stippled lines (in C,F,I,L) delineate the notochord (n). sm: slow muscle. Anterior left. (M–S) Embryos from TgBAC(hspb11:GFP) were treated with either azinphosmethyl (N,O), propoxur (P,Q) or galanthamine (R,S). The hspb11 transgene level was compared between two groups: one group treated with chemicals from 9–48 hpf (“not washed”; M,N,P,R), another treated from 9–24 hpf, followed by several washes in fresh fish water until 48 hpf (“washed”; O,Q,S). The intensity of hspb11 transgene is expressed in fold increase over control. Scale bars: (A,B,D,E,G,H,J,K,M–S) 100 μm; (C,F,I,L) 50 μm. (T) Quantification of the experiments exemplified in (M–S). The intensity of hspb11 transgene expression is shown as fold induction over DMSO control. There were significant effects of the washout of the compounds on the hspb11 transgene level (one-way ANOVA, F[1, 118] = 77.762, p = 1.228 × 10−14), with azinphosmethyl (TukeyHSD test from here onward, p = 0.0000000), propoxur (p = 0.0000429), and galanthamine (p = 0.0000087) treated embryos now showing lower hspb11 transgene expression. However, all washed groups showed still higher hspb11 transgene levels in comparison to DMSO solvent control (one-way ANOVA, F[3, 118] = 29.316, p = 3.09 × 10−14; TukeyHSD test, ***p = 0.0000000 for azinphosmethyl vs. control, p = 0.0507833+ with a substantial difference for propoxur vs. control, and *p = 0.0124261 for galanthamine vs. control).

Mentions: To test whether the transgene expression is responsive to compounds that cause muscle toxicity, we treated TgBAC(hspb11:GFP) embryos with azinphosmethyl, propoxur and galanthamine, which exert their toxic effects on muscle through inhibition of Acetylcholine esterase (AChE) activity20. Embryos were exposed to the three pesticides from the 90%-epiboly stage (9 hpf) to 48 hpf. Treatments with all three compounds caused an up-regulation of GFP expression in the slow muscle (sm, Fig. 2F,I,L) and also in the notochord (n, Fig. 2F,I,L) compared with a negative control treated with 0.1% (v/v) DMSO only (Fig. 2C). To assess muscle fibre arrangement or integrity under these treatments, we examined birefringence of the muscles. We observed that all chemicals also caused a reduction in muscle birefringence, consistent with their previously described effects and paralleling the transgene response. The magnitude of the transgene expression was roughly proportional to the loss of muscle integrity observed via the assessment of birefringence. We also assessed whether transgene activation is restricted to slow muscle by in situ hybridisation of azinphosmethyl treated and DMSO control embryos. An increase of gfp mRNA was detected in both slow and fast muscles in azinphosmethyl treated embryos but not solvent controls (data not shown).


Zebrafish biosensor for toxicant induced muscle hyperactivity.

Shahid M, Takamiya M, Stegmaier J, Middel V, Gradl M, Klüver N, Mikut R, Dickmeis T, Scholz S, Rastegar S, Yang L, Strähle U - Sci Rep (2016)

The hspb11 transgene responses to pesticides.Embryos from TgBAC(hspb11:GFP) were treated with either DMSO (solvent control; A–C and M), or the pesticides azinphosmethyl (APM; D–F), propoxur (PPX; G–I) or galanthamine (GAL; J–L). The embryos were treated from 90%-epiboly (9 hpf) to 48 hpf and examined for birefringence as a readout of muscle integrity (B,E,H,K; lateral view of the trunk region) and hspb11 transgene expression (C,F,I,L; dorsal view of the trunk region). Corresponding bright field images are given in (A,D,G,J; lateral view). The stippled lines (in C,F,I,L) delineate the notochord (n). sm: slow muscle. Anterior left. (M–S) Embryos from TgBAC(hspb11:GFP) were treated with either azinphosmethyl (N,O), propoxur (P,Q) or galanthamine (R,S). The hspb11 transgene level was compared between two groups: one group treated with chemicals from 9–48 hpf (“not washed”; M,N,P,R), another treated from 9–24 hpf, followed by several washes in fresh fish water until 48 hpf (“washed”; O,Q,S). The intensity of hspb11 transgene is expressed in fold increase over control. Scale bars: (A,B,D,E,G,H,J,K,M–S) 100 μm; (C,F,I,L) 50 μm. (T) Quantification of the experiments exemplified in (M–S). The intensity of hspb11 transgene expression is shown as fold induction over DMSO control. There were significant effects of the washout of the compounds on the hspb11 transgene level (one-way ANOVA, F[1, 118] = 77.762, p = 1.228 × 10−14), with azinphosmethyl (TukeyHSD test from here onward, p = 0.0000000), propoxur (p = 0.0000429), and galanthamine (p = 0.0000087) treated embryos now showing lower hspb11 transgene expression. However, all washed groups showed still higher hspb11 transgene levels in comparison to DMSO solvent control (one-way ANOVA, F[3, 118] = 29.316, p = 3.09 × 10−14; TukeyHSD test, ***p = 0.0000000 for azinphosmethyl vs. control, p = 0.0507833+ with a substantial difference for propoxur vs. control, and *p = 0.0124261 for galanthamine vs. control).
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Related In: Results  -  Collection

