Fish in our diet provides us with key nutrients such as marine omega-3 and -6 fatty acids, vitamins A and E, iodine and selenium that are vital for heart, brain, and bone health. But eating fish may also expose us to harmful substances in the environment such as persistent organic pollutants (POPs), pesticides, and heavy metals that fish ingest.
POPs detected in fish include legacy chemicals such as dioxins and polychlorinated biphenyls as well as more recent pollutants like brominated flame-retardants (BFRs). These tend to accumulate in fatty tissues (in fish and humans) potentially causing chronic exposure.
Dr Josef Rasinger, a nutritional toxicologist coordinating food and feed safety testing, and Dr Kai Kristoffer Lie, an ecotoxicologist, are using Qlucore Omics Explorer to study the effects of environmental contaminants throughout the fish food chain, including how these contaminants may interact with the nutrients in fish.
Based in Bergen at Norway’s National Institute of Nutrition and Seafood Research (NIFES) – a leading centre for research into fish nutrition and the effects of fish and seafood consumption on human health – Dr Rasinger and Dr Lie work on multi-level omic (molecular) analyses that combine gene expression profiles (transcriptomes), protein profiles (proteomes), and small molecule metabolite profiles (metabolomes).
Using these techniques, they are learning about the detailed mechanisms of nutrient and contaminant exposures and discovering novel biomarkers for health and disease.
“Compared to conventional measurements like organ-weights, clinical chemistry and microscopic histopathology, molecular marker studies enable us to explore mechanisms of toxicity at much lower doses. This allows us to discover earlier if an environmental contaminant is more or less of a problem than we thought. By seeing these trends, it means we can react faster and design focused toxicology experiments around the most problematic substances actually out there,” says Dr Rasinger.
Both scientists have a strong background in data analytics and a keen interest in the development and advancement of molecular, bio-statistical and bio-informatics tools. However, the sheer volumes and complexities of data generated at NIFES remain a challenge.
Simple and fast visualisation of data
With multiple projects in the pipeline, Qlucore Omics Explorer has proved to be a highly efficient way to carry out data exploration compared to other customised in house bio-informatics tools.
“Qlucore Omics Explorer has become our ‘go-to’ tool for getting a first glance of any multivariat data problem,“ explains Dr Rasinger. “It provides us with simple and fast visualisation of data, irrespective of the data source.“
He adds: “Typically we are looking at thousands of changing features and it’s great to have those summarised in a PCA [principal component analysis] plot or heat map. It means we can very quickly see if there is something interesting going on and if so whether we should dig deeper.”
An important benefit of the Qlucore tool, says Dr Rasinger (who is an expert in proteomics) is that it covers all levels of the omics hierarchy – proteins, RNA expression and metabolites. “It means I can look at correlations on a single platform and I don’t need to be an expert in RNA expression or metabolites to do this kind of analysis.“
Dr Rasinger and colleagues have used Qlucore Omics Explorer in a great many research studies. One early project that demonstrated the capabilities of the tool looked at the effects of a fish diet spiked with POPs on gene and protein expression in the brains of young female mice .
The POPs in the 2014 study were the non-dioxin-like BFR chemicals CB-153, BDE-47, HBCD and the dioxin TCDD. Children and high fish consumers have the highest intake of BFRs in general and polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) in particular.
“When we did this research, not much was known about these chemicals. Our interest was their effect on neurological diseases,” explains Rasinger.
“We had a huge amount of data: around 20 to 30k gene transcripts and around 2000 proteins and there were a lot features changing. We wanted to relate these changes to the exposure conditions, to find sets of proteins and genes that were changing at the same time, and to find out which changes are important,” he adds.
What made the analysis extra challenging is that the brain transcriptome is very stable so any detectable changes are tiny.
While the lab had specialised tools for separately analyzing proteomics and transcriptomics, these tools could not easily compare the two omics datasets to discover overlaps in toxicant features induced in the mouse brains.
Easy to compare data on the same platform
“With Omics Explorer, we could work together with the proteomics and transcriptomics data on the same platform so there was no need translate the outputs from one tool to another. It made it very easy to compare data, to communicate amongst the researchers, and to discuss the next steps,” comments Dr Rasinger. Exporting the data for downstream analysis was also very straightforward, he says.
The study concluded that all four POPs accumulated in the mouse brains. Based on the analysis of the omics data, the NIFES team hypothesised that the accumulation caused damage to neurons by the over activation of receptors for the neurotransmitter glutamate by dysregulation of the otherwise tightly controlled homeostasis of calcium and zinc.
