Applications of Omics Approaches to Decipher the Impact of Contaminants in Dolphins

Abstract

With the advent of omic technologies (genomic, transcriptomic, proteomic, metabolomic and lipidomic), it has been possible to identify global profiles of genes, proteins or metabolites in cells, tissues or organ systems at the same time. Key pathways can be identified associated with certain diseases, physiology processes or adverse effects in response to contaminants in marine organisms. This review focuses on underlining how the use of omics technology in dolphins has contributed to understanding its physiological responses and ambient stressors. They provide a basis for understanding dolphins’ physiology and a means for monitoring health conditions as well as furthering ecotoxicology studies.

1. Introduction

One of the concerns in environmental matters is the continuous discharge of countless numbers of chemicals derived from human activities into aquatic systems. These include a large number of contaminants, among these commercial and industrial products (e.g., metals, industrial additives, surfactants and pesticides), personal care products, pharmaceuticals and endocrine-disrupting compounds, among others [1]. The presence of relevant concentrations in the environment has dramatic consequences to the organisms that inhabit these systems (e.g., affecting reproduction and survival), which is reflected in the decline of their populations and accumulation of pollutants [2].

A major concern about contaminants in aquatic systems is the bioaccumulation and biomagnification that can result with all organisms present in these systems including harmful effects to human health [3, 4]. Mammalian organisms, especially dolphins, are considered sentinel species for monitoring the health of coastal marine ecosystems [5, 6]. The main reason for that is (1) they are at the highest trophic level of the food chain and due to their role as predators, they can bioaccumulate contaminants, and (2) they also can live for longer periods (more than 40 years). It makes them good organisms to show long-term accumulation characteristics from contaminants like heavy metals in the marine environment [7]. Recently, with the development of new technologies within the “omic sciences” such as genomics, transcriptomics, proteomics and metabolomics, great advances have been made in the biological science disciplines, particularly in human health. In environmental areas “omics” have begun to have a large impact [8], mainly in aquatic toxicology [9, 10]. Together, new genomic sequencing and postgenomic technologies make it possible to obtain detailed information on drugs, toxicants, pollutants, nutrients and physical and psychological stressors on an omic scale [11]. The use of these omic technologies has allowed the emergence of ecotoxicogenomic disciplines [12, 13].

With these technologies, it is possible to determine the effect of a particular event in the life of a cell, organ or organism in response to contaminants. Through the characterization of the transcriptome, proteome or metabolome, one can perform global analysis to determinate transcriptional/proteomic or metabolomic changes at the same time in many samples (cells, tissues, biofluids, etc.) and be able to make the comparison among them. Omics technologies in environmental matters can help to assess the health statuses of aquatic systems, understand the mechanisms of action of the contaminants, through profiling of genes, proteins or metabolites that may enrich key pathways (molecular or biochemical). It sheds light on how dolphins respond to contaminants while helping to predict adverse effects on other marine organisms (Figure 1). This review highlights the omics studies performed on dolphins to gather information regarding contamination levels and their effects on worldwide dolphin populations (Figure 2). Applications of the omics approach help to understand the dolphins’ physiology as a way to monitor dolphin health conditions and to further ecotoxicology studies. Conclusively, it might provide a method for developing regulations for chemical discharge as well as management and conservation strategies for these kinds of ecosystems.

Figure 1.

Integration of omics technologies in marine organisms.

Figure 2.

Number of studies related with omics approach in dolphin.

The three major omics technologies that have proven to have a tremendous impact include transcriptomics, proteomics and metabolomics [14].

2. Selection of bibliographic material

We reviewed relevantly and recently published studies on the applicability and usefulness of Omics in dolphins. The selection of scientific publications was made through the use of search engines from Google Scholar, PubMed and Scopus to locate studies of interest using the keywords “transcriptomic”, “proteomic”, “metabolomic”, “lipidomic” and “dolphin”. We excluded all repeated studies. It was inevitable that some omics research was bot captured due to them not included the keywords we used. For the selection of publications, only the research studies that were related to contaminants were included in the data set (Table 1).