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f2: The hspb11 transgene responses to pesticides.Embryos from TgBAC(hspb11:GFP) were treated with either DMSO (solvent control; A–C and M), or the pesticides azinphosmethyl (APM; D–F), propoxur (PPX; G–I) or galanthamine (GAL; J–L). The embryos were treated from 90%-epiboly (9 hpf) to 48 hpf and examined for birefringence as a readout of muscle integrity (B,E,H,K; lateral view of the trunk region) and hspb11 transgene expression (C,F,I,L; dorsal view of the trunk region). Corresponding bright field images are given in (A,D,G,J; lateral view). The stippled lines (in C,F,I,L) delineate the notochord (n). sm: slow muscle. Anterior left. (M–S) Embryos from TgBAC(hspb11:GFP) were treated with either azinphosmethyl (N,O), propoxur (P,Q) or galanthamine (R,S). The hspb11 transgene level was compared between two groups: one group treated with chemicals from 9–48 hpf (“not washed”; M,N,P,R), another treated from 9–24 hpf, followed by several washes in fresh fish water until 48 hpf (“washed”; O,Q,S). The intensity of hspb11 transgene is expressed in fold increase over control. Scale bars: (A,B,D,E,G,H,J,K,M–S) 100 μm; (C,F,I,L) 50 μm. (T) Quantification of the experiments exemplified in (M–S). The intensity of hspb11 transgene expression is shown as fold induction over DMSO control. There were significant effects of the washout of the compounds on the hspb11 transgene level (one-way ANOVA, F[1, 118] = 77.762, p = 1.228 × 10−14), with azinphosmethyl (TukeyHSD test from here onward, p = 0.0000000), propoxur (p = 0.0000429), and galanthamine (p = 0.0000087) treated embryos now showing lower hspb11 transgene expression. However, all washed groups showed still higher hspb11 transgene levels in comparison to DMSO solvent control (one-way ANOVA, F[3, 118] = 29.316, p = 3.09 × 10−14; TukeyHSD test, ***p = 0.0000000 for azinphosmethyl vs. control, p = 0.0507833+ with a substantial difference for propoxur vs. control, and *p = 0.0124261 for galanthamine vs. control).
Mentions: To test whether the transgene expression is responsive to compounds that cause muscle toxicity, we treated TgBAC(hspb11:GFP) embryos with azinphosmethyl, propoxur and galanthamine, which exert their toxic effects on muscle through inhibition of Acetylcholine esterase (AChE) activity20. Embryos were exposed to the three pesticides from the 90%-epiboly stage (9 hpf) to 48 hpf. Treatments with all three compounds caused an up-regulation of GFP expression in the slow muscle (sm, Fig. 2F,I,L) and also in the notochord (n, Fig. 2F,I,L) compared with a negative control treated with 0.1% (v/v) DMSO only (Fig. 2C). To assess muscle fibre arrangement or integrity under these treatments, we examined birefringence of the muscles. We observed that all chemicals also caused a reduction in muscle birefringence, consistent with their previously described effects and paralleling the transgene response. The magnitude of the transgene expression was roughly proportional to the loss of muscle integrity observed via the assessment of birefringence. We also assessed whether transgene activation is restricted to slow muscle by in situ hybridisation of azinphosmethyl treated and DMSO control embryos. An increase of gfp mRNA was detected in both slow and fast muscles in azinphosmethyl treated embryos but not solvent controls (data not shown).

Bottom Line: Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression.Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency.TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Postfach 3640, D76021 Karlsruhe.

ABSTRACT
Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

No MeSH data available.


Related in: MedlinePlus