1. INTRODUCTION
In human history, technological developments that have occurred from prehistoric times to the present have made life easier for people. With the discovery of metals, their use in animal husbandry and agriculture has continued in the industrial field. With the development of various chemicals, synthesized industrial products (such as plastics and pesticides) are used in different applications.
In the mid-twentieth century, one of the most important developments in human history was the discovery that industrially produced and agriculturally used 1,1′-(2,2,2-Trichloroethane-1,1-diyl)bis(4-chlorobenzene) (DDT) caused thinning of the eggshells of American bald eagles. Thus, it has become clear that chemical substances or products that facilitate human life can have adverse effects on natural life and therefore, on the environment. As a result, a new concept, ecotoxicology, has emerged.
In addition to these developments in human history, one of the scientific fields that emerged with technological developments is molecular biology. With the development of the field of molecular biology, the most basic structure of biological organization, the detailed examination of the cell has occurred. Technological developments have allowed the field of molecular biology to develop further, allowing the examination of nucleic acids, transcripts, proteins, and metabolites within the cell. Thus, omics technologies were born.
In this module, the toxicological effects of different stress sources from an ecological perspective will be explained together with the examination of omics technologies.
1.1. Definition and importance of ecotoxicology
Toxicology is a branch of science that deals with the sources of poisons, their physical, chemical, and biological effects on living beings (such as humans, animals, and plants), and their metabolism, clinical, chemical, biological, and pathological diagnosis of poisonings, treatment, and prevention.
Ecology is the science that studies the interaction of living organisms both with each other and with the environment in which they live.
Ecotoxicology is the combination of the sciences of toxicology and ecology. Ecotoxicology is an applied science whose aim is to identify the agents that cause environmental pollution and to analyze, evaluate, and predict the effects of these agents on the environment.
The information obtained through ecotoxicology guides remedial measures in ecosystem risk assessment studies. Ecotoxicological studies must be conducted at the level of biological organization. They perform environmental risk assessment studies by examining the effects of a stress condition (abiotic conditions of the environment such as temperature, oxygen, or environmental pollutants) on cells, tissues, organs, individuals, populations, communities, ecosystems, and the biosphere (Fig. 5.1).
Figure 5.1. The ecotoxicological pathway of toxic substance or environmental stress
1.2. Introduction of omics technologies
In environmental risk assessment studies, measurable levels such as adverse outcome pathways (AOPs) and source outcome pathways (STOs) at all levels of biological organization are of great importance (Fig. 5.2).
Figure 5.2. Omics in ecotoxicology (adapted from Zhang et el. 2018)
The starting point of omics technologies was genomics. With the analysis of the genome, which consists of deoxyribonucleic acids (DNA), the most basic building blocks in a cell, with technological devices, the study of cells or organisms’ genomes has become popular. Over time, different omics technologies have been developed that allow the examination of transcripts, proteins, and metabolites occurring in cell metabolism (Fig. 5.3).
Figure 5.3. The omics world in the cell
Omics techniques can be performed separately or as multiple techniques in the form of multiple omics. A single sample is sufficient for this. Using a commercial kit, DNA samples (genomics) and total RNA samples can be extracted, and transcripts can be obtained based on the creation of complementary DNA libraries from total RNA (transcriptomics). Total protein can be extracted and identified by mass spectrometry (proteomics), and metabolites can be extracted, and qualitative and quantitative analysis can be performed by mass spectrometry (metabolomics) (Fig.5.4).
Figure 5.4. The basic multi-omics principles of omics in the biological samples (adapted from Shi et al., 2024)
1.2.1. Genomics
DNA is the genetic material of almost all living organisms and is the basic chemical compound responsible for the functioning of the cell. The DNA molecule has a double-stranded structure. The entire DNA content of an organism is called the genome of that organism. For example, each cell in the human organism contains approximately 3 billion DNA base pairs. DNA consists of four organic bases (adenine, thymine, guanine, cytosine), and it has been stated that there are an estimated 20,000 to 25,000 genes in the human genome, and these genes code for an average of three proteins (Fig. 5.5).
