**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.

*Applications of Omics Approaches to Decipher the Impact of Contaminants in Dolphins DOI: http://dx.doi.org/10.5772/intechopen.102424*

#### **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 wholegenome 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 [27–29].

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 [40–43], and physiology [44–46] 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 [49–51]. 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

#### *Applications of Omics Approaches to Decipher the Impact of Contaminants in Dolphins DOI: http://dx.doi.org/10.5772/intechopen.102424*

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).