Specie	Omic approach	Type of sample	Contaminant/stressor	Genes/Proteins/Metabolites/Lipids	Contribution	Reference
Transcriptomic
Tursiops truncatus	Microarray (Custom 4x44K Agilent oligo array)	Cultures Skin	Ex vivo assay of BPA (0.1 or 1 μg/ml) and PFOA (0.1 or 1 μg/ml)	BPA: Genes involved response to stress (e.g., programmed cell death protein, tumor suppressor), immune system (e.g., complement factor H, class I histocompatibility alpha chain), lipid metabolism (e.g., adipogenesis regulatory factor, fatty aldehyde dehydrogenase isoform 2), and embryonic development and growth (e.g., titin, nuclear distribution protein nude-like 1).; PFOA: Genes involved response to stress (uv excision repair protein rad23 homolog a, heat shock protein 90), immune system (complement c5, acidic mammalian chitinase), embryonic development (vascular endothelial growth factor, Rho GTPase-activating protein)	Pathways affected: BPA: Fat (blubber)differentiation	[15]
T. truncatus	Microarray (Custom 4X44K Agilent oligo array)	Blood	55 PCBs congeners	Genes involved in cell cycle checkpoint and apoptosis, DNA damage and chromatin remodeling (e.g., DDB1, DCN and INO80). Pathway of cellular response to stress	PCBs could cause epigenetic response, DNA damage and chromatin remodeling	[16]
T. truncatus	Microarray (Custom 4X44K Agilent oligo array)	Blood from male and female	PCBs	Male: Genes involved in (1) development & differentiation (e.g., SOS1), signaling pathway to maintain cell growthand survival in thyroid cells (e.g., RAS genes), (2) Wound healing & anti-tumorigenic (e.g., DCN), (3) Inflammatory Response (IL23), (4) Xenobiotic metabolism (OXR1).; Female: Genes involved in (1) Transcription/Translation (Maf1 homolog), (2) Immune response (e.g., tyrosine–protein kinase JACK1, 3) Development/cell growth (PAR), BB1, (TRIP11), 4) Xenobiotic metabolism (OXR1).	The development and application of a microarray to monitor global gene expression in dolphin in response to contaminants	[17]
T. truncatus	RNA-seq	Skin from two ecotypes of dolphin: offshore and coastal	HOC	Genes: AHR, CYPIB1, IL16, ESR2, ESRRA, THRA. GO terms: xenobiotic metabolism, immune response, hormone metabolism, DNA repair, and metal binding.	It provides novel insight into contaminant exposure in two bottlenose dolphin ecotypes in the Southern California Bight and highlights potential relationships between HOC exposure and molecular biomarkers	[18]
T. truncatus	RNA-seq	Peripheral blood mononuclear cell (PBMC) from dolphin and human	PFOA y PFOS	In both species: Overexpression of genes linked to inflammation and autoimmune diseases. Difference between species: In human the interferon Signaling pathway is negatively regulated while in dolphin it is positively regulated. Dolphins lack Mx1 and Mx2, key proteins of the Interferon signaling pathway	This study provide a better understanding of the adverse effects of CECs (PFOA, PFOS) on both dolphin and human species	[19]
Metabolomic and lipidomic
T. truncatus	LC/MS	Exhaled breathe	Oil spill	Phosphatidic acid, phosphatidylethanolamine, and steroids (higher abundance or uniquely in dolphins of contaminated area). Phosphatidylglycerol.	Pathways affected: Cellular bilayer degradation; DNA and cellular damage processes; Immunological protection	[20]

Table 1.

Depict of the most relevant transcriptomic, proteomic and metabolomic studies performed in dolphin in response to the contaminants.

Based on these criteria, 59 publications were selected from >250 reviewed. Transcriptomics was the most frequently applied technique (38%) followed by proteomics (30%), metabolomics (21%) and finally, lipidomic (10.2%). In general searching the omic studies selected, we identified 8 topics including “contamination”, “physiology” and “health” among others based on the type of research described in the publications. More details about each topic are given in Figure 3. Contamination studies were dominant using the transcriptomic method (31%), compared to studies focusing on proteomics (0%), metabolomics (8%) and lipidomics (0%). We noticed that proteomics and lipidomics are less used in studies related to contamination. However, proteomics is the most frequent technology applied to identifying responses associated with the physiology of dolphins (28%), followed by lipidomics (33%), metabolomics (25%), and transcriptomics (13%). With respect to studies related to health, metabolomic tools (34%) were predominant, followed by transcriptomics (26%) and proteomics (11%). Interestingly, we noticed that the number of studies selected in omics and dolphins does not show an increase over time as we expected, it was diverse (Figure 1). After 2016, the selected literature showed an increase in the application of omics in dolphin research, notably, most studies focused on using metabolomics (LC/MS) and transcriptomic high throughput RNA sequencing (RNA-seq) tools as a diagnostic method for the detection of contaminants in oil spills and with contaminants of emerging concern (CECs). In general, it seems that there is a trend toward the increased use of transcriptomics, with studies dominating the literature from 2018 to 2019, and lipidomic applications from 2020 to 2021.

