Changes to RNA caused by environmental toxins and their links to disease

Recent studies on m6A modification have demonstrated its essential role in the control of post-transcriptional gene expression and many biological processes [24]. m6A is the most abundant internal modification in eukaryotic mRNA, influencing splicing, stability, and translation efficiency. As the predominant RNA epigenetic modification, m6A modulates mRNA metabolism via writers, erasers, and readers, impacting processes such as cancer and lipid metabolism [24]. M6A is deposited by methyltransferase complexes comprising METTL3, METTL14, and WTAP (writers), demethylated by FTO and ALKBH5 (erasers), and identified by proteins including YTHDF and IGF2BP families (readers) for dynamic control [25]. Research has revealed m6A’s dual function in influencing chromatin dynamics and transcriptional output, with m6A-modified chromatin-associated RNAs serving as molecular docking sites for histone modification proteins [26]. Exposure to heavy metals (e.g., arsenic and cadmium) and air pollutants (e.g., PM2.5), Aflatoxins, and cigarette smoking has been shown to alter m6A levels, disrupting normal RNA metabolism and contributing to diseases like cancer and neurodegeneration [27]. This alteration occurs in both coding and non-coding RNAs, significantly influencing cancer progression and therapeutic response [28]. The growing investigation into m6A alteration has revealed new possibilities in neuroscience, developmental biology, and oncology, underscoring its promise as a therapeutic target for cancer treatment [26, 28].

The mechanistic understanding of how environmental toxicants alter RNA modifications is of great importance, and it is important to move beyond general descriptors and discuss specific chemical constituents and their pathways of action. For instance, in tobacco smoke, benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon that forms DNA and RNA adducts. Studies show that BaP induces oxidative stress, which in turn impairs m6A methylation by suppressing METTL3 expression and altering reader protein (e.g., YTHDF2) localization, ultimately destabilizing oncogene transcripts. This contributes to the increased translational efficiency of oncogenic mRNAs in lung epithelial cells [29–31]. In the case of aflatoxins, particularly aflatoxin B1 (AFB1), the toxin interferes with the catalytic activity of RNA methyltransferases, including NSUN2 and METTL14. AFB1 exposure results in the reduction of m5C and m1A levels in both mRNA and tRNA, compromising RNA stability and translational accuracy [32]. Mechanistically, AFB1 binds to the catalytic site of methyltransferases, altering their conformation and reducing substrate binding affinity, which has been shown to lead to hepatic carcinogenesis in murine models [31].

