Metabolomic Characterizations of Liver Injury Caused by Acute Arsenic Toxicity in Zebrafish

Abstract

Arsenic is one of the most common metalloid contaminants in groundwater and it has both acute and chronic toxicity affecting multiple organs. Details of the mechanism of arsenic toxicity are still lacking and profile studies at metabolic level are very limited. Using gas chromatography coupled with mass spectroscopy (GC/MS), we first generated metabolomic profiles from the livers of arsenic-treated zebrafish and identified 34 significantly altered metabolite peaks as potential markers, including four prominent ones: cholic acid, glycylglycine, glycine and hypotaurine. Combined results from GC/MS, histological examination and pathway analyses suggested a series of alterations, including apoptosis, glycogenolysis, changes in amino acid metabolism and fatty acid composition, accumulation of bile acids and fats, and disturbance in glycolysis related energy metabolism. The alterations in glycolysis partially resemble Warburg effect commonly observed in many cancer cells. However, cellular damages were not reflected in two conventional liver function tests performed, Bilirubin assay and alanine aminotransferase (ALT) assay, probably because the short arsenate exposure was insufficient to induce detectable damage. This study demonstrated that metabolic changes could reflect mild liver impairments induced by arsenic exposure, which underscored their potential in reporting early liver injury.

Fig 1. Results of chemometric analysis and hierarchical clustering.

(A) PCA plot showing tight clustering of QC samples and inherent separation between control and treated groups. (B) PLS-DA plot with decent separation of the two groups. (C) Hierarchical clustering of samples using peak areas of the 34 potential metabolite markers. Peak areas were quantile-normalized and standardized to respective mean value of each metabolite among all samples. Hierarchical clustering was performed using Pearson correlation method without mean centering.

Fig 2. Effects of the metabolic alteration on Disease and Bio Functions and Tox Functions predicted by IPA (p<0.05).

(A) The top five entries in three aspects of biological impact: Disease and Disorders, Molecular and Cellular Functions, and Hepatotoxicity. (B) Top altered canonical pathways predicted by IPA (p<0.005). The p values were calculated using Fischer's exact test determining the probability that the association between the genes in the data set and the canonical pathway was due to chance alone. The ratios were calculated based on the number of genes from the data set that mapped to the pathway and the total number of genes in the pathway.

Fig 3. Histological examinations of liver sections.

(A-B) H&E staining revealed changes in cellular organization, cytoplasmic volume, and nuclei morphology in treated group (lower panel) as compared to control (upper panel). (C-D) ORO staining showed marked accumulation of lipid dropolets in As group. (E-F) Glycogen depletion in the As group was evident with PAS staining. (G-H) Increased apoptotic cells (stained with green fluorescence) were observed in the As group. Magnification: 400x. Arrow: binucleated cells. Scale bars: 50μm.

Fig 4. Liver function tests by Bilirubin assay (A-C) and ALT assay (D-F).

Total protein content (A) and total bilirubin (B) were significantly higher in plasma of As-treated fish as compared to control, with p = 0.008 and p = 0.037 respectively. Direct bilirubin (C) and plasma ALT activity (D) remained normal (p>0.3). No significant change was found in total protein concentration (E) or ALT activity (F) of liver (p>0.1). Five biological replicates were used for each assay and statistical test used was one-tailed student t-test.

Fig 5. Network of glycolysis, glycogen metabolism and fatty acid metabolism constructed based on metabolic changes found in this study and mRNA changes reported in our previous transcriptomic study [3].

Gene names: acaa2, acetyl-CoA acyltransferase 2; acsl5, acyl-CoA synthetase long-chain family member 5; aldob, aldolase b, fructose-bisphosphate; ehhadh, enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase; eno1, enolase 1, (alpha); gck, glucokinase (hexokinase 4); gpia, glucose phosphate isomerase a; hadh, hydroxyacyl-Coenzyme A dehydrogenase; ldhbb, lactate dehydrogenase Bb; mpc2, mitochondrial pyruvate carrier 2; pfkfb4b (pfkfb4l), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4b; pgam1a, phosphoglycerate mutase 1a; pgm3, phosphoglucomutase 3; pgk1, phosphoglycerate kinase 1; pkm2a, pyruvate kinase, muscle, a; pygb, phosphorylase, glycogen; brain; Pygl, phosphorylase, glycogen, liver; slc27a2, solute carrier family 27 (fatty acid transporter), member 2; taldo1, transaldolase 1.