Figure 5.5. A brief guide to genomics (taken from NIH, 2022)
The basis of sequencing analysis is the exact determination of the order of bases in the DNA strand. In sequencing by synthesis analysis, which is the most commonly used sequencing type today, DNA polymerase adds nucleotides labeled with fluorescent labels to the new DNA strand while producing a new DNA strand from the target DNA strand. During these events, nucleotides are excited by a light source and a fluorescent signal is emitted and detected. This basic technique is called Sanger sequencing because it was first performed by Dr. Frederick Sanger in 1977. Although Sanger sequencing has been used as the primary method in genomic studies for many years, in recent years, a large amount of DNA sequencing data has been obtained thanks to new generation sequencing technology, allowing faster and more economical studies (Fig. 5.6).
Figure 5.6. The basic principles of Sanger sequencing (taken from Hawkings, 2017)
Metagenomics, one of the fields of study of genomic technologies, is a field that analyses genetic material taken from environmental samples and shows microbial communities in the environment. In this way, samples taken from many places, such as water, sediment, soil, air, and even the human body, not only show the genetic diversity of microbial communities living in these habitats but also surpass traditional microbiological methods.
1.2.2. Transcriptomics
The transcriptome is the set of transcripts formed in a cell and their quantities. Transcriptomics is the technique that allows the examination of transcripts. Sequencing is performed using microarray-based technologies in this method. The transcriptome is composed of mRNA, rRNA, tRNA, and regulatory noncoding RNAs.
1.2.3. Proteomics
Genes are responsible for the enzymes or proteins’ production that make up messenger molecules in a cell. The genetic information in DNA is encoded on the messenger ribonucleic acid (mRNA), and the information in mRNA is converted into protein molecules by the amino acids combination in ribosomes. If a mutation occurs in a cell’s DNA, the cell’s natural function is disrupted, and abnormal proteins are produced (Fig. 5.7).
Figure 5.7. The central dogma in the cell
Proteomics analysis is a method that allows the identification and quantification of proteins using mass spectrometry. For this purpose, proteins are isolated from samples and then detected using chromatographic methods such as LC-MS (Fig. 5.8). Thanks to the advancement of technology, high-throughput molecular tools have increased the understanding of the effects of pollutants on organisms in ecotoxicology. Proteomic tools can analyse all proteins in a sample simultaneously. They also provide a holistic approach to the molecular processes and pathways that occur under stress conditions encountered by the organism. Protein analysis methods such as two-dimensional DIGE, iTRAQ, and label-free proteomics can be used to profile the modes of action of xenobiotics.
Figure 8. The workflow of the proteomics (taken from https://www.leibniz-fli.de/research/core-facilities-services/cf-proteomics)
1.2.4. Metabolomics
Metabolites are small molecules that are the end products or intermediates of cellular metabolic pathways. These molecules encompass a wide range of chemical compounds, including carbohydrates, proteins, lipids, organic acids, and nucleotides. As can be seen, these metabolites are the building blocks of a cell, having properties that drive and sustain the cell’s vital reactions (Fig. 5.9).
Figure 5.9. The metabolomics analysis (taken from Guijas et al. 2018)
1.2.5. Epigenomics
Gene expression regulation due to internal reactions in a cell or responses to signals from the environment and methylation formations in the DNA structure constitute the early stages of pathological changes in the cell and therefore, the organism (Fig. 5.10).
Figure 5.10. The epigenomics scheme (taken from NIH 2024b)
2. USING OMICS DATA IN ECOTOXICOLOGY
Many chemicals with toxic effects mix into different areas of the ecosystem, resulting from their intensive use. As a result of the necessity of deeply investigating the toxicity mechanisms of these chemicals, which are very diverse, such as metals, plastics, natural or synthetic organic substances in organisms, omics technologies have been included in the field of ecotoxicology. In this section, information will be given about the use of omics technologies in studies conducted in aquatic and terrestrial ecosystems, the examination of air pollution and the resulting health effects with omics, and the investigation of these chemicals that affect human health with omics (Fig. 5.11).
Figure 5.11. Omics in ecotoxicology (taken from Farrel 2022)
2.1. Aquatic ecosystems
In aquatic ecosystem health studies, examining aquatic organisms is essential for environmental change and pollution analysis. With the development of omics technologies, the number of water ecotoxicity investigation studies is increasing day by day. Aquatic organisms may exhibit mortality or adaptation when exposed to these pollutants, but before these situations, they generate global molecular responses. Thus, the effects of toxic substances can be studied with sequencing analyses using intact genome information obtained from these aquatic organisms. Single omics or multi-omics methods enable the depiction of multidimensional datasets for holistic interpretation of the molecular responses of biological systems. Thus, the health of aquatic ecosystems is examined.