A recent study has underscored the importance of m5C modification in RNA, especially in mRNAs [33]. This epigenetic modification affects RNA metabolism, stability, nuclear export, and translation [34]. The modification is essential in numerous physiological and pathological processes, including stress response, cancer, and embryogenesis [35]. m5C is subject to dynamic regulation by writers, erasers, and readers, and is present in several RNA types, including tRNAs, mRNAs, and rRNAs [34]. While TET enzymes (erasers) may help to demethylate, ALYREF and YBX1 proteins (readers) identify and control m5C-modified RNA function. DNMT2 and NSUN family enzymes (writers) catalyse m5C methylation [36]. Although less researched than m6A, m5C is gaining recognition as a significant epitranscriptomic marker with potential relevance for illness diagnosis, therapy, and monitoring Pesticides, industrial solvents, and endocrine disruptors like phthalates can dysregulate these enzymes, leading to aberrant m5C deposition [37]. Disruption of m5C impairs RNA export and translation and has been associated with immune suppression, metabolic dysregulation, and increased cancer risk [33]. For instance, phthalate-induced changes in m5C profiles in non-coding RNAs are linked to abnormal fetal development and adult-onset chronic diseases [38, 39]. Organophosphate pesticides such as chlorpyrifos and diazinon have been found to downregulate NSUN2, a key methyltransferase for m5C, especially in tRNAs and mRNAs [40]. This suppression leads to hypomethylation in detoxification-related genes, impairing translation fidelity and contributing to mitochondrial dysfunction, a central mechanism in neurodegenerative disorders. Organophosphates also induce A-to-I RNA editing errors through the inhibition of ADAR enzymes, which further destabilizes neuronal transcriptomes and synaptic plasticity [41]. Furthermore, the role of m5C in non-coding RNA regulation must be emphasized. Beyond mRNA, m5C is critical in small nuclear RNAs (snRNAs) and long non-coding RNAs (lncRNAs) [42]. Disruption of m5C in lncRNAs such as MALAT1 has been linked to altered chromatin structure and epigenetic regulation [43]. For example, phthalate exposure disrupts m5C in MALAT1 via downregulation of NSUN5, impairing its chromatin-binding function and thereby silencing tumor suppressor gene loci in urogenital tissues [44]. Moreover, m5C modifications in tRNAs affect codon–anticodon pairing, and their loss leads to ribosome stalling, increased frameshifting, and proteotoxic stress [44]. It is also essential to discuss the role of demethylases and erasers with specificity. For instance, ALKBH5 and FTO (Fat mass and obesity-associated protein) are the primary demethylases responsible for m6A removal [45]. Exposure to dioxins inhibits ALKBH5 activity through AhR (Aryl hydrocarbon receptor) pathway activation, leading to sustained m6A hypermethylation and aberrant gene expression profiles, particularly in immune cells. This contributes to T-cell dysfunction and heightened autoimmune risk [46].

Pseudouridine (Ψ) is a structural isomer of uridine that enhances RNA stability and ribosome function. Transcriptome-wide mapping approaches have identified Ψ sites in mRNAs and non-coding RNAs, broadening their recognised prevalence beyond tRNAs and rRNAs [47]. The Ψ alteration enhances RNA-RNA and RNA-protein interactions, hence affecting RNA stability and functionality [48]. It is associated with mRNA stability and translation efficacy, and Ψ-modified RNAs exhibit diminished innate immune responses, which is advantageous for mRNA vaccine development [48]. The pseudouridylation process, catalyzed by pseudouridine synthases (PUS), is not limited to structural RNAs like rRNAs and tRNAs but also extends to mRNAs, where it modulates splicing and translation. Tobacco-specific nitrosamines such as NNK (4-(methylnitrosamino)−1-(3-pyridyl)−1-butanone) reduce PUS1 expression and inhibit Ψ formation, destabilizing ribosomal RNAs and leading to inefficient translation [49, 50]. Experimental knockdown models confirm that this disruption contributes to defective ribosome biogenesis and proteostasis, key features in carcinogenesis [51]. Similarly, cadmium exposure significantly alters the activity of DKC1, a pseudouridine synthase that modifies both rRNA and snRNA. Inhibition of DKC1 compromises pre-rRNA processing and spliceosomal assembly, which is associated with ribosomopathies and myelodysplastic syndromes [52]. Thus, pseudouridine loss has cascading effects on both the transcriptome and proteome integrity [53]. Computational methods have been devised to effectively locate Ψ locations, augmenting experimental methodologies [54]. The identification of Ψ in mRNAs indicates a possible novel mechanism for proteome diversification and connects RNA pseudouridylation with cellular stress responses [47]. These findings underscore the increasing significance of Ψ modification in RNA biology and its prospective medicinal applications.

