Proteomic Analysis of Arsenic-Exposed Zebrafish (Danio rerio) Identifies Altered Expression in Proteins Involved in Fibrosis and Lipid Uptake in a Gender-Specific Manner

Abstract

The zebrafish (Danio rerio) was used to investigate protein expression in the liver following arsenic exposure. Several disorders have been linked to arsenic exposure, including cancer, diabetes, and cardiovascular disease. The mechanisms of arsenic toxicity are poorly understood. Prior studies have described altered gene expression, inflammation, and mitogenic signaling in acute or chronic exposure models. A proteomic approach was employed to investigate arsenic-induced alteration in the zebrafish liver proteome following a 7-day exposure to 50 ppb sodium arsenite. Over 740 unique proteins were identified, with fewer than 2% showing differential expression. Molecular pathway analysis software identified lipid metabolism and transport as potential molecular targets. Immunoblots were used to confirm protein expression changes, whereas qPCR was employed to investigate gene expression changes. Overall, 25 proteins were differentially expressed in a gender-specific manner, 11 in males and 14 in females. Of these 25, a single protein, hydroxysteroid dehydrogenase like 2, showed decreased expression in both males and females following arsenic exposure. These findings indicate that protein expression is altered following arsenic exposure. The changes presented here seem to be most prevalent in lipid transport and metabolic pathways, suggesting a potential increase in fibrosis in males and decreased lipid accumulation and uptake in females.

Development of a Convenient In Vivo Hepatotoxin Assay Using a Transgenic Zebrafish Line with Liver-Specific DsRed Expression

“…Interestingly, in an adult zebrafish acute exposure set-up, fatty acid binding proteins were found to be up-regulated by arsenate in male fish but not female fish. [44], [46]. It may suggest that the effect of arsenate on lipid metabolism is sex-dependent; however, our embryo assay evaluates toxicity without discrimination of sexes.…”

Zebrafish: An Important Tool for Liver Disease Research

“…126 The arsenic-metabolizing enzyme (encoded by as3mt ) is expressed in livers of mammals and zebrafish and makes the liver susceptible to toxic injury. 127 Transcriptional 124,128 and proteomic 129 analyses of livers from zebrafish exposed to arsenic identified lipid metabolic and carcinogenic pathways that were upregulated. Integration of high-throughput genomic, proteomic, and metabolomic analyses with studies of transgenic zebrafish could elucidate the mechanisms of arsenic-induced hepatotoxicity and predisposition to liver cancer, a goal that has not been achieved with any other animal model of arsenic toxicity.…”

Use of proteomics to detect sex-related differences in effects of toxicants: implications for using proteomics in toxicology

“…In addition other techniques have been applied, including 1D-SDS-PAGE (Chen et al 2016), Filter Aided Sample Preparation (FASP) (Schilling et al 2015) or gel-free proteomic approaches combined with (nano)-liquid chromatography (LC) in line with high resolution mass spectrometry analysis (Fogle et al 2011;Carlson et al 2013;Chen et al 2014;Fong et al 2014;Wang et al 2014;Gismondi et al 2015;Yamanobe et al 2015;Al-Daghri et al 2016;Le Manach et al 2016;Yang et al 2016). These methods often used 1D-SDS-PAGE gel separation and protein extraction from 3-10 slices of the gel, enabling identification of several thousands of proteins.…”

“…In addition, iTRAQ or TMT based methods have been developed. For the studies on sex-based differences, also use of iTRAQ (Carlson et al 2013;Fong et al 2014;Ji et al 2014;Wang et al 2014;Al-Daghri et al 2016;Ji et al 2016;Le Manach et al 2016;Yang et al 2016) or sometimes TMT (Tandem Mass Tag) (Chen et al 2014) was encountered (Table 1). Using these techniques, peptides are labeled with a stable isotope detection tag after trypsin digestion.…”

“…Although iTRAQ and TMT labeling seems promising, it is an expensive method and requires very good lab handling of the samples. iTRAQ or TMT labeling is mostly used in human studies in a medical setting (Al-Daghri et al 2016;Yang et al 2016), although in the overview presented in Table 1 the method was also applied for studies on mussels (Ji et al 2014;Ji et al 2016), zebrafish (Carlson et al 2013), medaka (Fong et al 2014;Le Manach et al 2016) or rodents (Fogle et al 2011;Wang et al 2014).…”