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Proc Natl Acad Sci U S A
2018 May 08;11519:E4416-E4425. doi: 10.1073/pnas.1721267115.
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Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline.
Regnault C
,
Usal M
,
Veyrenc S
,
Couturier K
,
Batandier C
,
Bulteau AL
,
Lejon D
,
Sapin A
,
Combourieu B
,
Chetiveaux M
,
Le May C
,
Lafond T
,
Raveton M
,
Reynaud S
.
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Despite numerous studies suggesting that amphibians are highly sensitive to endocrine disruptors (EDs), both their role in the decline of populations and the underlying mechanisms remain unclear. This study showed that frogs exposed throughout their life cycle to ED concentrations low enough to be considered safe for drinking water, developed a prediabetes phenotype and, more commonly, a metabolic syndrome. Female Xenopus tropicalis exposed from tadpole stage to benzo(a)pyrene or triclosan at concentrations of 50 ng⋅L-1 displayed glucose intolerance syndrome, liver steatosis, liver mitochondrial dysfunction, liver transcriptomic signature, and pancreatic insulin hypersecretion, all typical of a prediabetes state. This metabolic syndrome led to progeny whose metamorphosis was delayed and occurred while the individuals were both smaller and lighter, all factors that have been linked to reduced adult recruitment and likelihood of reproduction. We found that F1 animals did indeed have reduced reproductive success, demonstrating a lower fitness in ED-exposed Xenopus Moreover, after 1 year of depuration, Xenopus that had been exposed to benzo(a)pyrene still displayed hepatic disorders and a marked insulin secretory defect resulting in glucose intolerance. Our results demonstrate that amphibians are highly sensitive to EDs at concentrations well below the thresholds reported to induce stress in other vertebrates. This study introduces EDs as a possible key contributing factor to amphibian population decline through metabolism disruption. Overall, our results show that EDs cause metabolic disorders, which is in agreement with epidemiological studies suggesting that environmental EDs might be one of the principal causes of metabolic disease in humans.
Fig. 1.
ED exposure leads to metabolic impairments in Xenopus. (A) Glucose tolerance test. (B) Relative hepatosomatic indices of control and exposed animals. (C) Oil Red O staining for total lipid content measured in the livers of control and exposed animals. Lipid content is indicated by red staining. (Scale bars, 25 µm.) Histograms represent the relative Oil Red O area in the livers of control and exposed animals. (D) Relative blood triglycerides concentrations. (E) Relative mitochondrial state 2 respiration rate in liver of control and exposed animals. (F) Relative mitochondrial respiratory control indices in the livers of control and exposed animals. (G) Relative aconitase activity in control and exposed animals. (H) Relative citrate synthase activity in control and exposed animals. (I) Relative ALAT activity in control and exposed animals. (J) H&E stain of liver sections of control animals. (Scale bars, 70 µm.) (K) H&E stain of liver sections of BaP-exposed animals. The arrow indicates an area of ballooning hepatocytes. (Scale bars, 70 µm.) (L) H&E stain of liver sections of TCS-exposed animals. (Scale bars, 70 µm.) (M) Gomori’s Trichrom stain of sections of control animals. (Scale bars, 70 µm.) (N) Gomori’s Trichrom stain of liver sections of BaP-exposed animals. The arrow indicates a necrotic area with leukocyte infiltrates. (Scale bars, 70 µm.) (O) Gomori’s Trichrom stain of liver sections of TCS-exposed animals. The arrow indicates a dilated blood vessel. (Scale bars, 70 µm.) (P) Relative proteasome activity in the livers of control and exposed animals. (Q) Insulin immunostaining of the pancreas of control and exposed animals. Insulin content is indicated by red staining. (Scale bars, 25 µm.) The histograms present the relative insulin quantity estimated as area × intensity of islet stained for insulin in the pancreas of control and exposed animals. (R) Relative glycogen content in the muscles of control and exposed animals. (S) Relative glycogen content in the livers of control and exposed animals. The statistical analysis was performed using Dunnett’s test on log-transformed data. The asterisks indicate a significant difference from the control: *P < 0.05, **P < 0.01, ***P < 0.001; n = 4 per exposure group.
Fig. 2.
Liver transcriptome following ED exposure indicates a marked metabolic impairment typical of IR syndrome. (A) The genes showing a significant differential transcription from control in at least one condition were used for annotation enrichment using the Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tool (modified Fisher’s exact test with P < 0.05). Histograms indicate the level of enrichment of the pathways, whenever the levels were significant for BaP- or TCS-exposed animals. (B–D) Transcription of genes involved in the different pathways. The color scale indicates transcription ratios relative to the controls. The black boxes indicate the pathway highlighted by the gene found to be differentially transcribed compared with the control. Pathways have been grouped according to their parent root in the KEGG description. (E) Transcription of genes involved in the immune system or where a literature search yielded specific links with the NAFLD/NASH syndrome. The black boxes indicate the pathway highlighted by the gene found to be differentially transcribed compared with the control. NS indicates that the specific gene was not found to be differentially transcribed in the considered exposure with regard to the control. n = 4 per exposure group.
Fig. 3.
Persistence of metabolic impairments in Xenopus after 1 y of depuration. (A) Glucose tolerance test. (B) Relative hepatosomatic indices of control and exposed animals. (C) Oil Red O staining for total lipid content measured in the livers of control and exposed animals. Lipid content is indicated by red staining. (Scale bars, 25 µm.) Histograms present the relative Oil Red O area in the livers of control and exposed animals. (D) Insulin immunostaining of the pancreas of control and exposed animals. Insulin content is indicated by red staining. (Scale bars, 25 µm.) The histograms show the relative insulin quantity estimated from the product of area × intensity of the islet stained for insulin in the pancreas of control and exposed animals. The statistical analysis was performed using Dunnett’s test on log-transformed data. The asterisk indicates a significant difference from the control: *P < 0.05; n = 3 per exposure group.
Fig. 4.
Parental exposure to EDs leads to delayed metamorphosis and reduced size and weight of progeny. (A) Development curves of F1 individuals from hatching to metamorphosis. The distributions of F1 individual development times were compared between the control and each parental ED-exposed population using a Kaplan–Meyer test to evaluate global curve difference. The asterisk indicates a significant difference from the control: *P < 0.05 (n = 716, 1,020, and 72 for control, BaP, and TCS parental exposure). (B) Snout-vent length of F1 individuals at time of metamorphosis from each parental exposure group. (C) Weight of F1 individuals at time of metamorphosis from each parental exposure group. The statistical analysis was performed using Dunnett’s test on log-transformed data. The asterisks indicate a significant difference from the control: *P < 0.05, ***P < 0.001 (n = 46, 63, and 10 for control, BaP, and TCS parental exposure, respectively). (D) Development curves of F1 juvenile females from hatching to sexual maturity. The distributions of F1 individual development times were compared between the control and each parental ED-exposed population using a Kaplan–Meyer test to evaluate global curve difference. The asterisk indicates a significant difference from the control: *P < 0.05 (n = 10, 28, and 1 for control, BaP, and TCS parental exposure). (E) Percentage of successful amplexus 3 h poststimulation of F1 animals (n = 5, 5, and 1 for the control, BaP, and TCS parental exposures, respectively). (F) Number of hatched eggs per F1 female from each successful amplexus (n = 5, 5, and 1 for the control, BaP, and TCS parental exposures, respectively).
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