N1-Methyladenosine (m1A) is a common, reversible post-transcriptional alteration present in multiple RNA types, such as tRNA, rRNA, mRNA, and lncRNA [55]. (The alteration is dynamically controlled by “writers,” “erasers,” and “readers,” which are essential in RNA processing, structure, and function [56]. m1A has been demonstrated to influence gene expression regulation, cellular invasion, proliferation, and cell cycle regulation in cancer [56]. Research has concentrated on elucidating the role of m1A in cancer formation and progression, with prospective applications in diagnosis, therapy, and prognosis [56, 57]. Moreover, m1A levels in urine have been linked to illness development, indicating their potential as a biomarker for early detection and monitoring of numerous diseases, especially malignancies [58]. Exposure to bisphenol A (BPA) and other endocrine disruptors has been linked to altered m1A patterns, potentially affecting metabolic pathways [59]. Mechanistically, N1-methyladenosine (m1A) is regulated by TRMT6/TRMT61A complexes in mRNAs and by TRMT10C in mitochondrial tRNAs. Environmental endocrine disruptors such as Bisphenol A (BPA) directly impair TRMT6 expression, reducing m1A levels [58, 60]. This affects mitochondrial oxidative phosphorylation by disrupting mitochondrial tRNA folding and translation initiation [60]. This process results in ATP depletion and reactive oxygen species (ROS) accumulation, fueling inflammation and insulin resistance pivotal in the pathophysiology of obesity and type 2 diabetes. Moreover, m1A loss leads to reduced translation of anti-inflammatory mediators such as IL-10, which may contribute to the progression of chronic inflammatory diseases following environmental exposures [61]. Demethylases like ALKBH1 are also susceptible to toxicant inhibition, with exposure to mercury compounds shown to reduce their nuclear localization, leading to the buildup of aberrant m1A-modified RNAs [61].

Recent studies on 2’-O-methylation (Nm) in RNA underscore its ubiquity across diverse RNA types and its influence on physiological mechanisms. Writers such as fibrillarin catalyzes Nm modification. Nm changes impact RNA architecture, stability, and interactions, hence influencing translation, splicing, and the regulation of immunological responses [62, 63]. High-throughput detection techniques have progressed in NM research; nonetheless, hurdles persist in validating internal mRNA locations [62]. Nm has been associated with various diseases, including cancer, cardiovascular ailments, and neurological conditions [63, 64]. Research indicates that Nm can enhance the prevalence and duration of alternate RNA conformations, potentially modifying biological functions [65]. The therapeutic potential of Nm in RNA medicine is under investigation, with advancements in detection techniques like AI-based prediction and nanopore sequencing [64]. Viral infections and heavy metal exposure may disrupt Nm patterns, leading to aberrant immune signaling [66]. The 2′-O-methylation (Nm) process, primarily carried out by fibrillarin (FBL), is sensitive to heavy metals and air pollutants [63, 67]. Arsenic exposure, for example, reduces FBL expression, impairing Nm modification in rRNA and compromising translational fidelity. Nm loss has been associated with ribosome stalling at structured mRNA regions, triggering unfolded protein responses (UPRs) and subsequent apoptosis in lung epithelial cells [68]. Interestingly, oxidative stress generated by PM2.5 stimulates the nuclear translocation of small nucleolar RNAs (snoRNAs), which act as guides for Nm modifications. However, over-activation of this pathway can lead to aberrant Nm methylation in regions not normally modified, generating cryptic transcripts and oncogenic isoforms [69]. This suggests a dual-edged role of Nm under environmental stress, potentially contributing to adaptive but also pathological translational reprogramming.

Collectively, these disruptions show how environmental toxicants can reprogram the RNA modification machinery, compromising RNA function and contributing to disease susceptibility. These modifications are not passive targets but dynamic regulators of cellular responses to environmental cues. Their alteration precedes and predicts many of the downstream pathologies observed in environmental diseases. Understanding these interactions not only provides mechanistic insight into how toxins exert their effects but also opens up new possibilities for early detection of exposure, risk assessment, and targeted therapeutic interventions. As research advances, these epitranscriptomic marks may serve as sensitive biomarkers for environmental toxicity, offering a real-time window into how external insults translate into molecular dysfunction. Furthermore, the reversibility of many of these modifications offers therapeutic potential for modulating RNA responses and restoring cellular health in toxin-exposed individuals.