Figure 3.

Status of studies of omics in dolphins.

3. Omics technologies in marine organism: response to contaminants in dolphins

Omic approaches bring an integrated view of the molecules that compose a cell, tissue, or organisms in any target biological sample from a model or non-model organism. Notably, there is little information focused on proteomics, metabolomics, and lipidomics to investigate the impact of contaminants in dolphins species (Figure 2). We present a summary of the application of the three main omic technologies in dolphins associated with contaminants.

3.1 Transcriptomics

Transcriptomics has been the omic technique most used in biological areas because it represents all RNA molecules (e.g., miRNA, snoRNA), including the messenger RNA (mRNA) which constitutes the building blocks for translating DNA into amino acids to form proteins. The totality of mRNA is a reflex of the genes that are actively expressed in a cell or an organism at a given time and during a specific event. It permits deciphering how organisms respond to changes in the external environment or the presence of the contaminants [21]. The principal gene expression profiling methods used in transcriptomic are microarray and RNA-sequencing (RNA-seq). The difference between the potential of each method becomes apparent once the target sequences go beyond known genomic sequences. Hybridization-based techniques like microarray rely on and are limited to the transcripts bound to the array slides. Limitations of microarrays are due to the bioinformatic data available for the model organism’s genome and transcriptome. RNA-seq can detect annotated transcripts but also novel sequences and splice variants [22]. RNA-seq is considered a revolutionary tool for transcriptomics in non-model organisms and is powerful enough to explore the mammalian transcriptome which was not possible with microarrays [23].

With regard to the transcriptomic studies in dolphins and contaminants, there are few studies that have used microarray methods to identify genes and molecular pathways altered by bisphenol A [2,2 bis(4-hydroxyphenyl) propane (BPA), perfluoroalkyl substances (PFAS) and perfluorooctanoic acid (PFOA) in dolphin skin biopsies [15]. These contaminants can cause changes in key genes involved in pathways related to stress, immune response, development and lipid metabolism. Likewise, there are another two studies that describe the construction and validation of the use of microarrays in T. truncatus as well as using bioinformatic tools to detect polychlorinated biphenyls (PCBs) from dolphin blood during the monitoring of high-level contamination at Superfund sites on the Georgia coast in the US [16, 17], see Table 1. A limited range of sequencing data is available for dolphins from whole-genome assemblies to RNA-seq data [18, 19], however, two studies have documented the effects of halogenated organic contaminants (HOCs) at transcriptomic levels. For example, Trego and colleagues reported that 20 skin biopsys from T. truncatus dolphin collected on the Southern California Bight showed to have a positive correlation with the presence of HOCs and genes associated with the metabolism of xenobiotics and with the immune and endocrine pathways. Likewise, in another study also performed with T. truncatus, human peripheral blood mononuclear cells (PBMC) from both species were assessed to investigate the effects of contaminant exposures of CECs (PFAs; PFOA and perfluorooctane sulfonate (PFOS)) using RNA-seq. Transcriptomic analysis showed that in both human and dolphin pathways related with endocrine immune system that inflammatory responses increased (Table 1) [19].

3.2 Proteomics

The main focus of proteomics is to identify and quantify all protein content in a cell, tissue, or organism and understand their functions, structure and their modifications in response to external stimuli [24]. Based on proteomics, baseline studies have been conducted to characterize proteins from spermatozoa and seminal plasma in bottlenose dolphins [25] which has been used in zooarchaeology for species identification of cetaceans [26]. Other studies have been focused on developing bioinformatics tools or methods to obtain or analyze proteins from different samples [27, 28, 29].

