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BMC Genomics
2014 Aug 08;15:666. doi: 10.1186/1471-2164-15-666.
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Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence.
Regnault C
,
Worms IA
,
Oger-Desfeux C
,
MelodeLima C
,
Veyrenc S
,
Bayle ML
,
Combourieu B
,
Bonin A
,
Renaud J
,
Raveton M
,
Reynaud S
.
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BACKGROUND: Despite numerous studies suggesting that amphibians are highly sensitive to cumulative anthropogenic stresses, the role pollutants play in the decline of amphibian populations remains unclear. Amongst the most common aquatic contaminants, polycyclic aromatic hydrocarbons (PAHs) have been shown to induce several adverse effects on amphibian species in the larval stages. Conversely, adults exposed to high concentrations of the ubiquitous PAH, benzo[a]pyrene (BaP), tolerate the compound thanks to their highly efficient hepatic detoxification mechanisms. Due to this apparent lack of toxic effect on adults, no studies have examined in depth the potential toxicological impact of PAH on the physiology of adult amphibian livers. This study sheds light on the hepatic responses of Xenopus tropicalis when exposed to high environmentally relevant concentrations of BaP, by combining a high throughput transcriptomic approach (mRNA deep sequencing) and a characterization of cellular and physiological modifications to the amphibian liver.
RESULTS: Transcriptomic changes observed in BaP-exposed Xenopus were further characterized using a time-dependent enrichment analysis, which revealed the pollutant-dependent gene regulation of important biochemical pathways, such as cholesterol biosynthesis, insulin signaling, adipocytokines signaling, glycolysis/gluconeogenesis and MAPK signaling. These results were substantiated at the physiological level with the detection of a pronounced metabolic disorder resulting in a possible insulin resistance-like syndrome phenotype. Hepatotoxicity induced by lipid and cholesterol metabolism impairments was clearly identified in BaP-exposed individuals.
CONCLUSIONS: Our data suggested that BaP may disrupt overall liver physiology, and carbohydrate and cholesterol metabolism in particular, even after short-term exposure. These results are further discussed in terms of how this deregulation of liver physiology can lead to general metabolic impairment in amphibians chronically exposed to contaminants, thereby illustrating the role xenobiotics might play in the global decline in amphibian populations.
Figure 1.
Kinetics of benzo[a]pyrene (BaP) concentrations in water (grey curve) and metabolite concentrations in bile (black curve) as a function of exposure time during
Xenopus tropicalis
exposure. Values are mean ± SE for 3 replicates.
Figure 2.
Lipid metabolism disorder induced by BaP. A. Hierarchical clustering of cholesterol biosynthesis genes differentially transcribed compared to control. The color scale indicates transcription ratios relative to the control. Gene names or annotations are indicated. 7-DHCR, 7-dehydrocholesterol reductase; FDF1, farnesyl-diphosphate farnesyltransferase 1; 24-DHCR, 24-dehydrocholesterol reductase; CYP51A1, cytochrome P450-51A1. Stars indicate significant transcription variations (>1.5-fold in either direction and adjusted p < 0.05). B. Hierarchical clustering of genes involved in cholesterol biosynthesis regulation and depletion from the blood differentially transcribed compared to control. The color scale indicates transcription ratios relative to the control. Gene names or annotations are indicated. SREBP-TF2, sterol regulatory element binding transcription factor 2; LDLR, low density lipoprotein receptor, HMGCoAR, 3-hydroxy-3-methylglutaryl-CoA reductase. Stars indicate significant transcription variations (>1.5-fold in either direction and adjusted p < 0.05). C. Kinetics of cholesterol concentration in bile. Data represent mean ± SE values for 3 replicates. Statistical analysis was performed using the Mann–Whitney test, asterisks indicate a significant difference from the control: *, p < 0.05. D. Oil red staining for total lipid content measures in the livers of control and BaP-exposed animals. Lipid content was indicated by red staining. Bars = 25 μm. E. Percentage of oil red area in the livers of control and BaP-exposed animals. Data are mean ± SE values for 24 sections replicates. Statistical analysis was performed using the Mann–Whitney test (n = 3), and asterisks indicate a significant difference from the control: *, p < 0.05.
Figure 3.
Carbohydrate metabolism disturbances induced by BaP. A. Hierarchical clustering of genes found to be differentially transcribed compared to the control and involved in insulin, adipocytokine and gluconeogenesis pathways. Color scale indicates transcription ratios relative to the control. Gene names or annotations are indicated. Stars indicate significant transcription variations (>1.5-fold in either direction and adjusted p < 0.05). B. Glycemia kinetics in control and in BaP-exposed X. tropicalis. Data are expressed as mean ± SE of 3 replicates.
Figure 4.
Hepatotoxicity induced by BaP. A. Hierarchical clustering of MAPK genes found differentially transcribed compared to the control. Color scale indicates transcription ratios relative to the control. Gene names or annotations are indicated. HSP701A, heat shock protein 701A. Stars indicate significant transcription variations (>1.5-fold in either direction and adjusted p < 0.05). B. (a) TUNEL staining detecting apoptosis-induced DNA damage in hepatocytes in control and in BaP-exposed animals at 24 hours. Arrows indicate apoptotic hepatocytes. Bars = 50 μm. (b) Percentage of TUNEL-positive nuclei in the livers of control and BaP-exposed animals according to the exposure time points. Data are mean ± SE values for 21 sections replicates. Statistical analysis was performed using the Mann–Whitney test, and asterisks indicate a significant difference from the control: *, p < 0.05. C. (a) Hematoxylin-eosine-safran (HES) staining of liver sections from control and X. tropicalis exposed to BaP. Bars = 100 μm. (b) Percentage of pigment area in liver sections of control and BaP exposed animals. Data are mean ± SE values for 21 sections replicates. Statistical analysis was performed using the Mann–Whitney test (n = 3), and asterisks indicate a significant difference from the control: *, p < 0.05.
Figure 5.
Cellular pathways potentially involved in
X. tropicalis
hepatocyte responses to BaP exposure at sub-lethal concentration. This model, based on our transcriptome and liver phenotype dynamic analyses, suggests that BaP is responsible for the induction of an insulin-resistance-like phenotype in Xenopus. Insulin resistance is characterized by the over-transcription of gluconeogenic enzymes genes (PEPCK), sustained hyperglycemia, an over-transcription of glucose transporter (GLUT2) and severe liver steatosis. Consequently, hyperinsulinemia may lead to the marked induction of cholesterol synthesis pathways and to a decrease in cholesterol export to the bile. The accumulation of lipids and cholesterol in the hepatocytes thus induces both ER stress and lipid toxicity leading to apoptosis or necrosis. LDL, Low density lipoprotein; LDLR, Low density lipoprotein receptor; GLUT2, Glucose transporter 2; Glc, glucose; Chol, cholesterol; PEPCK, phosphoenolpyruvate carboxykinase 1; HMGCoAR, 3-hydroxy-3-methylglutaryl-CoA reductase; SREBP-TF2, sterol regulatory element binding transcription factor 2; ER, endoplasmic reticulum; FFA, Free fatty acid, TG, triglycerides; Insulin R, insulin resistance.
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