While our primary focus has been on five well-characterized RNA modifications, it is important to recognize that the RNA epitranscriptome is far more diverse. Several lesser-known modifications are increasingly gaining attention for their roles in cellular stress responses and disease progression. Among these, etheno (ε) derivatives such as 1,N6-ethenoadenosine (εA) and 3,N4-ethenocytidine (εC) are noteworthy [70]. These modifications are formed through interactions with reactive aldehydes generated during lipid peroxidation or through cytochrome P450-mediated metabolism of environmental toxins like vinyl chloride and acrolein [71]. Their formation has been associated with miscoding events that may contribute to genomic instability, cancer initiation, and neurodegeneration.

These etheno adducts are particularly relevant in the context of environmental exposure to pollutants found in cigarette smoke, industrial solvents, and combustion by products [72]. Their presence in RNA can disrupt normal translation and splicing processes, leading to altered gene expression patterns. Because they result from oxidative stress and persistent damage, εA and εC are increasingly being explored as biomarkers of toxic insult and early indicators of disease [72]. Including these modifications in environmental health research adds a valuable dimension to our understanding of how toxins affect RNA integrity beyond conventional epigenetic changes.

Another emerging class involves waiocine-type base analogues, which, although less well-studied, are hypothesized to arise under enzymatic stress conditions or toxic exposures [73]. Preliminary findings suggest they may be involved in adaptive cellular responses or pathological signaling in chronic exposure scenarios [73]. Advances in detection technologies such as mass spectrometry and nanopore sequencing are beginning to shed light on these rare modifications, opening new avenues for research. Expanding our view to include these non-canonical RNA modifications is essential for developing a more comprehensive understanding of RNA-mediated toxic responses and identifying novel biomarkers for environmental exposure and disease risk.

In addition to these rare modifications, some abundant RNA base modifications like methylguanosine (m7G) and 5-methylcytidine (m5C) have also been associated with pathological states [74]. These modifications occur naturally in transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and messenger RNAs (mRNAs), where they influence RNA stability, splicing, and translation. However, under abnormal physiological conditions or environmental stress, their levels and distribution may become dysregulated. For instance, elevated m5C levels have been linked to tumor progression and chemoresistance in various cancers, while aberrant m7G methylation patterns have been observed in neurological disorders and inflammatory diseases [75]. Their widespread presence and measurable alterations under stress make them promising epitranscriptomic markers of disease and exposure, further supporting the need to explore both rare and common RNA modifications in environmental health research.

This is one of the most commonly used techniques for detecting RNA modifications, particularly m6A [78, 79]. In this method, antibodies that specifically recognize modified bases (such as m6A) are used to immunoprecipitate the methylated RNAs, which are then subjected to high-throughput sequencing. This generates a transcriptome-wide map of RNA regions enriched in m6A [22, 80]. Techniques like MeRIP-Seq and m6A-Seq are widely applied in studies exploring environmental toxin exposure, as they allow for broad-scale identification of methylation patterns in response to stress [81]. However, one limitation is their resolution these methods typically pinpoint modified regions within ~ 100 nucleotides, but not exact sites. The technique is dependent on antibody specificity, which may affect sensitivity and reproducibility [81]. Nonetheless, due to its robustness and scalability, MeRIP-Seq has been extensively used to reveal changes in m6A distribution in diseases such as cancer and neurodegeneration caused by exposure to toxins like arsenic, PM2.5, or dioxins [82, 83]. The method is also compatible with both coding and non-coding RNA and can be used to assess dynamic changes over time. Its widespread use continues to support the discovery of toxin-responsive epitranscriptomic alterations and disease-linked m6A signatures.

