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Comp Biochem Physiol Part D Genomics Proteomics
2020 Jun 01;34:100658. doi: 10.1016/j.cbd.2020.100658.
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Sub-lethal effects of calcium dinonylnaphthalenesulfonate on Western clawed frog embryos.
Wallace SJ
,
Leclerc AJA
,
Prosser R
,
de Solla SR
,
Balakrishnan V
,
Langlois VS
.
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Naphthalene sulfonic acids (NSAs) are used as additives in lubricants, dyes, and greases and commonly act as surfactants in many industrial processes. The calcium salt of dinonyl NSA (calcium dinonylnaphthalenesulfonate; CaDNS) is listed among thousands of chemicals identified as priorities for assessment by the Government of Canada's Chemical Management Plan due to the limited toxicity data. The purpose of this study was two-fold: 1) to establish the toxicity of CaDNS to Western clawed frog (Silurana tropicalis) embryos and 2) to assess the sub-lethal effects and mechanisms of toxicity of CaDNS in amphibians through targeted gene expression and metabolite analyses. Frog embryos were exposed to water overlying sand spiked with a range of concentrations of CaDNS (17-1393 μg/g) over a 72-h period. Results indicated significantly higher mortality and presence of malformations in frog larvae exposed to over 672 μg/g CaDNS in the sand (14 ng/mL CaDNS in the water) compared to control treatments. An overall decrease in the glutathione redox cycle was observed, including decreases in relative mRNA levels of enzymes (glutathione S-transferase (gst), glutathione reductase (gsr), glutathione peroxidase (gpx)) and decreases in the glutathione (GSH) and glutathione disulfide (GSSG) metabolite concentrations. In addition, transcript levels of genes involved in antioxidant capacity and essential amino acid metabolites decreased significantly in embryos exposed to low levels of CaDNS. This is the first study to assess the toxicity of NSAs in amphibians, contributing important data to aid in the assessment of NSAs.
Fig. 1. Chemical structure of calcium dinonylnaphthalenesulfonate (CaDNS). It is composed of two identical sub-units, which each include a naphthalene molecule, carbon side-chains, and sulfonic acid components.
Table 2. Lethal and observable sub-lethal effects in Silurana tropicalis embryos exposed to CaDNS. Means are presented ± standard deviation (SD) except for the specific malformations are presented as a total count for that treatment. An asterisk (*) represents a significantly different value compared to the solvent control.
Fig. 2. The percent of Silurana tropicalis embryos with at least one visual malformation with exposure to increasing concentration (16.5â1393 μg/g) of calcium dinonylnaphthalenesulfonate (CaDNS). Data are presented as mean ± SEM. Data were analyzed using a one-way ANOVA (n = 3 replicates of 40â50 embryos). Significance compared to the solvent control is noted with an asterisk (*, p < 0.05). Regression was drawn using log(agonist) vs response â variable slope (4 parameters) constraining the bottom to 0% and the top to 100%.
Fig. 3. Relative mRNA levels in genes involved in oxidative stress defense including a) glutathione S-transferase (gst), b) glutathione peroxidase (gpx), c) glutathione reductase (gsr), d) superoxide dismutase (sod), and e) catalase (cat), and the f) tumor suppressing protein (p53) in Silurana tropicalis embryos exposed to calcium dinonylnaphthalenesulfonate (CaDNS) for 72 h. Data are presented as mean ± SEM. Data were analyzed using a one-way ANOVA (n = 10). Significance compared to the solvent control is noted with an asterisk (*, p < 0.05).
Fig. 4. Alterations to the glutathione redox cycle in Silurana tropicalis embryos exposed to a) 96 μg/g calcium dinonylnaphthalenesulfonate (CaDNS) compared to the solvent control and to b) over 672 μg/g CaDNS compared to the solvent control (solid arrows) or compared to lower exposure of CaDNS (dotted arrows). Black arrows represent significant difference (p < 0.05) and grey arrows represent an insignificant change. Capital letters represent metabolites measured and lower-case italics represent gene expression of the enzymes. Grey text was not measured in this study. Glutathione (reduced form, GSH), glutathione disulfide (oxidized form, GSSG), glutathione S-transferase (gst), glutathione peroxidase (gpx), glutathione reductase (gsr).
Table 3. Concentration of metabolites detected in Silurana tropicalis embryos exposed to solvent control, 96 μg/g, or 1393 μg/g calcium dinonylnaphthalenesulfonate (CaDNS) in the sand. Significant difference from solvent control is indicated with an asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001) and from 96 μg/g is indicated with a dagger (â p < 0.05).