Most of the proteomic studies in dolphins have focused on the physiology of proteins and peptides. These studies have provided valuable information, such as the case of the proline-rich antimicrobial peptides found in different cetacean species, where these peptides could provide useful insights for future antibiotics [30]. Through proteomics one can also identify peptides related to metabolic disorders [31] and biomarkers of infection for diagnosis of aspergillosis in dolphins [32]. Thanks to proteomics, it has been possible to identify stress proteins involved in apoptosis, proteotoxicity and inflammation on managed and wild dolphins and their relation with biological data such as serological, biochemical, hematological and endocrine variables [33]. In stressed cetaceans, 30 stress-activated proteins have been identified, where these proteins have an important role in cellular detoxification, stress response, cell growth and differentiation, apoptosis, immunologic, neurologic and hormonal signaling and oxidative stress response [34].

In toxicology, proteomic studies are important because the proteome is the link between effects at the molecular and the whole organism level and provide snapshot functional information of a cell under certain conditions, and it allows the identification of new biomarkers and pathways of toxicity [35]. However, studies related to contamination have not been reported yet.

Regarding the methods and tools used in proteomics, initially, the way to analyze variations of protein expression was by gel electrophoresis. Now the main tool used is mass spectrometry with their different techniques: LC/MS, MALDI TOF/TOF, ESI-QUAD-TOF, iTRAQ. Protein microarray has also been used for these kinds of studies and bioinformatic tools.

Proteomics generates a large amount of data that permit furthering one’s knowledge of mechanisms of action and toxicant effect of a contaminant in organisms and thus be able to understand biological processes [35]. However, the limitations in these kinds of studies are with peptide separations, identification and that many species lack of protein sequence information [14, 36].

3.3 Metabolomics

Metabolomics is responsible for identifying and quantifying all endogenous and exogen metabolites in an organism or biological sample [37]. Metabolites are all final products of cellular processes and knowing their levels permits one to understand the responses of a biological system to environmental changes [38].

This omic tool contributes to understanding of how environmental stressors can affect human and environmental health. However, these kinds of applications have not yet been explored as often in dolphins [39]. Most of the metabolomic studies in dolphins have been focused on establishing baseline information on health [40, 41, 42, 43], and physiology [44, 45, 46] with a few studies looking at the characterization of metabolites from exhaled breath and tears [47, 48].

Regarding pollution studies, just only a single work was discovered. After the spill of the Deepwater Horizon in the Gulf of Mexico, dolphin populations were severely affected, showing adrenal and lung diseases, poor reproductive success and higher mortality [49, 50, 51]. In bottlenose dolphins, Tursiops truncatus exhaled breathe metabolites had been studied [20] from a managed collection in San Diego, from a wild population in Sarasota Bay and Barataria Bay, the latter being the contaminated site. Several metabolites, such as yiamoloside B, diacylglycerol, leptomycin B, phosphatidylglycerol and phospholipids, were correlated with pulmonary disease. Cortisol and aldosterone levels were lower in Barataria Bay, also dolphins from this population presented thin adrenal gland cortices, supporting an impaired hypothalamus-pituitary-adrenal axis. Lower amounts of glucose in the contaminated area may represent a response to stress or feeding. Besides, metabolites as steroids, phosphatidic acid and phosphatidylethanolamine were unique or found in higher abundance in the contaminated area compared to the healthy reference dolphins which suggest cellular destruction. Many of the specific metabolites found in dolphins from Barataria Bay, were markers for arachidonic acid, lipid oxidation and lung surfactant breakdown. In addition, antibiotics, such as jadomycin B, leukomycin A1 and A7, lansonolide A, chivosazole E and mycolacton, were also found in dolphins from Barataria Bay. These compounds are products of fungi and bacteria suggesting that dolphins exposed to oil spill may have pneumonia.

In metabolomics, the main tools used for analysis are mass spectrometry with their different instrumentation: chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and in tandem (LC/MS/MS), high-performance liquid chromatography (HPLC), HPLC-MS/MS, reverse phase chromatography (RP)/UPLC-MS/MS, capillary electrophoresis time of flight mass spectrometer (CE-TOFMS), liquid chromatography/time-of-flight/mass spectrometry (LC-TOFMS) and the least used are nuclear resonance magnetic (NMR) and high-resolution magic angle spinning (HR-MAS) NMR spectroscopy.