Liquid Chromatography–Tandem: Mass Spectrometry (LC-MS/MS) is a powerful tool for the detection and quantification of multiple RNA modifications at the nucleoside level, including m6A, m5C, Ψ, and 1-methyladenosine (m1A) [84]. It works by enzymatically digesting RNA into its constituent nucleosides, which are then separated based on their mass-to-charge ratio [84]. This allows for the precise identification and quantification of modifications, even at low abundance. LC-MS/MS is especially valuable in environmental toxicology because it provides absolute quantification and can reveal global shifts in RNA modification levels in response to toxin exposure [85, 86]. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) is another mass spectrometry method offering high-throughput screening of RNA modifications [87]. These techniques are highly accurate and sensitive but do not provide sequence context; they cannot identify the exact nucleotide or transcript bearing the modification. Moreover, they require sophisticated equipment and trained personnel [88, 89]. Despite these challenges, MS-based tools are crucial for validating high-throughput sequencing results and detecting subtle changes in modification levels due to exposure to environmental pollutants like cadmium, bisphenol A, or lead [90]. The ability to quantify changes in RNA epigenetic marks makes MS indispensable in biomarker development and toxicological risk assessment.

This class of detection methods relies on selective chemical treatment of RNA to induce specific changes in modified bases, which can then be detected through reverse transcription and sequencing [91]. For example, bisulfite sequencing is widely used to detect m5C, as bisulfite converts unmethylated cytosine to uracil while leaving m5C unchanged. This enables base-resolution mapping of m5C sites [92]. Similarly, CMCT (N-cyclohexyl-N’-(2-morpholinoethyl)carbodiimide) treatment is employed for the detection of pseudouridine (Ψ) [93]. It modifies Ψ residues, resulting in characteristic reverse transcription stops that can be read out by sequencing. These chemical-based techniques are useful for modification-specific studies and offer high positional accuracy. However, they can be labor-intensive, require careful optimization, and are not amenable to all modification types [94]. In addition, chemical treatments may introduce artifacts or yield incomplete reactions, potentially affecting reliability. These methods have been effectively applied in studies where exposure to pollutants such as aflatoxins, phthalates, or mercury disrupts methyltransferase activity, leading to hypomethylation or pseudouridine loss [94]. Because these modifications affect RNA stability and translation, accurate detection is essential for linking environmental exposures to downstream disease outcomes. Therefore, chemical conversion techniques remain a cornerstone in mechanistic epitranscriptomic research.

Nanopore sequencing has emerged as a revolutionary tool in the field of epitranscriptomics by enabling direct RNA sequencing without the need for reverse transcription or amplification [95]. This technique works by passing native RNA molecules through a biological nanopore, where the electrical current changes based on the sequence and chemical modifications of the RNA. RNA modifications such as m6A, m5C, and Ψ alter the ionic current in distinct patterns, allowing them to be inferred computationally [96, 97]. The key advantage of nanopore sequencing lies in its ability to detect modifications in real time and at the single-molecule level. It can also sequence full-length RNA transcripts, preserving context and structural information. Despite its novelty, challenges include relatively lower base-calling accuracy compared to other sequencing platforms and the need for machine learning algorithms to improve modification detection [98]. This technology is increasingly applied in environmental epigenomics, where changes in RNA modifications can be detected in cells or tissues exposed to toxins like PM2.5, BPA, or heavy metals [98]. The portability, real-time capability, and single-molecule resolution make nanopore sequencing an invaluable asset in field-based environmental monitoring, as well as in clinical diagnostics [99].

Emerging genome-editing technologies such as CRISPR-Cas13 systems are being re-engineered to detect and modify RNA modifications with unprecedented precision [100]. Specifically, dCas13 (deactivated Cas13) fused with either methyltransferases or demethylases enables targeted editing or detection of RNA epitranscriptomic marks like m6A and m5C [101]. These CRISPR-based biosensors can be designed to recognize specific RNA sequences and their modified forms, allowing real-time monitoring of environmental toxin-induced epitranscriptomic changes in living cells. One major advantage of this system is its programmable nature, which enables researchers to direct the system to virtually any transcript of interest. It also allows for dynamic tracking and manipulation of RNA modifications without altering the underlying genome [102, 103]. Applications of this technique include detection of environmental pollutants such as pesticides, heavy metals, and endocrine-disrupting chemicals that modulate methylation enzyme activity [102]. Although still in its early stages, CRISPR biosensor platforms show promise for use in point-of-care diagnostics and field-deployable biosurveillance tools. However, challenges remain, including potential off-target effects, delivery limitations, and the need for enhanced specificity [104]. Nonetheless, the adaptability of CRISPR-Cas systems offers a powerful approach for environmental toxicogenomics, mechanistic studies, and even the development of epitranscriptomic-based therapeutics.

