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Sci Total Environ
2021 Jan 20;753:141940. doi: 10.1016/j.scitotenv.2020.141940.
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Developmental reproductive toxicity and endocrine activity of propiconazole in the Xenopus tropicalis model.
Svanholm S
,
Säfholm M
,
Brande-Lavridsen N
,
Larsson E
,
Berg C
.
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Environmental pollutants and especially endocrine disrupting chemicals (EDCs) are implicated as one of the drivers of the amphibian declines. To advance the understanding of the risks of EDCs to amphibians, methods to determine endocrine-linked adverse effects are needed. The aims were to 1) develop a partial life-cycle assay with the model frog Xenopus tropicalis to determine endocrine perturbation and adverse developmental effects, and 2) determine effects of propiconazole in this assay. Propiconazole is a pesticide with multiple endocrine modes of action in vitro. Its potential endocrine activity and adverse effects in amphibians remain to be elucidated. Tadpoles were exposed to 0, 33 and 384 μg propiconazole/L during critical developmental windows until completed metamorphosis. At metamorphosis, a sub-sample of animals was analysed for endpoints for disruption of estrogen/androgen (sex ratio, brainaromatase activity) and thyroid pathways (time to metamorphosis). The remaining individuals were kept unexposed for 2 months post-metamorphosis to analyze effects on sexual development including gonadal and Müllerian duct maturity and gametogenesis. At metamorphosis, brainaromatase activity was significantly increased in the high-dose group compared to control. In both propiconazole groups, an increased proportion of individuals reached metamorphosis faster than the mean time for controls, suggesting a stimulatory effect on the thyroid system. At 2 months post-metamorphosis, testis size, sperm and Müllerian duct maturity were reduced in the low-dose males, and the liver somatic index in males was increased in both propiconazole groups, compared with controls. In conclusion, our results show that propiconazole exposure caused endocrine perturbations and subsequent hepatic and reproductive effects evident at puberty, indicating persistent disruption of metabolism and male reproductive function. Our findings advance the development of methodology to determine endocrine and adverse effects of EDCs. Moreover, they increase the understanding of endocrine perturbations and consequent risk of adverse effects of azoles in amphibians.
Fig. 1. Schematic illustration of the kidney-gonadal complex and the localization of the histological section used for evaluating gonadal and Müllerian duct (MD, blue structures) maturation (dotted line) in Xenopus tropicalis two months post metamorphosis. The complex was divided (solid line) and imbedded in two parts. For assessment of MD length, cross-sections were taken at every 300 μm throughout the entire length of the complex, i.e. the two parts of the complex were sectioned from the cranial/caudal parts towards the centre (arrows). G = gonads, K = kidneys. Adapted from Jansson et al., 2016. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Proportion of X. tropicalis with shorter time to metamorphosis compared to control mean (47 days) after larval exposure to 0 (control), 33 (low) or 384 μg propiconazole/L (high). *Significantly different from control (p < 0.05), Fisher's exact test. Number of individuals (n) is shown within the parenthesis.
Fig. 3. Aromatase activity in the brain from metamorphosed (NF 66) X. tropicalis after larval exposure to 0 (control), 33 (low) or 384 μg/L (high) propiconazole. Each point represents one individual. âŽâŽsignificantly different from the control group pË‚0.01 (one-way ANOVA and Tukey's Multiple Comparison test). The number of individuals (n) is shown within the parenthesis.
Fig. 4. Results of histological analysis of the testes of Xenopus tropicalis at two months post-metamorphosis following exposure to 0 (control), 33 (low) or 384 (high) μg propiconazole/L during larval development. A) Testis maturation score (mean (SD)) based on the most mature sperm stage found in a histological cross-section of the testis; 1 Spermatogonium, 2 Spermatocyte, 3 Spermatid, 4 Spermatozoon. B) Proportion of males with a reduced testis area (≤194 mm2). âŽSignificantly different to control p < 0.05 (Fisher's exact test), âŽâŽSignificantly different to control p < 0.01 (Kruskal-Wallis test with Dunn's Multiple Comparison Test). Data are presented as a mean of both left and righttestis for each individual. The number of individuals (n) is shown within the parenthesis.
Table 2. Maturation stage and length of Müllerian ducts in X. tropicalis at two months post-metamorphosis after larval exposure to 0 (control), 33 (low) or 384 (high) μg propiconazole/L. Data are presented as mean (SD) value for both left and rightducts for each individual.
a Score based on criteria by Jansson et al., 2016. “1 - a small rounded bulge of irregularly packed mesenchymal cells at the lateral side of the kidney, 2– a small bud protruding from the kidney, 3 – a distinct structure attached to the lateral side of the kidney, 4 – a distinct tubular structure without a cavity, and 5 – a distinct tubular structure with a cavity lined by elongated epithelial cellsâ€.
b n = 11.
c n = 13.
d n = 10.
e n = 12.
f n = 9.
⎠Significantly different from corresponding control p < 0.05 (Kruskal-Wallis test with Dunn's Multiple Comparison Test).
### Significantly different from corresponding females p < 0.001 (Fisher's exact test).
^^ Significantly different from corresponding females p < 0.01 (Mann Whitney test).
^^^ Significantly different from corresponding females p < 0.001 (Mann Whitney test).
¤ Significantly different from corresponding high p < 0.05.
Table A.2. Growth and mortality in Xenopus tropicalis at metamorphosis after developmental exposure to either 0 (control), 33 (low) or 384 (high) μg propiconazole/L. Data is presented as mean (SD).
Table A.3. Sex ratio in Xenopus tropicalis after developmental exposure to either 0 (control), 33 (low) or 384 (high) μg propiconazole/L.
Table A.4. Aromatase activity in brain from X. tropicalis at 2 months post metamorphosis after larval exposure to either 0 (control), 33 (low) or 384 µg propiconazole/ L (high). The data are presented as a mean value per exposure group (SD).
Table A.5. Histomorphometrical data for testes in Xenopus tropicalis at two months post-metamorphosis following exposure to 0 (control), 33 (low) or 384 (high) µg propiconazole/L during larval development. Data are presented as a mean (SD) of both left and righttestis.
Table A.6. Distribution of oocyte stages in the ovaries of X. tropicalis 2 months post-metamorphosis after developmental exposure to 0 (control), 33 (low) or 384 (high) µg propiconazole/L. The data are mean of both ovaries and treatment.
Table A.7. Anti Müllerian hormone (AMH) and estradiol (E2) concentration in whole body homogenate from
females two months post metamorphosis exposed to either 0 (control), 33 (low) or 384 (high) µg propiconazole/L as tadpoles. Data is presented as mean (SD).
