Analysis of Sublethal Toxicity in Developing Zebrafish Embryos Exposed to a Range of Petroleum Substances.

The Organisation for Economic Co-operation and Development (OECD) test guideline 236 (fish embryo acute toxicity test; 2013) relies on 4 endpoints to describe exposure-related effects (coagulation, lack of somite formation, tail-bud detachment from the yolk sac, and the presence of a heartbeat). Danio rerio (zebrafish) embryos were used to investigate these endpoints along with a number of additional sublethal effects (cardiac dysfunction, pericardial edema, yolk sac edema, tail curvature, hatch success, pericardial edema area, craniofacial malformation, swim bladder development, fin development, and heart rate) following 5-d exposures to 7 petroleum substances. The substances investigated included 2 crude oils, 3 gas oils, a diluted bitumen, and a petrochemical containing a mixture of branched alcohols. Biomimetic extraction-solid-phase microextraction (BE-SPME) was used to quantify freely dissolved concentrations of test substances as the exposure metric. The results indicated that the most prevalent effects observed were pericardial and yolk sac edema, tail curvature, and lack of embryo viability. A BE-SPME threshold was determined to characterize sublethal morphological alterations that preceded embryo mortality. Our results aid in the understanding of aquatic hazards of petroleum substances to developing zebrafish beyond traditional OECD test guideline 236 endpoints and show the applicability of BE-SPME as a simple analytical tool that can be used to predict sublethal embryo toxicity. Environ Toxicol Chem 2019;38:1302-1312. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Hepatic gene expression in rainbow trout (Oncorhynchus mykiss) exposed to different hydrocarbon mixtures.

Traditional biomarkers for hydrocarbon exposure are not induced by all petroleum substances. The objective of this study was to determine if exposure to a crude oil and different refined oils would generate a common hydrocarbon-specific response in gene expression profiles that could be used as generic biomarkers of hydrocarbon exposure. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to the water accommodated fraction (WAF) of either kerosene, gas oil, heavy fuel oil, or crude oil for 96 h. Tissue was collected for RNA extraction and microarray analysis. Exposure to each WAF resulted in a different list of differentially regulated genes, with few genes in common across treatments. Exposure to crude oil WAF changed the expression of genes including cytochrome P4501A (CYP1A) and glutathione-S-transferase (GST) with known roles in detoxification pathways. These gene expression profiles were compared to others from previous experiments that used a diverse suite of toxicants. Clustering algorithms successfully identified gene expression profiles resulting from hydrocarbon exposure. These preliminary analyses highlight the difficulties of using single genes as diagnostic of petroleum hydrocarbon exposures. Further work is needed to determine if multivariate transcriptomic-based biomarkers may be a more effective tool than single gene studies for exposure monitoring of different oils.

Temporal patterns in the transcriptomic response of rainbow trout, Oncorhynchus mykiss, to crude oil.

Time is often not characterized as a variable in ecotoxicogenomic studies. In this study, temporal changes in gene expression were determined during exposure to crude oil and a subsequent recovery period. Juvenile rainbow trout, Oncorhynchus mykiss, were exposed for 96 h to the water accommodated fractions of 0.4, 2 or 10 mgl(-1) crude oil loadings. Following 96 h of exposure, fish were transferred to recovery tanks. Gill and liver samples were collected after 24 and 96 h of exposure, and after 96 h of recovery for RNA extraction and microarray analysis. Fluorescently labeled cDNA was hybridized against matched controls, using salmonid cDNA arrays. Each exposure scenario generated unique patterns of altered gene expression. More genes responded to crude oil in the gill than in the liver. In the gill, 1137 genes had altered expression at 24 h, 2003 genes had altered expression levels at 96 h of exposure, yet by 96 h of recovery, no genes were significantly altered in expression. In the liver at 10 mgl(-1), only five genes were changed at 24 h, yet 192 genes had altered expression after 96 h recovery. At 2 mgl(-1) in the liver, many genes had altered regulation at all three time points. The 0.4 mgl(-1) loading also showed 289 genes upregulated at 24 h after exposure. The Gene Ontology terms associated with altered expression in the liver suggested that the processes of protein synthesis, xenobiotic metabolism, and oxidoreductase activity were altered. The concentration-responsive expression profile of cytochrome P450 1A, a biomarker for oil exposure, did not predict the majority of gene expression profiles in any tissue or dose, since direct relationships with dose were not observed for most genes. While the genes and their associated functions agree with known modes of toxic action for crude oil, the gene lists obtained do not match our previously published work, presumably due to array analysis procedures. These results demonstrate that changes in gene expression with time and dose may be complicated, and should be characterized in controlled laboratory settings before attempts are made to interpret responses in field-collected organisms. Further, processes for analyzing microarray data need to be developed such that standardized gene lists are developed, or that analysis does not rely on lists of significantly altered genes before arrays can be further evaluated as a monitoring tool.

Identification of six differentially expressed genes in response to copper exposure in the aquatic plant Lemna gibba (Duckweed).

Aquatic plants are susceptible to metal pollution and provide an entry point for metals, such as copper, into the aquatic biosphere. Exposure of the aquatic plant Lemna gibba to copper has been associated with the production of reactive oxygen species (ROS) and oxidative damage, caused in large part by the ability of this metal to redox cycle. In particular, copper-mediated production of ROS, a known group of signaling molecules, triggers numerous defense responses in L. gibba. Therefore, the objective of the present study was to examine to what extent acute copper exposure alters gene expression. First, the kinetics of copper uptake was assessed to determine if assimilation occurred within the short exposures needed to induce gene expression. Subsequently, using differential display polymerase chain reaction, we identified six genes with expressions that were putatively altered in response to copper. Differential expression was confirmed by northern hybridization analysis and showed that copper causes an accumulation of transcripts that encode for callose synthase, heat shock protein 90, serine decarboxylase, and the biotin carboxylase subunit of acetyl-coenzyme A carboxylase. Conversely, copper caused a decline in transcript levels for genes encoding the HAP5 subunit of the heme-activated protein (HAP) transcription factor in addition to the chloroplast nucleoid DNA-binding protein CND41. Interestingly, the expressions of these genes are sensitive to cellular ROS levels. We believe that these gene products provide valuable information regarding the molecular mechanisms of copper toxicity.