Ribose-seq is a specialized chemical sequencing method designed to detect 2′-O-methylation (Nm), a key RNA modification that influences translation, splicing, and immunological responses [62]. This technique capitalizes on the resistance of Nm-modified nucleotides to alkaline hydrolysis or specific nucleases, enabling the mapping of Nm sites across the transcriptome [62, 105]. After hydrolysis, unmodified ribonucleotides are degraded, while Nm-modified residues remain intact and are sequenced to determine their exact positions. Ribose-seq, often used in combination with other tools like LC-MS/MS or nanopore sequencing, provides both positional and quantitative data [15]. Because Nm is often found in rRNAs, tRNAs, and snRNAs, its dysregulation by environmental toxins like arsenic, mercury, and PM2.5 has major implications for ribosome biogenesis, translational fidelity, and immune signaling. Importantly, under environmental stress, Nm modifications may become aberrantly distributed, leading to ribosome stalling, unfolded protein responses, and even oncogenic isoform formation. Ribose-seq and related assays such as Nm-Seq and RTL-P (Reverse Transcription at Low deoxy-ribonucleotide concentrations) are essential tools for understanding how such disruptions contribute to disease [106]. Despite technical challenges in validating internal Nm sites, these tools are advancing the field by uncovering nuanced RNA-level changes linked to environmental exposure.

Pseudouridine (Ψ) is the most abundant RNA isomerization modification and plays a pivotal role in RNA stability and function [107]. Techniques such as Pseudo-seq and Ψ-seq are developed to identify Ψ sites with high resolution across the transcriptome. These methods utilize chemical probes like CMCT (N-cyclohexyl-N’-(2-morpholinoethyl)carbodiimide) that selectively bind to Ψ residues [108]. Following treatment, the reverse transcription step results in characteristic termination or misincorporation events at Ψ sites, which are then detected through sequencing. These techniques are instrumental in identifying Ψ not only in rRNA and tRNA but also in mRNA and non-coding RNAs, revealing a broader regulatory landscape. Environmental toxins such as cadmium, NNK (a tobacco-specific nitrosamine), and heavy metals have been shown to suppress pseudouridine synthases (e.g., PUS1, DKC1), leading to global reductions in Ψ levels [109, 110]. These reductions impair ribosome function, spliceosome assembly, and protein synthesis, contributing to diseases such as cancer, neurodegeneration, and myelodysplastic syndromes. Pseudo-seq offers single-nucleotide precision and is particularly useful in evaluating toxin-specific transcriptomic disruption. Newer variations are incorporating high-throughput and machine learning approaches for more efficient data processing. As knowledge grows, Ψ mapping technologies will remain critical in evaluating how environmental exposures reshape the epitranscriptomic landscape.

One of the most significant advantages of epitranscriptomic biomarkers is their capacity for early detection of disease or cellular dysfunction. RNA modifications, such as m6A, Ψ, and m5C, are dynamic and sensitive to environmental changes [53]. They respond rapidly to various stressors, including environmental toxins, oxidative stress, and chemical exposures, even before phenotypic or physiological symptoms manifest [53]. This sensitivity enables researchers and clinicians to identify molecular alterations at the earliest stages of disease progression, well before traditional biomarkers such as protein levels or histopathological changes can be observed.