Metabolomics is relatively a new tool and captures more integrated information of the physiology of an organism than transcriptomics or proteomics [52] because it represents the final cellular signaling events, resulting from transcriptional and translational changes [39]. However, it presents some limitations such as targeting metabolites that are species specific as well as libraries and software programs that are not yet sufficiently extensive [52].

3.3.1 Lipidomics

Lipidomics is a specialized subfield of metabolomics. Through lipidomics, it is possible to characterize all lipids from a cell, tissue, fluid, etc. and understand how these lipids influence a biological system and participate in several processes as well as how they interact with other molecules and respond to environmental changes [53, 54]. Lipids represent a major component of the metabolome [54], have an important role as components of cell membranes and participate in many cellular pathways and due to these being involved in many physiological mechanisms, also are excellent candidates for monitoring the effects of stress [55].

One representative area in marine mammals is their blubber. This is the most important site of fat and energy storage and also participates in different processes such as insulation, thermoregulation and buoyancy and, it represents up to 50% of the body mass [56] and due to the great quantity of lipids, it makes it a good repository for contaminants that are lipophilic [57]. For these reasons, lipidomics makes an excellent tool for studying the effects of contamination in these sentinel species. Although lipidomic studies have been increasing in recent years, until now, there are no dolphin lipidomic studies related to contamination. Indirectly, one study focused on respiratory metabolites [20], where some lipids were detected, including phosphatidylethanolamine, from oil spill exposure. These lipids were found in higher concentrations in dolphins from the contaminated area.

Few lipidomic studies have been reported, with most focused on physiology [58, 59], and characterization of lipids from cardiac phospholipidome [59] of small cetaceans and lipids from the blubber of killer whales [60].

The main tool used for lipidomic studies is mass spectrometry. This analysis generally uses another instrument such as LC-electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF), liquid chromatography-high-resolution mass spectrometry (LC/HRMS/MS), GC-MS and LC-MS/MS and hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC-MS).

4. Omics and dolphin: future considerations

This review integrates the available information on the effects of pollution using an omics approach on dolphins and other cetaceans considered as ideal organisms to assess and monitor pollution in coastal or ocean systems. Although there are wide applications of omic approaches in other model and non-model aquatic organisms involving environmental matters, there are very few studies from an omic perspective in dolphins. There is much evidence in the literature of the analytical power that these tools have their contribution in providing relevant information on the MOA of contaminants in cells, tissues, organisms or populations to help to assess the health status of marine systems, to identify potential biomarkers of exposure and response to the contaminant as well to predict adverse effects on marine organisms. Information provided from this study may be useful for risk assesment analysis that may impact future environmental regulations. However, there are still several limitations that need addressing in their application in dolphins. (1) One of the main challenges is with sampling (non-invasive/biopsy). There are prohibitive costs and time delays associated with obtaining the permits required to obtain samples in wildlife organisms in some countries. A non-ideal but possible option is the sampling of strandings. (2) The application of omic studies in ecotoxicology still has many challenges. The increase of these studies at different omic levels has grown impressively thus requiring improved bioinformatics and computational tools for better analysis regarding environmental stressors, such as pollutants. (3) Likewise, the collaboration between academic government entities and industry still needs to be improved.

5. Conclusions

This review highlights the importance of omic studies in dolphins which have contributed greatly in recognizing the presence and effect of contaminants such as HOC, CECs (BPA and PFOs) and those associated with oil spills (summarized in Table 1). Omics technologies are important to study adverse effects of contaminants or environmental changes because they provide information on the alterations of genes, proteins, metabolites and phenotypic responses [14]. Transcriptomic-based investigations were used most frequently (31%); only a few studies used a metabolomic approach (8%). The principal tool used for transcriptomic is RNA-seq and for proteomics, metabolomics and lipidomics is mass spectrometry coupled to different types of spectrometers (Figure 1).

Some of the more likely applications for omics in dolphins are characterization and physiology. Although omics studies have been used for many topics, the number of studies concerning contamination is rather low. Studies of proteomics, metabolomic and lipidomic are still lacking; therefore, these findings may give insight for future studies. This type of study contributes greatly in establishing baselines for environmental health studies of coastal and marine systems, the health status of the dolphin reflects the status of their environment. Perhaps it may allow the local as well as the scientific community to be more aware of marine ecosystem conditions and to recognize the importance and possibilities of integrate omics studies regarding pollution.

Conflict of interest

The authors declare no conflict of interest.