This was an important result since disruptions during brain development and maturation have been previously linked with neurological disorders in adults. Indeed, the team’s functional pathway analysis integrating the transcriptome and proteome profiles also highlighted the functions ‘Behaviour’, ‘Neurological Disease’, and ‘Nervous System Development and Function’ to be significantly (p < 0.05) affected after dietary exposure to the four POPs.
Interestingly, subsequent studies [1, 2, 4] by Dr Rasinger and Dr Lie have shown that nutrients such as selenium, vitamin E and vitamin A in fish may counteract some of the adverse effects of otherwise harmful foodborne environmental pollutants.
While omics analyses are the main use of the Qlucore software, Rasinger and colleagues are also thinking of using the tool to analyse the fish monitoring data NIFES routinely collects and makes publicly available.
The NIFES database has hundreds of entries about pollutants and nutrients in different fish species, showing how levels are changing over time. For example, recent data shows that dioxins and PCBs have been decreasing in farmed salmon because their diets are more easily controlled than those of wild fish.
“We want to try to use Qlucore to give us a more visual representation of this data,” says Dr Rasinger.
1) Rasinger, J.D., A.-K. Lundebye, S. Penglase, S. Ellingsen, and H. Amlund. 2017. “Methylmercury Induced Neurotoxicity and the Influence of Selenium in the Brains of Adult Zebrafish (Danio Rerio).” International Journal of Molecular Sciences, 18(4), 725.
2) Dale, K., J. D. Rasinger, K. L. Thorstensen, S. Penglase, and S. Ellingsen. 2017. “Vitamin E Reduces Endosulfan-Induced Toxic Effects on Morphology and Behavior in Early Development of Zebrafish (Danio Rerio).” Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association, 101, 84–93.
3) Bernhard, Annette, Marc H. G. Berntssen, Anne-Katrine Lundebye, Anita Røyneberg Alvheim, Lene Secher Myrmel, Even Fjære, Bente E. Torstensen, Karsten Kristiansen, Lise Madsen, Trond Brattelid, and Josef D. Rasinger. 2016. “Marine Fatty Acids Aggravate Hepatotoxicity of α-HBCD in Juvenile Female BALB/c Mice.” Food and Chemical Toxicology: An International Journal Published for the British Industrial Biological Research Association, 97, 411–423.
4) Berntssen, Marc H. G., Robin Ørnsrud, Josef Rasinger, Liv Søfteland, Erik-Jan Lock, Kjersti Kolås, Mari Moren, et al. 2016. “Dietary Vitamin A Supplementation Ameliorates the Effects of Poly-Aromatic Hydrocarbons in Atlantic Salmon (Salmo Salar).” Aquatic Toxicology, 175, 171–183.
5) Olsvik, Pål A., Liv Søfteland, Ernst M. Hevrøy, Josef D. Rasinger, and Rune Waagbø. 2016. “Fish Pre-Acclimation Temperature Only Modestly Affects Cadmium Toxicity in Atlantic Salmon Hepatocytes.” Journal of Thermal Biology, 57, 21–34.
6) Lie, Kai Kristoffer, Karen Kvalheim, Josef Daniel Rasinger, Torstein Harboe, Andreas Nordgreen, and Mari Moren. 2016. “Vitamin A and Arachidonic Acid Altered the Skeletal Mineralization in Atlantic Cod (Gadus Morhua) Larvae without Any Interactions on the Transcriptional Level.” Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology, 191, 80–88.
7) Markhus, Maria Wik, Josef Daniel Rasinger, Marian Kjellevold Malde, Livar Frøyland, Siv Skotheim, Hanne Cecilie Braarud, Kjell Morten Stormark, and Ingvild Eide Graff. 2015. “Docosahexaenoic Acid Status in Pregnancy Determines the Maternal Docosahexaenoic Acid Status 3-, 6- and 12 Months Postpartum. Results from a Longitudinal Observational Study.” PloS One 10 (9): e0136409.
8) Rasinger, J. D., T. S. Carroll, A. K. Lundebye, and C. Hogstrand. 2014. “Cross-Omics Gene and Protein Expression Profiling in Juvenile Female Mice Highlights Disruption of Calcium and Zinc Signalling in the Brain Following Dietary Exposure to CB-153, BDE-47, HBCD or TCDD.” Toxicology, 321, 1–12.