Furthermore, epitranscriptomic changes often occur in a reversible and tightly regulated manner, allowing them to serve as real-time indicators of ongoing cellular processes [157]. Their presence can offer a window into disrupted gene expression patterns, aberrant cellular signaling pathways, and stress responses that precede irreversible damage or clinical symptoms. As a result, using RNA modifications as biomarkers has the potential to significantly improve diagnostic timelines, allowing for earlier therapeutic intervention and better patient outcomes. In contrast, conventional biomarkers frequently detect disease at later stages, when cellular damage may be more extensive and harder to reverse, limiting the effectiveness of treatment [158].

One of the key advantages of epitranscriptomic biomarkers is their compatibility with non-invasive sampling methods [159]. Unlike traditional tissue biopsies, which are often painful, risky, and require clinical settings, epitranscriptomic biomarkers such as chemically modified RNAs can be detected in easily accessible biofluids including blood, urine, and saliva [160]. This allows for routine and repeatable sampling, facilitating early diagnosis, disease monitoring, and treatment evaluation without causing discomfort to the patient [160]. For instance, extracellular vesicles and circulating RNAs in plasma or serum often carry distinct RNA modifications that reflect physiological or pathological conditions in real-time [161]. Moreover, these biofluids are relatively easy to obtain and store, making them ideal for longitudinal studies and large-scale screenings. The ability to track disease-associated RNA modification patterns through minimally invasive means represents a significant advancement in personalized medicine and public health, particularly for monitoring exposure to environmental toxins and early-stage disease processes [162].

High specificity and sensitivity

One of the most compelling advantages of epitranscriptomic biomarkers is their exceptional specificity and sensitivity in detecting environmental exposures. Certain RNA modifications such as m6A, m5C, and Ψ are selectively altered in response to specific environmental toxins like heavy metals, air pollutants, pesticides, and endocrine-disrupting chemicals [163]. These modifications act as unique molecular signatures, reflecting both the type and intensity of toxic exposure. Because these changes occur at the nucleotide level and often precede phenotypic alterations or disease onset, they offer a highly sensitive means of detecting early biological responses to toxic insults [164]. Additionally, the ability to link specific RNA modifications with particular chemicals or classes of toxins allows researchers and clinicians to trace the origin of exposure with great precision. This makes epitranscriptomic profiling a valuable tool not only for early detection but also for environmental risk assessment and personalized health monitoring [165].

One of the key advantages of epitranscriptomic biomarkers lies in the remarkable technological advancements in nanopore sequencing and mass spectrometry, which now enable real-time monitoring of RNA modification patterns [166]. Unlike traditional RNA sequencing methods that often overlook post-transcriptional modifications, nanopore sequencing can directly detect native RNA molecules without the need for reverse transcription or amplification [167]. This allows researchers to identify and quantify RNA modifications such as m6A, m5C, and Ψ in real time, across the entire transcriptome. Similarly, mass spectrometry-based approaches provide high-resolution, quantitative insights into the chemical structure of RNA modifications, offering precise identification and localization of modified nucleosides [168]. These methods have become increasingly sensitive and rapid, allowing dynamic assessment of how RNA modifications change in response to environmental stressors, toxins, or disease states. Together, these technologies empower scientists to capture transient and context-specific RNA modification events that are crucial for understanding regulatory mechanisms and disease pathogenesis. This real-time capability makes epitranscriptomic biomarkers highly promising for early diagnosis, monitoring of treatment efficacy, and personalized medicine [146].

Environmental Pollutants and Toxicology: Heavy metals, air pollutants, and endocrine disruptors exposure trigger epitranscriptomic changes in living organisms [183]. Monitoring these modifications in bioindicator species provides insights into environmental health. Microbial Community Analysis: Environmentally rich organisms with epitranscriptomic profiling of their microbiomes show adaptive trends that help scientists study both ecosystem modification research and pollution remediation efforts [184]. Agricultural and Food Safety: RNA modifications found in plants together with agricultural pathogens affect both crop resistance capabilities and pathogen resistance mechanisms [185]. Food quality assessment and health risks detection depends on detection techniques.