Antibiotic pollution from anthropogenic sources and the resulting antibiotic resistance is one of the most important problems we face in this era. As a result of the accumulation of antibiotic residues in areas such as water and sediment, changes can be observed in the structures of aquatic organisms, and antibiotic resistance can also occur in the microbial community structure in the aquatic ecosystem. Therefore, conducting metagenomic studies in aquatic ecosystems and encouraging the feasibility of these studies are essential. A metagenomic study conducted on the samples from the Ganges River (India) investigated the presence of antibiotic-resistance genes. In terms of the microbial community, Proteobacteria phylum was dominant in all locations where samples were collected, and the Pseudomonas genus was detected at the highest level in all samples. According to the antibiotic resistance genes examined with metagenomics, different genes were found in each sampled location, and resistance to antibiotics such as amoxicillin, cefoxitin, piperacillin, and penam was shown (Fig. 5.12).
In aquatic toxicology, there are studies on the effects of antibiotics on aquatic organisms, in addition to antibiotic resistance. In one of these studies, protein mapping was performed in the liver and brain tissues of sea bream (Sparus aurata) fish after chronic exposure to different application combinations of ciprofloxacin (CIP), sulfadiazine (SULF), and trimethoprim (TRIM) antibiotics.
Figure 5.12. An example of aquatic toxicology metagenomics study (adapted from Rout et al. 2024)
The number of proteins in the liver, which is an essential organ in the detoxification process, was found to be 39 in CIP exposure, 73 in SULF exposure, and 4 in TRIM exposure, while in the brain tissue, only nine proteins were affected in SULF exposure. The cellular functions of these proteins detected by proteomic approaches were found to vary in terms of liver and brain organs. As a result of the study, it was determined that antibiotics regulate the expression of important proteins in cellular functions such as energy metabolism, cytoskeleton formation stages, protein synthesis, DNA replication, and RNA synthesis (Fig. 5.13).
Figure 5.13. The proteomics analysis of sea bream (adapted from Fernandez et al. 2024)
In studies conducted with aquatic macrophytes (Vallisneria denseserrulata), the nanoplastics and arsenic substances effects were investigated separately and in combination. Metabolomics analyses identified metabolites in carbohydrate, amino acid, and lipid metabolisms. Transcriptomics analyses identified transcripts in photosynthesis reactions (Fig. 5.14).
Figure 5.14. The metabolomics and transcriptomics analysis of macrophytes (adapted from Tang et al. 2024)
2.2. Terrestrial ecosystems
The effects on terrestrial organisms affected by pollution in terrestrial ecosystems are examined with omics technologies. It is specially investigated with metagenomic and transcriptomic analyses in soil microbial communities (Fig. 5.15).
Earthworms are a commonly used model organism in soil ecotoxicology tests. These organisms are highly sensitive to environmental contaminants such as heavy metals. Antimony is one of the mineral sources that cause soil pollution. In a study examining its effects on soil ecosystems, it was determined that metabolites in lipid metabolism changed in chronic exposure using earthworms. In addition, it was shown that high-concentration exposure affected transcripts in functions such as antigen formation and synthesis and amino acid metabolism (Fig. 5.16).
Figure 5.15. Omics in terrestrial ecosystem (taken from Ge 2013)
Figure 5.16. Metabolomics and transcriptomics analysis of earthworm’s exposure to antimony (adapted from Chen et al. 2024)
Hydrocarbon pollution from petroleum is an important pollution agent to soil ecosystems. In a study examining its effects on soil, transcripts in the insect species Folsomiacandida were examined. Transcripts involved in different cellular reactions, including xenobiotic biotransformation and oxidative stress processes, were identified (Figure 5.17).
Figure 5.17. The transcriptomics analysis of insects (adapted from Pang et al. 2023)
2.3. Air pollution and ecotoxicological effects
Air pollution is the change of natural characteristics of the atmosphere caused by chemical, physical, and biological factors. Rapid urbanization and industrialization since the 19th century have caused air pollution to become harmful to all living organisms. Although air pollution levels have remained largely consistent since the turn of the millennium, particulate matter (PM) concentrations have varied considerably across regions. While PM concentrations have decreased in developed countries, PM concentrations have increased in developing and underdeveloped countries. Although air quality in European Union countries has steadily improved since 1990, 2021 data is above the maximum value of 15 μg/m3 set by the World Health Organization.
