Transcriptome Analysis Reveals That Naphthenic Acids Perturb Gene Networks Related to Metabolic Processes, Membrane Integrity, and Gut Function in Silurana (Xenopus) tropicalis Embryos

Abstract

Naphthenic acids (NAs) are oil-derived mixtures of carboxylic acids and are considered emerging contaminants with the potential to disrupt development of aquatic species. In the Oil Sands Region of Canada, NAs are components of the water released following processing of the bitumen-containing sand. The aim of this research was to identify potential mechanisms of toxicity of NA mixtures. Silurana (Xenopus) tropicalis embryos were raised in water spiked with commercial oil-derived NA extracts (S1 and S2) at a sub-lethal concentration (2 mg/L). The transcriptomic responses of the whole 4-day old embryos following exposure were assessed using a custom oligonucleotide microarray. Both NA mixtures induced embryonic abnormalities that included edema, and cardiac and gut abnormalities. Exposure to NAs also affected morphometric parameters and decreased total length, tail length, and interorbital distance of the embryos. Gene ontology analysis revealed that 18 biological processes, 5 cellular components, and 19 molecular functions were significantly enriched after both S1 and S2 exposures. Sub-network enrichment analysis revealed pathways that were related to phenotypic abnormalities; these included gut function, edema, and cartilage differentiation. Other notable networks affected by NAs included metabolism and cell membrane integrity. In a separate dose-response experiment, the expression of key genes identified by microarray (cyp4b1, abcg2, slc26a6, eprs, and slc5a1) was determined by Real-Time qPCR in S. tropicalis embryos exposed to the commercial NAs and to acid-extractable organics (AEOs) prepared from Oil Sands Process-Affected Water. In general, the RT-qPCR data agreed with the microarray data. In S. tropicalis embryos exposed to the AEOs, the mRNA levels of eprs (bifunctional glutamate/proline-tRNA ligase) and slcs5a1 (sodium/glucose cotransporter 1) were significantly decreased compared to

Petroleum-derived naphthenic acids disrupt hormone-dependent sexual behaviours in male Western clawed frogs

“…Toxicity assays in aquatic invertebrates comparing commercially available NA mixtures to NA fractions from tailings ponds found LC50s several times lower in the commercial extracts compared to those from tailing ponds ( Bartlett et al , 2017 ). Larvae of S. tropicalis exposed to commercial NAs showed lower LC50s and EC50s compared to larvae exposed to OSPW AEOs but experienced the same types of morphological abnormalities and disruption of gene networks involved in metabolic and developmental processes ( Gutierrez-Villagomez et al , 2019a ). Larval fathead minnows ( Pimephales promelas ) exposed to fresh and aged NA fractions from OSPW as well as commercial NAs all showed immunome disruptions ( Loughery et al , 2019 ).…”

“…Furthermore, these data suggest that NAs have multiple modes of action to induce developmental effects in amphibians. Based on this transcriptomic analysis, it can be concluded that some modes of action may be shared between commercial NAs and those extracted from OSPW ( Gutierrez-Villagomez et al , 2019a ). Similar conclusions were drawn when comparing transcriptomic responses of larval flathead minnows exposed to various NA sources ( Loughery et al , 2019 ).…”

“…From these studies, the LC50, EC50 and EC20 values for S. tropicalis embryos ( Gutierrez-Villagomez et al , 2019b ) are less than or within the concentration ranges reported in some OSPW and natural groundwater samples in Alberta. Global transcriptomic responses in whole S. tropicalis embryos indicated that numerous gene pathways related to gut function, edema and cartilage development are disrupted by sublethal levels of NAs ( Gutierrez-Villagomez et al , 2019a ). Furthermore, these data suggest that NAs have multiple modes of action to induce developmental effects in amphibians.…”

Frogs Respond to Commercial Formulations of the Biopesticide Bacillus thuringiensis var. israelensis, Especially Their Intestine Microbiota

“…The RNA integrity was assessed by the presence of two defined bands on an agarose gel. 35 The top band represents the 28S ribosomal RNA subunit (rRNA), and the second band represents the 18S rRNA. 36 However, smearing was observed in the liver samples of L. sylvaticus and therefore not included in the gene expression analysis to avoid false results.…”

“…Total RNA was isolated from the liver and tail using TRIzol reagent and phase maker tubes (Thermo Fisher Scientific) as described in the manufacturer’s protocol and adding 1 μL of 20 μg/μL glycogen (Thermo Fisher Scientific) to increase the RNA yield. The RNA integrity was assessed by the presence of two defined bands on an agarose gel . The top band represents the 28S ribosomal RNA subunit (rRNA), and the second band represents the 18S rRNA .…”