Air pollution not only causes many effects on living organisms but also can cause fatal diseases such as lung cancer in terms of human health. The effects of acute or chronic exposure to air pollution are examined with omics technologies. Although there are findings showing that air pollution affects the immune system in organisms and is an essential factor in chronic inflammation, oxidative stress, and DNA damage in cancer development, epigenetic mechanisms, and therefore epigenomics methods, have a vital place in the investigation of the effects that cause this.
Air pollution is one of the causes of cardiopulmonary disease in organisms. A study conducted with mice examined the acute effects of air pollution. According to the genomic results, 1247 oxidative stress genes were upregulated and 1383 were downregulated. In transcriptomic analysis, macrophage subtype Mox-like genes were found.
A study was conducted using multi-omics technologies to determine the biological responses and functions of different chemicals on the organism with tree swallows Tachycineta bicolor living in wastewater treatment plants and industrial areas. It was determined that there was a cluster of expressed genes associated with down-regulation in cell growth and cell division processes of liver tissues. It was determined that lipogenesis genes such as PPAR signaling, biosynthesis of unsaturated fatty acids, and lipogenesis-related metabolites were upregulated. The sources of these differences were determined to be polycyclic aromatic hydrocarbons and polybrominated diphenyl ethers in the areas where the birds were collected (Fig. 5.18).
Figure 18. Multi-omics in birds exposed to different pollutants (adapted from Tseng et al. 2023)
2.4. Human health and environmental impacts
In recent years, a new concept has emerged in human health: personalized medicine. This term or field optimizes the results by preparing personalized treatments by taking into account the genetic characteristics of the person and environmental factors. In this sense, toxicogenomic studies, which combine genetics with the pharmaceutical substances used, form the basis of the investigation of the toxicological aspects of these chemicals.
The Human Genome Project can be considered as the most important project or results of the field of genomics in terms of the human organism. The reference genome results of this project, published in 2003, allowed the characterization of variants in an individual’s genome. In the studies carried out after this project, the examination of variant types in many individuals enabled the understanding of the evolutionary mechanisms of these variants through disease and clinical diagnoses. In this context, it is important to examine the toxicological effects of drugs or drug combinations used in personalized medicine. Considering omics technologies separately or in multi-omics allows for defining the patient’s response to the disease and the toxicological effects of the drugs used on the individual (Fig. 5.19).
Figure 5.19. The risk assessments using omics technologies in the human health (taken from Singh et al. 2023)
Environmental toxicology can be examined in two parts: human health toxicology and toxicology of other living organisms. Since human toxicology focuses on a single species and usually an individual, it is easier than the toxicology of other living organisms. Therefore, it enables omics studies to be carried out quickly and easily.
In humans, biological age measurements are integrated with molecular processes in the aging process. Biological age can be estimated by epigenetic mechanisms such as DNA methylation and histone modification. For example, it can help establish a link between air pollution and aging. It has been stated that there are studies in which DNA methylation-based measurements were used in determining the biological age of air pollution. However, the adequacy of the findings has caused debate. The groups selected in the focus of these studies are non-Hispanic white populations (NHW) and do not include other races and ethnicities. In a survey conducted in the United States, black and NHW women with no history of breast cancer and at least one biological sister diagnosed with breast cancer in their family were studied. Black participants were exposed to higher levels of air pollution than NHW participants. Epigenomic studies conducted due to air pollution found differential methylation at 19 CpG sites in black women and one site in NHW women. These results suggest that air pollution is associated with higher epigenetic aging in Black women (Fig. 5.20).
Figure 20. The air pollution effect on the epigenetic age of NHW and black women (adapted from Koenigsberg et al. 2023)
3. CONCLUSIONS
The use of omics technologies in ecotoxicological studies has revolutionized the understanding of the effects of environmental pollutants on living organisms and ecosystems. Genomic, transcriptomic, proteomic, and metabolomic approaches allow for a multifaceted and detailed study of biological responses to pollutants. These technologies play a critical role not only in elucidating toxicity mechanisms but also in identifying biomarkers and increasing the sensitivity of environmental risk assessments. In the future, the integration of omics data with big data analytics and artificial intelligence will contribute to the development of more comprehensive ecological risk assessment models. However, the limitations of these technologies, such as cost and difficulty of analysis, as well as the complexity of environmental samples, should be carefully addressed. In this context, the integration of omics technologies in ecotoxicology will pave the way for scientific and practical advances in environmental management and contribute to the construction of a sustainable future.
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